E3^^^^^^^^^^^^^SI Marine Biological Laboratory Library Woods Hole, Mass. m I I I I I I I I i Presented by The American Association of University Professors August 1965 53 Plant Growth Regulation 1 ■■■ * - --A i - 1^ G^ Plant Growth Regulation Fourth International Conference on Plant Groivth Regulation sponsored by The Boyce Thompson Institute for Plant Research The New York Botanical Garden The Brooklyn Botanic Garden The low^a State University Press , .^^wies, Iowa, U.S.A. © 1961 by The Iowa State University Press. All rights reserved. Comiwsed and printed by The Iowa State University Press, Ames, Iowa, U.S.A. Reprinted 1963 Library of Congress Catalogue Card Xunihcr: 60-16603 COMMITTEES OF THE CONFERENCE Organizatioti Committee G. L. McNew, Boyce Thompson Insii- tute for Plant Research, Chairman J. M. Arthur, Boyce Thompson Institute for Plant Research G. S. Avery, Jr., Brooklyn Botanic Gar- den A. E. Hitchcock, Boyce Thompson Insti- tute for Plant Research R. M. Klein, Tlie Nezi' York Botanical Garden L. P. Miller, Boyce Thompson Institute for Plant Research W. J. Robbins, The New York Botanical Garden W. C. Steere, The New York Botanical Garden VV. R. Tulecke, Chas. Pfizer and Com- pany A. J. Vlitos, Department of Research. Caroni Ltd., Carapichaima, Trinidad, W.I. Program Committee for Scientific Sessions A. J. Vlitos, Department of Research, Caroni Ltd., Carapichaima, Trinidad, W.I., Chairman B. Aberg, Royal Agricultural College, Uppsala, Sweden VV. A. Andreae, Science Service Labor- atory, Lotidon, Ontario, Canada G. E. Blackman, Department of Agricul- ture, University of Oxford, Oxford, England J. Bonner, Division of Biology, Califor- nia Institute of Technology, Pasadena, California E. Bunning, Botanisches Inst i tut, Tii- bingen, Germany A. W. Galston, Department of Botany, Yale University, New Haven, Connec- ticut F. Skocg, Department of Botany, Uni- versity of Wisconsin, Madison, Wis- consin K. V. Thimann, Biological Laboratories, Han'ard University, Cambridge, Mas- sachusetts R. L. Wain, Wye College, Wye, Kent, England Housing and Transportation Committee P. H. Plaisted, Boyce Thompson Insti- tute for Plant Research L. H. Weinstein, Boyce Tliompson Insti- tute for Plant Research Luncheon and Dinner Committee L. P. jMiller, Boyce Thompson Institute for Plant Research, Chairman Clyde Chandler, Boyce Thompson In- stitute for Plant Research Florence Flemion, Boyce Thompson In- stitute for Plant Research S. E. A. McCallan, Boyce Thompson In- stitute for Plant Research Editorial Committee R. M. Klein, Neiu York Botanical Gar- den, Chairman G. S. Avery, Jr., Brooklyn Botanic Gar- den A. E. Hitchcock, Boyce Thompson Insti- tute for Plant Research L. P. Miller, Boyce Thompson Institute for Plant Research H. W. Rickett, New York Botanical Gar- den \V. R. Tulecke, Chas. Pfizer and Co. Dedication of Volume to Percy W. Zimmerman Early in its deliberations, the Organization Committee decided that Percy \V. Zimmerman would be designated as honorary chainnan of the Fourth International Conference on Plant Growth Rearulation. Fate intervened, and this opportunity to provide a small token of honor and appreciation for a lifetime of faithful senice to the study of plant growth was denied. He succumbed to an embolism following an emergency operation at Wenatchee, Washington, on August 14, 1958. The dinner meeting which he was to have addressed during the Confer- ence was changed to a memorial meeting in his honor, and Dr. ^V'. J. Robbins was asked to give the address. From Dr. Zimmerman's laboratory, in collaboration with Dr. A. E. Hitchcock, there came an ever-expanding series of discoveries over a period of a quarter of a century. They found the means of using indole- 3-butyric acid to root cuttings of species difficult to propagate and dis- covered the growth regidating properties of 2,4-dichlorophenoxyacetic acid, a-naphtlraleneacetic acid, and the halogenated benzoic acids. These and many other similar findings were not fortuitous accidents. They came from prolonged and diligent search that was guided by certain lines of provocative thinking. To know "Zim" was to respect him even when you differed with his ideas. He was unflinching in his examination of the validity of every new idea and every piece of data. He insisted on testing new concepts extensively on many kinds of plants under all sorts of conditions before accepting an observation as factual. He was just as relentless in question- ing the ideas of his colleagues in research and administration as he was in scrutinizing his own progress. His blunt questioning of all theories that were superficially drafted, or that left unanswered questions, did not necessarily endear him to his colleagues. He believed in the use of theory in the research lal)oi- atory but he was distressed that tenuous theories should be published as semi-facts where they might lead neophytes into the wrong channels of learning. The pages of history show that his skeptical attitude has been vindicated as the auxin a and auxin b concept has been laid to rest, the concept of the ubicjuitous role of "auxin" in all plant functions has been drastically changed, and the superficial theories on the relation of chemical structure to growth regulant aliility of molecules ha\e been disintegrated by wider knowledge. Such was the unwavering honesty and loyalty of the man to his dedicated purposes in life. He spent 32 years in the laboratories of the lioyce Th(jnipson Institute, breathing life into its ideal of acquiring use- ful new knowledge on plant life. It was a richer place for having had liini as an associate. The genial smile, the vvarni interest in everything and every person around him, and the ever youthful aj^proach and un- Percy W. Zimmerman wavering interest in all the problems of life gave him an enduring place in the hearts of his colleagues even as they recognized his oc- casional very human frailties. Dr. Zimmerman was born at Manito, Illinois, on February 23, 1884. He was trained at the University of Chicago from \vhich he received the B.S. degree in 1915, M.S. in 1916, and Ph.D. in 1925. His professional career included a period as public school teacher and Superintendent of Schools at Westville, Illinois (1910-13), Associate Professor of Botany and Dean of the College of Agriculture at the University of Maryland (1916-25), and Plant Physiologist at the Boyce Thompson Institute from 1925 until his death in 1958. This volume is afjeciionately dedicated to him as one of the pioneers luho opened the door on this exciting neiu area of plant re- search. Without the foresight, persistent effort, and dedication to re- search of men such as he, there would have been no assemblage in this conference of representatives from so many lands. No more fitting memorial could be offered to his memory than this volume loherein are assembled the best ideas of the distinguished leaders of today. George L. McNew Preface The Fourth International Conference on Plant Growth Regulation was held at the Boyce Thompson Institute for Plant Research in Yonkers, New York, from August 10 through August 14, 1959. The Conference was sponsored jointly by the Boyce Thompson Institute, The New York Botanical Garden, and the Brooklyn Botanic Garden through an Organization Committee headed by Dr. George L. McNew, Managing Director of the Boyce Thompson Institute. One hundred and forty scientists from eighteen countries were invited to participate through the presentation of formal papers, in organized discussion groups, or via informal question and answer periods. This volume reports the proceedings of the Conference. The program was arranged by a committee headed by Dr. A. J. Vlitos, who first suggested the Conference. Housing, transportation, and the memorable luncheons and dinners were handled by other committees. An international conference of this size is an expensive undertak- ing requiring funds to assist participants who came from abroad, to cover the costs of food, transportation, and the many other obliga- tions which had to be met. Our desire to hold the cost of this book to a reasonable figure meant it had to be subsidized. Financial sup- port was generously provided by The Rockefeller Foundation, the National Science Foundation, and the following corporations. Amchem Products Co., Agricultural Chemicals Division, Ambler, Pennsylvania. American Cyanamid Co., Stamford, Connecticut. Chas. Pfizer and Co., Brooklyn, New York. Diamond Alkali Co., Cleveland, Ohio. The Dow Chemical Co., Agricultural Chemicals Laboratory, Midland, Michigan. [ix] X Preface ,"■".. E. I. du Pont de Nemours and Co., Q'rasselli .G-heraicals Department, Wilmington, Delaware. Eli Lilly and Co., Agricultural and Research E^epartment, Greenfield, Indiana. Hercules Powder Co., Naval Stores Research Division, Wilmington, Delaware. Merck and Co., Product Development,,Plaht Chemical Section, Rahway, New Jersev. Monsanto Chemical Co., Agricultural Chemicals Section, "St. Louis, Missouri. Food Machinery and Chemical Corp., Niagara Chemical. Division, Middleport, New York. Rohm and Haas Co., Philadelphia, Pennsylvania. Shell Development Co., Agricultural Research Division, Modesto, California. United Fruit Co., Boston, Massachusetts. Upjohn Co., Research Division, Kalamazoo, Michigan. Many individuals from the sponsoring institutions were necessar- ily involved in making the Conference a success. Mrs. Bettie Brooks, Executive Assistant to the Managing Director, Dr. Clyde Chandler, and Mrs. Florence Flemion of the Boyce Thompson Institute and Mrs. Eileen Kene and Miss Bernice Winkler of The New York Botan- ical Garden did a great deal of the indispensable behind-the-scenes work that resulted in a smoothly running Conference. The excellent work of our recording engineer, Mr. W^illiam R. Begany, permitted the transcription of the discussion periods. Mrs. George L. McNew and Mrs. William C. Steere arranged programs for the wives and families of the participants. The heroic efforts of Dr. McNew for ob- taining the necessary financial support, for making special arrange- ments for just about everything, and for being, withall, a charming host, warrants a rising vote of thanks. Publication of these proceedings required considerable cooperation and forbearance from the many participants. The invaluable assist- ance of Dr. S. E. A. McCallan with text and figmes is most grate- fully acknowledged. Mr. A\'illiam G. Smith, Jr., head of the Illustra- tion Division of Boyce Thompson Institute, did yeoman service in preparing the figures for the book. Special thanks are due Mrs. Bettie Brooks for reading both the galley and page proof with painstaking thoroughness. The editors are also indebted to Dr. R. E. Buchanan for careful and conscientious preparation of a meaningful, compre- hensive index. The concerted efforts of the Editorial Committee and of Mr. Marshall Townsend, Manager of the Iowa State University Press, made the (iiainnnn's task almost too easy. Richard M. Klein Chairman, Editorial Committee Table of Contents INTRODUCTION George L. McNew The Broader Concepts of Plant Growth Regu- lation 3 William J. Robbins The Expanding Concepts of Plant Growth Regulation 13 NATURALLY OCCURRING PLANT GROWTH SUBSTANCES Joyce A. Bentley Some Investigations on Interconvertible Naturally Occurring Auxins 25 Poiil Larsen The Occurrence of Indole-3-acetaldehyde in Torbj^rn Aasheim Certain Plant Extracts 43 Donald G. Crosby New Auxins From 'Maryland Mammoth' To- A. J. Vlitos bacco 57 C. H. Fawcett Chromatographic Investigations on the Meta- R. L. Wain holism of Certain Indole Acids and Their F. Wightman Amides, Nitriles, and Methyl Esters in Wheat and Pea Tissues 71 P. F. Wareirig Growth Substance and Inhibitor Changes in T. A. Villiers Buds and Seeds in Response to Chilling .... 95 L. J. Audus On the Adaptation of Pea Roots to Auxins /. K. Bakhsh and Auxin Homologues 109 H. W. B. Barlow Some Biological Characteristics of an Inhibi- C. R. Hancock tor Extracted From Woody Shoots 127 H. J. Lacey THE MECHANISMS OF AUXIN ACTIVATION AND INACTIVATION P. L. Goldacre The Indole-3-acetic Acid Oxidase-Peroxidase of Peas 143 E. R. Waygood Inhibition and Retardation of the Enzymati- G. A. Maclachlan cally Catalyzed Oxidation of Indole-3-acetic Acid 149 [ xi ] xii Table of Contents P. E. Pilet Auxins and the Process of Aging in Root Cells 167 A. A. Bitancourt Pathways of Decomposition (Catabolic Lat- Alexandra P. Nogueira tice) of Indole Derivatives 181 Kaethe Schwarz Peter M. Ray The Interpretation of Rates of Indole-3-acetic Acid Oxidation 199 R. L. Hinman A Model Chemical System for the Study of P. Frost the Oxidation of Indole-3-acetic Acid by Per- oxidase 205 THE SYNTHETIC GROWTH REGULANTS Borje Aberg Some New Aspects of the Growth Regulating Effects of Phenoxy Compounds 219 G. E. Blackman A New Physiological Approach to the Selec- tive Action of 2,4-Dichlorophenoxyacetic Acid 233 Robert M. Muir Chemical Structure and Growth-Activity of Corwin Hansch Substituted Benzoic Acids 249 F. G. Teubner Relationship of Molecular Structure to Bio- S. H. Wittwer logical Activity in the A'^-Arylphthalamic Acids 259 Jane Y. Shen Michael K. Bach The Uptake and Fate of C^^-labeled 2,4-Di- /. Fellig chlorophenoxyacetic Acid in Bean Stem Sec- tions 273 V. H. Freed Some Physical-Chemical Aspects of Synthetic F. J. Reithel Auxins With Respect to Their Mode of /.. /•". Remmert Action 289 James Bonner On the Mechanics of Auxin-induced Growth 307 Daf^hne J. Osborne The Role of Auxins in the Control of Leaf Mary Hallaioay Senescence. Some Effects of Local Applica- tions of 2,4-Dichlorophenoxyacetic Acid on Carbon and Nitrogen Metabolism 329 5. M. Siegel Oxidants, Antioxidants, and Growth Regu- F. Porto lation 341 A. W. Galston The Intracellular Locale of Auxin Action: Ravindar Kaur An Effect of Auxin on the Physical State of Cytoplasmic Proteins 355 Kenneth V. Thimnnn Interrelationships Between Metallic Ions and Noriko Takaliashi Auxin Action, and the Growth Promoting Action of Chelating Agents 363 Peter M. Ray Problems in the Biophysics of Cell Growth . . 381 B. Kessler The Effects of Decapitation and (irowtli Z. ir. Moscicki Regulators on the Movement of Calcium in R. Bah Apricot Trees 387 William P. Jacobs The Polar Movement of Auxin in the Shoots of Higher Plants: Its Occurrence and Physio- logical Significance 397 A. C. Leopold S. L. Lam Bruce B. Stoioe Corivin Hansch Robert M. Muir J. xian Overheek B. B. Stoioe F. H. Stodola T. Hay as hi P. W. Brian Charles A. West Yusuke Sumiki Akira Kawarada Bernard O. Phinney Yusuke Sumiki Akira Kawarada M. J. Bukovac S. H. Wittwer C. Sironval M. Kh. Chailakhian James A. Lockhart H. R. Cams F. T. Addicotl K. C. Baker R. K. Wilson Roy M. Sachs Anton Lans: Takeshi Hay as hi William S. Hillman William K. Pitrves Jiro Kato A. W. Gals ton D. C. McCune Table of Contents xiii Polar Transport of Three Auxins 411 The Stinuilation of Auxin Action by Lipides 419 Electronic Effect of Substituents on the Ac- tivity of Phenoxyacetic Acids 431 New Theory on the Primary Mode of Auxin Action 449 - THE GIBBERELLINS The Early History of Gibberellin Research . . 465 ^ The Chemistry of Gibberellins From Flower- ing Plants 473 Occurrence of Gibberellin A^ in the Water Sprouts of Citrus 483 Dwarfing Genes in Zea mays and Their Rela- tion to the Gibberellins 489 Relation Between Chemical Structure and Physiological Activity 503 Biological Evaluation of Gibberellins Aj, A.,, Ag, and A^ and Some of Their Derivatives . . 505 Gibberellins, Cell Division, and Plant Flower- ing 521 Effect of Gibberellins and Derivatives of Nucleic Acid Metabolism on Plant Growth and Flowering 531 The Hormonal Mechanisms of Growth In- hibition by Visible Radiation 543 Acceleration and Retardation of Abscission by Gibberellic Acid 559 Shoot Histogenesis and the Subapical Meri- stem: the Action of Gibberellic Acid, Amo- 1618, and Maleic Hydrazide 567 The Effect of Gibberellin Treatment on the Photosynthetic Activity of Plants 579 Does Gibberellin Act Through an Auxin-me- diated Mechanism? 589 Physiological Action of Gibberellin With Special Reference to Auxin 601 An Analysis of Gibberellin-Auxin Interaction and Its Possible Metabolic Basis 611 xiv Table of Contents S. Housley The Influence of Gibberellic Acid on Indole- B. J. Deverall 3-acetic Acid Disappearance From Solutions Containing Pea Stem Tissues and Indole-3- acetic Acid Oxidase 627 P. W. Brian Interaction of Gibberellic Acid and Auxin H. G. Hemming in Extension Growth of Pea Stems 645 /. van Overbeek Inhibition of Gibberellin Action by Auxin . . 657 L. Dowding OTHER PLANT GROWTH REGULATORS G. Beauchesne Separation des Substances de Croissance d'Ex- trait de Mai's Immature 667 Louis G. Nickell Growth Substances and Plant Tissue Cultures 675 Walter R. Tulecke J. P. Nitsch Growth Factors in the Tomato Fruit 687 C. Nitsch Ulrich Naf On the Physiology of Antheridium Formation in Ferns 709 S. Tonzig- Ascorbic Acid As a Growth Hormone 725 E. Marre A. M. Mayer Coumarins and Their Role in Growth and A. Poljakoff-Mayber Germination 735 W. C. Hall Studies With C"-labeled Ethylene 751 C. S. Miller F. A. Herrero N. E. Tolbert (2-Chloroethyl)trimethylammonium Chloride and Related Compounds As Plant Growth Substances 779 IMPROVEMENT OF GROWTH REGULATOR FORMULATION A. S. Crafts Improvement of Growth Regulator Formu- lation 789 Donald P. Gowing Some Comments on Growth Regulators With a Potential in Agriculture 803 Leonard L. Jansen Physical-Chemical Factors of Surfactants in Relation to Their Effects on the Biological Activity of Chemicals 813 THE NEXT STEPS James Bonner The Probable Future of Auxinology 819 SUPPLEMENTARY INFORMATION Participants in the Conference 831 Index 837 Introduction GEORGE L. McNEW Boyce Thompson Institute The Broader Concepts of Plant Growth Regulation Men and plants have come a long way together down through the ages. The association has long since changed from the casual contact of a nomad with a quick meal to that of almost complete interde- pendence. One of the major goals of civilization has always been to improve the usefulness and reliability of plants in promoting human welfare. Those societies that have failed to achieve this improvement in proportion to the material needs of a growing population have crumbled and perished from the earth. THE PLASTICITY OF THE GROWING PLANT The plant scientist of the twentieth century has come to look upon the major crop plants as so many plastic materials of life that can be shaped and altered by skilled hands. Much of the altered design of plant development has been achieved by genetics — first by studying natural variants and selecting the preferred races and varie- ties, and more lately by selection of suitable building blocks for syn- thesizing new varieties with highly specialized attributes. This synthetic process of hastening or diverting the ordinary pro- cesses of evolution has come to be considered entirely inadequate. The natural processes of inherited growth regulation fail in so many respects that the geneticist has sought new tools such as gene muta- tion by irradiation or induction of polyploidy by chemicals or other means. The heritable processes of plant regulation are very desirable in that they are spontaneously self-reproducible and hence very eco- nomical to use once they are properly established. As men have gradually unravelled the mysteries surrounding nor- mal metabolism and growth processes in plants it has become clear that nearly all regulatory processes depend upon underlying chemical [3] 4 G. L. McNew activities of the cell. It has become increasingly self-evident that any- one who understands the chemical processes of the living cell has the potential power of regulating that cell's activities and its ultimate incorporation into a tissue and thence into a functional organ. The person who can control the activities of the living cell without de- stroying its life can determine the ultimate fate of the individual plant. This breath-taking concept has long since dropped from the realm of human fancy and daydreaming into the reality of agricultural practice. There is no really valid reason why the physiologists and chemists should not eventually design molecules that will duplicate, circumvent, block, or accelerate any and all the activities of the gene. The great problem is to design such a molecule so it will operate gently but specifically in the desired manner without disrupting major vital processes. Probably the greatest handicap to achieving this Utopia of chemi- cal control has been the simple difficulty of properly administering the material so its effects will be felt over a prolonged period of time. Exogenous chemicals have not yet duplicated the effect of genes be- cause they are applied crudely in massive doses that are dissipated or detoxified in very short order. What is needed is a relatively inert chemical that will generate the proper regulant over a long period of time as it is required in cell functions. The idea of a chemical that would generate a plant growth regu- lant over a sustained period is not an impossibility. Those people working with the dithiocarbamate fungicides, for example, have done this very thing. Relatively inert dithiocarbamates that can be piled on foliage in heavy doses without injury to the crop but which gener- ate highly fungitoxic isothiocyanates as they are needed are now being used to control fungous diseases of plants by the tens of millions of pounds each year. The protectants are so ephemeral they cannot readily accumulate in sufficient amount to injure plants as may occur with a more stable material such as a copper fungicide. There is sub- stantial evidence that several other organic sulfur molecules generate toxicants for fungi and nematodes in situ. There is good reason to believe that the entomologists have comparable tools in the organic phosphate insecticides and miticides that serve as systemic eradicants. THE NATURE OF PLANT REGULANTS NOW IN EXISTENCE Great achievements in plant growth regulation have come into being by the use of relatively simple chemicals that have been dis- Broader Concepts 5 covered and developed in the past quarter century. Weeds may be re- moved selectively from crops, the dormancy of buds can be prolonged or disrupted, the processes of abscission can be instigated or retarded, and the interconversion of starches and sugars can be directed one way or the other. Frequently the magnitude or even the direction of these interconversions can be regulated very specifically by the dosage applied and the maturity of the tissues at the time of application. It is of more than passing interest that a major proportion of the plant regulants discovered to date are organic acids. Many others contain strong electronegative or alcohol, ester, and ether groups that could be converted into carboxyl groups by relatively simple processes of hydrolysis or oxidation. The predominant presence of acidic moieties in the molecule raises serious questions as to the nature of their effect. One soon comes to suspect that they may be primarily involved in altering the nature of cell walls since they present won- derful possibilities for affecting the synthesis of cellulose, lignin, and pectin, the very materials that lead to restriction of cell expansion and define tissue integrity. If most of the mechanism of plant regulation by these acidic sub- stances is proven to be associated with cell wall deposition, then it becomes obvious that the science of auxinology is really in a very primitive state. Only the surface of the problem has been scratched, and the really great practical achievement must still be ahead of us. The great potential of the cell lies in the activities of the protoplast and especially in its nucleus rather than in the behavior of the cell membrane and its structural support in the wall. Therefore, it is not out of order to propose that attention must be directed to creating chemicals that will penetrate the living cell and enter into the vital processes of the natural cell-regulating sys- tem. A study of the analogues and homologues of nucleic acid com- ponents and those materials that will alter the processes of protein synthesis and activation should in due time provide fruitful leads to new plant regulants. Unfortunately this is a complicated area of cell function to attack. However, with the sweeping progress being made today in protein chemistry and the understanding of DNA and RNA synthesis it is not too much to expect that before long we will see chemicals that will generate a directed cytoplasmic structure that will regulate vital processes of metabolism on a more restricted self-sustaining basis. The closest analogy to this material known today is the plant viruses. At least one of these (breaking of tulips) has been associated with pro- duction of a desirable horticultural property. 6 G. L. McNew THE DESIGN OF A PLANT REGULATING MOLECULE Unfortunately, the golden era of the 1940's and 1950's in practical achievements of horticulture has not been matched by comparable progress in understanding the underlying principles. Too many people have developed pet theories without having first turned to the living cell to seek proper orientation of their ideas. The secret lies inside the living cell, and it will be unlocked only by careful analysis of the cell constituents on a quantitative basis or by tracing the metabolic fate of the plant giowth incitant. Fortunately, with the wonderful new tools that are coming into use for tracing minute quantities of chemicals and unravelling local changes in metabolism of a few cells, perhaps this neglected area will be investigated more fully, as it should be. There has been much written about the design requirements of a plant regulating molecule. These specifications have been modified from time to time — almost every time a new group of compounds has been introduced. Unfortunately, the theories have done very little to promote progress because they failed to analyze completely the various factors involved. Any change in chemical structure must be analyzed completely from the viewpoint of five effects on the chem- ical and physical attributes of the molecule; namely, selective solubility involved in cell permeation, translocatability, reactivity with specific cell metabolites, detoxication by extraneous reactions or physical or chemical binding to nonvital cell constituents, and type of degradation products formed during its metabolism by the cell. Very rarely have plant physiologists stopped, for example, to con- sider the effect of lengthening a carbon chain or adding a parachloro- phenyl group on lipide solubility. There are ample data on the effects of such changes in fungicides and bactericides on the partition coefficients between lipides and aqueous components of a mixed sys- tem. Undoubtedly any such change is going to affect the rate and completeness with which a molecule can penetrate cuticularized bar- riers from an aqueous spray dispersion and pass from the interstitial spaces in the cell wall through the lipoprotein barrier of the cell membrane. However, if its lipophylic-hydrophilic balance is not very carefully adjusted, it cannot pass from the lipide phase of the cell membrane into the aqueous substratum of the cytoplasm and thence to the site of enzyme activity, possibly by contact with the lipide phase of mitochondria. It is obvious that the first consideration in evalu- ation of any change in chemical structure on growth-regulant activ- ity must be upon the simple physical attribute of selective solubilities in a complex medium of various lipides and aqueous solutions. The potential capacity of the molecule to disperse through tissues Broader Concepts 7 and enter into the translocation streams becomes a major consider- ation. The very fact that so many vital processes of the living plant are concentrated in the apical meristem, the phloem parenchyma, and the root system which are relatively well protected from direct ex- posure to chemicals applied to plant surfaces is warning enough that this factor must be considered. There is very substantial evidence that the effective plant regulants do move readily in plant tissues and very often in the general direction of food translocation. To this extent they resemble the viruses that move strongly toward and into the roots of perennials in late summer or fall and upward into the growing shoots and expanding buds in the spring. The gen- eral rule for viruses, such as in the yellows disease of beets, is that they move from the areas of ample food reserves to deficient areas where assimilation into tissues is proceeding most rapidly. It has been shown that translocation of foreign bodies into and through the phloem can be accelerated along with sugar by addition of boron. The changes in chemical reactivity attendant to changes in chemi- cal structure have never been properly assayed because it is not clear what vital processes are changed by growth regulants. The materials are reactive and hence undoubtedly affect many enzyme systems. The great problem in perfecting better herbicides, growth stimulants, and retardants is to find methods of accentuating specific reactivities with- out promoting the indiscriminate reactions that are meaningless to cell regulation but exhaust and detoxify the regulant chemical be- fore it can reach the proper site of activity. Probably no area of research in the entire pesticide and plant reg- ulant field is more deficient than this one of the proper site of action for a molecule. There is no easy road to its solution because of the terrifically dynamic processes of the functioning and growing cell. However, the experimental approaches must take one of three direc- tions. The physiologist must do everything possible to measure the quantitative changes in cell activity in various tissues of the plant and to determine the change in biochemical activities to see where metabolic processes are accentuated or blocked. This will lead to many blind or false alleys, but eventually a pathway should be un- covered that has real fundamental significance. The second approach is to label or otherwise find the means of tracing the molecule of in- terest as it reacts with cell components or is metabolized into degrad- ation products. Already great progress is being made in this direc- tion. It is somewhat like looking for a needle in a haystack to follow the course of a few millions or thousands of millions of molecules through their various activities in the complex medium of the living cell but it must be done by one means or another. The third direction is to study the kinetics of the reaction of var- 8 G. L. McNew ious homologues and analogues in a class of active compounds with representative cell constituents in vitro. By defining types of reac- tions that molecules may be expected to enter into under specific con- ditions, a suggestion may be revealed as to what the investigator should be looking for in the living cell. This approach to the study of fungicides by Dr. Burchfield and Miss Storrs at Boyce Thompson Institute is revealing new analytical concepts. For example, one idea is that there probably are microenvironments within each cell which may very well determine whether the protein moiety of an enzyme may react through the amino or sulfhydryl group with an alkylating agent. It is only by prying into the living cell that the real answers to growth-regulant processes of a molecule can be determined reliably. Theories and reasoning from analogy are scarcely worth printing until the treated living cell has been examined to verify the new idea. A minor change in chemical structure in synthesizing a new member of a class of regulants modifies so many attributes simul- taneously that chances for error in reasoning are tremendous. Com- pound this error by exposure to the multitudinous environmental factors in the cell and one becomes most humble in recognition of his own ignorance. To be blunt, there is no easy road open to determining the nature of the relationship of chemical structure to activity. It is among one of the more complicated mental exercises of modern science, and more biologists should accept the situation in humility and determi- nation to obtain many divergent lines of evidence before being too positive in any one theory. THE GREAT NEED FOR BETTER PLANT REGULATION The opportunities to serve men better through science probably are greater in this field than in almost any area of scientific endeavor. VVe work to improve the material comforts of man, stabilize his so- ciety, and permit the continued growth and development of his econ- omy in a world where the physical resources for plant culture are drastically limiting. The things that can be done, and will be done, in this area are tremendous. Is there any harm to dream of what we may do in the years ahead? If we could free certain valuable crop plants from their depend- ence upon rigid photoperiod requirements, the barrier of geographi- cal latitude might be broken so many unused or poorly used areas could be sown to much needed crops. Furthermore, there are many areas where two seed crops might be grown in place of one each year Broader Concepts 9 if day length were not a dominating consideration. If we could but suppress the power of reproductive processes in plants, the useful life of forage crops and leafy vegetables could be prolonged by delaying senescence. The concentration of nutritious, palatable materials in the foliage and stems could be increased, and the yield of harvestable material could be multiplied by prolonged growth. The day men learn to regulate physiological activity of plants to match the ecological situation of that season will mark the first prac- tical great break-through in the age-old conquest of environment. There is no reason why drought resistance and winter hardiness, for example, should not be modified by chemical treatment so as to meet unusual local conditions and to expand plant culture out toward the desert, up the mountain, and over the tundra. Such regulation by chemicals should be more serviceable than the genetic modification that we now depend upon. No one has been able to predict all the fluctuations of environment or safeguard against them by breeding or selecting resistant sorts of crops. What is needed are treatments that can be applied to meet each season's development and whose intensity can be multiplied to match the fury or deficiency of the weather elements. This will never be done adequately by genetics alone but it could be done by combining the best in breeding and chemical treatment. The fight against crop pests has been grievously handicapped by modern genetics, standardized cultural practices, and repetitive inten- sive cultivation of crops in selected areas where they will thrive. The complete standardization of genetical composition and cultural con- ditions has given the parasitic fungi, bacteria, nematodes, insects, and viruses a field day at our expense. The pests have become perfectly adjusted to destroying every plant in a field each year, provided the weather does not hamper them. We cannot abandon the agronomic practices that have encouraged this situation because they are neces- sary for great efficiency in crop production. Neither can we tolerate the loss of 21 per cent of all crop productivity such as occurs in the United States now, in 1960. What is more important, we cannot afford, in a highly organized civilized society where factors of production are so delicately balanced with human need, to risk the possibility of a widespread outbreak of any major pest. For example, in the battle against black stem rust of wheat, there has been periodic development of new parasitic races that attack the prevalent disease resistant varieties. After half a cen- tury of breeding for disease resistance we are forced to conclude that we merely go from one crisis to another. In the past 50 years the 10 G. L. McNew wheat crop was reasonably protected only 20 years according to no less authority than Stakman and Harrar (Plant Pathology, Ronald Press, N. Y. 1957, page 507). The use of fungicides to meet these recurring crises comes to mind at once, but keeping all the wheat fields covered with a protective fungicide throughout the season is economically unsound. The real need is for a growth regulant that would alter the innate susceptibil- ity of a plant for 30 or even 10 days. Such material could be applied when the need for it was apparent, and only then. If the activity of a single gene will impart immunity to a specific race of rust fungus, is it too much to expect someone to find a chemical with equal resis- tance-regulation capacity? Looking toward other outlets for research in plant regulation, we become aware that the quality of plant products could be improved tremendously. For too many years the plant scientist has been overly concerned with quantity of produce rather than its quality. Most bota- nists are fully aware that by use of hybridization the average yield of maize was increased about 90 per cent in the United States within two decades. However, the yield of protein per acre held almost con- stant. The achievement was primarily in production of starch. There is no reason why chemical controls should not be available to increase nitrogen assimilation in proteinaceous crops, balancing of sucrose and other sugars with organic acids in fruits, or storage of starch or sugar in root crops. There is little need to dwell on these and other areas of potential service to agriculture. Great opportunities lie ahead if we can only develop sound concepts as to how cells function and grow. If we are to regulate plant growth in specific directions as indi- cated here, we must have a clear concept of what constitutes plant growth. In the broad sense it is the sum total of all cell activities that lead to normal expansion, differentiation, and multiplication of cells so that they may be incorporated into new, functional tissues and organs. Growth, therefore, starts in the processes of cell division, progresses with cell enlargement, and culminates in cell differentia- tion. The course and rapidity of any one of these stages of growth will be determined by the balance existing between cell constituents. These balances are both chemical and physical; for example, hydro- static pressure vs. strength of cell wall, pectic substances vs. lignin deposition, food reserve vs. water supply, etc. It would be unwise to believe that any one chemical that we ordinary mortals might syn- thesize will be all-powerful in determining the course of cell multi- plication, enlargment, or differentiation. The most it can do is to Broader Concepts 11 change the delicately adjusted balance that exists between thousands of components in the cell. Growth regulation, therefore, becomes a matter of modifying the balance between components of the cell. It is probably misleading to expect one massive disruptive force such as the blocking of a single enzyme system to regulate rate of flowering, abscission, stem elonga- tion, food conversion, etc. into specific desired channels. Many chemicals with many regulatory efl:ects must operate in cells to keep this balanced system functional. It is our purpose to locate these mul- titudinous natural coordinators of cell functions and gently re-enforce or retard their activities with specifically designed molecules. WILLIAM J. ROBBINS The New York Botanical Garden The Expanding Concepts of Plant Growth Regulation The Origin of Species by Means of Natural Selection or the Preserva- tion of Favoured Races in the Struggle for Life, written by Charles Darwin, was published in 1859 — one hundred years ago. The cen- tennial observances attest to the stature Darwin has attained. Charles Darwin was a botanist and, had he never written the Ori- gin of Species, would be remembered today as one of the major bo- tanical scientists of the nineteenth century. In fact, Charles Darwin's investigations of the sensitiveness of plants to light and gravity may justly be considered, in many ways, to have laid the foundations for our knowledge of plant growth regulators. His observations and conclusions were published in 1880 in a book entitled The Power of Movement in Plants (4). Chapter IX deals with the Sensitiveness of Plants to Light: Its Transmitted Effect. Chapter XI deals with Localized Sensitiveness to Gravitation and Its Transmitted Effect. In a series of ingenious experiments Darwin explored the responses to light of the coleoptiles of seedlings of Phalaris canariensis and Avena sativa, which had been decapitated or had been covered with caps of tinfoil; gold beater's skin, either transparent or painted so as to be impermeable to light; pipes of very thin glass or quills — some blackened; bandages of tinfoil applied to various parts of the coleoptile; coats of India ink and other procedures. The results of his experiments led irresistibly to the conclusion that the stimulus of light was perceived by the tip of the coleoptile and transmitted to the base where movement occurred. Darwin says, 'Trom these several sets of experiments, including those with the glass tubes, and those where the tips were cut off, we may infer that the exclusion of light from the upper part of the coty- [13] 14 W. J. Robbiyu ledons (coleoptiles) of Plialaris prevents the lower part, though fully exposed to a lateral light, from becoming curved. . . . We must, there- fore, conclude that when seedlings are freely exposed to a lateral light some influence is transmitted from the upper to the lower part, causing the latter to bend. . . . These results seem to imply the pres- ence of some matter in the upper part which is acted upon by light and which transmits its effects to the lower part." He concluded also that stimuli were perceived by the root tips and transmitted to the adjoining upper part where the bending oc- curred. To quote from his own words: "In the case of the radicles of several, probably of all seedling plants, sensitiveness to gravitation is confined to the tip, which transmits an influence to the adjoining upper part, causing it to bend toward the center of the earth. That there is transmission of this kind was proved in an interesting manner when horizontally extended radicles of the bean were exposed to the attraction of gravity for 1 or li/o hours and their tips were then am- putated. Within this time no trace of curvature was exhibited and the radicles were now placed pointing vertically downwards; but an influence had already been transmitted from the tip to the adjoining part, for it soon became bent to one side, in the same manner as would have occurred had the radicle remained horizontal and been still acted on by geotropism. . . . To see anything of the above kind in the animal kingdom, we should have to suppose that an animal whilst lying down determined to rise up in some particular direc- tion; and that after its head had been cut off, an impulse continued to travel very slowly along the nerves to the proper muscles; so that after several hours the headless animal rose up in the predetermined direction .... We believe that there is no structure in plants more wonderful, so far as its functions are concerned, than the tip of the radicle." If you have not recently read this volume on The Poxver of Move- ment in Plants, and especially the two chapters on light and gravity, I recommend them to you. You will feel as though you yourself were participating in the experiments as you note how he covered soil around the seedlings with black paper to prevent upward reflection of light, records the number of plants in many of the experiments which reacted or failed to do so, and tliscusses alternate explanations for his results. For example, he says, "When the upper halves of the cotyledons of Phalaris and Avena were enclosed in little pipes of tinfoil or blackened glass. . . . the lower and unenclosed part did not bend when exposed to lateral liglit, and it occurred to us that this fact might be due not to the exclusion of the light from the upper part, but to some necessity of the bending gradually travelling down Expandmg Concepts 15 the cotyledons, so that unless the upper part first became bent, the lower could not bend, however much it might be stimulated. It was necessary for our pmposes to ascertain whether this notion was true, and it was proved false; for the lower halves of several cotyledons be- came bowed to the light, although their upper halves were enclosed in little glass tubes (not blackened) which prevented, as far as we could judge, their bending. Nevertheless, as the part within the tube might possibly bend very little, fine rigid rods or flat splinters of thin glass were cemented with shellac to one side of the upper part of 15 cotyledons; and in six cases they were in addition tied on with threads. They were thus forced to remain quite straight. The result was that the lower halves of all became bowed to the light, but gen- erally not in so great a degree as the corresponding part of the free seedlings in the same pots, and this may be accounted for by some slight degree of injury having been caused by a considerable surface having been smeared with shellac." But before you read Darwin's account of his experiments, may 1 suggest that you visit his home, Down House, near the tiny village of Downe, some 20 miles from London where he and his sons, Francis and George, did the experiments about which he writes so engagingly. There you can see the remains of the simple greenhouses in which his plants were grown, his study, still much as it was when he used it, and the Sand Walk where he daily took his constitutional and re- viewed the experiments he and his sons had made, and planned new ones. Darwin did not express an opinion on how the stimulus of light was transmitted from the tip to the base. He was inclined to consider changes in turgescence as responsible for the movement but says, "In what manner light, gravitation, etc., act on the cells is not known." During the next 30 years investigators confirmed and extended the major observations and conclusions Darwin had made. The sensitiveness to light was defined in terms of light units, the sensitive area was more clearly limited, the effective wave lengths were studied, equipment for subjecting coleoptiles to light of known intensity and composition was devised, but the fundamental question — how was the stimulus transmitted from the tip to the coleoptile base — re- mained unanswered. Did light induce electric currents which, mov- ing downward, caused unequal growth and bending? Did light induce changes in electric potential, changes in turgor, in permeabil- ity, in sap reactions which were propagated downward? Or did it cause the formation of growth inhibitors, destroy growth acceler- ators, influence polarity, modify the movement of food, or water, or act in some ill-defined way? 16 W. J. Robbins In 1910 Boysen Jensen (1) of Copenhagen reported some simple but most illuminating experiments performed in a truly Darwinian manner which clearly indicated that phototropisin resulted from the movement of a water-soluble substance or substances from the illumi- nated tip. He made horizontal cuts about halfway through the coleop- tile tip 3 or 4 mm. from the apex. In some he inserted a thin piece of mica or platinum and then illuminated the tip; others were left with open cuts. If the cut was on the illuminated side, bending toward the light occurred. If the cut was on the shaded side, there was no response. The conclusion drawn was that something which would not penetrate the platinum, mica, or a dry cut passed from the illuminated tip down the shaded side, causing lengthening of that side and the bending toward light. In addition to these experi- ments, Boysen Jensen performed another still more critical and de- cisive. He severed the tip completely, covered the decapitated base with gelatin, and then replaced the tip. When the tip of such a plant was illuminated, the plant bent toward the light. This proved that the effect of the stimulus was transmitted over a discontinuity. It is worth noting that these experiments were performed, although Fitting (5) had reported 3 years earlier some similar experiments but with negative results, probably becatise the experimental plants were kept in too moist an atmosphere. Under such conditions the cut filled with exuded water through which the active material diffused. Paal (11, 12), Stark (22), Stark and Drechsel (23), Purdy (14), Sod- ing (21), Snow (20), Seubert (19), Boysen Jensen and Nielsen (2), and others confirmed and extended these significant observations. Although Boysen Jensen's experiments of 1910 now seem so con- vincing, not everyone was walling to accept them and the interpreta- tions placed upon them. Braimer (3) considered the process of bend- ing in response to light to involve: (1) increase in permeability and increase of growth inhibitors on the illuminated side, (2) movement of a growth inhibitor down the lighted side to the growth zones, (3) inhibition of growth on the illuminated side, (4) bending toward the lighted side. Priestley (13) said, "It may be permissible to point out what a pyra- mid of conceptions are struggling to maintain themselves upon one general experimental fact — the phototropic response of a coleoptile stump when its severed apex is replaced and alone laterally illumi- nated." Priestley then points out the frequency of the exudation of drops of water from coleoptile tips (guttation). He assumed the per- meability of the apical tissues of the coleoptile to be increased by light and, therefore, light falling on the apex to increase apical guttation. Lateral light increased guttation on the lighted side, decreased the Expanding Concepts 17 turgor, and caused in his opinion the bending toward light. The re- sults of the decapitation experiments he considered explainable on the basis that decapitation opened the veins and increased water loss. It is a little difficult to understand how so able a physiologist as Priestley could have read the results of Darwin, Boysen Jensen, and those who followed, and then could propose an explanation for the phototropism of coleoptiles which so obviously was inadequate and in error. The final and indisputable proof of the existence of potent growth regulators for plants was given by F. W. Went (25), son of the botanist F. A. F. C. Went, whose laboratories in Utrecht had for many years been concerned with a careful and extended study of phototropism. Went demonstrated that active material would diffuse from a coleop- tile tip into a block of gelatin which would then act as the tip itself did. From the diffusion rate he calculated the molecular weight of the compound to be in the vicinity of 376. It was thermostable and withstood drying. In addition to the final and convincing proof of the existence of a growth regulator, the great contribution made by Went was his method of quantitative determination of the growth regulator by using gelatin or agar blocks placed laterally on the de- capitated base of oat coleoptiles under controlled conditions. A preliminary report of the experiments of Fritz Went was made by his father in a notable address on Plant Movements at the Inter- national Congress of Plant Sciences at Ithaca, New York, in 1926 (24). I well remember hearing rumors that evidence would be presented for a thermostable, water-soluble substance involved in phototropism and the skepticism freely expressed by many of my colleagues. However, from this time on, a widening circle of investigators busied themselves with the problem. The discovery of a wide range of synthetic compounds which have effects similar to those of the na- turally occurring auxins has engaged the attention of many investi- gators, among others those at the Boyce Thompson Institute. The application of the auxins and similar compounds to the induction of root formation, weed control, prevention of preharvest drop of ap- ples and other fruits, increasing fruit set, inducing seedless fruit for- mation, thinning of fruit, regulating flowering, increasing fruit size, and hastening ripening has become increasingly important. Although the practical applications of the auxins are not to be underestimated, the great importance of their discovery was to give impetus to the concept that minute amounts of naturally occurring organic substances could profoundly influence plant growth and de- velopment. One of the few comforts of reaching what some charitably refer 18 IF. J. Robbins to as the more mature years is the privilege it gives of looking back and seeing the changes and advances which have occurred in a life- time. Plant physiology in my graduate student years, more than 40 years ago, consisted mainly of a consideration of mineral nutrition, transpiration, water requirements, osmosis and osmotic pressure, photosynthesis, nitrogen relations, especially nitrogen fixation, respir- ation, toxicity, antagonism, and balanced solutions, conduction of water and translocation of organic food, and similar fundamental pro- cesses. Any phenomenon not readily explainable on some other basis was assigned to that universal solvent of all problems, changes in permeability. That naturally occurring specific organic substances in minute amounts might materially influence the growth and de- velopment of a plant had little evidence to support it and received scant attention from teachers, students, or investigators. It is true that in 1858-1860 Pasteur had observed that the develop- ment of yeast and of lactic acid bacteria in a synthetic medium was favored by the addition of small amounts of natural products. Sachs (17) had proposed the concept but not the term hormone. Wildiers (26) reported that minute amounts of Bios, a concentrate of unknown composition, was of great importance for the growth of some races of yeast. Ludwig Jost, in his Plant Physiology (7) was quite prepared to account for the formation of insect galls by the action of some definite substance which diffuses out from the larva and stimulates the cells to hypertrophy. Loeb (8) had suggested that hormones produced by the leaf of Bryopliyllum played a role in the formation of new plants. But these reports and others like them could be and were ex- plained on some other basis than the action of specific compoiuids. It required something as decisive and dramatic as the auxins and their effects to change the viewpoint of plant physiologists interested in the problems of growth and development. For some years the auxins were the only growth regidators to which botanists devoted much serious attention. But new knowledge of the existence of vitamins, hormones, and similar substances, their chem- istry and their action in animal physiology, together with the discov- ery that there were some problems in the develo{)ment of plants not solved by auxins alone, led to a substantial expansion of our concepts of plant growth regulators. Schopfer's discovery (18) that Pliycomyccs blakesleeanus required for growth an external supply of small amounts of thiamin and in- vestigations on the growth retjuirements of yeast led to the inclusion of the vitamins, the purine and pyrimidine bases, and specific amino acids among the growth regulators. Kinetin (9) and the gibbercUins have been added more recently. Expanding Concepts 19 We have become convinced that growth regulators are involved in the flowering of plants even though their chemical nature still eludes us. The sex hormones of the water mold Achlya first reported by Raper (15) must also be admitted to the growing list of plant growth regulators, together with the antheridium-inducing factor from fern prothallia recently investigated by Niif (10). I would in- clude also among the plant growth regulators, acrasin, the autogenic chemical substance involved in the organization of some of the slime molds (16). To refer to all the plant growth regulators, that is, all the specific chemical substances which in minute quantities affect, even deter- mine, the rate and pattern of plant growth and also those for which there is convincing evidence, though they have not yet been isolated and identified, would make a long list indeed and one which is in- creasing rapidly. This is not the place to undertake such a review. How delicate the dynamic equilibrium of the metabolic systems of a living organism is. What profound effects on the rate, amount and character of growth can be produced by minute amounts of a specific organic compound. A few molecules of vitamin Bjo determine whether Euglena will grow and how fast. p-Chlorophenoxyacetic acid causes the apex of Kalanchoe to develop into a spathe-like organ which can be cut off and rooted. The ortho- and mefa-chlorophenoxy- acetic acids are inactive (Zimmerman and Hitchcock, 27). At the same time, how stable the systems are: like the treasures in a safe-deposit vault the course of metabolism which results in nor- mal growth and normal form is protected against all the agents which impinge upon it, except for those which have the right configuration, which are the keys capable of unlocking the vault door. This is the area which concerned Percy W. Zimmerman most. It is true that his botanical interests were broad. He developed hardy strains of Camellia japonica and of the American and English holly. He was a pioneer in the study of the effects of air pollution on plants, and because of long experience and extensive researches he was recog- nized as an authority in this field. But his great and abiding interest was the search for compounds which modified the normal growth pattern of a plant. Dr. Zimmerman was not a locksmith. He cared less for the mecha- nism of the lock and how the key turned the tumblers than he did for the key and the treasures in the vault. What keys he found, and what treasures were revealed! In cooperation with his close associate, Dr. A. E. Hitchcock, he investigated the properties of more than 500 compounds as growth regulators. (3-Indole-3-butyric acid, 1-naphthaleneacetic acid, substi- 20 W. J. Robbins tuted phenoxy acids, including 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4, 5-T), and chloro-substituted ben- zoic acids were some of those first tested in his laboratories. However, Percy Zimmerman was not a narrow botanical and hor- ticultural specialist. He served wherever his service would benefit his friends and neighbors and actively participated in a wide variety of community activities (6). We have come a long way since Darwin at his country home out- side London satisfied, so far as he was able, his curiosity about the response to light of the coleoptiles of Phalaris and Avena. He had the aid of tinfoil, India ink, flat splinters of glass, thread, and similar pieces of "complex apparatus," but above all, a clear, inquiring, and logical mind, and the serenity of a peaceful household managed by a devoted wife. A long succession of able and dedicated investigators, among whom we include Percy ^V^ Zimmerman, have answered some of the questions Darwin's investigations raised and carried on into areas of which Darwin never dreamed. Not the least of these areas is that of the effects of minute quantities of specific organic com- pounds — call them auxins, vitamins, hormones, growth regulators, or what you will — on the growth and development of plants. Investi- gation of these substances promises to be for some time to come one of the most important fields in plant physiolog). To those who have and are contributing to this subject, we might well say in the Avords of Swinburne: Tliy ivorks and mijic are ripples on the sea. Take heart, I say; xve knoiu not their end. LITERATURE CITED 1. Boysen Jensen, P. Uber die Leitung des phototropischen Reizes in Avena- keimpflanzen. Ber. Deutsch. Bot. Ges. 28: 118-120. 1910. 2. , and Nielsen, N. Studien iiber die hormonalen Beziehungen zwischen Spitze und Basis der Avenacoleoptile. Planta. 1: 321-331. 1925. 3. Branner, L. Lichtkrinnmung und Lichtwachstumsreaktion. Zeitschi. Bot. 14: 497-517. 1922. 4. Darwin, C. The Po^^e^ of M()\cmcnt in Plants. 592 pp. J. Murray, London. 1880. 5. Fitting, H. Die Leitung tropistischer Reize in parallelotropen Pflanzenteilen. Jahrb. Wiss. Bot. 44: 177-253. 1907. 0. Hitchcock, A. E. Percy W. Zimmerman (February 23, 1884 — August 14, 1958). Contr. Boyce Thompson Inst. 20: 1-5. port. 1959. 7. Jost, L. Lectures on Plant Pliysiology (transl. R. J. Harvey Gibson). 564 pp. Oxford. 1907. 8. Loeb, J. Rules and mechanism of inhibition and correlation in the regener- ation of Bryophyllum calycinum. Bot. Gaz. 60: 249-276. 1915. 9. Miller, C. O., Skoog, F., Saltza, M. H. von, and Strong. F. M. kinetin, a cell division factor from deoxyribonucleic acid. Jour. .\mcr. Chem. Soc. 77: 1392. 1955. Expanding Concepts 21 10. Naf, U. The demonstration of a factor concerned with the initiation of an- thcridia in polypodiaceous ferns. Growth. 20: 91-105. 1956. 11. I'aal, A. Ober phototropische Reizleitiuigen. Ber. Dcutsch. Bot. Ges. 32: 499-502. 1914. 12. tjber phototropische Reizleitung. Jahrb. Wiss. Bot. 58: 406-458. 1918. i.*?. Priestley, J. H. Light and growth. 111. An interpretation of phototropic growth curvatures. New Phytol. 25: 213-226. 1926. 14. Purdv, Helen A. Studies on the path of transmission of phototropic and geo- tropic stimuli in the coleoptile of Avena. Dansive Vid. Selsk. Biol. Meddel. 3 (8): 1-29. 1921. 15. Raper, J. R. Sexual hormones in Achlya. Proc. Third Internat. Cong. Micro- biol. 494. 1939. 10. Raper, K. B. Factors affecting growth and differentiation in simple slime molds. Mycologia. 48: 169-205. 1956. 17. Sachs, J. Stoff und Form der Pflanzenorgane. Arb. Bot. Inst. Wiirzburg. 2: 452-488, 689-718. 1882. 18. Schopfer, \V. H. Versuche iiber die Wirkung von reinen kristallisierten \'ita- minen B auf Phycomyces. Ber. Deutsch. Bot. Ges. 52: 308-312. 1934. 19. Seubert, E. Uber Wachstumsregulatoren in der Koleoptile von Avena. Zeit- schr. Bot. 17: 49-88. 1925. 20. Snow, R. Further experiments on the conduction of tropic excitation. Ann. Bot. 38: 163-174. 1924. 21. Soding, H. Werden von der Spitze der Haferkoleoptile Wuchshormone gebil det? Ber. Deutsch. Bot. Ges. 41: 396-400. 1923. 22. Stark, P. Studien iiber traumatotrope und haptotrope Reizleitungsvorgange mit besonderer Beriicksichtigung der Reiziibertragung auf fremde Arten raid Gattungen. Jahrb. Wiss. Bot. 60: 67-134. 1921. 23. Stark, P., and Drechsel, O. Phototropische Reizleitungsvorgange bei Unter- brechung des organischen Zusammenhangs. Jahrb. Wiss. Bot. 61: 339-371. 1922. 24. Went, F. A. F. C. Plant movements. Proc. Fourth Internat. (Bot.) Cong. Plant. Sci. (1926) 1: 1-12. 1929. 25. Went, F. W. Wuchsstoff und Wachstum. Rec. Trav. Bot. Neerl. 25: 1-116. 1928. 26. Wildiers, E. Nouvelle substance indispensable au developpement de la levure. Cellule. 18: 313-316. 1901. 27. Zimmerman, P. W., and Hitchcock, A. E. Formative effects of several substi- tuted phenoxy acids applied to KalancJiiJe. Contr. Boyce Thompson Inst. 15: 421-427. 1949. Naturally Occurring Plant Growth Substances JOYCE A. BENTLEY Marine Laboratory Aberdeen, Scotland Some InvestlgatLons on Interconvertible Naturally Occurring Auxins Modern techniques in plant hormone analysis, in particular the use of paper chromatography for the purification of extracts, followed by bioassay, have revealed a number of as yet chemically unidentified auxins in various plant species. This paper is concerned with one particular group, which has come into prominence recently and about which very little is known — that is, the group of so-called intercon- vertible auxins. A group of three interconvertible auxins was first reported in the ether-insoluble fraction of tomato roots (8), followed by a report of similar substances in pea roots (1). The present investi- gation deals with a group of interconvertible auxins located in vari- ous species of algae. The algae were chosen as a field of investigation for two reasons. Firstly, there is an ecological problem; there is con- siderable evidence that the growth and distribution of both marine and freshwater algae may be governed, at least in part, by minute traces of metabolites in their aqueous environment; these metabo- lites may range from toxins to vitamins and hormones (8, 9). It is of interest to investigate whether the algae do in fact produce hormones of the auxin type and excrete them to the media in which they are growing. Secondly, the single-celled algae are free from the prob- lems of differentiation encountered in the higher plants. Since the auxins affect fundamental aspects of growth, for example cell elonga- tion and cell division, their primary effect appears to be on the indi- vidual cell, and it is therefore of interest to investigate their occur- rence in single-celled organisms. MATERIALS The following materials have been examined. I am grateful to the various people, indicated in the text, who generously supplied material. [25] 26 J. A. Bentley 1. Oscillatoria spp. (Cyanophyceae) from Dr. A. J. Brook, Fresh- water Fisheries Laboratory, Pitlochry. A sample of approximately 150 g. fresh weight was collected from a small Scottish lake, where it occurs as a persistent bloom and as practically a unialgal culture. 2. Chlorella pyrenoidosa (Chlorophyceae). 118 g. fresh material was received frozen from Dr. G. E. Fogg, University College, London. This was an 11-day-old culture and was extracted immediately. 3. Ochromonas malhamensis (Chrysophyceae). Two samples, con- sisting of 3.3 and 2.1 g., respectively, of freeze-dried cells, were re- ceived from Dr. J. E. Ford, National Institute for Research in Dairy- ing, Reading. EXPERIMENTAL TECHNIQUES Extraction 1. Oscillatoria spp. The inaterial was frozen ( — 30° C), then thawed, acidified to pH 5.0, and extracted with ether. The dried ether extract was partitioned between 95 per cent methanol and pe- troleum ether (40° to 60° C), and the petrol fraction rejected. The methanol fraction was separated into acidic and neutral components. 2. Chlorella pyrenoidosa and Ochromonas malhamensis. The ma- terial was extracted at pH 4.0 by stirring with 70 per cent aqueous ethanol. After neutralization, the alcohol was distilled in vacuo and the re-acidified residue extracted with petroleum ether (40° to 60° C.) and then ether. The ether extract was separated into acid and neutral fractions. The aqueous residues were saponified with A'' NaOH (15 min. at 15 lb. pressure), extracted with ether and the ether extract separated into acid and neutral fractions. Chromatography Extracts were usually purified by preliminary paper chromatogra- phy using water as eluant; the pigments remained on the starting line, which was rejected. The remainder of the paper was flushed with ethanol and the recovered material chromatographed usually in isopropanol-ammonia-water (10:1:1) or water. Chromatograms were examined under filtered ultraviolet light (2537A) and used either for color tests [usually Ehrlich, Salkowski and nitrous-nitric acid reagents, using the techniques of Jepson (6)] or for bioassay. Chromatograms to be bioassayed were cut into ten equal portions and the portions eluted with water for testing. Assay Technique The Avena straight-growth method was used (2), with some modi- fications which give greater sensitivity. It is possible to use 10 sec- Interconvertible Naturally Occurring Auxins 27 tions (10 mm.) with only 1 ml. of test solution in 3.5 cm. petri dishes, instead of 10 ml. in 5 cm. petri dishes, as used previously. Under these conditions, best growth is obtained with the addition of 1 per cent sucrose and phosphate-citrate buffer (10- M K2HPO4 and 0.5 X 10- M citric acid at pH 5.0. With these modifications, the sensi- tivity of the test can be increased 10 to 100-fold over that previously obtained. RESULTS Oscillatoria spp. Chromatography and bioassay of the acidic ether fraction showed clear-cut activity in two zones, which have been called zones X and Z (Figure lA). These zones are still clearly defined even on diluting the extracts 10 times. There is a spot giving a purple Ehrlich, pink Sal- kowski, and pink nitrous-nitric acid reaction in zone Z. When zone X is eluted and rechromatographed in either alkaline or neutral conditions [?i-butanol-ethanol-1.5 N NH3 (6:2:2) or water. Figures IB and IC, respectively], zone Z appears in addition to X. There is also evidence of an intermediate zone (Y) in Figure IB. This nomencla- ture of the zones is considered further in the discussion. Similarly, when Z is eluted and rechromatographed in isopropanol-ammonia, X appears (Figure ID). Thus, a mutual interconvertibility between X and Z can be demonstrated, similar to the interconvertibility demon- strated in tomato roots (3) and pea roots (1). It should be noted that the conversion of one zone to the other is never complete — both zones can be picked up on the paper and there appears to be an equi- librium between them. Zone Y is more transient in the algae and not so easily demonstrated. Chlorella pyrenoidosa All fractions received preliminary purification by chromatography in water, and rejection of the pigment zone. Color tests of the acidic ether fraction chromatographed in iso- propanol-ammonia gave a spot with unusual color reactions, a rapid dark-blue Ehrlich reaction and faint yellow nitrous-nitric acid re- action, at Rf approximately 0.8. This spot, which is also found in corresponding fractions of dried Chlorella, is referred to in the dis- cussion as Chlorella 5. Solutions from this zone rechromatographed after bioassay gave a faint pink Ehrlich reaction in the same region. Bioassay of the acidic ether fraction after chromatography in iso- propanol-ammonia showed that there was activity in this region and also in the lAA region (Figure 2A). Rechromatography of zone Z in le 16- 14 12- lO I 1 XIO 4iln lAA I 1 ^ r""^ ( pu Ehr I" ( pk No 2/3 •2 ■4 lAA 4^ 10 16- 14' 12- lO B HjO X IAN IAN — I — l-O X H (9 Z u 16- 14- 12- lO H,0 -I— •4 lA A — I — 10 16- 14- 12- lO H^ LZ —I— O -T— •2 Rf lO Fig. 1. Acidic ether fraction of Oscillatoria spp. A. ChromatograpliccI in isopropa- noi-ammonia: extract = 10 g. fresh wt. of original material. B. Zone Rf 0.3 to 0.5 of lA elated and rechromatographed in Ji-butanol-cthanol-1.5 X NH, (6:2:2). C. Zone R, 0.3 to 0.5 of lA eluted and rcchromatograpiicd in water. D. Zone R, 08 to 1.0 of lA eluted and rechromatographed in isopropanol-ammonia. Controls of lAA are 1 = 5 X 10"" g/™^ ^^^ successive x 10 dilutions. [28] 16 15 14- 13- lAA IAN H,0 — H X 10 diln. 1 z X 1 1 1 1 , j -I 1 blue Ehr iu- < < 18- E 16- V. r^ 1 1 c^ 14- O O o O X X X X in «r> m m r Rf Fig. 2. Acidic ether fraction of Chlorella pyrenoidosa. A. Chromatographed in isopropanol-ammonia; extract = 7.5 g. fresh wt. of original material. B. Zone Rf 0.2 to 0.7 of 2A treated with N HCl, extracted with ether and rechromatographed in ammoniacal isopropanol. C. Zone Rf 0.8 to 1.0 of 2A eluted and rechromato- graphed in water. D. Zone Rf 0.8 to 1.0 of 2A rechromatographed in water after hioassay. Controls of lAA are as in Figure 1. [29] 30 J. A. Bcntlcy water showed high activity, with the production of peaks which are possibly zones X and Y (Figure 2C). Rechromatography of zone Z (from Figure 2A) in water after bioassay clearly showed two peaks of activity (Figure 2D). If it is assumed that X and Z behave similarly to lAA and IAN respectively on chromatography in the solvents used here, then the peak near the solvent front in Figures 2C and 2D will be X, released from Z, which is running at an Rf of approximately 0.4. Treatment of zone X (Figure 2A) with A^" HCl and rechroma- tography in isopropanol-ammonia gave a large peak in zone Z (Fig- ure 2B) and a hint of zone Y, Thus, a mutual interconversion of X and Z has again been demonstrated as in Oscillatoria, and X, Y, and Z appear to be stable to treatment with N HCl. The possibility was considered that the active substances wxre not completely separating during chromatography, and that dips in the histograms between peaks of activity might be due to the presence of inhibitors, although true inhibition (below the level of the water controls) is rarely obtained. Frequently, however, bioassay of dilu- tions of the original solutions {e.g., Figures lA and 2A) show the same general pattern of distribution of activity. Inhibitory effects generally disappear very much more quickly on dilution than growth-promot- ing effects, and if an inhibitor had been present at Rj 0.6 to 0.8 in Fig- ure 2A in sufficient quantities to cause a dip in the level of activity, one would expect that bioassay of tenfold dilutions would show over-all activity along the length of the paper from X to Z. This does not happen, and the original promoting peaks are still clearly de- fined. It is thus more than likely that peaks X and Z represent true peaks due to discrete substances. The over-all activity sometimes ob- tained on papers such as those illustrated in Figure 2B and Figure 2C is more likely to be due to the activation of precursors on the paper during the various operations to which it is subjected. Evidence is con- tinually turning up that there are inactive precursors on the papers. Ochromonas malhamensis The petroleum ether fractions were inactive and were rejected. The acidic ether fractions of both samples contained active substances, which were concentrated in zone Z near the solvent front in the first sample (Figure 3A), but which separated into three well-defined /ones in the second sample (Figures 3B and 3C, zones X, Y and Z). Ochrofnonas 1 extracts were chromatographed directly in isopro- panol-ammonia, but Ochromonas 2 fractions were first purified by line-loaded chromatography in water, because of the large amounts of pigments in the extracts. The base line portion where most of the 18 16- 14- 12- < < fV ro H^O h^ IAN I 1 2 ^ V No Ehrlich Color ho 20 18 - I6H X z w -J \M -i 4 Z iZ 12- B lAA ^ m H^O X ' — 1 "H z 1 Y ■■ f 1 •4 ■6 8 0 -i lO 18 16- 14. 12- (V ^ IAN ()un(l. Interconvertible Naturally Occurring Auxins 41 Dr. Fawcett: I'd just like to say that from some advice that Dr. Bennet-Clark gave a year or two ago we switched from using ether to ethyl acetate for the extraction of indole compounds. Although we ran into some problems with emulsions, we found that we got a much better and wider spectrum of the indole compounds. I wonder if Dr. Bentley had any experience in using ethyl acetate with the algae. Dr. Bentley: No, I haven't used ethyl acetate and I couldn't say anything about it really. Dr. Nitsch: May I ask Dr. Bentley a point of technique which is very important? Could you tell me if you leave the solvent in the tank and use it over and over again? Dr. Bentley: No, we mostly renew it. We found that the percent- age of ammonia changes. Dr. Nitsch: Do you use fresh solvent every time? Dr. Bentley: No, not necessarily every time. POUL LARSEN and TORBJ0RN AASHEIM University of Bergen Tke Occurrence of lndoie-3-aceialdehyde in Certain Plant Extracts In 1939 (19) the senior writer detected a neutral growth substance in extracts of etiolated epicotyls of Pisiim sativum and Vicia faba and in hypocotyls of Helianthus. Subsequent investigations of this sub- stance (20, 21) led to the conclusion that it was identical with indole- 3-acetaldehyde (lAAld) . Since the active material was not isolated in the chemically pure state, its identification rested on indirect evidence. Similar evidence for the occurrence of lAAld in extracts of other plants was subsequently provided by Hemberg in 1947 (16), Gordon and Sanchez Nieva in 1949 (11, 12), Yamaki and Nakamura (31), and others. The literature concerning the occurrence and possible function of lAAld in plants has been reviewed by Larsen, 1951 (22) , and Gor- don, 1954 (8) . Among the more recent studies of these problems may be mentioned the ones by Wiedow-Patzold, 1955 (30) , Gordon, 1956 (9, 10), and Clarke and Mann, 1957 (7). The only other natural auxin that had been claimed to play a role in plant growth regulation was Kogl's auxin a lactone. As it became more and more unlikely that this compound (or auxin a or b) oc- curred in plants, it became customary to ascribe auxin activity found in nonacidic fractions of plant extracts to the action of lAAld. In 1952, however, lAAld was synthesized by Brown, Henbest, and Jones (6) , and in the same year, indole-3-acetonitrile (IAN) was isolated from cabbage in the chemically pure state by Jones, Henbest, Smith, and Bentley (18). The biological activities of the two neutral syn- thetic auxins were studied by Bentley and Housley (4) and by Bentley and Bickle (3) . Two neutral auxins had now to be considered, and since the occur- rence in plants of the lAAld still rested on indirect evidence only, it was natural for some workers to suspect that in the past the aldehyde [43] 44 P. Larsen and T. Aasheim might sometimes have been confused with the nitrile (1, 2) . In certain cases, however, the nitrile can be excluded as the active material in the neutral preparations, namely when the auxin activity is de- termined in the Avena coleoptile curvature test and found to be in- creased several-fold by conversion of the active material to an acid. The nitrile is already as active as indole-3-acetic acid (lAA) or even more so (3, 24, and our own data) ; preparations containing it cannot be made more active by any change in the nitrile molecule. Only a concomitant removal of a growth inhibitor coidd explain the increase in activity if the nitrile were the active, neutral material in such cases. In the early work by the senior writer (20, 21), only extracts of etiolated pea epicotyls were thoroughly investigated, and the evidence for the occurrence of lAAld actually pertained only to Pisum. \\^hen the presence of a neutral growth substance in extracts of cabbage could be demonstrated (21) , it was only by analogy that the identity of this with lAAld was inferred. No attempts were actually made to establish the aldehyde nature of this material. In light of the work of Bentley and her co-workers, it would now be highly desirable to reinvestigate the status of lAAld as an auxin. For several years we were discouraged from resuming the work on lAAld, however, primarily on account of the reported extreme lability of the compound, not to mention the fact that in spite of its synthesis in 1952 it remained inaccessible for research purposes. Recently, a number of findings have been reported which should be favorable for a successful attempt to demonstrate the presence of lAAld in plant extracts. First, a simplified method for obtaining chemically pure lAAld was published by Gray in 1958-59 (13, 14) utilizing Langheld's method in treating tryptophan with sodium hypochlorite, but intro- ducing the refinement of trapping the aldehyde in benzene before it would become destroyed in the reaction mixture. According to Gray, the aldehyde is not nearly as labile as had been believed; and its sodium hydrosulfite addition product proved to be stable for many years. In the meantime, Nitsch and Nitsch, 1955 (28), and Nitsch, 1956 (27) , had been searching for a chromatographic solvent system that would separate various neutral auxins on filter paper. As a result of tlieir extensive investigations they recommended 7?-hcxane or a prac- tical grade hexane to be used in a water-saturated atmosphere. Such a system, which contains no acid or alkaline component and very little water, should be considerably less harmful to an inistablc compound like lAAld than most of the systems j)reviously preferred in chroma- tograj)hic work on auxins. \i\ addition, the hexane gives a much better separation of known neutral auxins. Indole-3-acetaldehyde in Playit Extracts 45 Finally, a new, sensitive spray reagent for indole derivatives was described by Harley-Mason and Archer in 1958 (15). This reagent is p-dimethylaminocinnamaldehyde (DMCA) , a vinyl compound corre- sponding to Ehrlich's reagent, p-dimethylaminobenzaldehyde (DMBA) . DMCA, possessing an additional double bond, was expected to give more intensely colored condensation products with the indole compounds and is reported to be about ten times as sensitive as Ehr- lich's reagent as a detector of indole and tryptophan. Basing our procedures on the work of Gray, Nitsch, and Harley- Mason and Archer, we hoped to be able to clarify the situation with respect to the occurrence of lAAld and IAN in ether extracts of etio- lated pea epicotyls and heads of cabbage. MATERIAL AND METHODS The compound, lAAld-NaHSOs, was prepared from tryptophan and NaOCl, following exactly the directions given by Gray (14) , ex- cept that all quantities of material were reduced to one-half (starting with 1.5 g. of tryptophan instead of 3 g., etc.) . Free lAAld, lAA, and IAN were chromatographed (descending) on filter paper in the same system as was used by Gray, namely 7i-butanol saturated with 2.8 per cent aqueous ammonia. After 7 hrs. the paper was dried and sprayed with a 1 per cent solution of dimethylaminobenzaldehyde in 1 N HCl. lAAld and lAA gave practically the same Rf values as reported by Gray who used the ascending technique, namely 0.92 to 0.95 and 0.23 to 0.25, respectively (Gray: 0.93 and 0.28 to 0.29). IAN, not tested by Gray, ran at Rf 0.89. The color reaction of lAAld with DMBA is bluish purple, changing to brownish. Weller, Wittwer, and Sell (29), who obtained a sample of lAAld-NaHSOg from Gray, report that lAAld runs at Rf 0.88 in 1-butanol saturated with 5 per cent NH4OH and in 1-propanol: concentrated NH40H:HoO (60:30:10). The free aldehyde was obtained by adding 4 ml. water and 1 ml. 0.5 M NaoCOg to 5 ml. of an aqueous solution containing 0.5 or 1.0 mg. of lAAld-NaHSOg. The alkaline solution was shaken three times with 8 ml. of peroxide-free ether; the ether, containing the aldehyde, was adjusted to a volume of 25 ml. It is not yet known whether all of the original lAAld is secured in the ether fraction by this procedure, so quantities of lAAld are given as relative values only in the follow- ing: IAN. This compound was purchased from the Aldrich Chemical Company, 3747 N. Booth St., Milwaukee 12, Wisconsin. It was used without further purification. p-Dimethylaminocinnamaldehyde (DMCA) (Aldrich, see above). The reagent was prepared as directed by Harley-Mason and Archer 46 P. Larsen and T. Aasheim (15) : 2 g. of DMCA were dissolved in 100 ml. 6 N HCl plus 100 ml. 96 per cent ethanol. Plant Extracts Extracts were made as described by Larsen (21) . Pisum. 15 to 50 g. samples of the upper 10 to 15 cm. portions of epicotyls of etiolated 7- to 9-day-old pea seedlings 'Alaska' were ex- tracted at room temperature with 25 to 100 ml. portions of peroxide- free ether. The ether was renewed twice after 24 or 48 hr. periods. The 3 portions of ether were reduced in volume and combined. A suitable number of such extracts were combined, adjusted to a volimie of about 12 ml., and shaken three times with 10 ml. HoO -j- 1 ml. 8 per cent NaHCOg. The aqueous, alkaline fraction was discarded. The ether fraction, now containing only the nonacidic materials, may be tested in the Avena curvature test, but contains fatty materials in quan- tities too high for a satisfactory application of concentrated droplets to the starting line of a paper chromatogram. Most of these fatty mater- ials were removed by the following method. The ether was evaporated to dryness in a 50 ml. Erlenmeyer Hask. The residue is a smooth layer, covering the bottom of the flask. This residue was dissolved in 2.5 ml. 96 per cent ethanol which was then evaporated off at about 45° C. Probably on account of the water present in the ethanol, the residue is now in the form of small flakes, which can be suspended in water. Eight ml. water were added to the flask which was then shaken for 10 min. at 45° C. and cooled to 5 to 7° C. The suspension was filtered through a coarse Pyrex sintered glass filter, using suction. The flask was rinsed three times in this manner. The combined slightly opaque filtrates, about 24 ml., were shaken with three 12 ml. portions of ether, and the combined ether fractions were adjusted to a volume of 25 ml. The content of fat in this ether extract caused no difficidties in chromatographic work. Brassica. 50 g. of the inner, etiolated leaves of a head of cabbage were extracted and treated as described above for Pisntii, except that removal of fat by carrying the neutral fraction through water was omitted. These extracts were used at much lower concentrations than those of Pisum. Avena Coleoptilc Curvature Tests These tests were carried out as described by Larsen (23) . Avena seedlings were grown individually in soil in vials and decapitated twice. Ether extracts were transferred to 0.1 nil. agar platelets bv the ether-dropping mctliod. Indole-3-acetaldehyde in Plant Extracts 47 Paper Chromatography All the chromatography was carried out on Whatman No. 1 filter paper. Except for a few control experiments, the plant extracts and synthetic samples were chromatographed in hexane (27, 28) . The de- scending technique was used. The solvent (7z-hexane, Merck) was shaken with water before use, but no water was placed in the upper trough. The atmosphere in the tank was kept saturated with water vapor by means of a sheet of filter paper dipping into a separate trough containing water. Nitsch and Nitsch (27, 28) emphasize that the water conditions during the experiment have a pronounced influence on the Rf values of IAN and ethyl indole-3-acetate (lAEt) . Our Rf values for IAN and lAAld were low, but reproducible. They were 0.10 and 0.055, respec- tively. Rf values for lAEt were variable (0.38 to 0.57) , and more streak- ing occurred behind the spot. In order to get good separation of lAAld and IAN, the chromatogram was run for 18 hrs. or more. Under these conditions, of course, the front would run off the paper and for a direct determination of Rf values, chromatograms had to be run for a shorter time, e.g., Si/o hrs. IAN and lAAlcl were used as reference markers in the establishment of Rf values for other compounds in the 18 hr. runs. RESULTS Synthetic compounds. CJiromatography. A list of Rf values in water- saturated n-hexane and color reactions with DMCA given by lAA and a few neutral indole derivatives is given in Table 1. It will be seen that the two compounds with which we were most directly concerned, IAN and lAAld, can be distinguished without difficulty. These two auxins became well separated when applied in mixture to the same starting point. Table 1. Rf values in water-saturated 7z-hexane (Merck) and color reactions with dimethylaminocinnamaldehyde (DMCA) given by lAA and some neutral indole derivatives. Compound Rf Color Reaction With DMCA Tndolp-S-acetic acid ^lAA") 0.0 variable, 0.38 to 0.57 0.055 0.04 0.0 0.10 Bluish purple Ethyl-3-indoleacetate (lAEt) Indole-3-acetaldehvde HAAld") Bluish purple Clear blue Indole-3-ethanol ( — trvDtoohol^ Bluish purple Indole-3-acetamide Reddish purple Indole-3-afetonitrile ^IAN"i Purple 48 P. Larsen and T. Aasheim Bioassay One of the findings that indicated the aldehyde nature of the neutral auxin in Pisimi extracts in earlier work was the fact that it could be converted to lAA by treatment with soil (21) . The activity of a soil-treated preparation was many times higher than that of an untreated, neutral fraction. Using the Avena coleoptile ciuvature test, we therefore determined the auxin activity of synthetic lAAld and IAN both directly and after treatment with soil. The soil treatment was carried out as described by Larsen (21) : The auxin was transferred to a 1-mm. thick agar platelet (1 sq. cm.) by the ether-dropping method. The 1-mm. platelet was covered with an agar platelet of the same area, but only 0.5 mm. thick. A suitable amotint of soil was placed on top of the cover platelet. The soil was always taken from one of the vials in which the day's test plants were grown. This means that the physical and biological conditions in the soil samples were standardized. After the lapse of 90 min. the cover platelet with the soil was removed and the lower 1-mm. platelet cut into test blocks, 2 X 2 X 1 mm., which were later applied to twice-decapitated test plants. The curves marked lAAld in Figure 1 represent the results of tests with this compound. The solid line is the concentration-activity curve obtained directly, without soil treatment, and the broken line, the corresponding curve for soil-treated material. It will be noted that the activity of the preparation was increased by several hundred per cent by the soil treatment. In this respect the synthetic lAAld re- UJ q: o UJ o (r 3 o 1 -1 1 1 1 1 1 40 Pisum ' / / / / / lAAId. / / lAAId^^ - 20 -^ 0 I' ^ ^ ^^ i 1 r_ i__ 1 /^ Pisum ' 1 1 12 CONCENTRATION Fig. 1. EfTctt of soil ircaimciits on auxin activity of Visum extracts and of iiulolc- 3-acelaldchyde. Solid lines represent activity obtained directly and the broken lines activity after treatment with soil. Indole-3-acetaldehyde in Plant Extracts 49 sembles the neutral auxin from Pisum and Vicia faba and the auxin prepared from isatin and tryptophan (21) . Synthetic IAN, when tested at low concentrations, proved to have about the same activity per microgram as lAA in the Avena coleoptile curvature test. This confirms the results of Bentley and Bickle (3) . The standard soil treatment (see above) almost completely removed the activity of the preparation. Examples: IAN, 80 jxg/l, 30.4° curv- ature; soil-treated, 0° curvature; IAN, 8 /xg/1, 8.5° curvature; soil- treated, 0° curvature. IAN and lAAld can thus be distinguished by their different reactions to the standard soil treatment. Plant Extracts Pisum. Samples of neutral, defatted fractions of pea epicotyl ex- tract, each representing 11 to 12 g. of fresh plant material, were evap- orated as spots on the starting line of the chromatographic paper. Samples of synthetic lAAld and IAN (8 to 10 /xg.) were applied at sepa- rate spots to serve as markers. The chromatograms were developed by spraying with DMCA after about 18 hrs. A colored spot at the starting line w-as reddish purple and a 40 mm. long spot around Rf 0.055 (lAAld location) was clear blue. Faint blue streaking was visible behind the blue spot at Rf 0.055, both in the pea extract and in the synthetic lAAld preparation. No other coloration was detected on the chromatogram. Suitable zones (see Figure 2) were cut out of two parallel, unsprayed strips of the paper. Each of these zones was cut to smaller pieces and extracted with three changes of ether over a period of 3 days. Each of these eluates was made to a volume of 25 ml. and tested at various concentrations in the Avena coleoptile curvature test. The results are given in Figure 2. Auxin activities are expressed in rela- tive units, based on the amount of plant material needed. The con- centration yielding 10° curvature was interpolated on the ascending part of concentration activity curves (example in Figure 1) . The solid lines in Figure 2 represent direct tests without soil treat- ment. By far the highest activity is present in zone III containing the clear blue spot at Rf 0.055. Considerably less was present in zones I and II, and no activity was detected in the other parts of the chroma- togram down to Rf 0.31, although concentrations representing up to 110 kg. fresh plant material per liter of agar were tested. The broken lines (Figure 2) indicate the activity of the eluates after soil treatment. In zone III the auxin activity was increased several-fold by the soil treatment (see also Figure 1). Also in zone I the activity was increased, but to a smaller extent. The eluate from zone II was not treated with soil. Soil treatment did not produce any activity in zones IV and V. 50 P. Larsen and T. Aasheim From these results, the identity of the neutral auxin in etiolated pea epicotyls with lAAld is indicated by R^ (0.055) , color reaction with DMCA (clear blue) , and reaction toward soil treatment (several- fold increase in auxin activity). Obviously, this auxin cannot possibly be identical with IAN. Brassica. Samples representing 0.5 to 0.6 g. fresh weight were ap- plied as described above for Pisum, and the chromatograms were treated and eluted in the same manner. Results are shown in Figure 2. 8 0 0.8 0.4 0 - 1 I Brassica 1 1 1 I 1 >- 1- > t- o < X < UJ > -1 \\ 1 - Pisum I n 1 12 iz: VI 1 r - cr IE -\ O 1 1 ® lAAId. (D IAN 1 ® 1 0.05 0.10 0.15 Rf Fig. 2. .'Vuxin activity of eluates of chromatograms of extracts of Pisum and Brassica. Solid lines represent activity olitaincd directly and broken lines after treatment with soil. Areas corresponding to the indole-3-acetaldehvde in Pisum show a considerable increase in activity after treatment in soil. For further expla- nation see text. Colored spots dc\el()ped in the follow iiig places after spra)ing Avilh DMCA: starting line (reddish purple), Rf 0.10 (purple), Rf 0.135 (pur- ple) . In addition to these spots a faint, reddish-pinple spot was de- tected at Rf 0.4 on a chromatogram which was run for Si/ hrs. instead of 18 hrs., and in which the front had moved to 32.7 cm. Bioassays were made on zones III and IV in the IS lir. chromato- gram (Figure 2) and very high activity was fouiul in botli. (Note the different scales for Brassica and Pisum.) Supramaxinuini cmvatines were obtained from /one IV at the concentrations tested, but no at- Indole-3-acetaldehyde in Plant Extracts 51 tempt was made to determine the activity in this zone more accurately. The auxin present here is undoubtedly IAN. Zone III, which includes the locus of lAAld, contained no definite colored spot, but only a very faint purple streaking. The auxin activity found here was much higher than expected from the faint coloration, and it seems probable that a nonindole compound is responsible for this activity. Such nonindole auxins were also reported to be present in members of the Cruciferae by Linser and Machek (26) and by Housley and Bentley (17). Since this activity occurred in the zone containing the locus of lAAld, we first thought that it was due to this compound, but soil treatment proved definitely that this was not the case. The activity was reduced to less than one-tenth by the soil treatment, whereas it would have been increased several-fold had it been due to lAAld. These results, however, do not exclude the occurrence of lAAld in cabbage since the starting material on the chromatogram represented less than 1/20 of the amount of plant material used in the case of Pisum. Linser, Kiermayer, and Youssef (25) studied the auxins in Brassica napus and three varieties of Brassica oleracea. Extracts of seeds, leaves, stems, and roots of these plants were chromatographed in propanol, water, ammonia (80:15:5) on filter paper. In stems of one of these plants, B. oleracea var. sahauda (Wirsingkohl), auxin activity coincided with the locus, giving a yellow color reaction with ferric chloride and perchloric acid at Rf 0.77. The authors report that the color reaction and position of this locus agree with those of synthetic lAAld, un- fortunately, however, without mentioning the origin of their sample of synthetic lAAld. As regards our ether extracts of heads of cabbage, it can be concluded that, if present at all, lAAld occurs. The occurrence of IAN in members of the Cruciferae has been established beyond doubt by isolation and chemical characterization. As regards extracts of other plants, the identification of IAN rests ex- clusively on chromatographic data. A survey of the literature on chromatographic separation of auxins shows that IAN and a number of other neutral auxins, now also including lAAld, run rather close together in the majority of systems containing an aliphatic alcohol, water, and ammonia. [The statement by Blommaert (5) that lAAld runs at Rf 0.40 to 0.42 in n-butanol saturated with 2 A^ ammonia is probably erroneous since IAN ran at Rf 0.87 to 1.0 in Blommaert's system. Blommaert does not mention the source of his sample of lAAld. ] Biological activity in the "IAN zone" of such systems does not unequivocally indicate the presence of IAN. The activity may as well have been due to other neutral auxins unless more specific tests for IAN have also been carried out. 52 P. Larsen and T. Aasheim SUMMARY Preparations of synthetic indole-3-acetaldehyde (lAAld) and in- dole-3-acetonitriIe (IAN) were chromatographed (descending) on Whatman No. 1 filter paper in water-saturated n-hexane. Chromato- grams were developed by spraying with dimethylaminocinnamalde- hyde (DMCA) . The colors obtained with this reagent are clear blue for lAAld and purple for IAN. Rf values, determined after Bi/o hrs. at 20° C, were 0.055 for lAAld and 0.10 for IAN. Good separation was obtained by running the chromatograms for 18 hrs., letting the front run off the paper. Ether extracts of etiolated epicotyls of pea (Pisum) and of the etiolated, inner leaves of a head of cabbage (Brassica) were chroma- tographed under the same conditions as the synthetic compounds. Pisum yielded a clear blue spot at the same location as synthetic lAAld, but no spot at the IAN locus. Brassica yielded a purple spot at the same location as IAN, but no spot at the lAAld locus. \Vith Brassica, however, colored spots were also observed at Rf 0.135 (pur- ple) and Rf 0.4 (reddish purple) . The auxin activity (Avena coleoptile curvature test) of synthetic lAAld and of the material from the zone containing the lAAld locus in Pisum could be increased several-fold by treatment with soil, whereas the activity of IAN almost disappears by the same treatment. \Vith Brassica, auxin activity was found both in the IAN zone and in the zone corresponding to the locus of lAAld. No colored spot, hoAV- ever, was developed in the latter, and the auxin activity in this zone was reduced very considerably by treatment with soil. It is concluded that the Pisum extracts contain lAAld, but very little, if any, IAN, and that the Brassica extracts contain large amounts of IAN, but considerably less lAAld, if any. LITERATURE CITED 1. Bentley, Joyce A. The naturally-occurring auxins and inhibitors. Ann. Rev. Plant Physiol. 9: 17-80. 1958. 2. .Chemistry of the native auxins. //;.■ H. Burstrom (ed.), Encyclopedia of Plant Physiology. 14. Springer-Verlag. Heidelberg. (In preparation.") 3 Bentley, Joyce A., and Bickle, ,'\. S. Studies on plant growth hormones. II. Further biological properties of 3-indolylacetoniirile. Jour. Exper. Bot. 3: 406- 423. 1952. 4. Bentley, Joyce A., and Housley, S. Studies on plant growth hormones. I. Bio- logical activities of 3-indolylacetaldehyde and 3-indolylacetonitrile. Jour. Exper. Bot. 3: 393-405. 1952. 5. Blommaert, K. L. J. Growth promoting and inliiljiting substances in relation to the rest period of the potato tuber. Nature. 171: 970-973. 1951. 6. Brown, J. B., Henbest, H. B., and Jones, E. R. H. 3-Indolylacetaldehvde and 3- indolylacetone. Jour. Chem. Soc. London. 3167-3172. 1952. Indole-3-acetaldehyde in Plant Extracts 53 7. Clarke, A. J., and Mann, P. J. G. The oxidation of tryptamine to 3-indolylacet- aldehyde by plant amine oxidase. Biochem. Jour. 65: 763-774. 1957. 8. Gordon, S. A. Occurrence, formation, and inactivation of auxins. Ann. Rev. Plant Physiol. 5: 341-378. 1954. 9 . The biogenesis of natural auxins, pp. 65-75. In: R. L. Wain and F. Wightman (eds.). The Chemistry and Mode of Action of Plant Growth Sub- stances. Butterworth Sci. Publ., London. 1956. 10. . Auxin biosynthesis — a cytoplasmic locus of radiation damage, pp. 44_47. /„: J. s. Mitchell, B. E. Holmes, and C. L. Smith (eds.). Progress in Radiobiology. OUver and Boyd, Edinburgh and London. 1956. 11. Gordon, S. A., and Sanchez Nieva, F. The biosynthesis of auxin in the vegetative pineapple. L Nature of the active auxin. Arch. Biochem. 20: 356-366. 1949. 12. The biosynthesis of auxin in the vegetative pineapple. IL The precursors of indoleacetic acid. Arch. Biochem. 20: 367-385. 1949. 13. Gray, Reed A. Preparation, chemical and biological properties of pure 3-indole acetaldehyde. Plant Physiol. 33 (suppl.) : v. 1958. 14. Preparation and properties of 3-indoleacetaldehyde. Arch. Biochem. Biophys. 81: 480-488. 1959. 15. Harley-Mason, J., and Archer, A. A. P. G. Use of p-dimethylaminocinnamalde- hyde as a spray reagent for indole derivatives on paper chromatograms. Biochem. Jour. 69: 60 P. 1958. 16. Hemberg, T. Studies of auxins and growth-inhibiting substances in the potato tuber and their significance with regard to its rest-period. Acta Horti Berg. 14: 133-220. 1947. 17. Housley, S., and Bentley, Joyce A. Studies in plant growth hormones. IV. Chro- matography of hormones and hormone precursors in cabbage. Jour. Exper. Bot. 7: 219-238. 1956. 18. Jones, E. R. H., Henbest, H. B., Smith, G. F., and Bentley, Joyce A. 3-Indolylace- tonitrile: a naturally occurring plant growth hormone. Nature. 169: 485-487. 1952. 19. Larsen, Poul. Skototenin, ein neuer Streckungswuchsstoff in hoheren Pflanzen. Naturwis. 27: 549, 550. 1939. 20. Beta-Indolyl-Acetaldehyd als Streckungswuchsstoff in hoheren Pflanzen. Bot. Tidsskr. 46: 146, 147. 1943. 21. 3-Indole acetaldehyde as a growth hormone in higher plants. Dansk. Bot. Ark. 11 (9): 1-132. 1944. 22. . Formation, occurrence, and inactivation of growth substances. Ann. Rev. Plant Physiol. 2: 169-198. 1951. 23. . Growth substances in higher plant, pp. 565-625. In: K. Paech and M. V. Tracy (eds.), Modern Methods of Plant Analysis. 3. Springer-Verlag, Heidel- berg. 1955. 24. Linser, H. Chemical configuration and action of different growth substances and growth inhibitors: new experiments with the paste method, pp. 141-158. In: R. L. Wain and F. Wightman (eds.). The Chemisti^ and Mode of Action of Plant Growth Substances. Butterworth Sci. Publ., London. 1956. 25. , Kiermayer, O., and Youssef, E. Der Wuchsstoffgehalt verschiedener Brassica-Pflanzen in Abhangigkeit von ihrem Entwicklungszustand. Planta. 52: 173-186. 1958. 26. , and Machek, F. Kolorimetrische und biologische Bestimmung sowie chromatographische Trennung von Wuchsstoffen aus Pflanzen. Planta. 41: 567-588. 1953. 54 P. Lay sen and T. Aasheim 27. Nitsch, J. P. Methods for the investigation of natural auxins and grouth in- hibitors, pp. 3-31. In: R. L. Wain and F. Wightman (eds.), The Chemistry and Mode of Action of Plant Growth Substances. Butterworth Sci. Publ., London. 1956. 28. , and Nitsch, C. The separation of natural plant growth substances by paper chromatography. Beitr. Biol. Pfl. 31: 387^08. 1955. 29. Weller, L. E., Wittwer, S. H., and Sell, H. M. The detection of 3-indoleacetic acid in cauliflower heads. Chromatographic behavior of some indole compounds. Jour. Amer. Chem. Soc. 76: 629, 630. 1954. 30. Wiedow-Patzold, Hanne-Lore. Studien iiber die "neutrale Phase" von Wuchsstof- fextrakten. Ber. Deutsch. Bot. Ges. 68: 219-222. 1955. 31. Yamaki, T., and Nakamura, K. Formation of indoleacetic acid in maize embryo, Sci. Pap. Coll. Gen. Educ. Univ. Tokyo. 2: 81-98. 1952. DISCUSSION Dr. Thimann: How do you account for the disappearance of activity in the various fractions? What happens in the soil? Dr. Larsen: As regards lAAld, I imagine that it diffuses from the agar platelet into the soil where we know it becomes oxidized to lA.A.. lAA will diffuse from the soil to the agar platelet, thereby increasing the auxin activity of the agar. But if we leave the soil in contact with the agar for a time considerably longer than 90 mintites, the auxin activity decreases again, indicating that lAA is being inactivated by the soil. The inactivation must be assumed to take place also during the time when the auxin activity in the agar is increasing, but we have a steady state (lAAld-^ IAA-> inactive products) for some time. Plotted against time, the auxin activity shows a broad optimiun around 90 minutes after application of the soil. As regards IAN, there are two possibilities. (1) If we assume that IAN is converted to lAA by the soil, this conversion Avill not be mani- fested as an increase in activity, because these two substances are equally active in our test. On the contrary, as soon as some IA.\ has been formed, it will be subject to inactivation, thus loAsering the total auxin activity of the agar-soil system. (2) IAN may be inactivated ^\•ithout a preceding conversion to lAA. In both cases the auxin activity will be steadily decreasing. Dr. Bcnnet-Clark: Why did you use soil in preference to one of the conventional oxidizing agents such as alkaline iodine solutions or hydrogen pcroxitle? Dr. Larsen: Because the soil treatment was a simple procedinc. and because we wanted to show that the synthetic lAAld reactetl in the same way as the material in plant extracts which we had studied in the past. But of course it will be important to accomplish the oxida- tion also by other means, such as aldehyde dehydrogenase or inorganic oxidizing agents. Gray has shown that his synthetic I.\Ald can be Indole-3-acetaldehydc in Plant Extracts 55 converted to lAA by oxidation with permanganate, silver oxide, or Inclrogen peroxide, but the lAA is subject to break-down by the chemicals employed. In my own previous work with neutral pea ex- tracts, inorganic oxidants have failed to yield lAA in quantities that could be demonstrated, but the chances may be better now that we have a method to isolate lAAld from plant material by means of paper chroma tography . DONALD G. CROSBY Union Carbide Chemicals Company A. J. VLITOSi Boyce Thompson Institute New Auxins From 'Maryland Mammoth.' Tobacco Indole derivatives have held a position of pre-eminence in the field of natural auxins for many years. In fact, they have been regarded by many workers to constitute probably the only group of growth regulators to occur in nature. At the present time, the only two in- dole derivatives possessing pronounced growth-promoting activity to have been isolated from plants are indole-3-acetic acid (lAA) and in- dole-3-acetonitrile (IAN), although there is evidence for the occurrence of a variety of other 3-substittited indoles. Paper chromatography has emerged as a versatile method for the resolution of the complex chemical mixtures occurring in plant ex- tracts. With the development of sensitive and rapid techniques for the bioassay of growth substances, this has resulted in our ability to measure "hormone profiles" which present an indication of the extent to which different growth-regulating substances occur in extracts of plant material. Considerable effort has been made to ascribe the areas of growth stimulation of such profiles to lAA, IAN, and the other indoles. An increasing number of hormone profiles has been determined for different plant species growing under a wide variety of conditions. However, continued refinement of technique has caused their inter- pretation in terms of the known indolic growth substances to become increasingly difficult. Many investigations have failed to show the presence of the known indoles, while others have revealed growth activity in chromatogram areas which do not correspond to classical growth substances (1). The work described in this paper, which started with an investigation of one such case, has led to the discovery of two ^ Subsequently: Central Agricultural Research Station, Caroni, Ltd., and Ste. Madeline Sugar Company, Ltd., Waterloo Estate, Carapichaima, Trinidad, W.I. [57] 58 D. G. Crosby and A. J. Vlitos new types of naturally occurring nonindolic growth substances ^vhich easily might be confused with lAA and IAN on paper chromatograms. AUXINS IN MARYLAND MAMMOTH' TOBACCO During the period of 1954 to 1956, Vlitos and co-workers (10, 11) studied, with the aid of paper chromatography, the relationship of naturally occurring indole compounds to flowering in photoinduced plants. It was found that a substantial increase in the levels of lAA and indole-3-pyruvic acid occurred after photoinduction of a short- day soybean (Glycine max, 'Biloxi') . However, extension of the in- vestigation to a short-day variety of tobacco (Nicotiana tahacum, 'Maryland Mammoth') revealed that neither lAA nor IAN could be detected in leaves and apical tissue of these plants. A material similar to, but not identical with, IAN in its chromatographic and colorimetric characteristics was observed, and bioassay of chromatogram areas con- taining this substance indicated the presence of a growth stimulant. Several factors were responsible for the decision to attempt isola- tion of the growth-promoting substances of 'Maryland Mammoth' to- bacco. The unusual vigor and rapid growth rate of this variety have long been intriguing. Inability to detect the classical auxins, and the presence of the unidentified indole, suggested that an unusual type of hormonal growth regulation might be in operation. In order to obtain sufficient material for the isolation, seedling tobacco plants were transplanted to a farm near Winfield, VV^est Vir- ginia. After 3 months of growth, the leaves and apical tissue were harvested, frozen in solid carbon dioxide, and transported to the re- search laboratories of the Union Carbide Chemicals Company. The frozen material was ground in a large, cooled mill, extracted with absolute alcohol, and the extract was treated by a modification of the methods reported previously (9,11). The total fresh weight of the tobacco used was 2,300 lbs. The bioassay method of Nitsch and Nitsch (7) was used for meas- urement of growth-promoting activity. Sections of first internodes of dark-grown oat seedlings (Avenn sativa, 'Brighton') , 4 nun. in length, were rotated for about 20 hours in citrate-phosphate buffer (pH 5.0) containing 2 per cent sucrose. The final length of the sec- tions was measured with the aid of a photographic enlarger, and the resulting data were subjected to statistic al analysis — a step which was found to be very important in obtaining significant hormone pro- files. The shaded areas on histograms drawn from these data indicate responses significant at the 1 per cent level. Hormone profiles of 'Maryland Mammoth' tobacco were obtained by extraction of the homogenized leaves and apical tissue in the usual Neio Auxins From 'Maryland Mammoth' Tobacco 59 way with ethanol, followed by removal of the alcohol, extraction of the residual solution with ether, and chromatography of the two layers separately on paper using isopropanol-ammonia-water (80:5: 15) as solvent. Figure 1 reveals the presence of ether-soluble growth substances having Rf values identical with those of lAA and IAN. However, the area of greatest growth-promoting activity (R^ 0.5 to 0.6) failed to give the typical indole color reactions, while the areas above Rf 0.9 gave only a very faint color, as described previously (11). o o o o UJ o QL UJ QL x" t- o q: CD 320 240 160 80 lAA N. TABACUM IAN 0.2 0.4 0.6 0.8 1.0 Rf Fig. 1. Ether-soluble growth substances from N. tabacum L., 'Maryland Mammoth/ as detected by the Avena first internode assay. The shaded areas indicate responses significant at the 1 per cent level. 60 D. G. Crosby a?id A. J. Vlitos The profile of the water fraction (Figure 2) is very simple. Based on results to be described later in this paper, there is reason to believe that the activity observed at Rf 0.5 to 0.6 is due to the same substance responsible for that at the same place on chromatograms of the ether fraction. The cause of the activity at Rf 0.3 is not yet known. No in- dolic compounds could be detected on the chromatograms. The materials responsible for activity in these t^vo areas Avere con- centrated on paper chromatograms, eluted with methanol, and re- chromatographed in both water and isopropanol-ammonia-water. Rj values of the active zones still corresponded to those of lAA and IAN. The material having an Rf above 0.9 (called A) could be isolated on Whatman No. 1 paper by ascending chromatography, and on a Grycksbo filter paper column by descending chromatography. The ma- terial at Rf 0.5 to 0.6 was further concentrated on Whatman No. 17 paper. o o o o cc UJ CL o cc CD 240 160 80 « I ■ I ■ fl M lAA IAN N. TABACUM J L J L J I I L 0 0.2 0.8 1.0 0.4 0.6 Rf Fig. 2. Water-soluble growth substances from N. tabacum L., 'Maryland Mammoth. New Auxins From 'Maryland Mammoth' Tobacco (■)! After repeated fractional crystallization from anhydrous ether, con- centrate A provided a small amount of waxy white solid which melted at 70 to 72° C. and contained only carbon, hydrogen, and oxygen. Its infrared and ultraviolet spectra were those characteristic of a long- chain unbranched fatty alcohol such as 1-docosanol (melting point 73° C). Color tests for the indole nucleus were negative. Figure 3 shows the results of bioassays of the tobacco isolate and several of the common naturally occurring alcohols. All together, more than 60 long-chain compounds were examined, and Figure 4 shows the activity of one of these, 2-heptadecanol, at several concentrations. With two exceptions, activity was found to be restricted to alcohols containing from 17 to 22 carbon atoms and their acidic inorganic esters (5). Repeated precipitation of concentrate B from methanol solution with absolute ether resulted in a white crystalline material which pos- sessed pronounced auxin activity, but which rapidly became dark and gummy, even in the cold, with complete loss of activity. The crystal- line material did not melt below 300° C, and also contained no nitro- gen, phosphorus, sulfur, or halogen. Chemical studies and data from infrared, ultraviolet, and emission spectra revealed that the active material was the sodium salt of a long-chain unsaturated fatty acid. A variety of other fatty acid salts has now been bioassayed, and some 1 1 1 1 1 1 1 TOBACCO ISOLATE 1 MG /L ■f< MMMMlii^iifAMm* I-DOCOSANOL 100 MG./L. PHYTOL 100 MG./L. 1 1 1 1 1 1 80 160 240 GROWTH, PER CENT OF CONTROL 320 Fig. 3. Growth-promoting activity of several naturally occurring long-chain alcohols. 2-HEPTADECANOL 100 MG./L. 2-HEPTADECANOL 10 MG/L. 2-HEPTAOECANOL I MG./L. 2-HEPTADECANOL 0.1 MG./L. _L 0 80 160 240 GROWTH, PER CENT OF CONTROL Fig. 4. Growth -promoting activity of 2-heptadecanol. 320 ISOLATE 100 MG/L. ISOLATE 10 MG/L. ISOLATE I MG/L. ISOLATE 0.1 MG./L. ISOLATE 0.01 MG./L. lAA 0.1 MG./L. (I0"^M) _L J_ _L _L -L. 100 200 300 400 GROWTH, PER CENT OF CONTROL 500 600 FiR. 5. Growtli promoting activity of the long-chain fatty acid from N. tabacum L., 'Maryland Mammoth." New Auxins from 'Maryland Mammoth' Tobacco 63 show significant growth promotion. Figure 5 indicates the activity of the tobacco compound, although the values are only relatively quan- titative due to the probable presence of impurities in the sample. The identity of the substance which lay behind all this effort — the elusive indolic material similar to IAN — has not yet been determined. So far, all attempts to isolate it have failed, and, in fact, it frequently has even escaped detection. The continued search for this substance may yield an exciting story in itself some day. DISCUSSION A number of instances have been reported (1) in which the pres- ence of lAA and IAN was suspected on the basis of bioassay and chromatographic data, but in which the characteristic color reactions of the indole nucleus could not be detected. Undoubtedly, many more such cases have gone unreported. The fact that the substances re- sponsible for growth promotion on these occasions were not exten- sively investigated indicates the dominating influence which knowl- edge of the naturally occurring indoles has had on auxin studies. It is quite possible that the substances detected in previous examples are similar to, or perhaps even identical with, the growth promoters in 'Maryland Mammoth' tobacco. The similarity of the chromatographic characteristics of these non- indolic compoimds to those of lAA and IAN certainly is coincidental. However, such similarity cannot be considered unlikely, since the pre- cision of resolution in the usual hormone profile is only 0.1 to 0.2 Rf units. Despite the great value of paper chromatography to the plant hormone field, other severe limitations of this method for the deter- mination of hormone profiles have become apparent in the present investigation. Since a biologically active area may contain more than one growth-stimulating substance, extensive efforts must be made to separate the chemical factors in each case. Certainly, Rf data cannot be used as proof, nor generally even as critical evidence of the chemi- cal structures involved. "Specific" color tests also may be deceiving. For example, an in- active indole may have the same Rf as a growth-promoting nonindolic compound, or, in the case of an active indole, the two biological effects may be superimposed. Although a great deal of valuable in- formation obviously can be obtained by thorough chromatographic analysis of plant materials, rigorous proof of the structure of the com- pounds present probably can come only through actual isolation and chemical study. It is apparent from the examination of 'Maryland Mammoth' to- bacco that nonindolic growth-promoting compounds may be iso- 64 D. G. Crosby and A. J. Vlitos lated from plant extracts. Since it has not been possible to demon- strate the presence of either of the two principal known indolic auxins, this tobacco variety may present one example of a hormone system in which indoles are not involved, or in which at least they do not play a major role. Other examples have been found in rapidly growing species of bamboo. Figure 6 presents a hormone profile of Bambusa multiplex obtained in the same w^ay as those of tobacco. In this case, the ether fraction showed neither activity nor the presence of indolic com- pounds. The aqueous fraction represented in the figure exhibited a high level of activity, again at about Rf 0.5, although chromogenic reactions suggested the presence of an indole at Rf 0.41. The hor- mone profile of an aqueous acetone extract of the bamboo Sinocala- miis oldJiami was very similar to that of B. 77iultiplex. 240 o on o o u. o z UJ o q: UJ Q. o o 160 80 I: 1 1 • ••**!*!*1*/I' J L _L lAA B. MULTIPLEX J I L AN J L 0.2 0.4 0.6 Rf FiR. 6. Water-soluble growth substances from Bambusa iiudtiplex 0.8 1.0 New Auxins From 'Maryland Mammoth' Tobacco 65 The possibility that the growth-promoting substances isolated from 'Maryland Mammoth' tobacco are artifacts cannot yet be ex- cluded. The extensive work of Chibnall et al. (4) and others has shown that long-chain alcohols occur widely in both the cuticle and cellular waxes of plants (12) , although the wax of tobacco was found to be associated primarily with the cell (4). The occurrence of a great va- riety of unsaturated fatty acids in plant tissues also is well established. However, these two types of substances are interesting in their own right. The comparatively narrow range of activity in the series of long-chain alcohols is noteworthy, since there exists very little differ- ence in solubility, chemical reactivity, and other physical and chemi- cal properties between the inactive l-hexadecanol and the active 1- octadecanol. The physiological effects of long-chain fatty acids have been re- ported previously by several workers. Haagen-Smit and Viglierchio (6) found that several of these compounds, such as myristic acid and linoleic acid, were active in the Wehnelt bean test for wound hor- mones. Stowe (8) reported that several long-chain fatty esters, includ- ing a preparation isolated from 'Alaska' peas, were active in stimu- lating growth in pea epicotyl sections, although they were inactive on oat coleoptiles. However, the degree of activity of the tobacco iso- late is unusual and suggests that it may possess peculiar structural features not now appreciated as being important to growth-regulatory activity. The importance of long-chain unsaturated fatty acids such as lino- lenic acid, arachidonic acid, and docosahexaenoic acid in animal nutri- tion is becoming increasingly evident upon continued investigation. In recent months, two similar compounds important to the lives of insects have been reported: Butenandt et al. (2) have shown that the sexual attractant of the silk worm (Bombyx mori) is the unsaturated, primary alcohol 10,12-hexadecadiene-l-ol, while the active constituent of the royal jelly of the honey bee has been identified as 10-hydroxy- decenoic acid (3). The results of the investigation of 'Maryland Mam- moth' tobacco strongly indicate that this type of compound may prove to hold an equally important place in the plant world. SUMMARY A paper-chromatographic study of growth-promoting substances from 'Maryland Mammoth' tobacco (Nicotiana tabacum L.) revealed the presence of two compounds which, although they exhibited chro- matographic behavior similar to that of indole-3-acetic acid and in- dole-3-acetonitrile, were found to be nonindolic. Isolation and char- acterization provided evidence that one active material consisted 66 D. G. Crosby and A. J. Vlitos principally of the long-chain alcohol I-docosanol, while the other was the sodium salt of a long-chain unsaturated fatty acid. These find- ings emphasize the necessity for actual isolation and chemical study of natural growth substances and the importance of long-chain com- pounds to plant life. ACKNOWLEDGMENT The authors wish to express their gratitude to the many people in the Research Department of the Union Carbide Chemicals Com- pany and the Boyce Thompson Institute for Plant Research Avho contributed to this effort. In particular, the skillful services of Messrs. R. V. Berthold, H. G. Cutler, I. D. J. Phillips, and Werner Meudt are gratefully acknowledged. LITERATURE CITED 1. Bentley, J. A. The naturally-occurring auxins and inhibitors. Ann. Rev. Plant Physiol. 9: 47-80. 1958. 2. Butenandt, A., Beckmann, R., Stamm, D., and Hecker, E. Uber den Sexual- Lockstoff des Seidenspinners Bombyx mori. Reindarstellung und Konstitution. Zeitschr. Naturforsch. 14b: 283, 284. 1959. 3. , and Rembold, H. Uber den Weiselzellenfuttersaft der Honigbiene I. Isolierung, Konstitutionserraittlung und Vorkommen den IO-Hydio\y-A--decen- saure. Zeitschr. Physiol. Chem. 308: 284-289. 1957. 4. Chibnall, A. C, Piper, S. H., Pollard, A., Williams, E. F., and Sahai, P. N. The constitution of the primary alcohols, fatty acids and paraffins present in plant and insect waxes. Biochem. Jour. 28: 2188-2208. 1934. 5. Crosby, D. G., and Vlitos, A. J. Growth substances from Maryland Mammoth tobacco: long chain alcohols. Contr. Boyce Thompson Inst. 20: 283-292. 1959. 6. Haagen-Smit, A. J., and Viglierchio, D. R. Investigation of plant wound hormones. Rec. Trav. Chim. 74: 1197-1206. 1955. 7. Nitsch, J. P., and Nitsch, C. Studies on the growth of coleoptile and first in- ternode sections. A new, sensitive, straight-growth test for auxins. Plain Phys- iol. 31: 94-111. 1956. 8. Stowe, B. B. Growth promotion in pea cpicotyl sections by fattv acid esters. Science. 128. 421-123. 1958. 9. Vlitos, A. J., and Meudt, W. The role of auxin in plant flowering. II. Meth- ods for the extraction and quantitative chemical determination of free 3-in- doloacetic acid and other indole compounds from plant tissues. Contr. Boyce ihompson Inst. 17: 401-Ul. 1954. 10. , and Meudt, W. The role of auxin in plant flowering. III. Free indole acids in short-day plants grown muler photoinductivc and nonphotoinductive daylengths. Contr. Boyce Thompson Inst. 17: 413—117. 1954. 11. , Meudt, W., and Bcimlcr, R. The role of auxin in plant flowering. IV. A new unidentified naturally occurring indole hormone in normal and gamma irradiated Maryland Mammoth tobacco. C^ontr. Boyce Thompson Inst. 18: 283- 293. 1956. 12. Warth, A. II. The Chemistry and Technology of Waxes. 2nd ed. 910 pp. Reiiiliold Publ. Corp., New York. 1956. Neiu Auxins Fiuin 'Maryland MammoUi Tobacco 67 DISCUSSION Dr. Stowe: I am interested in this rejjort because we obtained much the same results in pea straight-growth assays, testing a number of higher alkyl lipide compounds. In the pea section-growth assay, it is necessary for auxin to be present if growth acceleration is to be found. May I ask if lAA or other auxins were introduced in your assay? Dr. Crosby: No exogenous lAA was ever introduced. Dr. Wain: Reference has been made to the occurrence of plant waxes. We all know that these are in the main esters of long-chain fatty acids and long-chain monohydric alcohols. Obviously, with this large amount of material a good deal of wax would be extracted from the tobacco. It is rather significant that one gets here not only an alcohol but an acid. Does the possibility exist that these two com- pounds have arisen from the cuticular wax? Dr. Crosby: From its configuration, our long-chain fatty acid must be an extremely unstable one. It is possible, but I would be sur- prised if this were the case. We would never say that these materials positively had an importance to the plant, but they are of interest to us in that they stimulate growth. Dr. Kefford: My particular interest in the auxins of 'Maryland Mammoth' tobacco is the presence or absence of lAA. The most spe- cific auxin test is the Avena curvature test and, to date, of the natu- rally occurring auxins only lAA has been found to give this test. I have been able to detect activity with this test in extracts of 'Mary- land Mammoth' tobacco following repeated chromatography. But the activity is very small - about 1/40 of the activity found in other to- bacco varieties. Dr. Vlitos: I would like to say that we have not tested the fatty alcohols in the Avena curvature test, although Dr. Kefford, through correspondence, has many times urged us to do so. I feel that w'e have used the most rigorous type of chemical evidence to identify a naturally occurring auxin, and I have not felt it necessary to rely on what is perhaps a nonspecific biological assay. Since we were using the Avena first internode test which seemed to be responsive to the alcohols, and it looked as though we were getting a classical growth dosage response in that particular assay, we relied entirely on chemi- cal identification. I would say I feel that there are very few instances where growth substances have been isolated in crystalline form and shown by a series of rigorous chemical methods to be a particular substance. We relied neither on a bioassay nor on a colorimetric test. 68 D. G. Crosby and A. J. Vlitos Dr. Galston: We have been interested in the differences in grow th characteristics between completely etiolated pea sections and those which have received a prior exposure to morphogenically active red light. It has been reported that the surface characteristics of peas and of other plants could be markedly affected by the degree of prior ex- posing to red light. By an ingenious carbon-casting technique, flakes and scales were seen on the surface of plants which were waxes. They may contain some of these growth-active materials. Are we, by virtue of prior red light exposure, inducing the synthesis of more cuticular wax on the surface of these sections? Are we then producing, in a sense, artifactual growth promotions or inhibitions? Dr. Crosby: Dr. Galston, I think this is likely. One problem which we have had in dealing with compounds such as docosanol and octa- decanol is that we have no way to measure their aqueous solubility. They are extremely insoluble; even surface tension measurements can- not determine their solubility. How, then, can they act as growth stimulants? In our bioassay, we rotate the sections for 20 hrs. and, certainly, each section must become evenly coated with the alcohol as it comes to the surface. It is interesting that we get a very definite change of activity with concentration. This has been repeated many times and on different occasions over a period of many months. \Vhat is the cause of this growth stimulation? Perhaps the thickness of the waxy material influences directly the growth of the sections. We don't know. Dr. Thimann: Would any of these active materials be present in lanolin that is normally used in the laboratory? Lanolin has long been known to produce some small amount of growth and cell divi- sion. Dr. Crosby: The esters of the long-chain fatty acids and straight- chain fatly alcohols would be present as minor constituents in lanolin. Dr. Bitancourt: Is there any interaction between the fatty acids and lAA? 1 ask because in attempts to get solutions of lAA that would not decompose over a long period, we used air-free water cov- ered by a layer of paraffin oil. Instead of getting the stability that we expected, we found that our solutions decomposed more rapidly or as rapidly as those that hadn't been protected. I am wondering whetlier there coufil f)e some chemical change in lAA induced by the hytlrocarbons from jjaraflin oils. The decomposition was clearly dif- ferent from that wfiufi occurred in aerated solutions where we got a brownish coloration, whereas we got a beautiful red coloration in tlic- sohuion iliat was piotected f)y paraffm oil. Dr. Crosby: W'c have not tried such experiments on a chemical basis. Ohl- would not c\p(.( I a long-chain fatty alcohol such as doco- sanol U) stabili/c 1A.\. On the other hand, salts of our long-chain Nexu Auxins From 'Maryland Mammoth' Tobacco 09 unsaturated fatty acid might be expected to do so. These compounds, particularly in the configuration we believe ours has, do act as anti- oxidants. Dr. Housley: Is it possible that this type of compound influences cell division and that the growth you have been getting has reflected this action rather than an effect on cell elongation? Dr. Crosby: We have not carried out microscopic examination of these materials. We do note that the sections elongate; they do not seem to grow in bulk. Dr. Osborne: Do the substances which have been isolated from 'Maryland Mammoth' tobacco have any effect on accelerating abscis- sion? In the Agriculture Department in Oxford we are trying, with Prof. E. R. H. Jones, to isolate this abscission-accelerating factor. It seems likely, from what we know so far of the compound, that it might fit in with your findings. As a subsidiary question, may I ask if there was a large number of very old leaves in the ton of material you extracted? Dr. Crosby: The plants were close to the flowering stage. We took only the younger, bright green leaves and the apical tissue. WVt have not investigated the effects of these materials on abscission. Dr. Fawcett: Activity in the wheat coleoptile test is shown by certain compounds which do not possess a ring structure (Nature. 178: 972. 1956). Ethanol, ethylenediaminetetraacetic acid, certain xan- thates, and chloroalkanecarboxylic acids were cited as examples. All these compounds had the same low order of activity and their opti- mum activity was given at a concentration just below the level at which toxic symptoms were observed. Since they were inactive in the pea curvature and tomato leaf epinasty tests and showed some other common features, their growth-regulating activity was regarded as nontypical — possibly resulting from subacute toxicity. S-carboxy- methyl N.N-dimethylaminodithiocarbamate, however, was highly ac- tive in the wheat, pea, and tomato tests, and we think this type of nonring structure can induce a typical auxin response. Have you tested the lower members of this homologous series of alcohols? Dr. Crosby: No, we started our examination with the C-10 alco- hol. I think the physical and chemical properties of the alcohols be- low C-10 fall somewhat into one class, and the properties of those greater than C-10 fall into a different class. Dr. Thimann: It was shown recently that many algae are ex- tremely sensitive to ethanol and respond to quite low concentrations. The indication was that it perhaps acts as a nutrient but I would say in several instances, effects on algal growth ascribed to lAA were really due to the ethanol in which it was dissolved. C. H. FAWCETT R. L. WAIN F. WIGHTMAN^ Wye College, University of London Chromatographic InvestLgations on the Metabolism, of Certain Indole Acids and Their Amides, Nitriles, and Methyl Esters in Wheat and Pea Tissues'" Research at Wye has been concerned for several years with the metab- olism in various plant tissues of homologous series of chloro- and methyl-substituted phenoxyalkanecarboxylic acids (4, 5, 9). Chemical and biological evidence has been obtained that the side-chain of such co-substituted fatty acids can be degraded by (3-oxidation, and although the capacity to effect this breakdown is common to many plants, spec- ificity has been observed in some species to the members of certain series. In this connection, the results indicate that the number and orientation of the nuclear substituents affect the ease with which ^- oxidation occurs at the shorter side-chain lengths, and these findings provide an explanation for the different patterns of growth-regulat- ing activity shown by the many homologous series examined. In a related study the growth-regulating activity and metabolism in wheat and pea tissues of the first six members of the homologous series of co-(2,4-dichlorophenoxy)alkanecarboxylic acids and their cor- responding amides and nitriles were investigated (6,7) . The pattern of activity shown by the acid and amide series was found to be simi- lar, and chromatographic examination of the treated tissues showed that the amides, after hydrolysis to the corresponding acid, were de- graded by (3-oxidation in a manner identical to that observed with ^Subsequently: Prairie Regional Research Laboratory, Saskatoon, Saskatche- wan, Canada. ^This paper was read at the Conference by F. Wightman. [71] 72 Fawcett, Wain, and Wightman members of the acid series. The nitriles, however, showed exceptional behavior in both the tissues employed. In pea tissue, only the first member of the series (i.e., 2,4-dichlorophenoxyacetonitrile) showed evidence of hydrolysis to the corresponding carboxylic acid, whereas in wheat tissue, all homologues produced not only the corresponding acid, but also the next lower carboxylic acid. In the case of the higher nitriles, subsequent ^-oxidation of one or other of these acids resulted in the production of the highly active 2,4-dichlorophenoxyacetic acid. This behavior of the nitriles in wheat tissue is explicable in terms of an initial modification of the -CHoCN group by two mechanisms, namely, either hydrolysis to the corresponding acid or conversion to the lower carboxylic acid with the loss of a one-carbon fragment. The latter type of breakdown was referred to as a-oxidation of nitriles, and further evidence for this reaction in plant metabolism has been obtained from studies on the degradation of indole-3-acetonitrile in wheat, pea, tomato, maize, and celery tissues (8). a-Oxidation of this nitrile yields indole-3-carboxylic acid as end product, and the occur- rence of indole-3-aldehyde as an intermediate has been established (7) . In view of the importance of indole compoinids and in particular indole-3-acetic acid and indole-3-acetonitrile in relation to the hor- monal control of plant growth, it was logical to extend the above in- vestigations by examining the growth-regulating activity of a homolo- gous series of indole-3-alkanecarboxylic acids together with the cor- responding amides, nitriles, and methyl esters. This has been carried out using the wheat cylinder, pea segment, and pea curvature tests, and a study has been made of the metabolism of these compoimds in pea and wheat tissues. It is with the results from the latter aspect of the work that this paper is mainly concerned. Briefly the metabolism experiments involved exposing solutions of the various indole com- pounds to wheat coleoptile or pea stem tissues with subsequent ex- traction and paper chromatographic separation of the metabolic products present in the tissues and in the residual solution. After de- velopment, the chromatograms were examined by chromogenic and biological methods. MATERIALS AND METHODS The compounds examined are given in Table 1 together with their uncorrected melting points, Rf values in two different solvent systems, ultraviolet fluorescence characteristics, and chromogenic reac- tions with three reagents. The growth-regulating activity of each of these substances was as- sessed in the wheat cylinder, pea segment, and pea curvature tests. ■a c o u a u bD a T3 • ^ o ea CO 03 3 C O V u bjp +-J V ;z; V V V V V ^■ir b€ tJD tuD b€ bJD ^^ C G C C C T3 "H t^ c^ c^ c^ c^ 0^ CJ I-l J-H U I-( U pioiOOOOO U / — - u E • nj ™ ' — " — ~ ■ £ be E G i^ ej V , bo u !-. -C I be.22 JJ e tie .boJi S c a c c ^ ^ ^ ^ 0000 U ^ U i-. J2X1 J2 XJ till OJ iSSSM ,-~, s_ U !-c W5 ' — — ^^T^ — ' D U OJ I 'a.'H.'a O •„ i- u 33 3 3 3 jj a cu a '-' XI -3 .3 ■^^^^^ Pnpa WCQP3 3 "a XJ ^ eft ■■ — ' ^— ^ v^'-v ^'cii i:iii O IJ Q._2 D W U — ^ IJ '" 3 -J 3 3 O '- 3 Xl ^ -3 — S-c Sh ;i3 o ID 3 m U U I; U W _3 _3 _3 _3 _3 _3 3 3 3 3 3 3 4-1 ■|-> •(-•-«-> '4-' -W i> X -c j3 X j= x: 3 bo bo 5f:.^''.5p.Sf' S J J J J ^ J 3 X «J bp_3 _^ _ 3 be _3 3 ■ X •"c _3 3 x: 3 ^ 5 ,2 ^ ^ X2 JD OScqS u x; X X X 3 be bo bo bo 6 JJ > 6 3 J J J J c > i—H O O 3 3 P5 Cn lO ^— I 00 10 rO O ■^ CN CO rO ^ tn vO CM lO 00 CM O t^ r~- r- 00 00 o CM m -^ ■* CTn C^ CN Cn CN •^ CM CO -^ m o vo CN CN C^ CS C^ CN CN 0000000 00000 00000 0000000 _ I c ) fS o 00 o o ■* ■^ O Ln CM CO ■* lo o r-- r- -o O CM o Cn r~- r-- 00 00 00 -o r^ 00 o ■^ CO 00 00 CN CN IT) vO I~~ C^ '— I cO ■^ 00 00 00 00 CN O CN 0000000 00000 00000 0000000 c o Ph . beO .2 13 bo X 3 LT) in CO O ■* '^ o ■^ CM vO CO (M ^ CA I 7 7 T 7 u-i CM to CO CO lO CN CM O CO CM O CO O (M ^ ^ ^ ^ ^ as !■ CM CM ■^ •^ CN ■^ 10 LO O ^ CN 1 't r- I LO uo r--- LO CO I -11 •rj- ^- 00 CO \0 ^- LO r-~ r~ lo o 00 I be bo s a s s 0 0 O LO O ^ O "- CM . 'O . . a_L a. a LO O 00 ■ CO \0 Cv I I r~- "^ f^ « ■ ^ r-, ■^ CO \0 J2 LO X XJ T3 c 3 o a B o O I— I < < M^-O << 5^ " -T-l O 03 u ■1-' a a O J2 I I I I I CO CO CO CO CO I I I I I 1; gj aj a; C; 3 3 3 3 3 "O -a "O -O T3 C C 3 C 3 X o u < 3 ^ 3 ^ O XI S-( P3 X X-o^i^ 03 -. .-. 3 3 3 O =« § O n— - u 4J 1) cfl 03 o ti — i:; S <« (U o X2 u ■•^ ^ 3 03 t^ CLX > CO CO-7; -S-; O O -0 -a c a _ M oa c^-o '^ K^ V I I ^ O CO CO rO Cfl I ■ I 1 1 lU lU OJ CO CO "tS -:; --5 1 1 O O O V V -a ~a -a S'o G c 3 -a -^ " !ri.!r:^ 3 3 I 1 I !_, _i '53. (>-'« 4J 1 11' O rO CO cO 03 I < I , V V V CO CO -7; -7; — 1 I o o o U U T3 TJ TJ 3 3.S S c .S .S 'Q- ?-'« X X X x; -3 .«— I -4— > ■!>-> 4->* -t-* 4J QJ 1^ flj "U ►7 CO CO "S -= -5 -= — 2SSSS .S^ci^^ o 3 3 3 CO CO CO CO CO I I I 1 I V V V p 3 3 T3 T3 T) C 3 3 V V o3 -a -a 3 3 o c 3 3 (U 6 a 0 t) > V TJ u 0 0 0 T3 a. 03 L.C eft , ^ (U 0. 3 3 a -a 0 , — ^ ■0 u ^— ' 3 • » c« eft X U •^ X u ■*-< hn !-! 3 i; 0 03 • ' a -«-• 3 3 T) a D 0. 03 0 0 0) T3 > 11) C T3 eft e<-i 0 eft 4J 3 hr R3 i to u 0 u eft > rt 0 X 4; rt 0 1; (-■ X (1) -n ^^ 03 u he a 0 0 rt CO a 0 0 u 0 J3 (J 4_, he 3 n t; '>- E ffl a u c:i a en > u 9J 0 3 a T3 w w -0 a u n 03 0 0 e^ n 0 3 1) u ■t-' -0 t« « a X "^ — ^ H ^ CM 74 Fawcett, Wain, and IVightmati The compounds were dissolved in water containing 0.1 per cent ace- tone and were examined at five concentrations ranging from lO'^M to 10-8M. Wheat Cylinder Test Ten-mm. sections excised from 2-cm. coleoptiles of 3-day-old wheat seedlings ('Eclipse') were used as the experimental material. The sections were threaded on glass capillaries and floated on the test solutions contained in petri dishes, ten sections per dish. After 24 hrs. treatment at 25° C, the length of the test sections was determined and the results are presented as a percentage of the final length of water controls. Pea Segment Test Five-mm. segments excised from the second internode of 6-day-old pea plants ('Alaska') were used as test material. The seedlings were grown in sand under red light at 25° C, and the second internode was approximately 2 cm. in length when the test segment was re- moved. Batches of ten segments were placed in petri dishes on filter- paper bridges supported by two glass rods, the ends of the filter paper dipping into the test solution and serving as a wick for supplying solution to the segments. After 24 hrs. treatment at 25° C, the seg- ments were measured under a microscope and the results are again presented as a percentage of the final length of water controls. Pea Curvature Test Three-cm. segments were excised from the second internode of young pea plants ('Alaska') grown for 7 days as described above. Each segment was split longitudinally with a razor blade for approximately 2 cm. through its upper elongating region, and after washing in water for 2 hrs., five split segments were placed in each test solution for 24 hrs. at 25° C. The curvatures induced were assessed by a numerical scale from 0 (inactive) to 6 (highly active) similar to that suggested by Went and Thimann (10). In the metabolism experiments, solutions of each compound were exposed to wheat coleoptilc or pea stem tissue with subsccjuent ex- traction and paper chromatograj)hic analysis of the products present in the tissue and in the residual solution. For each treatment, 100 1-cm. coleoptile segments or 50 I-cm. pea stem segments were placed in a petri dish containing 1,000 y^ig. of the compound in 50 ml. of distilled water, the solutions being then incubated in the ilark for 48 hrs. at 25° C. It was usual to metabolize 4,000 /xg. of each compound Chromatographic Investigations of Indole Compoimds 75 in one experiment, this amount being evenly distributed among four petri dishes. A tissue-in-water treatment and a solution of each com- pound untreated with tissue were included as controls in each experi- ment. Bacterial contamination was negligible in all treatments since streptomycin at a concentration of 10-*M was included in all solu- tions. After 48 hrs. the solutions together with tissue were frozen overnight at — 10° C. On the following day the solutions were thawed and the four identically treated solutions of each compound com- bined. During this process the tissue was removed and rapidly ground to a fine paste and then recombined with the residual solutions. Com- pounds present in this homogenate were removed by acidifying the system to pH 2.5 and extracting three times with 200-ml. quantities of ethyl acetate. The combined extract was dried over anhydrous so- dium sulfate and concentrated for analysis by paper chromatography. Chromatographic analysis of each extract was carried out by the ascending method using Whatman No. 1 paper in all glass tanks. The solvent used in most instances was a mixture of 77-butanol, am- monia (0.880) , and water (100:3:18 v/v) , although occasionally isopro- panol, ammonia (0.880), and water (10:1:1 v/v) was employed for comparative purposes. Temperature was controlled at 20° C. and each chromatogram was developed for 12 hrs. Chromatograms for chromogenic analysis were spotted with amounts of ethyl acetate ex- tract equivalent to 1,000 /xg. of the compound in the original solution. After development, the papers were dried in air and in most cases sprayed with Ehrlich's reagent applied as 1 per cent p-dimethyla- minobenzaldehyde dissolved in 50 per cent alcoholic HCl. Other sprays, such as the Salkowski reagent and nitrous acid reagent which were prepared by conventional methods, were also used. When preparing chromatograms for biological examination, ethyl acetate extract equivalent to 2,000 fxg. of the original compound was evenly distributed over 20 spots on a 10-inch wide sheet of Whatman No. 1 paper. After development, a longitudinal strip containing two spots was removed from one side of the chromatogram and sprayed with Ehrlich's reagent to establish the position of indole compounds. The rest of the sheet was cut transversely into twenty strips of equal size each corresponding to one-twentieth of the distance travelled by the solvent front. Each strip was thus equal to half an Rf unit, i.e., 0 to 0.05, 0.05 to 0.1, 0.1 to 0.15, etc. The strips were placed in petri dishes, one per dish, containing 10 ml. of distilled water, and the bio- logical activity of any compound present in each strip was then de- termined by the wheat cylinder test. The activity of a control strip taken from above the starting line of the chromatogram was deter- 76 Fawcett, Wain, and Wightman mined in each experiment. The results obtained are recorded in his- togram form which shows clearly the position on each chromatogram of compounds with growth-promoting activity. EXPERIMENTAL RESULTS AND DISCUSSION The activity shown by members of the series of acids, amides, ni- triles, and methyl esters in the wheat and pea tests are shown in Table 2. For convenience, the results for each series will be discussed below in separate sections together with the evidence obtained in chro- matographic studies on the metabolism of these compounds in wheat and pea tissues. Chromatographic analysis of the extracts of solutions of all the compounds incubated for 48 hrs. at 25° C. in the absence of tissue showed that with the exception of certain methyl esters, all were ef- fectively stable under these conditions. In the case of the ester series, indications were obtained to show that several of these compounds will hydrolyze to a slight extent to the corresponding carboxylic acid when solutions are left in an incubator for 48 hrs. The production of the corresponding carboxylic acid in untreated solutions, however, was in no way comparable with the amount produced when the solu- tions were exposed to wheat or pea tissues. a)-(Indole-3-)alkanecarboxylic Acids With the exception of indole-3-carboxylic acid, all members of this series are highly active in the wheat cylinder, pea segment, and pea curvature tests (Table 2) . The acetic, butyric, and caproic homo- logues are the most active members of the series, and this result is consistent with the probable degradation of the side-chain of the latter two acids by j3-oxidation to yield the highly active acetic deriva- tive. The activity shown by the propionic, valeric, and heptanoic derivatives appears, at first, to be inexplicable in terms of (3-oxidation of the side-chain within the test tissues since the expected end-product, namely indole-3-carboxylic acid, has no growth-promoting activity. When solutions of all these acids were exposed to wheat coleop- tile or pea stem tissues, the chromatograms obtained using extracts of the residual tissues and solutions provided clear evidence that (3- oxidation of the side-chain of the higher homologues had occurred ill Ijoth tissues employed (Figure 1). Thus both chromatograms showed evidence of a blue spot corresponding to the acetic acid in extracts from treated butyric and caproic acid solutions, and further- more, the caproic derivative also yielded the butyric acid presum- ably as an intermediate degradation product. Similarly, a blue spot corresponding to the propionic acid was obtained in both tissues from the valeric and heptanoic acids, the latter acid also producing the (-" ^ .-H f/1 V , — V r^ u V j2 o D « s " > D .-H 1 c; C o 03 '^ ^— < C^ c 1 ■*~> o V J2 t— " w U c o < a ai ^ OvO\OOOOvO OcOcOvO'^ OvOvO\0~0 Of^"— 'OOO'* o-^LOo-'i-om Ot-notO' O'^lDld-* O'^OOOOCM OCNcO-'t^'^O OOOcOO OcOCMcOCM OOOOOO'^ O^-hOCMOCMO OOO'—O OCMOtNO ooooooo ooooooo ooooo ot-^Ot-ho ooooooo -0 V o '-3 c c w o c o u "3 o c o u 4- O o f^2 I O cofMc^iLTiooo ocN-^fvitn OrOrOmrOmrO Oc<^CJt^(N r- o o (M o CN CO m CO cN >-<■<*■ so O 00 CN o o ^ o o CN CN ■^ — Tl- O NO CM NO -r- NO (M O ^^ CO o ■* O ■* CM NO CM T— CM O 00 lO o o o ■* o CM NO CO •^ CM O LTl lO lO lO O ■* 00 00 NO O NO u^ "^ lO u-1 NO NO -^ 00 o o O NO CM NO -"t NO NO r- O 00 -^ LO •* CM o r- CN r-- o r- NO o NO r^i o cv) o CM O "^ O NO O lO o ooooo .-■ NO ■* LO NO O LO T— NO ^-' O i~~- 00 CM o 00 un o NO CN lo o ^ CO CO CM CO in CM LD CN O ■* O CO O CO CN CM O CN] I~~- CO LO o O (N O ^ O ^ O •^ CO 00 •^ CN o o CN o o O CM 00 O •^ o o CN o o CM r~- 00 un o O •* CN in O o NO CN in 00 O CM CN •^ CN CN O CN to CN LO CO CN NO CN ' — ^ CN lO T— I CN .^ CN o CM ■r- r-- CN CO CN O O CM o u to D. c "c bo lO o a a bo V w o a > c a J2 CO > c o u II ex C o bO 2 " V ^-H 3 O c« ^ rt 00 U O -4-* ^— • ■o ■n a c c« -4—' () m HI rl u ^ CJ C3 n r- H-( o -t-* c w o T3 C 3 O a a o U -o c«.^T).ii t« o u onic ic ac c aci ^.y .y§ (^ >- ^ O P3 o ti-i a a •; 3 cfl ci3 u 0-Xl > U J3 T3 U u CCS O KJ COcO-S — — --■-> COCO CO CO CO CO CO 1, 1 1 1 1 1> t; 5j i; ^ O "o "o "o "o V f "O -a -a "O -a "O'O C C C C G c a Si ■" cfl y CO CO CO o c3 • ■ n S =^ V C O (« ^ O -^ J; i- 3 nj . . "o "o "o aj l; T3 "O "O 'o'o C C C C C - coco^-^ C G rO CO ro ■ I I V V T3 -a T3 C C G •"^ ^ — ii h |_(l— iQi^-'O "J^^ h-ll— i«i^- -4-) -M ■)-' i3j CJ CJ IJ QJ • -. O CO CO CO G rt ,' I 1 I I U 4J qj U O "O T3 T3 'o'o C G G ^ G .ti o c c o a a. a o s: CO CO 'o'o TI ID C G G G ' 1 I (—1 1— ( lO o J L J L J L J_ J L 4i J I ^ J L ooo ooo ooo ooo ooo ooo ooo (D)00 U>IOO (orOO (OIOO i0K>O <0K>O (OIOO (nOdiNOD iO %) HlMOaO N0ISN31X3 ri ■- 'X: X ^ '.J o c A 5 ;^ (T 3 3 o -;-.£ «! a- ■:3 -Ts CE r^ CO ca c 2 ^ < u < o 00 q: < < bJ U < u < a o tr a. < >- 3 u IT < > < o IT Q. < U U < u o z < I- Ol (iJ X CM .5* o CInomatographic Investigations of Indole Compounds 81 acetic acid showed that the compound occurring at low Rf possessed growth-promoting activity (Figure 2), a result which is consistent with the activity previously reported for authentic indole-3-acetylaspartic acid (1) . With the ethyl-acetate extraction technique used in this se- ries of experiments, it has been possible to demonstrate the presence of one or more of these aspartic acid condensation products in the extracts of all the pea-treated indole acids, except in that obtained from indole-3-carboxylic acid. Thus, in addition to the metabolites produced in the (3-oxidative degradation of these acids in pea tissue, all the higher homologues yielded small blue spots with low Rf val- ues which corresponded chromatographically with either indole-3- acetyl- or indole-3-propionylaspartic acids (Figure 1). co-(Indole-3-)alkanecarbonaniides With the exception of indole-3-carbonamide, all members of this series showed growth-promoting activity in the three tests employed (Table 2). Such activity is consistent with hydrolysis of these amides with the test tissue to the corresponding carboxylic acid, which may then be active per se or converted to an active product by (3-oxida- tion. Although no conversion of amide to acid occurred in the ab- sence of tissue, clear evidence for this was found on the chromato- grams obtained from metabolism experiments with both wheat and pea tissues (Figure 3) and from the bioassay results of comparable chromatograms (Figure 4). Thus, the chromatogram of metabolized acetamide and jjropionamide showed distinct blue spots (at Rf 0.24 and 0.30) when sprayed with Ehrlich's reagent, which corresponded respectively to indole-3-acetic and (3-(indole-3-) propionic acids. Each chromatogram also showed a second spot with a high Rf value which represented the unchanged amide present in the extract. The metab- olized butyramide and valeramide yielded three large blue spots on the chromatograms which corresponded with three major peaks of activity in the bioassay results. In the case of the butyramide, the first two spots correspond respectively to indole-3-acetic and -butyric acids, whereas the third spot near the solvent front was the residual amide. Similarly, on the valeramide chromatograms, the first two major spots correspond respectively to indole-3-propionic and -valeric acids and the third spot was again due to the residual amide. This amide also yielded appreciable quantities in the extracts from both wheat and pea-treated solutions of the unknown acidic compound at Rf 0.25 which gave a distinct greenish-orange spot with Ehrlich's reagent. The presence of aspartic acid conjugation products with low Rf val- ues was again observed on the chromatograms from pea metabolism experiments (Figure 3). i CONW, ANH. PnHj BnHj Vl^H, w 11 ; 0 i 1 $ • 1 • A * 1 # COMIJ, "• -_ =>NH, BNHj V^jWJ ' wm • ■V • B 5.:. VEMT F90MT Fig. 3. ClnomatojTiams of extracts of (A) uheat-trcatcd and (B) peaiicated solu- tions of w(indole-3-)alkanecaibonamidcs sprayed \vitli Ehrlich's reagent. The au- thentic acids chromaiographcd in the water plus tissue extract (W) arc, in descend- ing order, indole-3-acet\iaspartic, indolc-3carboxvlic, indolc-3-acctic. and /i- (indole- 3-)propionic acids. See I able 1 for cliemical names corresponding to the al)brevia- lions. r 82 o UJ I- < UJ or < UJ O UJ \- < UJ oc I- < UJ X J L < UJ o o o (O lO o en a: o o o (O lO o o o o (O lO o ^ IS O JD to aj ^^ B so a i ■51 o -^ - I- ■n CO UJ o < UJ -I < > bc5 [83] 84 Fawcett, Wain, and Wightman From the quantities of acids detected on these chiomatograms, as revealed by their spot size and intensity of color, it is apparent that the members of this homologous series of amides vary in their suscep- tibility to hydrolase action in wheat and pea tissues. The results in- dicate that the differences observed are related to the distance be- tween the ring system and the terminal amide group. "When these are directly attached to each other, as in 3-indolecarbonamide, only slight production of the corresponding carboxylic acid occurred, whereas when a comparatively long side-chain separates the amide group from the indole ring, as for example in S-(indole-3-)valeramide, hydrolysis of -CONHo > -COOH readily occurred. Methyl Esters of co-(indole-3-)alkanecarboxylic Acids The pattern of biological activity shown by this homologous se- ries of esters is similar to that already observed with the corresponding acids and carbonamides. All members of the series, except methyl indole-3-carboxylate, showed high growth-promoting activity in the three tests employed (Table 2). The activity of the acetate and the higher homologues is consistent with their probable conversion within the test tissue to the corresponding carboxylic acid which is followed, in the case of the butyrate and valerate, by (3-oxidation of this acid to yield respectively indole-3-acetic or (3-(indole-3-)propionic acids. The chromatograms (Figure 5) and the bioassay results (Figure 6) obtained from metabolism experiments show clear evidence for this type of degradation in wheat and pea tissues. Furthermore, the chromatographic results indicate that methyl indole-3-carboxylate is also hydrolyzed to the corresponding carboxylic acid in both these tissues. The results from pea metabolism experiments again pro\ide evi- dence for the formation of aspartic acid derivatives in this tissue. For example, in extracts of the metabolized acetate, a large blue spot was obtained at Rf 0.05 which was chromatographically identical with authentic indole-3-acetylaspartic acid. Other substances produced in metabolism experiments with this series of esters include the green- ish-orange compound at Rf 0.25 which appeared in extracts of both wheat and pea-treated propionate and valerate and also indole-3-car- boxylic acid, which occurred not only on the chroma togram from the treated carboxylate, but was also apparent in slight (]uaiititics in ex- tracts from metabolized indole-3-acetate. The chromatographic results (Figure 5) obiainctl with these esters indicate that conversion of -COOCH3 > -COOH occurs more readily as the series is ascended. When the side-chain is short, only a small amount of the corresponding carboxylic acid is produced. Chromatographic hivestigations of Indole Compounds 85 Cotv* ^y., PM. BM. VM. W^ COK. Am. -- :■■ /M, n # • 1P» Fig. 5. Chromatograms of extracts of (A) wheat-treated and (B) pea-treated solu- tions of the methyl esters of w-(indole-3-)alkanecarboxylic acids sprayed with Ehr- lich's reagent. See page 73 for chemical names corresponding to the abbreviations. whereas when the side-chain is comparatively long, hydrolysis to yield the corresponding acid readily occurs. This situation, like that al- ready observed in the corresponding homologous series of carbona- mides, clearly suggests that enzymatic hydrolysis of the terminal ester grouping is influenced by the proximity of the indole ring. ca-(Indole-3-)alkanenitriIes All of the nitrile series, except indole-3-nitrile, showed activity in the wheat cylinder test (Table 2, p. 77) . This result suggests that hydrolysis of -CN > -COOH, followed in the case of higher members of the series by (3-oxidation to yield either the highly active acetic or propionic acids, is the most probable degradation pathway for members of this series in wheat tissue. The chromatographic and bioassay results (Figures 7 and 8) obtained in wheat metabolism ex- periments support this view. Thus, in extracts of the metabolized nitrile, acetonitrile, and propionitrile, the corresponding carboxylic, acetic, and propionic acids were clearly apparent on the chromato- gram (Figure 7). Similarly, the butyro- and valero-nitriles were shown to produce not only the corresponding butyric and valeric acids, but also, respectively, the acetic and propionic acids by p-oxidation. With the capro- and heptano-nitriles, however, no indication of the pres- ence of the corresponding caproic and heptanoic acids was observed on the chromatogram, but the presence of their lower alternate homo- logues, which could have arisen from the (3-oxidation of these two o UJ I- < UJ < iij < UJ < UJ X < UJ 0^ o o o «0 lO o o o o «) ro c\j O O O «) fO o o o o iff to O o o o U> f o p ;r V X £ o o o «) lo o ("lOdlNOO io %) HlMOyO N0ISN31X3 cc UJ »- < UJ X o OD < UJ < »- UJ u < Ui < Q. O a. iij < >- »- n CD Ul I- < UJ _l < > tc- §i U V. u ^ C 5J -J-. ■5 "c <^ re 2 — ' 5/3 Ct TO *^ So re 2 S X \ to £ CInomatographic Investigations of Indole Cotnpoiinds 87 ACN PCM VCN CCM w cm Acw bCvj VCM Hew B Sou\ £NT FUCKIT Fig. 7. Chromatograms of extracts of (A) wheat-treated and (B) pea-treated solu- tions of a;-(indole-3-)alkanenitriles sprayed with Ehrlich's reagent. See Table 1 for chemical names corresponding to the abbreviations. higher acids, was clearly revealed. It is of further interest to note that the chromatographic and bioassay results with the wheat-treated aceto-, butyro- and capro-nitriles revealed traces of a compound at Rf 0.04 which from its Rf, color reactions and biological activity would appear to be indole-3-acetylaspartic acid. As previously observed (7, 8) , indole-3-acetonitrile is converted in wheat tissue not only to the corresponding acetic acid but also to in- dole-3-carboxylic acid. The latter reaction, which involves the conver- llJ J L M J L J L or ~ ■c''^ ■^ ic ■*' ~ ~ o rz Si y o ? ? V '^ _^ oo i£ "-* i) C w - UJ J L < UJ H J \ L_ J L i J I i_ J L '^-^: J L J L J L ClOdlNOO ^0 %) HlMOaO N0ISN3iX3 U < UJ _J K £ liJ _l O a U) Q. O q: Q. J L OOO OOO OOO OOO OOO OOO OOO OOO (DlOO (Oroo (flroo loioo loioo (OioO u>K>0 (OIOO O q: >- m a: O < > UJ _j CE O q: a. < UJ Z O z < I- Q. Ui X c - « c *J r. i a o " <« (T o m - <« J- X — r- ~ S 2 P Sou T~ O r^ =ii -C W "^ O W5 .^ ^ £^ cA •— £ S b .!i t« ■" w _ ■,Z « r: o T o > J •^ u ? — 00 4^ i> CO . y •£ cj Clnomatographic Investigations of Indole Compounds 89 sion of -CHoCN > -COOH and is considered an example of the a-oxidation of nitriles, might be expected to occur with all the higher homologues of the nitrile series when metabolized in wheat tissue. In each case, the -CHoCN grouping would be converted to -COOH and the resulting acid would then be subjected to (3-oxidation. The chro- matographic results (Figure 7) support this possibility and indicate that the higher indole nitriles are metabolized in wheat not only by a sequence involving initial hydrolysis of the nitrile to the corre- sponding carboxylic acid and subsequent p-oxidation, but also by a sequence involving an initial a-oxidation of the nitrile to the next lower carboxylic acid which then undergoes [5-oxidation. Evidence for these two distinct pathways is most clearly revealed on chromato- grams of the metabolized capro- and heptano-nitriles. Thus, for ex- ample, the extract of wheat-treated capronitrile yielded spots at Rf 0.26 and 0.40 which were identified as the acetic and butyric acids respectively and presumably arose via the hydrolysis followed by (3- oxidation pathway. Distinct blue spots were also evident on the chro- matogram at Rf 0.32 and 0.49, and from their Rf value, color reac- tions, and biological activity the compounds were identified as the propionic and valeric acids, respectively. The latter acid could arise from a-oxidation of the capronitrile, and the propionic acid would then be produced by |3-oxidation. A similar explanation could ac- count for the degradation products observed on the chromatogram obtained from the metabolized heptanonitrile. In the extracts of both of these higher nitriles a further compound was apparent which from its Rf value (0.20), characteristic color re- actions, and absence of biological activity was identified as indole-3- carboxylic acid. Although this acid is the logical end product of the heptanoic acid > valeric acid > propionic acid (3-oxidation sequence, earlier metabolism experiments using the authentic acids produced no evidence that this final stage occurred in wheat tissue (Figure 1). In the present experiments, however, in addition to its appearance in appreciable quantities during the metabolism of the capro- and heptano-nitriles, a small amount of indole-3-carboxylic acid was also observed on the chromatogram of the metabolized valeronitrile. Hence there is a possibility that in wheat tissue these higher nitriles are degraded by another mechanism which involves oxidation of the methylene group adjacent to the indole ring. This type of degradation reaction may be referred to as co-oxidation. The following are the results obtained with this series of nitriles in the pea segment and pea curvature tests (Table 2). Only indole-3- acetonitrile and l;-(indole-3-)heptanonitrile were active, suggesting that hydrolysis of -CN > -COOH does not readily occur with all mem- 90 Fawcett, Wain, and IVightman bers of this series in pea tissue. The chromatographic (Figure 7) and bioassay results (Figure 8) from metabolism experiments with pea tis- sue sustain this conclusion, only the acetonitrile and heptanonitrile being found to yield acidic compounds with high growth-promoting activity. The metabolized acetonitrile gave rise to three major spots on the chromatogram at Rf 0.21, 0.26, and 0.93 which were identified as indole-3-carboxylic acid, indole-3-acetic acid and indole-3-acetoni- trile respectively. Production of the acetic acid confirms the view that the activity of indole-3-acetonitrile in the pea tests is related to its conversion to this highly active acid, and the appearance of appreci- able quantities of indole-3-carboxylic acid indicates that the conver- sion -CHoCN > -COOH also readily occurs in pea tissue. Of the higher nitriles, only the heptanonitrile yielded evidence of acidic degradation products, and from their Rf value, color reaction, and bio- logical activity these compounds were identified as indole-3-acetyl- aspartic acid (Rf 0.05) , indole-3-carboxylic acid (Rf 0.20) , indole-3- acetic acid (Rf 0.25), and y-(indole-3-)butyric acid (Rf 0.38) . The acetic and butyric acids presumably arose by [3-oxidation of <,-(indole- 3-)caproic acid produced by a-oxidation of the heptanonitrile. The appearance of indole-3-carboxylic acid may be due to co-oxidation of this nitrile as occurred in wheat tissue. SUMMARY It would appear from the metabolism experiments that the growth-regulating activity shown by all the higher indole acids in wheat and pea tissue is due to the breakdown of the side-chain of each acid by (5-oxidation to yield either the highly active acetic or propionic acids as end product. The activity shown by the higher amides and methyl esters can be similarly explained, except that ^viih these compountls hydrolysis of the amide or ester grouping to the (orresjionding carboxylic acid precedes the (3-oxidative degradation reactions. The contrasting behavior of the nitrile series in the three standard tests is evidently due primarily to the different abilities of wheat and pea tissue to convert the higher nitriles either by hydroly- sis to the corresponding acid or by a-oxidation to tiie next lower carboxylic acid. Except in the case of the heptanonitrile, neither of these reactions appears to proceed in pea tissue with the higher mem- bers of this series, and in consecjuence further degradation of the side-ciiain does not occur. On the other hand, in wheat tissue the higher nitriles are subject to two and possibly three distinct degrada- tion pathways, namely (a) hydrolysis to the corresponding acid fol- lowed by (3-oxidation, (b) a-oxidation to the next lower carboxylic ;icid followed by ^-oxidation, and (c) co-oxidation to yield in all instances inclole-3-carboxylic acid. Chromatographic Investigations of Indole Compounds 91 All these results provide further evidence that, although the tis- sues of different plants can often carry out the same degradation re- actions, thus suggesting the presence of similar enzyme systems, these enzymes can nevertheless show different behavior towards a specific substrate. Studies of this type are of use in defining some of the bio- chemical reactions which are important in plant metabolism and which may be involved in the regulation of plant growth by synthetic chemicals. LITERATURE CITED 1. Andreae, W. A., and Good, N. E. The formation of indoleacetylaspartic acid in pea seedlings. Plant Physiol. 30: 380-382. 1955. 2. , and Good, N. E. Studies on 3-indoleacetic acid metabolism. IV. Con- jugation with aspartic acid and ammonia as processes in the metabolism of carboxylic acids. Plant Physiol. 32: 566-572. 1957. 3. Fang, S. C., Thiesen, P., and Butts, J. S. Metabolic studies of applied indole- acetic acid-I-C" in plant tissues as affected by light and 2,4-D treatment. Plant Physiol. 34: 26-32. 1959. 4. Fawcett, C. H., Ingram, J. M. A., and Wain, R. L. The /3-oxidation of w-phe- noxyalkylcarboxylic acids in the flax plant in relation to their plant growth- regulating activity. Proc. Roy. Soc. B. 142: 60-72. 1954. 5. , Pascal, R. M., Pybus, M. B., Taylor, H. F., Wain, R. L., and Wight- man, F. Plant growth-regulating activity in homologous series of w-phenoxyal- kanecarboxylic acids and the influence of ring substitution on their break- down by j8-oxidation within plant tissues. Proc. Roy. Soc. B. 150: 95-119. 1959. •6. , Taylor, H. F., Wain, R. L., and Wightman, F. The degradation of cer- tain phenoxy acids, amides, and nitriles within plant tissues, pp. 187-194. hi: R. L. Wain and F. Wightman (eds.), The Chemistry and Mode of Action of Plant Growth Substances. Butterworth Sci. Publ., London. 1956. 7. , Taylor, H. F., Wain, R. L., and Wightman, F. The metabolism of certain acids, amides and nitriles within plant tissues. Proc. Roy. Soc. B. 148: 543-570. 1958. 8. Seeley, R. C, Fawcett, C. H., W^ain, R. L., and Wightman, F. Chromatographic investigations on the metabolism of certain indole derivatives in plant tissues, pp. 234-247. In: R. L. Wain and F. Wightman (eds.). The Chemistry and Mode of Action of Plant Growth Substances. Butterworth Sci. Publ., London. 1956. 9. Wain, R. L., and Wightman, F. The growth-regulating activity of certain w- substituted alkyl carboxylic acids in relation to their /3-oxidation within the plant. Proc. Roy. Soc. B. 142: 525-535. 1954. 10. Went, F. W., and Thimann, K. V. Phvtohormones. 294 pp. Macmillian Co. New York. 1937. DISCUSSION Dr. Thimann: Your bioassay pictures appear to settle the ques- tion as to whether indole-3-butyric and indole-3-caproic acids are active per se or only on conversion to indole-3-acetic acid. They are apparently active per se. Would you agree with that? There is clearly more than one peak. Dr. Wightman: We have often discussed this question at Wye, par- ticularly with regard to whether the activity of indole-3-butyric acid 92 Fawcett, Wain, and Wightman is due solely to its conversion to indole-3-acetic acid in the test tis- sues or whether it is due partly to the production of the acetic acid and partly to the fact that the butyric acid molecule itself possesses growth-promoting activity. I favor the view that indole-3-butyric acid is active per se, because I find it difficult to explain the high ac- tivity of this acid in the wheat and pea tests in terms of the small amount of lAA produced in metabolism experiments. Dr. Thimann: It would mean 100 per cent conversion. Dr. Wightman: Yes, it would, because if you examine the activity of indole-3-acetic and indole-3-butyric acids over a wide range of con- centrations, you find that the butyric acid is more active than the acetic acid at lower concentrations. Unless you assume that the bu- tyric acid penetrates the tissue more readily and is then converted al- most 100 per cent to the acetic acid, it is difficult to explain the high activity of the butyric acid at low concentrations, solely in terms of its conversion to lAA. Dr. Thimann: But still you do have two peaks in the bioassay of the chromatogram of metabolized indole-3-butyric acid. Dr. Wightman: That is correct. The first peak is due to the small amount of indole-3-acetic acid produced from the metabolism of the butyric acid and the second peak is due to the residual, unchanged butyric acid. Now, as we have already discussed, the activity shown by the residual butyric acid may be due either to the compound it- self or the result of its conversion to lAA in the tissue used in the bio- assay. Dr. Andreae: We tried solutions and could never find indole-3- acetylaspartic acid. Do you analyze solutions only, not the tissues? Dr. Wightman: No, we do analyze both the residual solutions and the treated tissue. In our extraction procedure we first remove the segments of tissue from the residual solution and immediately grind them uj) with acid-washed sand. The macerated tissue is then added back to the residual solution and after acidification the homogenate is extracted several times with ethyl acetate. One point I would like to make is that our pea metabolism experiments differed from yours in that we used much less tissue. According to your papers, you used 100 g. of pea stem tissue per treatment, whereas we used only approxi- mately 1 g. of tissue per treatment. The reason why we extracted both the treated tissue and the residual solution was because we found in earlier metabolism experiments with phenoxy compounds that we obtained metabolites in the solution, and since we were anxious to obtain as much of these metabolites as possible, we extracted not only the tissue but also the residual solution. This, of course, resulted in Chromatographic Investigations of Indole Compounds 93 our including quite a considerable amount of the unchanged com- pounds in the extract. Dr. Andreae: Whenever we applied the material to our chroma- tograms we used aliquots not exceeding 3 g. Did you ever get the amide? Dr. Wightman: No, we found no evidence at all on our chroma- tograms for the formation of indole-3-acetamide in wheat tissue. This may be due to the fact that we did not use sufficient tissue in our treatments to produce enough indole-3-acetamide to give a color re- action on the chromatogram when sprayed with Ehrlich's reagent or a peak of activity in the bioassay. Certainly this amide is not as sen- sitive to Ehrlich's as many of the other indole compounds used in this investigation and is only active at fairly high concentrations in the wheat cylinder bioassay technique. Our results, therefore, do not preclude the possibility that indole-3-acetamide is formed during the metabolism of lAA in wheat tissue, but they do indicate that it is certainly not formed to any appreciable extent. Dr. Thimann: We can confirm that too, because our work on barley tissue and also with the extracted enzyme shows not only that the amide is not formed in any appreciable amount but that if it were formed it is not acted on by the enzyme at any appreciable rate. Dr. Wain: I think the most important thing that Dr. Wightman has mentioned is this conversion of the CHoCN group to COOH, which is quite a new reaction. As he said, this is an alpha-oxidation of nitrile and involves the breaking of a carbon carbon bond with the loss of one carbon fragment. It's not strictly analogous to the break- down of the cyanhydrin, as for example, the ones produced from glyco- sides. Recently we have shown that you can take cell-free extracts from pea tissue and effect alpha-oxidation of nitriles very readily. Here is the same type of breakdown which occurs in the animal since com- pounds like p-chlorobenzylnitrile fed to dogs are excreted as deriva- tives of p-chlorobenzoic acid. Dr. Jepson: We must not forget that the whole of this indole story as related to plants sprang from investigations that weren't really on plants at all, but on human beings - the indole-3-acetic acid isolated by Kogl and Haagensmit from the urine of their laboratory assistant came not from the plants he ate but from the metabolism of his dietary tryptophan. I want to suggest that further information on plant indoles may well be obtained from studies of indoles ob- tained directly from animals, though of course in general they come initially from plants via tryptophan. For example, one is able to find in human urine two of the compounds that Dr. Wightman wanted to 94 Fawcett, Wain, and Wightman find in the experiments that he's just related to us — indole-3-acrylic acid and indole-3-carboxylic acid. Indole-3-acrylic acid is a normal component of human urine as its glycine derivative. The small amount in all human urine from subjects on a normal mixed diet disappears on fruit-free and plant-free diets. It must be derived from some unknown indolic component, probably with a 3-carbon side chain, present in plant or fruit products. It is not derived from tryp- tophan, because a single subject can take as much as 10 g. of oral tryp- tophan (a procedure I don't recommend) without causing any indole- 3-acrylic acid or its derivative to appear in the urine. But it is found in urine if Dr. Wightman's compound indole-3-propionic acid is fed, as reported by Decker. Indole-3-propionic acid has never been found, as far as I'm aware, in plants, but it may have some precinsor which may give rise to it on ingestion. Another possibility, currently being investigated with Dr. K. N. F. Shaw, is that indole-3-pyruvic acitl gives rise to the acrylic acid. Indole-3-pyruvic acid has had a check- ered history in this connection, and it may be that there is some plant component other than tryptophan which gives rise to indoIe-3- pyruvic acid, say through the action of bacteria in the gut, and from that to indole-3-acrylic acid. These are probably acetic materials. They may be auxins, we do not know. There are a lot of indoles which occur in plants which may well have important influences on animal metabolism. Serotonin is found in many plants, in banana fruit for example, in tremendous amounts ])hysiologically speaking, together with smaller amoiuits of related amines; tomato contains a large amount of tryptamine, which wdll normally give rise to indole-3-acetic acid. At the National Institutes of Health, w^e are giving drugs to pa- tients to prevent or divert the normal oxidative metabolism of amines including tryptamine and serotonin. If patients on these drugs eat a lot of bananas, the accumulated indolic amines will materially affect their physiology. So we may find tliat this topic of indole auxins, which is branching out so rapidly into all fields of plant and agridd- tural jjhysiology, may well find itself in human physiology as well. Dr. Fawcett: There are two papers wliich give evidence that in- dolcpropionic acid occurs in Brassica species (Planta 44: 103, 1954; Planta 50: 557, 1958) . Althougli conversion of indolepropionic acid to indoleacrylic acid occurs in human metabolism, there is no evi- dence from our work that this occurs in pea or wheat metabolism. I have syntiiesi/ed indoleacrylic acid, and when it is chromatographed on paper with ;ni acjueous 20 per cent potassiimi chloride solution, it has an Rf of 0.1 1. Hie imknown comj)ound we found in the metabo lism experiments has a much higher R, in this solvent (about 0.67) so thai it appaicMitlv is not indolcac i ylic acid. p. F. WAREING and T. A. VILLIERS^ University College of Wales Growth Substance and Inhibitor Changes in Buds and Seeds in Response to Chilling During the past 50 years, a number of hypotheses have been put for- ward to account for the phenomena of dormancy in plant organs. Since the discovery and isolation of plant growth hormones, however, many workers have attempted to explain dormancy in terms of these substances. Since the ability for growth is in some way arrested in dormant tissues, it is clear that dormancy is closely linked with gen- eral problems of growth control, and it is reasonable to consider how far the alternating cycles of growth and dormancy shown by many plants are controlled by specific growth substances. Now dormancy can be envisaged as being due either to the lack of certain essential growth factors or to the presence of active growth inhibitors. Certain earlier authors suggested that the inability of dormant tissues to grow may be due to lack of auxin, and indeed sev- eral workers found that auxin appears in buds only in the latter part of the winter (2, 4, 24). It seems unlikely that dormancy is controlled primarily by auxin deficiency, however, since application of exoge- nous auxin is generally not effective in breaking dormancy. On the other hand, others have concluded that dormancy of buds is caused by the presence of supraoptimal concentrations of auxin (7, 17), but this view is difficult to reconcile with the fact that auxin levels are very low during the early stages of bud dormancy. The view that bud dormancy may be due to specific growth-inhibit- ing substances was first put forward by Hemberg (10 to 14), who showed that the peel of dormant potatoes and the bud scales of Frax- imis excelsior contain growth-inhibiting substances, and that when dormancy is broken by chilling or treatment with ethylene chlorhy- ^ Subsequently: Botany Department, Makerere College, Kampala, Uganda. [95] 96 P. F. Wareing and T. A. VilUers drin, there is an associated decrease in the level of the endogenous inhibitors. Various workers have since investigated the changes in inhibitor content of buds during chilling, and most of these have con- firmed that the inhibitor level gradually decreases during the winter, reaching a minimum in the spring when the buds are expanding (5, 9, 15). Phillips and Wareing (20) investigated the changes in inhibi- tor level of buds of Acer pseudoplatanus throughout the year and ob- served that the amount of inhibitor gradually increased in develop- ing buds in the late summer, reaching a maximum in October. The inhibitor level decreased gradually during the winter, reaching a min- imum in April when the buds were expanding. There is no doubt, therefore, that in this species there is a marked annual variation in inhibitor level which is correlated with the state of dormancy of the buds. Such a correlation does not, of course, necessarily imply a causal relationship, and studies of this sort on tree buds imder natural conditions do not readily lend themselves to experimental techniques designed to elucidate whether the changes in inhibitor level control the changes in states of dormancy. For this latter purpose, seeds are much more suitable objects of study since they can readily be main- tained under controlled conditions and can be more easily exposed to various chemical and other treatments. Studies on changes of inhibitor levels in seeds in response to chill- ing have been very few. Barton and Solt (3) and Luckwill (19) ob- served some reduction in the inhibitor content of seeds of Sorbus au- cuparia and apple, respectively, in response to chilling. Lasheen and Blackhurst (18) observed that ether-soluble inhibitors disappeared from seeds of Rubiis during chilling, and the disappearance of the inhibitor was correlated with the breaking of dormancy and ability of the seeds to germinate. On the other hand, there was little corre- lation between the inhibitor content of the embryos and (heir state of dormancy. Several workers have studied dormancy in seeds of Frax- inus in relation to inhibitors. Ferenczy (8), using crude extracts of the various parts of the fruit of F. excelsior, concluded that most of the inhibitory material is present in a mucilaginous layer surroimd- ing the seeds. He found a decrease in this inhibitory material during moist storage at both 20° and 5° C. Using ether and aqueous ex- tracts of F. spaetliiana, Asakawa (1) found that there was some in- hibitory activity in the pericarps, but little in the seeds. The peri- carp inhibitors decreased during moist storage at both 2° C. and lab- oratory temperatures, probably by leaching. Studies With Seed of Fraxinus excelsior During the past 3 years we have carried out a detailed study of doiniaucy in seeds of F. excelsior, particularly in relation to growth Changes ??i Buds and Seeds in Response to Chilling 97 substances and inhibitors. Ripe fruits of this species are in a very deep state of dormancy, and neither intact fruits nor seeds will ger- minate over a period of 16 months if they are maintained moist at laboratory temperatures. When the seed is shed, the embryos are morphologically complete, but they need to undergo a further period of maturation, during which there is considerable growth, involving both cell division and cell extension. This maturation takes place more rapidly at laboratory temperatures than at chilling temperatures. The embryos themselves are found to be dormant, when dissected out of the seeds. In order to obtain germination of the intact seeds or fruits, a period of chilling is required, and this is only effective after the embryo has undergone maturation at a higher temperature. The minimum treatment times for the seeds are 1 month at laboratory temperatures, followed by 5 to 6 months of chilling. If the dry seed is extracted, first with ether and then with water, then a well-marked growth promoter is found in the ether fraction at Rf 0.8 to 0.9. This is found to be a neutral substance and it ap- pears to be indole-3-acetonitrile (IAN), on the grounds of its Rf value in several solvents and color reactions. This promoter is gradually reduced as the embryo undergoes maturation at laboratory tempera- tures (Figure 1). There is evidence in some extracts (especially of the endosperm) of a second ether-soluble promoter, which may be lAA from its Rf value. The aqueous extracts contain certain promoting regions at low z -I UJ < o a: ^, LJ — ' > o ^ o o 18 - 16 14- 12 ■ I 1 1 1 A 1 - ■Till B ' 1 1 — 1 — 1 r - . ' — 1 1 1 ^ — 1 - i_ 1 1 1 1 1 1 .--J — 1 1 1 1 0.4 0.8 0 Rf 0.4 0.8 Fig. 1. Ether extracts of seeds of Fraxinus excelsior assayed by the Avena coleop- tile test. A — fresh (untreated) seeds; B — seeds imbibed 3 months (embryo full size). Running solvent 99 parts 80 per cent aqueous isopropanol: 1 part ammonium hydroxide (S.G. 0.88); descending chromatography. Solid horizontal line indicates water control. 98 P. F. Wareing and T. A. ViUiers 18 X Z 16 ■D Z oJ -1 cn lij _j < h- O 14 IT 1 UJ n ^ o lid 10 1 A 1 1 1 LJ I — I B 1 [ 1 1 r ■- ■ I 1 1 1 - r — 1 1 _ - -- \ . 1 - _ - 1 ' 1 1 , .. 1 . 0.4 0.8 0 Rf 0.4 0.8 Fig. 2. Aqueous extract of F. excelsior seeds assayed by the Avena. coleoptile test. A — extract of dry seeds; B — extract of seeds imbibed for 48 hrs. Solid horizontal line indicates water control. (Running solvent as for Figure 1.) Rf values, but aqueous extracts of the dry seed contain no inhibitors. If, however, the seed is allowed to imbibe water at laboratory tem- peratures for 24 hrs. or more, then an inhibitory region appears on the chromatograms at Rf 0.7 to 0.9 (Figure 2). Evidently this inhib- itor is metabolically produced, since it does not appear during moist storage at 0° C. This inhibitor, which is water soluble and ether in- soluble, not only inhibits Avena coleoptile sections, but also cress roots, lettuce seeds, and embryos of F. excelsior (Figure 3). On ger- mination the level of this water-soluble inhibitor appears to be de- pressed slightly and an ether-soluble inhibitor of the same R,- as the (^-inhibitor of other workers appears. The presence of this growth inhibitor raises two questions, viz. (1) Does the inhibitor play any significant role in the dormancy of the seed? (2) Does chilling-treatment remove the dormancy by bring- t- z o — z -1 UJ Q. o UJ _l o o 14 12 - III' A B I 1 1 T |— ^~"~^~"-^^ ^ , 1— , 1 1 1 1 1 1 — 1 1 — 04 08 04 0.8 Rf 6 I o z - 4 Z UJ - _l o UJ Q O) < < cc UJ cc o u. z o Fig. 3. Assay of a- J- a: O CD < ^' 2 0 UJ LJ Q oc UJ UJ 1- QD < S Z Z) ^ tr UJ CD 8 - 6 - 4 - 0 A ' I 1 1 C — r / I T I ^ — - \ \ ^ ^-J-J B " - - - - - - - - - - - n"""n""" 1 1 1 i.. r 0 0.4 0.8 0.4 0.8 Rf Fig. 5. Effect of aqueous embryo extracts of Fraxinns excelsior seeds on germina- tion of unchilled embryos. A — extract of unchilled embryos. B — extract of chilled, nongerminating embryos. C — extract of chilled, germinating embryos. Dotted horizontal line indicates water control. 102 P. f- Wareing and T. A. Villiers chilled seeds. The subsequent growth of unchilled embryos stimulated to germinate in this way appears to be normal. It seems clear that the extracts of chilled embryos contain certain substances not present in unchilled embryos, which are capable of inducing germination in the latter. It would seem, therefore, that the effect of chilling is not to reduce the level of inhibitor but to increase the level of an endogenous germination stimulator which overcomes the effect of the inhibitor; some evidence in support of this hypothe- sis was obtained in the following experiment. The region of a chromatogram containing the inhibitor (Rf 0.65 to 0.80) was cut into four strips and each strip moistened with one of the following: (1) eluate from Rf 0.2 to 0.3 of a chromatogram of extract of 10 unchilled embryos; (2) eluate from Rf 0.2 to 0.3 of a chromatogram of extract of 10 chilled embryos; (3) distilled water (control); (4) 0.25 per cent thiourea. A further control of filter paper moistened wuth distilled water only was set up (i. e., no inhibitor). Leached (nondormant) embryos were then planted on the paper. It was found that there was little germination either on the paper containing only inhibitor or inhibitor plus extract of unchilled embryos (Table 1) . On the Table 1 . Interaction between inhibitor and germina- tion promoter in embryos of F. excelsior. Germination Medium* Percentage Germination Water onlv 95 Inhibitor only 30 Inhibitor -f eluate from zone Rf 0.1 to 0.3 of extract of unchilled embryos .... Inhibitor + 0.5 per cent thiourea Inhibitor + eluate from zone Rf 0.1 to 0.3 of extract of chilled embryos 45 85 100 * Leached, unchilled embryos weie placed on filter paper moistened with solutions indicated — 20 embryos per dish. other hand, the presence of extract from cliillcd embryos enabled the test embryos to overcome the effect of the inhibitor, and thiourea was almost equally effective. Embryos receiving only water produced stunted seedlings, those exposed to the inhibitor did not grow further, while those exposed to the eluate from chilled embryos produced normal seedlings. It would seem, therefore, that the effect of chilling is to lead to the acciunulation of a germination promoter ^vhirli en- ables liie embryo to overcome the effects of the inliiljitor. DISCUSSION Fxom the foregoing evidence, it would seem that both promoters and inhibitors are involved in the control of dormancy in embryos of /'". excelsior. Although it appears that the changes resulting from chill- Changes in Buds and Seeds in Response to Chilling 103 ing involve primarily the germination promoter, nevertheless there is good evidence that an inhibitor plays an important part in the dormancy of the seed. Indeed, the responses of the seed appear to in- volve interaction between the promoter and the inhibitor, and the hypothesis is suggested that dormancy is due to the presence of the inhibitor and that emergence from dormancy involves the accumula- tion of the promoter to a level which overcomes the effect of the in- hibitor. Since the greater part of the inhibitor present in the seed is contained in the endosperm, whereas the promotor is confined to the embryo, it would seem that a build-up of promoter is necessary to enable the embryo to overcome the inhibitory effect of the endosperm. Since gibberellins are able to break the dormancy of various rest- ing organs, including the unchilled embryos of F. excelsior, the ques- tion arises whether the germination promotor present in the chilled embryos is a gibberellin. Several pieces of evidence suggest that this is not the case; for example, the very small amount of tissue required to be extracted for its detection contrasts markedly with the relatively large amounts of tissue generally required for the detection of gibber- ellins. Moreover, the embryo promoter is capable of removing the stunting of seedlings derived from unchilled embryos, whereas gibber- ellic acid does not have this effect. Whatever the nature of this promoter may prove to be, it would seem very probable that it functions as such in the intact seed, for the great merit of the work with Fraxinus seeds is that detection of the germination promoter was carried out with embryos of the same spe- cies, so that there is strong presumptive evidence that the in vitro ex- perimental results are equally applicable in vivo. If these results with seed of F. excelsior prove to be of more gen- eral application to buds and other resting organs, then it would seem that the reduced inhibitor level, reported to occur in the buds of sev- eral species in response to chilling, is not the only or even the pri- mary cause of emergence from dormancy of these buds. It is thus de- sirable to investigate whether there is any accumulation of dormancy breaking substances during the chilling of buds. The existence of such substances in buds of Tilia and Fraxinus has, in fact, already been reported (22). SUMMARY Dormancy can be envisaged as being due either to a lack of cer- tain essential growth factors or to the presence of active growth inhib- itors. Studies of the changes in the levels of growth inhibitors in rest- ing buds have shown a correlation between the state of dormancy and the level of inhibitors, and suggest that the effect of winter chilling in removing dormancy is to reduce the level of inhibitor. 104 P. F. Wareing and T. A. Villiers A study of dormancy in seeds of Fraxinus excelsior, in relation to growth substances and inhibitors, is reported. In order to remove the dormancy of these seeds, a period of maturation for 4 to 6 weeks at warm temperatures is required, followed by chilling treatment for 5 to 6 months. The dry seeds contain no inhibitors, but after they have been permitted to imbibe water for 24 hrs., a water-soluble in- hibitor is present in the endosperm and embryo, and is apparently metabolically produced. The unchilled embryos themselves are dor- mant, but their dormancy can be removed by leaching the excised embryos for 48 hrs. Seedlings derived from leached, unchilled em- bryos show stunted growth. Application of inhibitor to leached em- bryos restores their dormancy. Thus, it appears that dormancy in these seeds is due to the presence of the inhibitor in the endosperm and embryo. Chilling treatment results in no appreciable reduction in the level of inhibitor. On the other hand, it is found that extracts of chilled embryos contain a germination promoter which is capable of over- coming the dormancy of unchilled embryos. The promoter is able to overcome the effects of the inhibitor when both substances are added to the germination medium. It appears that dormancy in Frax- inus seed is controlled by interaction between the inhibitor and the promoter, and that chilling results in an increase in the concentration of promoter to a level which overcomes the effect of the inhibitor. The promoter is also effective in removing the stunting of seedlings from unchilled embryos. LITERATURE CITED 1. Asakawa, S. On the growth inhibitors in Fraxinus fruits. Bui. Gov. For. Exper. Sta. 83: 29-38. 1956. 2. Avery, G. S., Jr., Burkholder, P. R., and Creighton, H. R. Production and distri- bution of growth hormone in shoots of Aexculus and Malus, and its probable role in stimulating cambial activity. Amer. Jour. Bot. 24: 51-58. 1937. 3. Barton, L. V., and Solt, M. L. Growth inliibitors in seeds. Contr. Boyce Thompson Inst. 15: 259-278. 1948. 4. Bennett, J. P., and Skoog, F. Preliminary experiments on the relation of growth-promoting substances to the rest period in fruit trees. Plant Physiol. 13: 219-225. 1938. 5. Blommaert, K. I.. J. Growth- and inhibiting-substances in relation to the rest period of the potato tuber. Nature. 174: 970-972. 1954. fi. Donoho, C. \\., Jr., and Walker, D. R. Eflect of gibberellic acid on breaking the rest period in Elberia peach. Science. 126: 1178, 1179. 1957. 7. Eggert, F. P. The auxin content of spur buds of apple as related to the rest period. Proc. Amcr. Soc. Hort. Sci. 62: 191-200. 1953. 8. Fcrenczy, L. The dormancy and germination of the seeds of the Fraxinus ex- celsior l^. Acta Biol. 1: 17-24. 1955. 0. Guiienhcrg, H. von, and Leike, H. Untersuchungen iiber den Wuchs- und Ikiumstoligchalt ruhender und triebender Knospen von Svrin[ra imlgaris L. Pianta. 52: 96-120. 1958. Changes in Buds and Seeds in Response to Chilling 105 10. Hemberg, T. Studies of auxins and growth-inhibiting substances in the po- tato tuber and their significance with regard to its rest-period. Acta Hort. Berg. 14: 133-220. 1947. 11 . Significance of growth-inhibiting substances and auxins for the rest- period of the potato tuber. Physiol. Plant. 2: 24-36. 1949. 12. . Growth-inhibiting substances in terminal buds of Fraxinus. Physiol. Plant. 2: 37-i4. 1949. 13. . The occurrence of acid inhibitors in resting terminal buds of Fraxi- nus. Physiol. Plant. 11: 610-614. 1958. 14. . The significance of the inhibitor j8 complex in the rest period of the potato tuber. Physiol. Plant. 11: 615-626. 1958. 15. Hendershott, C. H., and Bailey, L. F. Growth inhibiting substances in ex- tracts of dormant flower buds of peach. Proc. Amer. Soc. Hort. Sci. 65: 85-92. 1955. 16. Kahn, A., Goss, J. A., and Smith, D. E. Effect of gibberellin on germination of lettuce seed. Science. 125: 645,646. 1957. 17. Kassem, M. M. The seasonal variation of hormones in pear buds in relation to dormancy. Thesis, Univ. Calif. 1944. [Cited by Eggert (7)]. 18. Lasheen, A. M., and Blackhurst, H. T. Biochemical changes associated with dormancy and after-ripening of blackberry seed. Proc. Amer. Soc. Hort. Sci. 67: 331-340. 1956. 19. Luckwill, L. C. Growth-inhibiting and growth-promoting substances in rela- tion to the dormancy and after-ripening of apple seeds. Jour. Hort. Sci. 27: 53- 67. 1952. 20. Phillips, I. D. J., and Wareing, P. F. Studies in dormancy of sycamore. I. Seasonal changes in the growth-substance content of the shoot. Jour. Exper. Bot. 9: 350-364. 1958. 21. Rappaport, L. Growth regulating metabolites; gibberellin compounds derived from rice disease-producing fungus exhibit powerful growth regulating prop- erties. Calif. Agr. 10(12): 4, 11. 1956. 22. Richter, A. A., and Krasnosselskaya, T. A. A contribution to the knowledge of the breaking of winter dormancy in buds of woody plants. Dokl. Akad. Nauk. SSSR. 47: 218,219. 1945. 23. Wareing, P. F., and Foda, H. A. Growth inhibitors and dormancy in Xanthium seed. Physiol. Plant. 10: 266-280. 1957. 24. Zimmermann, W. A. Untersuchungen iiber die raumliche und zeitliche Ver- teilung des Wuchsstoffes bei Biiumen. Zeitschr. Bot. 30: 209-252. 1936. DISCUSSION Dr. Torrey: Is it possible to say from your assays whether your in- hibitor affects cell elongation or cell division in the embryos? You gave evidence to suggest that the accelerator affected the acceleration of cell enlargement in roots. Can you distinguish between the two processes? Dr. Wareing: I am afraid not. We have looked at cell division and cell extension at certain stages, but I am afraid I can't specifically answer that question with regard to the promoter on root growth. Dr. Tukey: Dr. Wareing, you spoke about the inhibitor zone. This is the endosperm, is it? [Yes.] There is a very interesting situation in peach that falls right in line with what you are suggesting. Of course, the peach seed must be after-ripened before it will germinate, but if the integuments are removed, the naked embryo 106 P. F. Wareing and T. A. VilUers will germinate without after-ripening. Now, if just the chalazal re- gion of the integuments is removed, the seed will not germinate. In fact, if all the integuments are removed, excepting for 2 or 3 mm. at the micropylar end, the seed will not develop. On the other hand, if this 2 or 3 mm. portion of the integuments at the micropylar end is removed, the seed will germinate even though the remainder of the integuments is left intact. If you examine the peach seed mor- phologically, you will observe that there are rudiments of endosperm tissue immediately adjacent to the radicle, or the hypocotyledonary axis in the micropylar region. When the integuments are removed, this endosperm tissue comes away with it. From all of this, one might speculate that an inhibitor lies in the endosperm of the peach seed at the micropylar end, and he might be prompted to look for it there. Dr. Wareing: In the case of ash, of course, the endosperm com- pletely surrounds the embryo, but what Dr. Tukey said ties up also with observations on Iris. Randolph and Cox (Proc. Amer. Soc. Hort. Sci. 43: 284, 1943) showed that the endosperm has a very strong in- hibitory effect which is probably due to a specific inhibitor. Dr. Evenari: I would like to compare this situation with the case of lettuce seeds. Dry lettuce seeds are full of inhibitors and there is apparently no promoter present. After root growth has started, the inhibitors disappear and a number of promoters (the chemical nature of which is imknown) appear. This disappearance of inhibitor and appearance of promoters occurs quite late insofar as the germination process is concerned and occurs only after germination has, in reality, finished and root growth has already started. In this case, at least, it will be difficult to correlate the so-called dormancy of the seeds with the presence of the inhibitor. I think wc have to be careful here in differentiating between what we call germination inhibitors and growth inhibitors, as apparently these two are different from each other. Dr. Wareing: First of all, when Professor Evenari speaks of pro- moters, he's talking of promoters revealed in the Avena coleoptile test which presumably means auxins and, therefore, I would not be surprised to find no particular correlation between dormancy in let- tuce seed and changes in auxins. On the question of correlations be- tween inhibitors and dormancy, the whole subject is fraught with pit- falls because so many things will stop Avena coleoptile growth or, for that matter, will inhibit germination, and the real crux of this problem is to sort out purely toxic substances from functional inhib- itors. Professor Evenari said that in lettuce seed he cannot get a cor- relation between the inhibitor content and the stale of dormancy. Changes in Buds and Seeds in Response to Chilliyig 107 On the other hand, in the seed of cocklebur, we were able to get a very marked correlation. For example, oxygen will break the dor- mancy of dormant cocklebur, but the inhibitor disappears 30 hrs. after you have put them into oxygen, before there are any visible signs of germination. There, the inhibitor disappears before germi- nation. Dr. Burstrom: Do your results imply that your promoters and in- hibitors are active in the soluble state, since you have only studied the soluble fractions, contrary to auxins which probably are active in some bound form or other? Dr. Wareing: Well, it's very difficult to answer Professor Bur- strom's question in the present state of our knowledge. In fact, I would almost be inclined to throw it back at him and ask him how he would demonstrate this. Dr. Wain: Just a very small point on technique here. In the early part of your report, the dry seeds were extracted with water and no inhibitor was found. Then when you soaked the seed and ex- tracted with water, you found an inhibitor. It does seem to me that this might be explicable in terms of ease of extraction. Dr. Wareing: I don't think so. Professor Wain. If you extract the dry seed at 0° C, you get no inhibitor. If, however, you keep the seeds soaking for 24 hrs. at laboratory temperature, you get an inhib- itor. On the other hand, if you keep them soaking for 24 hrs. at 0° C. no inhibitor appears. We were very conscious of the possibility that these effects were simply the result of extraction technique, but fur- ther consideration seems to leave no doubt that the inhibitor is met- abolically produced shortly after the seed is soaked at laboratory tem- peratures. L. J. AUDUS and J. K. BAKHSH Bedford College University of London On the Adaptation of Pea Roots to Auxins and Auxin Homologues The phenomenon of adaptation whereby enzyme balance in an actively functioning cell may be modified or even transformed by a change of exogenous metabolite or the administration of a growth repressant has been widely studied in microorganisms. It now seems likely that enzyme induction by endogenous substrates or by struc- turally related molecules in the cell is a universal phenomenon whereby the adjustment of constitutive enzyme levels is normally accomplished (5). The fundamental significance of these new con- cepts for the biochemical, and hence structural, differentiation of the organism needs no stressing. Recently, these ideas from microbial behavior have been applied to higher plants in which, it has been suggested by Galston (6) , indole-3-acetic acid-oxidase may be induced to form in this way by its own substrate. A certain amount of ex- perimental evidence supports this claim, and the implications, in terms of the auxin control of plant growth, are, as Galston has pointed out, far-reaching. But Burstrom (4), who has demonstrated that without doubt growing root cells show progressively adaptive changes in their response to indole-3-acetic acid (lAA) when grown continuously in dilute solutions of that substance, does not think that the induced augmentation of lAA-oxidase activity can explain those changes. He visualized a switch in the mechanism of cell ex- tension involving, as we interpret his meaning, other enzyme sys- tems, probably those concerned in the incorporation of cellulose into the growing wall. This being so, it might be expected that changes in growth response to other homologous plant growth regulators, which could be expected to act in the same cell wall system, but which are not metabolized by lAA-oxidase, might result from lAA adaptation. Furthermore, the system might also be expected to adapt [109] 110 L. J. Audus arid J. K. Baklisli directly to these lAA homologues, either with or without changes in lAA-oxidase, depending on whether Galston's or Burstrom's interpre- tation of adaptive growth changes are correct. The purpose of the present investigation therefore was to in- vestigate adaptive changes in pea roots, not only to lAA but also to its synthetic homologue, 2,4-clichlorophenoxyacetic acid (2,4-D), and to two other compounds, both suspected of interfering with growth via the auxin system. One was 2,3,5-triiodobenzoic acid (TIBA), which, among other things, is claimed to increase lAA-oxidase activ- ity in pea stem tissue (7), and lowers lAA concentrations in pea roots (3). The other was 2,4-dichIoroanisole (DCA), which has cer- tain claims to being an auxin antagonist by direct competition at the growth centers (8). The changes studied ^vere of two kinds: (a) growth responses to depressive concentrations not only of the adapt- ing molecule but also of the other substances; and (b) changes in lAA-oxidase activity during adapting treatments. METHODS Seeds of 'Meteor' pea were germinated and grown from tiie second to the fourth day with their roots in a dilute solution of the chosen growth substance at concentrations that induced small inhibitions of elongation. Tests of the sensitivity of these roots to lAA and other growth substances were made by the excised-segment technique (2). Segments 1.7 to 2.0 mm. long were cut 1 mm. behind the apex of both treated and normal roots of the same age and then grown in aerated solutions of 0.5 per cent sucrose. Total extension of these segments was determined over the subsequent 48 hrs. Complete fac- torial experiments involving growth substances at different concentra- tions allowed the growth responses of segments from treated roots to be compared with those from normal roots. In each factorial ex- periment samples consisted of 10 root segments each and were repli- cated once. Furthermore, each experiment was exactly repeated a number of times on different occasions and the data thus acquired were subjected to an analysis of variance to determine the con- sistency of the responses and of their dependence on the adaptation treatment. Residual errors from these analyses were used to tleter- mine the least significant differences to be used as a basis of com- parison in the results to follow. Since the length of the meristem of adapted roots usually differs somewhat from that of normal roots (e.g., with lAA adaptation it is slightly shorter, as shown by Burstrom in wheat roots), it is clear that by taking the same length of segment at the same distance from the apex, the segments from normal and adapted roots will have Adaptation of Pea Roots to Auxins and Hornologucs III somewhat different cellular constitutions; indeed there will be some variations even within groups of segments cut from the same root sample. Obviously it is not possible to allow for such differences in the cutting of the segments and so, to supplement observations on the over-all growth, studies were made of the extension of individual epidermal cells of representative segments, so that the behavior of comparable cells in segments from normal and treated roots could be studied. Such observations were made on 8 ^ sections cut from segments fixed in Navashin's fixative and stained in hematoxylin. Samples of five segments were taken for each treatment and lengths of individual cells measured from basal to apical end. Taking the cells in successive groups of 10 (i. e., groups of 50 cells for the sample of five segments), mean lengths and their standard errors were de- termined for the construction of cell length distribution curves. Finally lAA-oxidase content of seedling roots similar to those used for the cutting of segments was determined from enzyme extracts by methods described by Galston and Dalberg (7). Average rates of lAA destruction, eliminating the small inconsistent initial lag and the enzyme inactivation which sets in as the reaction proceeds, were determined from a number of replicate samples by a statistical meth- od, which also gave estimates of over-all errors for the evaluation of confidence limits. RESULTS Segments From Roots Grown in 10^ G/Ml of Indole-3-acetic Acid Responses to inhibiting concetrations of lAA (10^^ and 10-'' gjin^)- In Figure lA are plotted the mean percentage extensions of samples from eight identical factorial experiments. In sucrose, lAA-grown segments extend less (i.e., about 15 per cent) than normal segments. On the other hand, they extend very much more than normal seg- ments exposed directly to the adapting concentrations. If the re- duced extension of these adapted segments is due to a carry-over of lAA from the adapting solution, then either its concentration must be much lower than in the adapting medium or cell sensitivity to lAA has been lowered by the adaptation treatment. Inspection of the cell extension graphs (Figure IC, D) shows that this reduced growth is not a property of all cells in the adapted segment. In normal segments in sucrose all cells extend, but the less mature cells at the apical end of the segment, where extension had not com- menced at the time of excision, do not attain the same final length as the more mature cells at the basal end of the segment. In lAA- grown segments, these more mature basal cells extend as much as the corresponding cells in normal segments while the growth of the 150 100 50 -00 73 n m Z z (A o z 150 100 50 -00 _l_ -8 LOG lAA G/ML. -7 -7 0^ o tr o LU O ' vO\|^^- LOG 2,4 -D G/ML. Fig. 1. Growth responses of segments from roots grown in indole-3-acetic acid solutions (10-" g/ml). A and B. Total extension in 48 hrs. of normal (open circles) and lAA-grown (solid circles) segments. (A) Responses to lAA. (B) Responses to S.^l-D. Vertical lines indicate the least signilicant dilference (5 per cent) between means. C and D. Epidermal cell length distribution before and after 48 hrs. of extension. (C) Normal segment responses to I.\A. (D) L\A-grown segment responses to I A A. Open parallel lines — segments inunediatcly after excision. Blacked-in parallel lines — segments after 48 hrs. in 0.5 per cent sucrose solution. These lines are plotted at distance of ^^ O u u o 13 PQ S « be be 7 X > « CM bc I o X bo Adaptation of Pea Roots to Auxins and Homologues 115 Responses to inhibitory concentrations of 2,-f-D (10^^ g/»^^ ^""^ 70 « g/'ml) . Here only total segment extension has been measured, and results are recorded in Figure IB. Growth of normal segments is not significantly reduced by the lower but is reduced by about 48 per cent by the higher concentration. lAA-grown segments, on the other hand, are not significantly affected by these 2,4-D concen- trations, the total extension being the same as in sucrose alone. Un- fortunately, time has not permitted analysis of growth on a cell basis for these 2,4-D-inhibited segments, but it is clear from these results alone that lAA adaptation has markedly reduced the sensi- tivity of cells to 2,4-D inhibition. It might be suggested that the lack of response to 2,4-D is due to the domination of the growth centers by lAA carried over from the adapting solutions, but this would certainly not be so if lAA-oxidase were at a high level in the cells. It would be much more logical to suppose that here we are dealing Avith a change in the growth centers themselves involv- ing a great loss in sensitivity not only to 2,4-D but also most prob- ably to lAA itself. It is very unlikely that lAA-oxidase induction plays any direct part in these 2,4-D sensitivity changes. Segments From Roots Grown in 2,4-D (3 X 10 § g/ml) In this series of nine identical factorial experiments we tested the responses of normal and 2,4-D-grown segments to three lAA concen- trations (10-11, 10-9, and 10-' g/ml) . The pooled results of these ex- periments are drawn in Figure 2A. Normal control segments show, contrary to earlier experiments by Audus and Das (1), no stimulation in 10-11 g/ml lAA but a progressive inhibition in higher concentra- tions, reaching about 27 per cent in 10^ g/ml- The pattern of cell extension in these normal segments (Figure 2C) closely resembles that already described, with restricted growth of the cells at the apical end. lAA (10 " g/ml) reduces the extension of all cells in the normal segment, with a proportionately greater effect on the more rapidly extending maturer basal cells. Root segments grown in 2,4-D extend much less (about 27 per cent) in sucrose than normal segments (Figure 2A) . The analysis of cell length distribution (Figure 2D) shows that this restriction of extension applies to all cells, with perhaps a slightly greater effect (as for L\A adaptation) on the immature apical cells. Such a reduced extension could undoubtedly be explained in terms of residual 2,4-D left associated with the growth centers after excision and exerting, at least in part during subsequent extension, a continuing growth in- hibition. But this reduction in extension is much less than that pro- duced by the direct action of the adapting concentration (3 X 10-s 116 L. J. Audiis and J. K. Bakhsh 250 -CO LOG lAA G./ML, UJ o UJ a 150 100- 40' to o cc o z llJ Fig. 2. Growth response of segments from roots grown in 2,4-dichlorophenoxyacetic acid solution (3 x 10-° g/ml). A and B. Total extension in 48 hrs. of normal (open circles) and 2,4-D-grown (solid circles) segments. (A) Responses to \\\. (B) Responses to 2,4-D. Vertical lines indicate the least significant difference (5 per cent) between means. C and D. Epidermal cell length distribution before and alter 48 hrs. of extension. (C) Normal segment responses to IA.\. (D) 2,4-D-grown segment responses to lAA. Open parallel lines— segments immediately after exci- sion. Blacked-in parallel lines — segments after 48 hrs. in 0.5 per cent sucrose solu- tion. These lines are plotted at distance of cr/VN above and below tlie respective mean of successive groups of cells. g/ml) on normal segments, which is of the order of 60 per cent. This suggests that either 2,4-D is rapidly lost from the segments inuler growth conditions in sucrose, about 80 per cent loss being necessary to explain the much lower inhibition, or that there might have occurred a direct loss of sensitivity to the persistent 2,4-D, of a nature reminiscent of bacterial adaptation to drug action. The latter altern- ative is somewhat favored by observations on the growth of such Adaptation of Pea Roots to Auxins and Homologucs 117 adapicd segments in a range o£ 2,4-D solutions (Figure 2B) . In these experiments concentrations up to 10-s g/ml had only slight addi- tional depressive effects and the growth of these segments was slight- ly (although not signiEcantly) greater than normal segments in the same concentration (10-^ g/^il)- Convincing evidence of a change in auxin sensitivity comes from the response of 2,4-D-grown segments to lAA. Here, instead of re- duction, there is a progressive increase in segment extension which in 10 "^ g/ml reaches 19 per cent over those in sucrose, a difference which is highly significant (Figure 2A) . The cell length distribution results of Figure 2D show a surprising feature: the mature basal cells of the adapted segment are completely insensitive to these high lAA concentrations, but in the immature cells at the apical end extension is stimulated, their growth equaling that of corresponding normal cells and being at least twice that of corresponding adapted cells in sucrose. In the first place, it is very unlikely that changes in lAA-oxidase activity of the 2,4-D-grown cells have any part to play in these changed responses. Direct measurement of lAA inactivation by ex- tracts of adapted roots gives mean rates only 12 per cent above those of normal roots, the difference being completely nonsignificant (Table 1). This is supported by previous observations by Audus and Thresh (3), which showed that such 2,4-D treatments had no effect on the internal levels of lAA in pea roots. It seems possible that the lack of sensitivity of the more mature basal cells could be caused at least in part by the retention of 2,4-D at the growth centers of the treated cells. If all these centers were occupied by 2,4-D to the exclu- sion of the lAA penetrating from the medium, then one would ex- pect the results obtained. Even if lAA were to replace 2,4-D, no great change in growth would be expected since, molecule for molecule, they have the same order of inhibiting effect on root cells. But the almost normal growth of the younger apical cells is very much more puzzling. The following speculations may be worth con- sideration. First, there might be a mutual, and therefore chemical neutralization of these two substances in apical meristematic cells. This would mean that 2,4-D adaptation involved the generation of an enzyme catalyzing this reaction and would necessitate that this enzyme should disappear as soon as extension commenced; this would explain the persistence of inhibition (either by 2,4-D or lAA or both) in cells just beginning to extend at the time of excision and lAA treatment. From what is known at present of the chemical proper- ties and biochemical behavior of these two substances, this would seem most unlikely. Alternatively, we could explain this virtually 118 L. J. Audus and J. K. Bakhsh normal growth by invoking changes at the growth centers in both the sensitivity to and the affinity for growth substances in these imma- ture adapted cells. It would be necessary to postulate that residual 2,4-D is easily pushed off these cells by the lAA entering from the medium but, at the same time, the sensitivity of the growth centers to lAA is so reduced that it has virtually no effect and normal exten- sion ensues. Furthermore, there would have to be a rapid increase in 2,4-D affinity at the commencement of extension in order to explain the lack of lAA effect in the basal cells starting to extend at the time of excision. Segments From Roots Grown in TIBA (5 X 10^ g/ml) A series of five identical factorial experiments has been carried out to test the responses of these roots to IQii, 10 «, and 10 ' g/ml lAA solutions. The pooled treatment means are drawn in Figure 3A. The behavior of control segments is as in previous experiments. lAA at 10'^ g/ml giving an inhibition of extension of about 48 per cent. TIBA-grown segments extend about 19 per cent less than normal segments in sucrose. The cell length distribution curves of Figure :^B and C show that this is due, as in the case of lAA-adapted seg- ments, to a marked restriction of the extension of the youngest apical cells. Although, for some unknown reason, the mature basal cells of this particular sample of control segments grow less than usual, thus emphasizing these differences, corresponding cells of the TIBA-grown segments appear to extend more than normal, reaching a significantly higher final length. The responses of these treated segments to lAA are very slight (Figure 3A), amounting to a reduction of only 9 per cent in 10 "^ g/ml, as compared with those of corresponding segments in sucrose only. Whereas lAA inhibition is exerted along the whole length of the normal segment, in TIBA-treated segments, the much reduced inhibition is exerted mainly on the maturer basal cells. In all re- spects the growth behavior of TIBA-adapted segments closely re- sembles that of lAA-adapted segments and the similarity also extends to augmented lAA-oxidase activity. Enzyme extracts of TIBA-treated roots gave mean lAA destruction rates well over twice those of normal roots, the difference being very highly significant (Table 1). TIBA in low concentrations appears to have a direct activating effect on lAA oxidase in vitro. But such small effects (12 per cent at 10*5 g/ml) of TIBA in the extracts of treated roots could hardly ex- plain these large activity increases. It would seem that here ^se are dealing with lAA-oxidase induction by TIBA. The lowered sensitivity of TIBA-treated segments to ai:)plied lAA 60r CO z o a: o 200 Fig. 3. Growth responses of segments from roots grown in 2,3,5-truodobenzoic acid solutions (5 x 10-" g/ml). (A)Total extension in 48 hrs. of normal (open circle) and TIBA-grown (solid circle) segments. Response to lAA. Vertical Imes indicate least significant difference (5 per cent) between means. B and C. Epidermal cell length distribution before and after 48 hrs. of extension. (B) Normal segment responses to lAA. (C) TIBA-grown segment responses to lAA. Open parallel lines- segments immediately after excision. Blacked-in parallel lines— segments after 48 hrs. in 0.5 per cent sucrose solution. These lines are plotted at distance of a. O CO UJ UJ q: ui Q 120 - -120 -80 - 0.003 0.01 0.03 0.1 0.3 CONCN. SODIUM INDOLE-3- ACETATE, RRM. Fig. 4. Average curvature per side of slit pea stems in response to NalAA with (broken lines), or without (solid lines), inhibitor at 10 ints/ml. The curve shows the average response from two separate experiments, the individual values being shown as open and closed circles for sections with and without inhibitor resj^ec- tively. The method of measuring the curvature is shown in the insets. (15); they were slit to within 1 cm. of the base, washed in running tap water for a few hours, and then dispensed six to a petri dish con- taining 3 ml. solution. After ca. 20 hrs. tracings were made of the segments, and the amount of curvature measured as shown in the inset on Figure 4 (14). The graphs show the steadily increasing in- ward curvature with increasing concentration of NalAA, and an aug- mentation of this effect by inhibitor at 10 ints/ml except at the high- est auxin level used (3 p.p.m.). Interpretation of the results of the slit pea stem test is difficult (15), and without further work it is impossible to say whether the inhibitor has had an "auxin-sparing" effect, making each auxin con- centration in effect higher, or has inhibited the inner tissue more than the outer, so resulting in a greater net inward bending for a given elongation of the outer tissues. Straight grotvth of pea stem sec- tions is certainly inhibited, but only a few tests have been performed and comparable data for inner and outer tissues — the critical point in this connection — have not been obtained. Pollen-Tube Extension The germination of apple pollen was not greatly affected by even quite high concentrations of inhibitor, but the length of the tubes 134 Barlow J Hancock^ and Lacey Table 2. The effect of different inhibitor concen- trations on the length {n) of pollen tubes of two apple varieties growing in 10 per cent sucrose; average of solutions containing 0, 1, and 5 p. p.m. 1-naphtha- leneacetic acid. Inhibitor Ints/Ml 'Edward Vir 'Lord Lambourne' 0 35.4 40.3 1 34.3 24.7 5 21.0 16.6 50 18.2 14.3 was reduced. Table 2 shows the results of two experiments averaged together. It is of interest that the highest inhibitor concentration, which would have a drastic effect on coleoptile section extension, has al- lowed quite an amount of growth by pollen tubes. These test ob- jects were too delicate to wash and transfer to inhibitor-free solution to find out if recovery could occur. Extension of Cress Roots Cress seeds were grown on damp filter paper at 25° C. for 2 days, and those with radicles 5 to 7 mm. long selected (11), and placed, five in a line, on filter paper in petri dishes sloped at ca. 45°; 2 ml. solutions were applied to the papers, and the root length measured after 48 hrs. at 25° C. In another method, 20 seeds were sown di- rectly onto filter paper wetted with the solutions, an extra ml. being added to allow for the water taken up by the gelatinous seed coats. Results with either method have always been very variable, but from a large number of tests it is possible to say with certainty that the inhibitor does not function as an anti-auxin, neither promoting root growth at any concentration, nor relieving the inhibition due to NalAA. Fungal Growth The inhil)itor, even at 100 ints/ml, had no effect on the growth of Pulularia puUulans (a saprophytic ascomycete), J'ryticilliinn albo- atnim (a pathogenic ascomycete), or an imidentified yeast, in tests kindly carried out by the Plant Pathology Section of this station. Ab.scission Three experiments, with three clones of Coleus sp. in each, failed to detect any effect of inlnbitor on the abscission of debladed petioles Characteristics of an Inhibitor From Woody Shoots 135 on detached half nodes in a humid chamber (10), either treated with various concentrations of a-naphthaleneacetic acid or not. Some tests on bean explants^ similarly showed no definite effect of the inhibitor. Respiiation of Coleoptile Sections No differences could be detected either in oxygen uptake or car- bon dioxide output after tipping inhibitor into the Warburg flasks, although growth was greatly reduced; the sections were in K2HPO4 buffer at pH 4.6 at 20° C. This inhibitor evidently does not affect growth by a general reduction in metabolic activity. Permeability of Cells to Water The short-term effect of inhibitor on water exchange, which was independent of growth, was studied by observing the rate of loss of fresh weight by potato discs in 0.5 M mannitol solution with or with- out inhibitor at 10 ints/ml, and the rate of recovery of plasmolyzed discs on their return to water with or without inhibitor. In the former case a loss of 32 per cent of the initial weight, and in the lat- ter a regain of 29 per cent, were unaffected by the presence of inhib- itor, indicating that the inhibitor does not operate by altering the permeability of the protoplast to water. Transport of NalAA and Inhibitor Through Coleoptile Sections Agar plates 12 X 9 X 1 mm. were prepared containing NalAA at 0.25 p. p.m., inhibitor at 10 ints/ml, both, or neither. Coleoptile sec- tions 1 cm. long were cut, and stored on a klinostat at 3° C; at appropriate times samples were taken, the leaf removed, and three sections of 2 mm. cut from the middle region of each 1 cm. section. Three sets of three of these rings were placed lower ends downward onto an agar plate on a glass slide, and a second plate applied on top of the rings; this upper plate was covered by a thin sheet of polyethy- lene, and the whole system stored under humid conditions at 20° C. for times varying from 2 to 23 hrs. Either the upper or the lower plate was a "donor," containing the substance to be transported, and the op- posite one a "receptor" of plain agar; at the end of the period the plates ^\ere separated, cut into three strips, and placed in the stand- ard section test vials w^ith 0.25 ml. water for testing with five coleop- tile sections as usual. The rings used for transport w^ere fixed, and subsequently measured under the microscope. Figure 5 shows the response of sections to the material present in donor or receptor plates after periods of transport of 0, 2, 4, 8, 15, or 23 hrs. The apical plate is shown on the left of each pair of histo- grams; the donor plate is shaded and the receptor plain. It is clear that the XalAA has behaved "classically," the donor plate in row ^Kindlv carried out for us by Dr. D. J. Osborne at Oxford. 136 Barlow, Hancock, ajid Lacey I losing promoter and the receptor gaining it \vith increasing time of transport; in row 2, with the donor plate at the base of the section, there has been no movement of promoter. Inhibitor has not moved either way (rows 3 and 4), nor has it prevented the basipetal move- ment of promoter; with the mixture of auxin and inhibitor giving a response about the same as the controls to begin with, when applied apically (row 5) the promoter moves out of the donor block, so that it becomes more and more inhibitory, and into the receptor block which becomes more and more promoting. Basal application of the mixture allows no movement so that the net effect of the plate re- mains at about the control level. It is of great interest, however, that the sections used for transport have responded equally to promoter applied at apex or base, the length of the rings after 23 hrs. transport time being 108.5 per cent and 109.5 per cent, respectively, of the length of rings between plain agar plates; they also responded similarly to apical or basal supplies of inhibitor (90.5 and 90.6 per cent) or the mixture of inhibitor and promoter (94.0 and 94.6 per cent), implying that these substances can enter the coleoptile at either surface, but only the promoter can leave it, and then only from the basal end. The entry at either end to produce the expected promotion or inhibition of growth has been demonstrated in another series of ex- periments with 1 cm. coleoptile sections. Short lengths of polyvinyl chloride tubing of a bore slightly larger than a coleoptile were held in two parallel strips of plasticene on 3 inch x 2 inch glass slides so that pairs of ttibes were in line, with their ends 8 mm. apart; a coleop- tile section was held between the tubes with 1 mm. inserted in each. There were ten pairs of tubes per slide, the left-hand row always re- ceiving the apical end of the section; solutions were dispensed into the appropriate tubes from a micrometer syringe, and after loading with coleoptile sections the slides were placed on a klinostat so that the sections rotated horizontally about their long axes (to keep them straight) . Table 3 shows the results of two experiments averaged to- gether, and indicates that inhibitor at either end reduces growth about equally. Because NalAA is rather more effective supplied api- cally than basally (d vs. b, g vs. c) [confirmed by other experiments here, and with other techniques by Housley, Bcntley, and Bickle (7) and Choudhuri (5)], only strictly comparable treatments must be con- sidered, e.g., compared with water, inhibitor at either entl causes a reduction of 8 per cent (e — f , e — h), but in the presence of promo- ter the real effects of inhibitor are represented not by a — c, and a — g. but by the difference between water and inhibitor at a given end, i.e., b — c = 12 per cent for apically supplied inhibitor, and d — g = 13 per cent when supplied basally. 40 I" < 20 b^ 1 -i 1 "1 1 X 40 - I- o U 20 60- R I I I I E5 1 UJ 20 -'^^^ cr o o a: iij a. m a s 40 - 20 -oa — a 40 -^ 20 40 - 20 X 4 8 15 TRANSPORT PERIOD IN HOURS 23 Fig. 5. The response of coleoptile sections (percentage increase over original length) to materials transported through coleoptile tissue from a "donoi" agar plate (shaded histograms) to a "receptor" plate (unshaded); the donor plate contained NalAA, inhibitor, or a mixture of the two. The response to the apically applied plate is shown on the left of each pair of histograms, that to the basal application on the right. Transport periods were 0, 2, 4, 8, 15, and 23 hrs. (For further details see text.) 138 Barlow, Hancock, and Lacey Table 3. Percentage increase over initial length (10 mm.) of coleoptile sections given separate apical and basal supplies of NalAA 1 p. p.m., inhibitor 20 ints/ml, or water. Mixture of NalAA and inhibitor at both ends = 17. Apical Supply Basal Supply NalAA Water Inhibitor NalAA a. 48 b. 38 c. 26 Water d. 50 e. 24 f. 16 Inhibitor g. 37 h. 16 J- 12 DISCUSSION Although the concentration response curves differ according to the test objects employed, this inhibitor reduces extension in the fol- lowing: coleoptile sections with or without an exogenous source of auxin, pea epicotyl sections, wheat leaf bases, with or without added auxin or GA, cress roots, and pollen tubes; it also reduces the auxin- induced curvature of Avena coleoptiles. It increases the effect of auxin in causing inward curvature of slit pea stem sections, but its action here may not necessarily be truly synergistic. It has no effect in our tests on abscission, respiration, or permeability of the protoplast to water; the inhibitor does not move out of coleoptile sections into agar in either direction; transport of NalAA through coleoptile sec- tions is not affected. How then does this material effect a reduction in cell extension? These experiments do not provide an answer, but they do indicate the desirability of testing substances for biological activity in a variety of ways before calling them "inhibitors" or "promoters," or at least of keeping the definition of their activity continually in mind. Had we assayed our chromatograms by the slit pea stem test, ^\•e should have found a "synergist of lAA"; by an abscission or respiration test the same region would have been "inactive," while by many other tests it would have appeared as "inhibitory." Which of these roles, if any, does this material play in the physiology of the plant from which it has been extracted (Cf. 4) ? This is the basic and disturb- ing question which applies to many growth substance studies, and which has seldom been answered imequivocally, particularly lor these enigmatic "inhibitors ' of coleoptile section extension. It is obvious that the nature, mode of action on the cell, and func- tion in the plant, of such substances, warrant intensive study by chem- ists and biochemists, at least to the same extent as that devoted to auxins and gibbcrellins. Characteristics of an Itihibitor From Woody Shoots 139 SUMMARY Shoots of the plum rootstock 'Myrobalan B' extracted with ether yielded material which, when separated on paper chroma tograms, was inhibitory to coleoptile extension. This material was tested in various ways, with the following results. Inhibition: coleoptile section extension with or without added promoter (sodium salt of indole-3-acetic acid = NalAA) and when supplied to either end of the section; coleoptile curvature due to unilaterally applied auxin (but less sensitive than section growth); wheat leaf-base extension in the presence or absence of NalAA or GA (gibberellic acid); pea stem section extension; apple pollen tube exten- sion; cress root extension. Augmentation of auxin effect: slit pea stem inward curvature due to auxin was increased by inhibitor. No effect: abscission of Coleus and Phaseolus explants; respiration of coleoptile sections; water loss during plasmolysis of potato discs or water uptake after plasmolysis; transport of NalAA basipetally through coleoptile sections was not hindered by inhibitor. Recovery of coleoptile sections is considerable, and depends on the severity of the inhibitor treatment before releasing the inhibi- tion by washing in water. Our lack of knowledge on the nature, mode of action, and func- tion in the plant of such inhibitors is stressed, and study of these sub- stances by chemists and biochemists called for. LITERATURE CITED 1. Barlo\v, H. W. B., and Hancock, C. R. Studies on extension growth in cole- optile sections. III. The interaction of temperature and /3-indolylacetic acid on section growth. Jour. Exper. Bot. 10: 157-168. 1959. 2. , Hancock, C. R., and Lacey, H. J. Some observations on growth in- hibitors extracted from woody shoots. Rep. E. Mailing Res. Sta. 1954: 115-121. 1955. Hancock, C. R., and Lacey, H. J. Studies on extension growth in coleoptile sections. I. The influence of age of coleoptile upon the response of sections to lAA. Ann. Bot. II. 21: 257-271. 1957. 4. Burton, W. G. Some observations on the growth substances in ether extracts of the potato tuber. Physiol. Plant. 9: 567-587. 1956. 5. Choudhuri, S. H. Auxin synthesis in embryos. (Auxin production by rye embrvos, and the effect of external auxin supply on excised rye coleoptiles.) Ph.D. Thesis, London University. 1957. 6. Hancock, C. R. Studies on extension growth in coleoptile sections. II. The effects of high concentrations of /3-indolylacetic acid on section growth. Ann. Bot. II. 23: 107-119. 1959. 7. Housley, S., Bentley, J. A., and Bickle, A. S. Studies on plant growth hor- mones. III. Application of enzyme reaction kinetics to cell elongation in the Avena coleoptile. Jour. Exper. Bot. 5: 373-388. 1954. 8. , and Taylor, W. C. Studies on plant-growth hormones. VI. The na- ture of inhibitor-^ in potato. Jour. Exper. Bot. 9: 458-471. 1958. 140 Barlow, Hancock, and Lacey 9. Larsen, P. Conversion of indole acetaldehyde to indolenrctic acid in ex- cised coleoptiles and in coleoptile juice. Amer. Jour. Bot. 3G: 32-41. 1949. 10. Luckwill, L. C. Two methods for the bioassay of auxins in the presence of growth inhibitors. Jour. Hort. Sci. 31: 89-98. 1956. 11. Moewus, F. Die Wirkung von Wuchs- und Hemmstoffen auf die Kresse- wurzel. Biol. Zentralbl. 68: 58-72. 1949. 12. Radley, M. The distribution of substances similar to gibberellic acid in higher plants. Ann. Bot. II. 22: 297-307. 1958. 13. Rawes, M. Rosalie, and Hatcher, E. S. J. A method for estimating hormone activity in the plant. Rep. E. Mailing Res. Sta. 1948: 157-159. 1949. 11. Thimann, K. V., and Schneider, C. L. Differential growth in plant issues. Amer. Jour. Bot. 25: 627-641. 1938. 15. Van Overbeek, J., and Went, F. W. Mechanism and quantitative application of the pea test. Bot. Gaz. 99: 22-41. 1937. l(i. Van Steveninck, R. F. M. Abscission-accelerators in lupins (Liipinus hiteiis L.). Nature. 183: 1246-1248. 1959. The Mechanisms of Auxin Activation and Inactivation p. L. GOLDACREi C.S.I.R.O., Division of Plant Industry, Canberra, Australia The I ndote-3 -acetic Acid Oxidase-Peroxidase of Peas I want to give a brief outline of the structure and properties of the enzyme known as indoleacetic acid oxidase from peas. This enzyme system has suffered somewhat from the publication of experiments which are ahnost relevant, and from unsympathetic comparisons of preparations from diverse tissues, with the result that the literature is conflicting, both in substance and in detail. I want to try to recon- cile some of these divergences, and present a generalized picture. Firstly, indole-3-acetic acid (lAA) is inactivated by plant tissues. Secondly, macerates from many plant tissues can inactivate lAA by oxidation (9, 17, 19) . Whether such preparations have real signifi- cance for the growth of the plant or are artifacts of maceration (1,2) is a question discussed elsewhere. Thirdly, it seems likely that there exists a number of possible ways in which lAA is enzymatically destroyed. Evidence is accumu- lating that there are several alternative systems. These may vary from tissue to tissue or may coexist to varying degrees in the same tissue (8, 9). Any postulated role assigned lAA oxidase for a particu- lar tissue must take into account the properties of those enzyme sys- tems present in that tissue. The lAA oxidase system of etiolated pea epicotyls or roots is pres- ent entirely in the nonparticulate fraction (8). Each mole of lAA disappearing is accompanied by the consumption of one mole of oxy- gen and the production of one mole of carbon dioxide (17, 19). The stepwise reduction of oxygen, with the intermediate formation and subsequent utilization of hydrogen peroxide, is inferred from the total inhibition of lAA destruction by peroxide-consuming addenda. ^Deceased April 16, 1960. [143] 144 P. L. Goldacre such as catalase (4, 6), certain peroxidase substrates (6, 13), or manga- nese (4) in the presence of the peroxidase of the preparation. The no- tion that peroxidase is obligatorily implicated in the process is sup- ported by the total inhibitions produced by cyanide, azide, and other agents which bind heavy metals (17, 19). Thus, we may picture that lAA is subject to two successive oxida- tive steps, one mediated directly or indirectly by oxygen, the other by hydrogen peroxide: lAA -\- HviOo > Pi (peroxidase) V Pi + O2 > Po + H2O2 (oxidase) The peroxide must be produced in one of the steps in the oxida- tion of lAA itself, for if the oxidation of an exogenous metabolite were involved obligatorily, two and not one mole of oxygen per mole of lAA would be consumed. Since catalase can inhibit oxygen up- take completely, we may infer that the peroxidase moiety deals -with lAA itself and the oxidase with the reaction product. Otherwise we would expect a maximum inhibition of 50 per cent oxygen uptake while the oxidase step continued independently. Dialysis (8) or ultrafiltration (13) of enzyme preparations reveals that activity is enhanced by the presence of a diffusible cofactor. However, exhaustive dialysis may not reduce the activity to zero, and for a particular tissue, there remains a consistent residue of activity which is not cofactor-dependent (8). This residue may amount to about 40 per cent in macerates from the epicotyls of peas grown in weak red light, 10 per cent for those grown in darkness, and zero for pea roots. The pineapple enzyme appears to have no cofactor require- ment (9). This suggests that there are at least two systems for lAA destruction, acting in parallel. However, as oxidation of lAA is totally inhibitablc by catalase, cyanide, or guaiacol, the total activity depends on peroxidation. Activity lost by dialysis can be re]:)laced, or even increased above the original level, by adding a monohydric phenol, e.g., 2,4-dichlorophenol (DCP), optimum concentration 3 X 10-5 M. As DCP can overcome the inhibition by catalase (6, 7), it is concluded that it stimulates production of peroxide or promotes its more effective utilization. A property of great interest in the pea epicotyl preparation is its activation by light. Though there is considerable activity in dark- ness, up to fourfold promotion may be obtained by 250 foot candles of \\)n(c i; MnO + O. + H,.0 MnO + MnO. > MuoOg Mn.O;, + H.O, > 2MnO + Oo + H.O Promotion occurs in the presence of natural cofactor or DCP by fa- cilitating utilization of peroxide (13, 20). Thus, it seems that purified peroxidase can mediate the peroxi- dation of lAA and the oxidation (by oxygen) of its product. This is not an unique instance, highly purified peroxidases acting similarly toward dihydroxymaleic acid (16), tryptophan (15), phenylacetalde- hyde (12), and certain dicarboxylic acids (14) . It is now established (11, 18) that plant tissues contain several sep- arable peroxidases, differing in substrate specificities. It would be desirable to compare the peroxidase components of tissues under study, determine wliich can mediate lAA oxidation, and examine whether these separated components show any differential response to light or DCP. The total lAA oxidase activity of pea epicotyl brei may be con- tributed to by a number of enzymatic components, and modified by the presence of manganese, natural cofactors and inhibitors, and light. The important task facing us is not only to resolve the descrip- tive biochemistry of the macerates, especially for each tissue under consideration, but to interpret the meaning of this lAA-destroying activity for the plant. LITERATURE CITED 1. Uoiincr, \V. D., Jr. Soluble oxidases and their functions. Ann. Rc\ . IMant I'hysiol. 8: 427-452. 1957. 2. Briggs, \V. R., Sleeves, T. A., Sussex, I. M., and Wetmore, R. H. .\ compari- son of auxin destruction by tissue extracts and intact tissues of the fern Os- munda cinnamomca L. Plant Physiol. 30: 148-155. 1955. 3. Galston, A. W., and Baker, R. S. Studies on the physiology of light action. II. The phoiodynamic action of riboflavin. Amcr. jour. Rot. 36: 773-71^0. 1949. 4. , and Baker, R. S. Studies on the physiology of light action. III. light activation of a flavoprotein enzyme by reversal of a naturally occurring inliibi- tion. Amer. Jour. Bot. 38: 190-195. I95I. 5. , Bonner, J., and Baker, R. S. Flavoprotein and peroxidase as com- ponents of the indoleacetic acid oxidase system of jJcas. Arch. Biochem. Bio- phys. 42: 456-470. 1953. 6. Goldacre, P. L. Hydrogen peroxidase in the enzymatic oxidation of hctcro- auxin. Austral. Jour. Sci. Res. Ser. B. 4: 293-302. 1951. lAA Oxidase-Peroxidase of Peas 147 7. , and Galston, A. W. The specific inhibition of catalase by substituted phenols. Arch. Biochem. Biophys. 43: 169-175. 1953. 8. , Galston, A. W., and Weintiaub, R. L. The elTect of substituted phenols on the activity of the indoleacetic acid oxidase of peas. Arch. Bio- chem. Biophys. 43: 358-373. 1953. 9. Gortner, W. A., and Kent, M. Indoleacetic acid oxidase and an inhibitor in pineapple tissue. Jour. Biol. Chem. 204: 593-603. 1953. 10. Hillman, W. S., and Galston, A. W. Interaction of manganese and 2,4- dichlorophenol in the enzymatic destruction of indoleacetic acid. Physiol. Plant. 9: 230-235. 1956. 11. Jermyn, M. A., and Thomas, R. Multiple components in horse-radish per- oxidase. Biochem. Jour. 56: 631-639. 1954. 12. Kenten, R. H. The oxidation of phenylacetaldehyde by plant saps. Biochem. Jour. 55: 350-360. 1953. 13. . The oxidation of indolyl-3-acetic acid by waxpod bean root sap and peroxidase systems. Biochem. Jour. 59: 110-121. 1955. 14. , and Mann, P. J. G. The oxidation of certain dicarboxylic acids by peroxidase systems in presence of manganese. Biochem. Jour. 53: 498-505. 1953. 15. Knox, W. E. The action of peroxidases with enzymatically generated per- oxide in the presence of catalase. Biochem. Biophys. Acta. 14: 117-126. 1954. 16. Swedin, B., and Theorell, H. Dioximaleic acid oxidase action of peroxidases. Nature. 145: 71, 72. 1940. 17. Tang, Y. W., and Bonner, J. The enzymatic inactivation of indoleacetic acid. I. Some characteristics of the enzyme contained in pea seedlings. Arch. Biochem. 13: 11-25. 1947. 18. Theorell, H. Reversible splitting of a peroxidase. Ark. Kem. Min. Geol. 14B(No. 20): 1-3. 1940. 19. Wagenknecht, A. C., and Burris, R. H. Indoleacetic acid inactivating enzymes from bean roots and pea seedlings. Arch. Biochem. 25: 30-53. 1950. 20. Waygood, E. R., and Maclachlan, G. A. The effect of catalase, riboflavin, and light on the oxidation of indoleacetic acid. Physiol. Plant. 9: 607-617. 1956. E. R. WAYGOOD and G. A. MACLACHLAN^ University of Manitoba I nhibit'Lon and Retardation of the Enzymatlcally Catalyzed Oxidation of I ndole-3 -acetic Acid From studies described previously (5,6,9) on the enzymatic and nonenzymatic breakdown of indole-3-acetic acid (lAA) in vitro, a chain oxidative reaction sequence has been proposed for the peroxi- dase catalyzed decarboxylation and oxidation of lAA. The chain reaction is initiated by a system consisting of either per- oxidase or catalase and a specific type of phenolic cofactor which oxi- dizes manganous (Mn^^) ^q manganic ions (Mn^^). propagation of the chain is brought about by a reaction between lAA (S-COOH) (S — skatole) which results in its spontaneous decarboxylation and the consumption of one equivalent of oxygen to form a skatole peroxy- radical as follows: Mn-3 + SCOOH > Mn-- + H^ + CO, + S- s. + Oo > so,- The skatole peroxy-radical is stabilized by an enzyme-controlled peroxidation involving the phenolic cofactor (ROH) as hydrogen donor, resulting in the latter's oxidation and the formation of an end product of empirical formula (SO,H); viz. so... + ROH i«I^> SO,H + RO. catalase The phenolic radical (semiquinol) is capable of oxidizing manga- nese and regenerating the reduced cofactor by the Kenten-Mann re- action (4) until the supply of lAA is exhausted, e.g., Mn-2 _^ H- + RO- > Mn-3 _^ rqH In the present communication the inhibitory and retarding effects of various substances on lAA oxidation are interpreted in terms of ^Subsequently: Department of Botany. ITniversity of All^erta, Edmonton. Canada. [149] 150 E. R. IVaygood and G. A. Maclachlan a chain-stopping or chain-transferring mechanism. It should be noted that the destruction of lAA differs from standard chain oxidations (7) by being dependent on peroxidase. Consequently, substances that inhibit peroxidase or catalase, e.g., cyanide, also inhibit LA^\ oxida- tion. Such enzyme poisons are to be distinguished from inhibitors and retarders of the chain oxidation of lAA and are not considered in this paper. METHODS The preparation of wheat leaf extracts and horseradish peroxidase, the reaction conditions, and the experimental techniques used in the present study have been described previously (6, 9). Unless otherwise stated, standard systems with resorcinol or dichlorophenol (DCP) as cofactor have been used to catalyze the oxidation of lAA. These contain the following components: 0.50 ml. wheat leaf en- zyme (ca. 0.2 mg. protein N) ; 3.0 fjii MnCL; 1.5 fJM resorcinol or 2,4-dichlorophenol (DCP) ; 150 fjuM orthophosphate, pH 6.0; 6.6 fuM lAA, ammonium or sodium salt, pH 6.0 z= 158 ^1. Oo: \ol. 3.0 ml.; 29.5° C. EXPERIMENTAL RESULTS Typical progress curves of the oxygen uptake during L\A oxida- tion catalyzed by extracts from winter-grown wheat leaves exhibited 80 120 TIME. min. Fig. 1. Retardation and inliihiiion of indole-3-acetic acid oxidation. .S\sttins standard with: A, no additions; B, 0.03 fiM (10"* M) catechol; C, 0.63 mM (2.1 x ^^~* M) riboflavinphospliatc; D, 0.015 fiM (1.5 x 10"'' A/) livthocjninonc. Inhibition and Retardation of the Oxidation of lAA 151 Table 1. Inhibition and retardation of indole-3-acetic acid oxidation in the presence of catechol, hydroquinone, and riboflavinphosphate. * Induction Period in the Presence of Catechol, (Minutes) Percentage of Control Rate Hydroquinone Riboflavinphosphate Cofactor Dark Light Resorcinol Dichlorophenol. . Phenol 46.5 24.0 26.5 30.0 41.0 55 59 45 53 42 35 28 31 33 40 100 97 91 Maleic hydrazide. Natural factor. . . . 94 147 ■ Concentrations: Catechol 1.5 X 10-^ fiM; hydroquinone 6 X 10/ ^Mj ribo- flavinphosphate 3.3 X 10-1 f,M; resorcinol, DCP, phenol, 1.5 ^M; maleic hydrazide 30 txM, natural factor 0.15 ml. Vol. 3.0 ml. blue light, 220 foot candles. a short induction period and a subsequent rapid rate of oxidation that gradually decelerated until theoretical molar equivalence was attained (Figure lA). The rate of oxidation was influenced by sub- stances that either extended the induction period, e.g., catechol at 10-5 M (Figure IB), or retarded the rate of oxidation from the out- set, e.g., riboflavinphosphate at 2.1 X 10"' M (Figure IC) or hydro- quinone at 1.5 X 10-^ M (Figure ID). The above experiments were performed using resorcinol as co- factor of the oxidation, but the effects of these inhibitors and re- tarders were the same in the presence of other cof actors (Table 1). Catechol resulted in an extension of the induction period the length of which depended on the cofactor used. On the other hand, the degree to which hydroquinone and riboflavinphosphate retarded the oxidation of lAA was almost independent of the nature of the co- factor. The systems retarded by riboflavinphosphate, but not those retarded by hydroquinone, were rendered fully active by illumination (Table 1). Extension of the Induction Phase (Inhibitors) The relationship between the concentration of catechol in the system and the length of the induction period is shown in Figure 2. The data show that the oxidation of lAA would never occur above a catechol concentration of 1.9 X 10-^ M. However, this value can be regarded only as approximate since the age of the solutions and the enzyme used were found to influence the length of the lag phase induced by catechol (9). A similar inhibition was caused by pyrogallol and the flavonoid pigment rutin, both of which extended the lag phase by 25 min. at 152 E. R. Waygood and ('.. A. Maclnrhlan Induction Period, nnin. Fig. 2. Induction period in indole-3-acetic acid oxidation caused l)y catechol. Sys- tems standard, catechol added prior to lAA. conceiuiaiions of 1.7 X 10-^ M and 0.5 X 10-^ M respectively. Cate- chol and pyrogallol were reported also to inhibit the oxidation of lAA catalyzed by horseradish peroxidase (3). Ascorbic acid is another inhibitor that extended the lag by 45 min. at 3.33 X 10"^ ^^- ^^ is noteworthy that an appreciable lag phase was induced by these in- hibitors at concentrations as low as 10 •'* M. Rctarders Typical of the progress curves of oxygen uptake at varying con- centrations of retarder are those shown in Figure 3, where riboflavin- phosphate was progressively more inhibitory in darkness as its con- centration was increased above 10-^ M. The progress curves for hy- droquinone retardation were similar except that the systems at- tained a low equilibrium (Figure 1). This was not due to enzyme or cofador destruction, but apparently to a decline in the conren- TIME, min. Fig. 3. Progress of indole-3-acetic acid oxidation in the presence of riboflavin- phosphate in darkness. Systems standard, riboflavinphosphate concentration: A, 0; B, 0.126 ixM; C, 0.188 /xM; D, 0.375 ,iM; E, 0.63 /mM; F, 3.65 fiM. 154 E. R. Waygood and G. A. Maclachlan 40r-- Concn. Retarder, M. Fig. 4. Relative rales of iiidole-3-acetic acid oxidation in the presence of various concentrations of retarders. Systems standard containing: A, hydroqninonc; B, ^-quinone; C, riboflavinphosphate; D, scopoletin. tration of lAA, since the further addition of lAA at equilibrium caused a resumption of oxygen uptake. Both hydroquinone and its oxidation product p-benzoquinone retarded the oxidation to the same extent. The coimiarin derivative scopoletin, already shown to be a competitive inhibitor of lAA oxidation (1), had effects re- sembling riboflavinphosphate. The maximum initial rates of lAA oxidation in the presence of each of these retarders are compared as a function of retarder con- centration in Figure 4. Unlike the inhibition induced by catechol, etc., inhibition caused by retarders did not appear to depend on the age of reagents, enzyme, etc., but only on the concentration. Irrespective of the absolute rate of the unretarded control, close to 50 per cent retardation occurred at the following concentrations: hydroquinone 3.0 X ^0'^ ^t'> p-quinone 3.5 X 10'^ ^^^; riboflavin- l)hosphatc 7.5X10"^"' ^^J', scopoletin 1.25x10^ ^^^- As indicated Inhihition and Retardation of the Oxidation of lAA 155 Table 2. Retardation of lndole-3-acetic acid oxidation* by ribollavinphosphate. The effect of enzyme, manganese, dichlorophenol, and indoIe-3-acetic acid concentra- tions. Concentration of Varying Component Percentage Retardation Enzvme, ml. Mn+2, DCP, lAA, mA/ Enzyme Mn+2 DCP lAA 0.1 1.5 0.5 1.66 10 75.5 24 42 0.25 3 1.0 3.33 44 75 39 53.5 0.50 6 1.5 5 52 76,5 59 60.5 0.75 30 4 6.66 60 77 70 67.5 1.0 60 6 10 58 69 77 75.5 * Standard DCP-horseradish peroxidase systems containing 0.6 iiM riboflavin- phosphate. Components were varied individually. previously (Table 1) the extent to which systems were retarded by hydroquinone and riboflavinphosphate was almost independent of the nature of the cofactors. In order to elucidate the mechanism by which riboflavin or its phosphate exerts a retarding effect on the system in darkness, kinetic studies were undertaken to determine the effect of the concentration of the individual components on the degree of retardation. The data in Table 2 show the effects of varying the concentration of enzyme, substrate, manganese, and dichlorophenol on the retardation brought about by riboflavinphosphate at a concentration of 0.6 fM. Standard DCP-horseradish peroxidase systems were employed. The data indi- cate that under these experimental conditions an increased concen- tration of any of the components was unable to overcome the re- tardation by riboflavin. However, it has been shown previously (6) that it would be more accurate to describe the optimum concen- tration of the phenolic cofactor relative to the substrate concentra- tion since in this delicately balanced system its activity is dependent on the concentration ratio, e.g., lAA/resorcinol = 2.2, and lAA/ DCP =z 4.4. In subsequent kinetic experiments it was shown that when this ratio was maintained in the case of lAA/DCP and the concentration of lAA increased as in the experiments in Table 2, then the retardation by riboflavinphosphate could be partially over- come. In these standard DCP systems with 6.6 /JVf of lAA the rate was retarded 60 per cent by 0.5 jjlM of riboflavinphosphate. When the concentration of lAA was increased to 13.2 /xM and 16.5 fxM and the concentration ratio lAA/DCP maintained at 4.4, the retardation was 42 per cent and 24 per cent, respectively. 156 E. R. Way good and G. A. Maclachlan The Effect of Light and Riboflavin As was demonstrated previously with the catalase-dichlorophenol system (8), blue light, absorbed by riboflavin, overcame the retarding effect of riboflavinphosphate on the wheat leaf system in the presence of any one of the cofactors (Table 1). The rate of oxidation of illu- minated systems containing riboflavin was no greater than the rate in darkness without riboflavin except when the natural factor (9) was used. In the absence of riboflavin, light had no effect on the system. The augmentation of the dark rate in the illuminated system con- taining the natural factor and riboflavin may have been due to the combined effect of riboflavin and light in overcoming an inhibitor present in the partially purified extract of the former (cf. 8). The alleviation of the riboflavin effect depended on the quality 140 -^CVJ Fig. 5. EfTect of quality and iiuciisily of light on ribofUu in inhibited indole-3- acetic acid oxidation. Standard system plus 6 /iM riboflavinphosphate. Color and intensity of light (in foot candies at surface of reaction vessel) indicated in diagram. Inhibition and Retardation of the Oxidation of lAA 157 and intensity of illumination. Figure 5 shows the progress ol the oxidation with various kinds of illumination. As would be expected from the absorption spectrum of riboflavin, blue light was most effective, whereas red light was totally ineffective and white light was required at a higher intensity to give the same effect as blue. In systems containing 0.33 fxM riboflavinphosphate, light saturation was attained at an intensity of 110 foot candles, under which conditions the rate of oxidation attained a maximum equal to the dark rate without riboflavin. Oxygen uptake always proceeded well past the theoretical oxygen equivalence for lAA (Table 3). It is noteworthy that after the light had been switched off (Figure 5), rapid oxidation of lAA continued for some time, indicating a residual effect of light except when lAA became limiting. The oxygen consumed in the breakdown of lAA by catalase systems was observed to exceed the theoretical molar equivalence of lAA when illuminated in the presence of riboflavin (8). This also occurred with the wheat leaf system (Figure 6A and Table 3). In the experiment (Table 3) carried on for 400 min., 199 fx\. of oxygen were consumed by an illuminated standard system containing 6.0 /JVf riboflavin and the equivalence of 3.3 jxM or 79 /xl. lAA. The rate of oxygen uptake showed no signs of abating, and similar results were obtained when DCP or maleic hydrazide was present in place of resorcinol. The amount of riboflavin used in this experiment was approximately 30 times greater than that required to produce 50 per cent inhibition. In the absence of lAA (Figure 6C), oxygen was consumed by these systems at a slower initial rate, but at the same final rate as with lAA (Figure 6A). The difference between the oxygen consumed with and without lAA (Figure 6B) always exceeded at equilibrium the molar oxygen equivalence of lAA. The difference was greater at Table 3. Oxygen consumed by illuminated systems con- taining riboflavin.* Oxygen Uptake (;ul/400 min) Cofactor Plus lAA Minus lAA Difference Resorcinol 199 181 149 70 53 32 129 Dichlorophenol Maleic hydrazide 128 117 * Standard wheat leaf systems containing: 3.33 ixM lAA; 6 . 0 /iA/ riboflavinphosphate; blue light, 220 foot candles. Molar oxygen equivalence of lAA = 79 ^il.; riboflavinphosphate = 133 ix\.; resorcinol and dichlorophenol =35.5 ^1- maleic hydrazide = 712 m1- 158 E. R. Waygood and G. A. Maclachlan TIME, min. Fig. 6. Oxygen uptake of systems containing excess riboflavin with and without inclole-3-acetic acid: A, standard system phis 6 fiM riboflavinphosphate in bUie light (220 foot candles) indole-3-acetic acid = 3.33 jxhl = 79 /^l. O2 (molar equiv- alence); B, oxygen uptake of A minus uptake of C; C, same as A minus indole-3- acetic acid; D, same as C in darkness. higher light intensities or when the riboflavin concentration was increased, e.g., to twice the molar concentration of lAA. DISCUSSION In order to inhibit such an autoxidation sequence a substance must interrupt a chain of interdependent steps by reacting with an essential intermediate of the system. Inhibition may be expressed in either of two ways: (1) as an extension of the induction period which occurs in all autoxidations; or (2) as a retardation of reaction velocity. As pointed out by Waters and Wickham-Jones (7) these ef- hiliibition and Retardation of the Oxidation of lAA 159 fects are caused by agents which interfere in the reaction sequence in fundamentally different ways and which may be described as chain- stopping and chain-transferring agents respectively. In the presence of an inhibitor or chain-stopping agent the ini- tiation of autoxidation is prevented. At low inhibitor concentrations the reaction may commence abruptly after a long induction period and attain a rate equal to that of the control in the absence of in- hibitor. Such temporary inhibition or lag-extension indicates that the inhibitor is irreversibly changed during the induction period to a product that is not inhibitory to the reaction. Its destruction must occur at the expense of an essential intermediate of the system, but it also may be aided by side reactions, e.g., by autodestruction. Thus, the system would never operate in the presence of excessive amounts of the inhibitor or if the latter were not destroyed. On the other hand, in the presence of retarding agents, the re- action may proceed to completion at a reduced rate without an in- duction phase. This could occur only if the retarder acted as a chain- transferring agent by substituting a sequence of slow reactions within the rapid sequence of the control. In order to exert its retarding effect continuously, the substance must be reformed following re- action with an essential intermediate. Thereby the retarder inserts a shunt into the normal reaction sequence which slows down the speed of propagation. Mechanism of Inhibition by Chain-stopping Agents According to the foregoing definition, it may be validly assumed that the catechol-type inhibitor is a chain-stopping agent and is de- stroyed by an intermediate prior to or during the first reaction step of lAA oxidation. The length of the catechol-induced lag period is dependent on its concentration, and when an amount of catechol that would normally cause a 45 min. lag phase is added during the oxidation only a brief staggering of the rate resulted. This was also true of the inhibitor found in boiled undialyzed preparations of commercial catalase (8). Such effects w^ould be expected if the de- stroying agent is an essential intermediate present at a higher con- centration after the oxidation has commenced. Catechol, pyrogallol, ascorbic acid, and probably rutin are all readily oxidized and could temporarily inhibit the oxidation of lAA by reducing manganic ions, a cofactor radical, or possibly a skatole radical. \A'e have suggested previously (9) that manganic ions arising dur- ing the initiation and propagation reactions preferentially oxidize catechol. This is supported by the findings that MnOo, PbOo, HoO^, or an oxygen atmosphere may partially or completely overcome catechol inhibition since these are known to lead to the formation 160 E. R. Way good and G. A. Maclachlan of Mn+3 from Mn^^. Manganiversene is rapidly reduced by catechol, and pre-incubation of catechol with manganiversene or even manga- nous ions also destroys its inhibitory effect, presumably by promoting oxidation of catechol. Tiie evolution of carbon dioxide is also in- hibited by catechol, antl this lends support to the hypothesis that catechol exerts its inhibition in the initiation reactions or in the first step of propagation in which reactions manganic ions are involved. Nevertheless, the possibility cannot be excluded that the inhibition by catechol is caused by stabilization of a cofactor or skatole radical which in turn results in its destruction. The fact that the catechol- induced lag period varies in length with the cofactor used (Table 1) suggests that the cofactors or their radicals may compete at different rates for catechol, but this could also be interpreted in terms of their varying reactivity in manganigenesis. If, on the other hand, catechol were involved in a reaction with skatole radicals which are involved solely in propagation, it would be more likely to exert a retarding effect rather than an inhibition at least at lower concen- trations. The most probable explanation is that catechol reacts with manganic ions produced during the initiation reactions and tem- porarily blocks the decarboxylation of lAA. The same argument would apply to pyrogallol, ascorbic acid, and possibly rutin. Mechanism of Retardation by Chain-transferring Agents The retarding effects of hydroqtiinone or its oxidation product, p-benzoquinone, riboflavin, or its phosphate, and scopoletin persisted even though several himdred moles of oxygen were consimied per mole of retarder present. This persistence indicates that although these retarders are changed by interfering in the reaction sequence they must also be reformed probably by participating in reversible redox system, a property that they wotdd all share. Hydroquinone p-benzoquinone. Systems containing hydroqtiinone differed from the others in the lower final equilibrium attained (Figure 1). This was due to a decline in lAA concentration and indi- cates a side reaction of the retarder with lAA. The oxygen uptake of systems containing either hydroquinone or p-benzoquinone dif- fered only in the initial velocity, which was always slightly greater with the latter. Thereafter the progress curves continued parallel as would be expected if a steady state equilibrium was established between the oxidized and reduced forms. Since hydroquinone rapidly reduces manganiversene it is prob- able that it competes successfully with lAA for Mn^"^ in tlie same way as catechol, but differs in that the oxidation product p-quinone is not without effect on the system, but enters into another reaction Inhibition and Retardation of the Oxidation of lAA 161 in which it is reduced back to hydroquinone and indirectly causes the further production of Mn^'. The only readily oxidizable compo- nent that could take part in such a reversible reaction is the phenolic cofactor. Resorcinol forms an insoluble oxidation product merely on standing with p-quinone. Dichlorophenol and maleic hydrazide did not show any visible reaction, probably because their oxidized radi- cals (RO-) do not condense readily to colored products. Neverthe- less, when p-quinone was incubated with pyrophosphate or citrate, the addition of either maleic hydrazide or dichlorophenol resulted in the oxidation of manganese. Manganic ions were rapidly produced on warming and were detected as pink manganipyrophosphate or orange manganicitrate. This is essentially the Kenten-Mann reaction for the oxidation of manganese (4), but differs in that quinone re- places peroxide plus peroxidase. Assuming that hydroquinone [ O (—OH) 2 ] competes with lAA for Mn+3, then the mechanism of this retardation may be considered due to the partial substitution in the chain of the following series of slower reactions, where ROH repre- sents the monohydric phenolic cofactor. Q (—OH), + Mn-' -^ Q (=0). + Mm^ + 2H- O (=0)o +2ROH ^ O (—OH), + 2RO- RO- + Mn-2 ^ H- ^ ROH + Mn-^ Riboflavin. In contrast to hydroquinone, riboflavin (Rb) is added in the oxidized form only. Consequently, if it were not involved in a reversible redox system, it would have no effect on the system, but in this respect it resembles p-benzoquinone. We have implied pre- viously that the redox system (Rb ^ Rb-2H) is established and that most probably the oxidized form indirectly generates Mn^^ by reacting with the phenolic cofactor and reduced riboflavin (Rb-2H) would compete with lAA for M.\Y'\ This would have the effect of retarding the oxidation from the outset by a chain transfer mecha- nism. The evidence in support of the manganigenic properties of riboflavin is as follows. Riboflavin partially overcomes the catechol- induced lag period of lAA oxidation in the same manner as man- ganiversene, MnOo, PbOo, HoOo, and oxygen which effects have been interpreted as manganigenic. Riboflavin, as well as these other agencies, also overcomes the inherent lag period of resorcinol oxida- tion catalyzed by Mn-peroxidase system (6). This oxidation appears to be dependent on the generation of Mn+s since it is completely inhibited by pyrophosphate and citrate as is lAA oxidation when catalyzed by Mn-peroxidase-resorcinol systems (9). The only readily oxidizable component of the lAA oxidation system is the phenolic 162 E. R. Waygood and G. A. Maclachlan cofactor, e.g., resorcinol or DCP, etc., and presumably riboflavin oxidizes resorcinol, for example, to its semiquinol which can in turn oxidize Mn+-' to Mn^^ by the Kenten-Mann reaction (4). Quite apart from this work, Andreae (2) has demonstrated that light-activated riboflavin generates Mn^^ from Mw- in the presence of monohydric and polyhydric phenols which do not readily form quinones on oxi- dation, and he has postulated a similar reaction. The ease with which riboflavin forms free radicals w'hen illuminated suggests that this would occur not too infrequently in the presence of metal ions in darkness or in the dilfuse light of the Warburg bath. In the absence of lAA, standard Mn-phenol-peroxidase systems containing 6 ixM of riboflavin consumed oxygen in the dark, a phenomenon that was accelerated by illumination. The oxygen con- sumed by these systems could not have been due to irreversible co- factor oxidation entirely since it appeared to be capable of contin- uing indefinitely and by 400 min. had exceeded the molar oxygen equivalence of the cofactors. Since no oxygen uptake occurred in the absence of a cofactor or manganese it is suggested that the prod- uct of the reaction in light or darkness must be oxidized manganese formed by reversible cofactor oxidation. From the work of Kenten and Mann (4) it is known that manganic ions are to some extent stable in orthophosphate, and there is also the possibility that MnO^ could be produced. A mechanism whereby riboflavin (Rb), a redox catalyst (ROH), and peroxidase could interact to oxidize manganese is summarized as follows: Rb + 2ROH -^ Rb-2H + 2RO- (A) Rb-2H + Oo -^ Rb 4- HoOo (B) peroxidase HoO., + 2R0H' > 2H..O + 2RO- (C) or catalase RO- + Mn-2 ^ H- ;?:± ROH + Mn-^ ) ) (D) RO- + Mn*=^ -f H^ -^ ROH + Mn^^ ) Reaction (B) is a well-known spontaneous reaction, (C) is normal peroxidation, and (D) the Kenten-Mann reaction. This reaction sequence is similar to that proposed by Andreae (2) with the excep- tion that peroxidase and catalase are considered here to perform a peroxidatic reaction (C) and thus dispose of hydrogen peroxide which would otherwise speed the decomposition of oxidized man- ganese. In the absence of enzyme some oxygen uptake occurred (6) and manganic ions were formed (2), though to a lesser extent. It is important to note that light absorbed by riboflavin is not an absolute requirement, but merely an accelerator of these reac- tions. In darkness, manganic ions were still produced by Andreae's hihibition and Retardation of the Oxidation of lAA 163 system and oxygen was consumed at a slow rate by the system described here. When these systems were illuminated, the production of manganic ions was increased (2) as was also oxygen uptake in our system. With respect to the mechanism by which light activates the re- action sequence, tests were made on the effect of light on the oxygen uptake of riboflavin-resorcinol systems (reaction A), and on the spon- taneous oxidation of reduced riboflavin in air (reaction B). In the absence of manganese and enzyme, very little reaction was found to occur between riboflavin and resorcinol in light or darkness, and hence an activation by light of reaction A as proposed by Andreae (2) appears unlikely. On the other hand, white light (980 foot candles) almost doubled the rate of oxygen consumed by solutions of riboflavinphosphate that had been reduced by dithionite. There- fore, we suggest that light activated Andreae's system and the system described herein at the stage of reaction B. Oxygen uptake in this system appeared to continue indefinitely and in all probability could exceed the combined theoretical molar equivalence of riboflavin and cofactor. This is possibly due to the cyclical nature of the reaction in which the over-all reaction is an oxidation of Mn+" to Mn+4, but the intermediate valence stage Mn+^ may compete with oxygen for reduced riboflavin as follows: Rb-2H + 2Mn-3 _^ Rb ^ 2Mn-2 ^ 9h- When the system is ilkmiinated, reduced riboflavin reacts prefer- entially with oxygen owing to light activation at this stage (reaction B). This would explain the increased production of manganipyro- phosphate as found by Andreae (2) and the increased oxygen uptake of our system under illumination. The significant points emerging from the experiments of Andreae (2) and those reported here are that riboflavin can indirectly generate manganic ions in this system and that reduced riboflavin may pos- sibly react with Mn+3. This would provide an explanation not only for the retardation of the indole-3-acetic acid system by riboflavin, but also for the alleviation of this retardation by light. Evidently the reduced form of the retarder which is a necessary product of manganigenesis must be the immediate cause of retardation by be- ing involved in a second redox system with an essential oxidized intermediate. The degree of retardation would depend on the equilibrium established between the oxidized and reduced forms of riboflavin, the latter diverting an oxidized intermediate from its normal function as a chain reactant in indole-3-acetic acid autoxida- tion. The intermediate with which reduced riboflavin reacts is most 164 E. R. Way good and G. A. Maclaclilan probably the manganic ion. This is supported by the toUowing evi- dence. Riboflavin suppresses oxygen uptake and carbon dioxide evo- lution equally; therefore, it must compete ^\ith an oxidized inter- mediate in the initiation reactions or in the first step of propaga- tion. Reduced riboflavin decolorizes manganiversene instantaneous- ly, and kinetic evidence suggests that no reaction occurs with other oxidized intermediates. For example, any reaction of reduced ribo- flavin with oxidized cofactor radical (reverse of reaction A) could occur only at the expense of the normal reaction of the cofactor radical with Mn+2 since an increase in Mn^^ concentration failed to counteract the retardation by riboflavin in darkness, then manganese and riboflavin probably do not compete for the cofactor radical. Similarly riboflavin does not compete with the cofactor for the skatole peroxy-radical; otherwise different cofactors would have a significant effect on the degree of retardation and an increased con centration of cofactor by itself should counteract the retardation. The alternative explanation is that reduced riboflavin competes with lAA for Mn+*^, but increased concentrations of lAA alone do not overcome the retardation. However, because the critical optimum concentration of the cofactor is dependent on the concentration of lAA, the unbalance caused by varying the concentration of lAA in- dependently of the cofactor may be the cause of the ineffectiveness of lAA in overcoming the retardation by itself (Table 2). \\lien the concentration ratio lAA/DCP was maintained constant at 4.4, in- creasing concentrations of lAA were able to overcome the inhibi- tion. Such evidence makes it appear most probable that Mn^-' is implicated in a reaction with reduced riboflavin. Accordingly it is possible to formulate a hypothesis for the mechanism of retardation as follows: In darkness (retardation). Rb + 2ROH -^ Rb-2H + 2RO- RO- + Mn-2 _^ H- ;e± ROH + Mn^^ Rb-2H -f 2Mn^3 _^ Rb _|- 2Mn*2 ^ 2H- This series of slow reactions would be substituted in part for the normal sequence of reactions. Some Rb-2H possibly cannot escape reaction with oxygen, but it would more likely be preferentially oxi- dized by Mn^3 In light. The retardation is overcome since Rb-2H reacts prefer- entially with Oo (light activated) and the HoOo produced supjjorts the production of cofactor radicals for manganigcnesis in tlic Ken- ten-Mann reaction as follows: Inhibition and Retardation of the Oxidation of lAA 165 Rb + 2ROH > Rb.2H + 2RO- Rb-2H + O. — > Rb + HoOo H.,Oo + 2ROH P!I^^^!£^> 2RO- + 2HoO " " catalase In this illuminated system riboflavin would be maintained in the oxidized state by oxygen rather than by Mn+-^; thus it would not interfere in the reaction sequence of lAA oxidation and cause a transfer of the chain reaction. Also oxygen consumption would con- tinue well past the theoretical for lAA oxidation since it would re- vert to a slower cyclical reaction in which Mn+4 (MnOo) may be the eventual end product accompanied by the uptake of oxygen as dis- cussed earlier. Implicit in the interpretation of the mechanism of action of in- hibitors and retarders in this paper is the essential correctness of the scheme proposed by the present authors to explain the catalytic action of wheat leaf enzymes, horseradish peroxidase, and beef liver catalase on the oxidation of lAA in vitro. The fact that all com- ponents of the system exist or are readily available in vitro justifies consideration of the physiological significance of the system in the control of plant growth, in which lAA apparently plays a central role. From a biochemical viewpoint, the enzymically catalyzed free radical mechanism proposed for the oxidation of lAA represents a departure from classical interpretations of reaction mechanisms in plant physiology. Such phenomena as the photoreversible inhibition of lAA oxidation by riboflavin, as well as providing a useful system to study the kinetics and mechanism of photochemical reactions, may also prove, in their interpretation, to strengthen our knowl- edge of probable biochemical mechanisms underlying the photo- control of plant growth. ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of the Canada Department of Agriculture under Contract EMR-14 and also the able technical assistance of Miss Sally Dangerfield. LITERATURE CITED 1. Andreae, W. A. Effect of scopoletin on indoleacetic acid metabolism. Nature. 170: 83, 84. 1952. 2. . The photoinduced oxidation of manganous ions. Arch. Biochem. Biophys. 55: 584-586. 1955. 166 E. R. Waygood and G. A. Maclachlan CI. Kenlen, R. H. The oxidation of indolyl-3-acetic acid by uaxpod bean root sap and peroxidase systems. Biochem. Jour. 59: 110-121. 1955. 1. , and Mann, P. J. G. The oxidation of manganese by peroxidase sys- tems. Biochem. Jour. 46: 67-73. 1950. 5. Maclachlan, G. A., and Waygood, E. R. Catalysis of indoleacetic oxidation by manganic ions. Physiol. Plant. 9: 321-330. 1956. 6. , and Waygood, E. R. Kinetics of the enzymically-catalyzed oxidation of indoleacetic acid. Canad. Jour. Biochem. Physiol. 34: 1233-1250. 1956. 7. Waters, W. A., and Wickhani-Jones, C. The retardation of benzaldehyde autoxidation by p-cresol. Jour. Chem. Soc. 812-823. 1951. 8- Waygood, E. R., and Maclachlan, G. A. The effect of catalase, riboflavin and hght on the oxidation of indoleacetic acid. Physiol. Plant. 9: 607-617. 1956. 9. , Oaks, A., and Maclachlan, G. A. The enzymically catalyzed oxida- tion of indoleacetic acid. Canad. Jour. Bot. 34: 905-926. 1956. p. E. PILET University of Lausanne Switzerland Auxins and the Process of Aging in Root Cells The relationship between the aging of plant tissues and auxin metabolism has been discussed in a number of papers by several investigators (5, 14, 18, 30). Among these, the work of Galston and Dalberg (6) is of special interest, for they have shown that the ability of pea seedling cells to destroy the native auxins increases as the cell ages. The purpose of this paper is to analyze various biochemical properties of root cells in relation to indole-3-acetic acid (lAA) destruction. The advantage of working with root sections is that they possess, simultaneously, both very young tissues (meristem) and older ones (root tip). MATERIALS AND METHODS The experimental material used was Lens culinaris Med. The seeds were selected and nearly 100 per cent germination occurred after 38 hrs. Seeds were first soaked in de-ionized water for 12 hrs., then washed, and finally placed on wet filter paper in petri dishes in darkness at 22 ± 0.5° C. The first selection was made after 24 hrs., and only seedlings measuring 3 ± 1 inm. were kept and replaced in the above conditions. The seedlings were removed for treatment when they had reached a length of 18 rb 2 mm., for this has been found to be the period of optimal growth (11,29). A series of cyto- logical analyses has shown that the tip, composed of older cells, is located between the extreme point and 0.5 mm. from it while the meristem, containing the young cells, is situated in the region from 0.5 to 3.0 mm. The work on the process of aging was done ex- clusively on these two types of fragments. In order to prepare these two root sections, a small guillotine (22) was developed (Figure 1); it is a modified version of a design by Linser and Kiermayer (10). [167] 168 P. E. Pilet ___-AM I I I — I 0 6 CM ,— L PM Fig. 1. Guillotine. With the use of this apparatus, the exact root sections (0-0.5; 0.5-3.0 mm.) can be easily obtained; the roots, 18 mm. in length, are placed on a grooved (G) plate (PO) with the tips just touching a vertical upright. .\ quick depression of the handle (AM, R) lowers two blades (L) which cleanly cut the roots to size, producing the desired fragments. BIOLOGICAL ANALYSES Auxin Content The extraction of auxins from the roots (an acid fraction of ex- tracts obtained with peroxide-frcc CIICl.,) was carried out as de- scribed elsewhere (11), with subsequent measurements of activity by the Avena test. The auxin content was determined in the two re- gions previously mentioned and expressed (in terms of equivalent lLig. lAA) as a function of the age of the roots (length). Table 1 shows that the concentration of auxins in the meristem is consistently greater than that in the tip. Furthermore, the level of auxins in- creases as the roots age. These facts, together with many other observations (12), suggest Auxins and the Process of Aging in Root Cells 169 an interpretation concerning the phenomenon of root growth and aging in relation to the strength of native auxins. Figure 2 gives the essential points of this theory. The speed of growth passes a maxi- mum at the moment when the level of auxins, which increase with increasing age, becomes supra-optimal. When the root is young, lAA treatment induces an increase in elongation, whereas exposure to light inhibits growth. These facts can be explained if, at this moment, the roots possess only very low levels of auxins. If the roots are old, however, this phenomenon is reversed, for auxin treatment inhibits the elongation while illumination stimulates the growth. Needless to say, the period of stimulation is extremely brief and it can be supposed that in certain cases it is practically non-existent (4,32). Auxin Destruction Data on lAA destruction, measured by a colorimetric technique (15,26), are plotted in Figure 3. They indicate that lAA destruction is greater in old than in young tissues. The suggestion can be made that lAA-oxidases determine the endogenous auxin level: high en- zyme activity meaning low auxin content (old cells) and vice versa. Meanwhile, recent observations do not exactly confirm this hypothesis. If the lAA destruction is expressed in terms of growth gradients, simple relations between auxin catabolism and endogenous auxins are not so evident. [ See P. E. Pilet, Gradients de croissance et problemes auxiniques. Bui. Soc. Bot. Suisse. 70: (in press) 1960. ] In both regions, this lAA destruction increases with the in- creasing age of the roots. The fact, however, that lAA destruction increases with age would seem at first sight to contradict the material discussed above (which shows that the auxin content increases with increasing age). Nevertheless, it can be supposed that these two Table 1. Auxin content (acid fraction free auxins) in ng IAA/100 mg fresh weight of root segments. Auxin Content Length of Roots, Mm. Old cells Young cells Total 2 ± 0.5 0.07 6.84 6.91 6 ± 0.8 0.10 17.12 17.22 10 ± 1.0 0.14 20.04 20.18 18 ± 2.0 0.13 21.00 21.13 32 ± 3.0 0.15 22.07 22.22 o cr o AGE, LENGTH Fig. 2. Auxin content and growth (control, with lAA and light) of Lens roots in relation to their age (length). o 8 a. d o Q UJ >- O cc t/) ui 2 o < a. 0 YOUNG CELLS OLD CELLS 10 20 30 LENGTH IN MM. 40 50 Fig. 3. lAA destruction for the two types of fragments (root tip: nlil cells as filled circles; merislem: young cells as open circles) of roots from dilicrciil lengths. Auxins and the Process of Aging in Root Cells 171 OLD CELLS --0-- YOUNG CELLS CONCENTRATION OF lAA, /Ig/mL Fig. 4. lAA destruction for the two types of fragments pretreated by lAA at different concentrations for 20, 60, and 100 seconds. processes operate simultaneously. In other words, the tissues greatly increase their ability to destroy native auxins in proportion to the age of the roots at the same time as the accumulation of auxins is increasing. Even if the destruction is greater, the final auxin content rises because auxins are produced faster than they are destroyed. These observations suggest a process of enzyme adaptation, or in- duction, and work of a similar nature (6) performed on a different material clarifies this phenomenon. If, before making the enzyme extracts, the tissues are treated with lAA of increasing concentra- tions and increasing time of incubation, the destructive power of the extracts (which remains stronger for the tissues of the root cap in relation to those of the meristem) increases slightly more for the young than for the old cells (24, Figure 4). Several substances have been used which induced stimulation or inhibition of the in vitro lAA destruction by Lens root tissues: 2,4- dichlorophenol, 2,4-dinitrophenol, 2,4-dinitro-o-cresol and Mn^2 (jg)^ maleic hydrazide (16), gibberellic acid (17), glutathione (20), and in- Table 2. Stimulation or inhibition of lAA destruction in Lens roots, 18 mm. in length. Molar Concentration lAA Destroyed/60 Min/100 Mg Fresh Weight Old cells Young cells Treatment mG. Per cent* ;.G. Per cent Control 75 0 21 0 2,4-Dinitro-o-cresol 2,4-Dinitrophenol 2,4-Dichlorophenol Maleic hydrazide Mn+^ (MnCla) 1 X 10-6** 1 X 10-6** 5 X 10-5** 1 X lo-n 2 X 10-6 97 88 84 89 73 +29 + 18 + 12 + 19 - 3 84 81 76 44 27 +300 +285 +262 + 105 + 29 Gibberellic acid 5 X 10-5 1 X 10-" 4 X 10-5 1 X 10-" 57 52 48 31 -24 -31 -36 -59 18 17 18 15 - 14 Glutathione - 19 Indole - 14 3-Methoxy-4-hydroxy- cinnamic acid - 29 Treated-Control * Per cent = X lO^. Control ** Optimal concentration. t No effect up to 5 X IQ-" M. Table 3. Biochemical gradients in Lens roots, 1 8 mm. in length. Determination Made Expi-essed As: Old Cells Young Cells Fresh weight Total N Mg/100 fragments /ig/lOO mg. fresh \vt. Mg/108 cells Mg/10 mg. fresh \vt. Mg/108 cells 28.4 185.3 10.08 89.7 8.12 163.7 97.9 Protein N 3.58 47.4 1.22 Auxin in equivalent micro- grams lAA Per 100 mg. fresh wt. Per mg. N total N Per mg. protein N In 108 cells 0.13 0.12 0.16 1.26 21.00 24.10 70.61 86.38 Auxin destruction Per 100 mg. fresh wt. Per mg. total N Per mg. protein N In 108 cells 75 73 90 73.3 21 24 71 8.6 Auxins (1)1(1 the Process of Aging in Root Cells 173 12 o ^ 10 O YOUNG CELLS • OLD CELLS 80 40 60 TIME IN MINUTES Fig. 5. lAA destruction for two types of fragments with or without 2,4-dichloro- phenol (DCP) at 1 x 10''^ M, in relation to time. dole (19). It is a general observation (Table 2) that the stimulation is stronger when the activity is stronger. Study of the progress of lAA destruction (Figure 5) shows that in the case of the tip tissues, the decomposition of lAA is almost immediate, although, for the meristem, there is a certain time lag (6). Treatment with 2,4-dichlorophenol (DCP) alters this process, since it induces an immediate stimulation of lAA destruction in the two root fragments. This seems to indicate that lAA-oxidase in- duction cannot be expressed in terms of adaptive formation of en- zyme since, in young cells, DCP (substance without action on non- enzymatic lAA destruction) produces immediately a dramatic stim- ulation of lAA-oxidase which was already present in the tissues but probably bound. BIOCHEMICAL ANALYSES It was essential to establish, for the analyzed tissues, the nature and importance of some biochemical gradients: first, to see if a cor- relation existed between these gradients and the aging of cells (19, 28); secondly, to express more logically and to compare on the same basis (23, 30) the native auxin content and lAA-destroying activity. Data are shown in Table 3, and it can be seen that, regardless of 174 P. E. Pilet the criterion used, (1) auxin concentration is greater in the meristem than in the root tip, and (2) auxin destruction has a higher value for the tissues of the root tip than for those of the meristem. BIOCHROMATOGRAPHIC ANALYSES The study of the active factors has been undertaken on the acid fractions of free auxins extracted by peroxide-free ether (8) . For the two types of tissues, the biological measurements (21) were obtained by two different tests: sections of Lens root (R) and fragments of Leyis stem (T) (25). It seemed best to use equivalent material and, thus, the roots utilized for the measurements (the 5 mm. root tips from the 18 mm. roots) were identical to those which were used to prepare the extracts. Examination of the resulting histograms (Fig- ure 6) clearly shows the existence of lAA in a concentration which produces an inhibition of the R test and a stimulation of the T test. Further, the level of lAA is, according to the previous results, greater in the meristem than in the root tip. Additionally, it shows the pres- ence of two factors already described (2, 3, 9, 21) and discussed (1) elsewhere: an inhibitor and an accelerator (probably an artifact) (7). 120 80 40 I AA ^^X^^-B^-^ 1^=°^ : li- -40 "— O S^ 40 T TEST L "T TEST OLD CELLS -40 -80 ^ ^ ^^J~Lp L R TEST I AA ftp YOUNG CELLS '''''' I I ' I I I I I I I I I I I I I I I I I I I I I . I , I I I I ° 0-5 10 0.5 I Rf Fig. 6. Histograms [biochromatograms: root (R) and stem (T) tests] for two types of fragments. Auxins and the Process of Aging in Root Cells 75 t o X CO z o _I 0- 6 - 0"- 2 h 0 4 I- 0 0 OLD CELLS b 60 YOUNG CELLS r YOUNG CELLS 0 0.5 0 0.5 Rf Fig. 7. Radiochromatograms (lAA labeled with C" in the 2-carbon atom of the ring, applied to the enzyme extracts) for two types of fragments. Collections made at 0, 60, and 120 min. RADIOCHROMATOGRAPHIC ANALYSES The use of lAA labeled with C^* in the 2-carbon atom of the heterocyclic ring has made it possible to determine the nature of lAA destruction (31) in the two types of tissue. Examination of the radiochromatograms (obtained with extracts corresponding at differ- ent times of incubation) indicates (Figure 7) that the speed of lAA destruction is faster for the tissues of the tip. Furthermore, the deg- radation products are rather different. Table 4 furnishes some quantitative relations (27) on this subject. The study of the chemical nature of the products resulting from the lAA (labeled with C^^) de- struction by the root extracts will be presented in detail elsewhere. [See P. E. Filet, Physiol. Plantarum. (in press) I960.] ID O Si T3 u X/1 CQ W Si D, X u c _o '■*-> u 3 Si 4-> X/i lU T3 s N c I— I O «J u u he 5P "» ui (U Si rs CO ■» t) .t; be ^.£ ■*-* -T3 C "•- 2 ^■^ T3 O ti -t-j U C s- o . C T3 O « 3 ») u :S< 6 E o h 0.8 O 10 00 O CO 00 (N m 00 ^1 ITI sC ■ 0-8 ■ Cv -^ ■ 00 - 00 c^ CO 10 -a o Cv 00 CO 1^ CO CM ■ T- -^ < < CM 00 t-- 00 10 r^ O 10 CO O CO T-H O VD 'O O O CO (N -< Auxins and the Process of Aging in Root Cells 177 LITERATURE CITED 1. Audus, L. J., and Gunning, B. E. S. Growth substances in the roots of Pisuvi sativum. Physiol. Plant. 11: G85-697. 1958. 2. Bennet-Clark, T. A., and Kefford, N. P. Chromatography of the growth suli- stances in plant extracts. Nature. 171: 645-647. 1953. 3. Bentley, J. A., and Housley, S. Growth of Avena coleoptile sections in solu- tions of 3-indolylacetic acid and 3-indolylacetonitrile. Physiol. Plant. 6: 480- 484. 1953. 4. Burstrom, H. Physiology of root growth. Ann. Rev. Plant Physiol. 4: 237- 252. 1953. 5. Galston, A. W. Some metabolic consequences of the administration of indole- acetic acid to plant cells, hi: R. L. Wain and F. Wightman (eds.), The Chem- istry and Mode of Action of Plant Growth Substances, pp. 219-233. Butter- worth Sci. Publ., London. 1956. 6. , and Dalberg, L. Y. The adaptative formation and physiological sig- nificance of indoleacetic acid oxidase. Amer. Jour. Bot. 41: 373-380. 1954. 7. Housley, S., and Bentley, J. A. Studies in plant growth hormones. IV. Chro- matography of hormone precursors in cabbage. Jour. Exper. Bot. 7: 219-238. 1956. S. Larsen, P. Growth substances in higher plants, hi: K. Paech and M. V. Tracey (eds.), Moderne Methoden der Pflanzenanalyse. 3: 565-625. Springer Verlag, Berlin. 1955. 9. Lexander, K. Growth-regulating substances in roots of wheat. Physiol. Plant. 6: 406-411. 1953. 10. Linser, H., and Kiermayer, O. Methoden zur Bestimmung pflanzlicher Wuchsstoffe. 181 pp. Springer Verlag, Wien. 1957. 11. Pilet, P. E. Repartition et variations des auxines dans les racines de Lens cul- inaris Medikus. Experientia. 7: 262-264. 1951. 12. . Physiologic des racines du Leiis cuUnaris Medik. et hormones de crois- sance. Phyton Austria. 4: 247-262. 1953. 13. . Variations de I'activite des auxines-oxydases dans les racines du Lens. Experientia. 13: 35-37. 1957. 14 . Activite des auxines-oxydases et vieillissement des tissus. Compt. Rend. Acad. Sci. Paris. 244: 371-373. 1957. l.">. . Dosage photocolorimetrique de I'acide /3-indolyl-acetique: application a letude des auxines-oxydases. Rev. Gen. Bot. 64: 106-122. 1957. 16. Action of maleic hydrazide on in vivo auxin destruction. Physiol. Plant. 10: 791-793. 1957. 17. . Action des gibbcrellines sur I'activite auxines-oxydasique de tissus cultives in vitro. Compt. Rend. Acad. Sci. Paris. 245: 1327, 1328. 1957. 18. . Aspect biochimique du vieillissement des tissus vegetaux. Bui. Soc. Vaud. Sci. Nat. 66: 473-476. 1957. 19. . Action de I'indole sur la destruction des auxines en relation avec la senescence cellulaire. Compt. Rend. Acad. Sci. Paris. 246: 1896-1898. 1958. 20. . Action du glutathion sur la morphologic et I'activite auxines-oxy- dasique de tissus cultives in vitro. Physiol. Plant. 11: 747-751. 1958. 21. . Analyse biochromatogiaphique des auxines radiculaires: techniques et resultats. Rev. Gen. Bot. 65: 605-633. 1958. 22. . Une methode de preparation de fragments de tissus ou d'organes vegetaux. Bui. Soc. Vaud. Sci. Nat. 67: 133-138. 1959. 23. . Activite auxines-oxydasique et expression cellulaire. Compt. Rend. Acad. Sci. Paris. 248: 1573-1576. 1959. 24. . Un cas d'adaptation auxines-oxydasique (racine). Rev. Gen. Bot. 66: 450-460. 1959. 178 P. E. Pilet 25. Pilet, P. E., and Collet, G. fitude de rallongcment de sections d'epicotyles (comparaison de tests auxiniques). Bui. Soc. Bot. Suisse. 69: 47-57. 1959. 26. , and Galston, A. W. Auxin destruction, peroxidase activity and per- oxide genesis in the roots of Lens culinaris. Physiol. Plant. 8: 888-898. 1955. 27. , and Lerch, P. Utilisation d'auxines marquees par du C". Methodes et premiers resultats (auxines-oxydases). Mem. Soc. Vaud. Sci. Nat. (In preparation.) 28. , and Siegenthaler, P. A. Gradients biochimiques radiculaires. I. Auxines et reserves azotces. Bui. Soc. Bot. Suisse. 69: 58-74. 1959. 29. , and Went, F. W. Control of growth of Lens culinaris by temperature and light. Amer. Jour. Bot. 43: 190-198. 1956. 30. Ray, P. M. Destruction of auxin. Ann. Rev. Plant Physiol. 9: 81-118. 1958. 31. , and Thimann, K. V. The destruction of indoleacetic acid. I. Action of an enzyme from Oniphalia fJavida. Arch. Biochera. Biophys. 64: 175-192. 1956. 32. Torrey, J. G. Physiology of root elongation. Ann. Rev. Plant Physiol. 7: 237-266. 1956. DISCUSSION Dr. Andreae: In our studies on lAA metabolism, we became particularly interested in the fate of lAA applied to intact tissues. Studies during the past few years have shown that part of the applied lAA accumulates in the tissues as indoleacetylaspartic acid; this has been confirmed by other workers (Fang et ah. Plant Physiol. 34: 26. 1959). Fawcett et al. (Nature 181: 1387. 1958) were unable to find any evidence for this reaction. The failure of the latter workers can be ascribed to the inadequate extraction procedure used in their studies. Conjugation of lAA with aspartic acid has only so far been ob- served in intact tissues. Cell-free breis entirely lose their ability to conjugate JAA with aspartic acid although such breis may retain lAA-oxidase activity. We found that indoleacetylaspartic acid accumulates in pea tis- sues as the major Salkowski-reactive lAA metabolite, but could find no evidence of a metabolically produced, Salkowski-reactive, lAA- protein complex reported by Siegel and Galston (Proc. Nat. Acad. Sci. 39: 1111. 1953). These workers found that dining the initial hours of incubation, lAA is rapidly bound to the pea root proteins and precipitated by trichloroacetic acid. \n oin- studies, the Salkow- ski-reactive material in pea roots can be extracted with ethanol and consits entirely of lAA and indoleacetylaspartic acid (Figure 1). We therefore believe that the lAA-protein complex of Siegel and Galston is not a product of plant metabolism, but an ariifad of the iri- chloroacetic acid precipitation procedine. The time course of lAA conjugation with aspartic acid in pea roots is shown in Figure 1. Roots were removed from solution, washed, homogenized, and extracted with ethanol. The entire extract Auxins and the Process of Aging in Root Cells 179 1 1 1 1 ^ lAA lAA-Amide • « • •. ^B 1 AA 1 f Ifftf 1 1 1 lAA-Asp A t • 0 4 8 12 16 Standard HOURS Fig. 1. Chromatogram of the Ehrlich-positive substances extracted by ethanol from pea root tips following incubation in auxin for different periods of time. Five mm. root tips from 2-day-old seedlings incubated in solution containing lAA, 1 X 10"* ^t] sucrose, 0.5 per cent; Ca(N03)2, 1 x 10"^M; potassium phosphate, 5 x 10-^ M; streptomycin, 30 p.p.m.; penicillin, 15 p.p.m.; pH 5.2. was chromatographed in wo-propanol-ammonia-water (80:10:15). Three phases can be differentiated. During the initial phase, lasting about two hours, free lAA rapidly accumulates in the tissues. Quan- titively, it has been found that the initial concentration of lAA reaches ten times that of the external solution and that during this time all the lAA lost from solution can be accounted for as accumu- lated lAA. During the second phase, lasting from about the second to the twelfth hour, the lAA content declines and conjugation com- mences. In the third phase, free lAA is no longer detectable in the tissues, while the indoleacetylaspartic acid content continues to in- crease. The formation of indoleacetylaspartic acid appears to be a detoxication process and not a physiological activation of the lAA molecule. A. A. BITANCOURT ALEXANDRA P. NOGUEIRA KAETHE SCHWARZ Plant Cancer Research Center, Institute Biologico, Sao Paulo, Brazil Pathways of DecomposltLon (CataboUc Lattice) of Indole Derivatives Plant tumors induced by Agrobacterium tiimefaciens usually have a higher content of free auxin than the corresponding normal tissue (4, 13, 16, 17). This may be due to increased auxin synthesis in tumors or more active auxin destruction in the corresponding normal tissue (3). The formation and destruction of auxins in general, and of in- dole-3-acetic acid in particular, have therefore an important bearing on the etiology of plant cancer and in 1954 we started a study of the metabolism of indole derivatives (30). We soon noticed that destruc- tion of indole substrates occurred in our blanks as well as during extraction and chromatographic procedures, due to spontaneous de- composition. We concluded that a knowledge of the spontaneous process was necessary for the interpretation of metabolic studies. Since 1956 we have studied the spontaneous or induced decomposi- tion, by ultraviolet radiation, of 14 indole derivativesi, most of which are involved in the classical scheme of indole metabolism (8, 11,20) represented in the central part of the diagram of Figure 1. ^The follo^ving indole derivatives were used in these studies: tryptophan (TRPH), 5-hydroxytryptophan (5-HTRPH), indole-3-pyruvic acid (IPA), trypta- mine (TRAM), 5-hydroxytryptamine (5-HTRAM), indole-3-acetaldehyde (lAAL), indole-3-acetic acid (lAA), 5-hydroxyindole-3-acetic acid (5-HIAA), N-hydroxyin- dole-3-acetic acid (N-HIAA), indole-3-glycolic acid (IGCA). indole-3-glyoxylic acid (IGXA), indole-3-aldehyde (lA), skatole (SK), and indole (IN). After the meetings in 1959, we were able to compare the sample of indole-3- aldehyde used in these studies (Bios Laboratories, Inc.) with one purchased from Aldrich Chemical Co., Inc. The two samples are obviously different, although both are aldehvdic indole derivatives. The Aldrich sample is the only one that agrees with the physical and chemical properties of indole-3-aldehyde reported m the literature. It is therefore obvious that the substance mentioned under the initials of lA in this paper is not indole-3-aldehyde, but it is possibly one of its hydroxy derivatives. [181] 182 Bitancourt, Nogueira, and Schwarz POLYMERS INDOLE DERIVATIVES Benzene ring Cltdvage Oxidation Pyrrole ring Oxiddhon Cledvdge P ♦■ Y R- R O L 4- E 0 E „- R I ♦■ V A I V ♦- E 6-. 7- H Y 0 - R X Y ^ D R I ^ V A ♦ T . ' ♦ V . E ♦ S TRYPTOPHAN INDOLEPYRUVIC ACID INOOLEACETALDEHYDE 1 ■INDOLE ACE TIC ACID INDOLECLYCOLIC ACIO INDOLECLYOXVLIC ACIO INDOLEALOEHYOE I INOOLECARBOXYLtC ACIO i INOOLE Fig. 1. Catabolic lattice of indole derivatives. In the course of these studies we had the opportunity to establish or improve a number of useful chromatographic methods, notably that of double chromatography (27) which, in the case of eminently labile substances like some indole derivatives, furnish valuable evi- dence on the nature of the decomposition products and their path- ways of decomposition. The complete studies are published or arc in jneparation for publication (7, 23, 26, 28, 29), and this paper presents a condensation of the principal results. Fig. 2. Chromatogram of ultiaviolet decomposition products of indoIe-3-acetic acid. Ascending chromatography. Acetone and water 8:2. Citric acid-disodium piiosphate bufler solutions (0.1 M) at pH 3, 5, 7, and 8 were applied along the vertical lines before chromatograpliy. The decomposed solution was applied at I. The short-wave ultraviolet absorjjtion of sonic portions of zone X (lAA) is indicated i)y stippling. Tlie coniplele oulliiie of this zone as revealed by Ehrlich's reagent, is indicated by two internipleil lines. Dots and crosses indicate the center of zone II at the level of pH lines and in between lines, respectively. Conventional hatching indicates approximate fluorescence colors as in Figure 7. Pathways of Decomposition of Indole Derivatives 185 MATERIAL AND METHODS Chemicals Some of the indole derivatives were purchased, others were gen- erously supplied by Drs. J. A. Bentley, D. Von Denffer, A. W. Gal- ston, R. A. Gray, Q. Mingoia, G. F. Smith, and K. V. Thimann. N- hydroxyindole-3-acetic acid was synthesized according to Houft et al. (14). Decomposition of aqueous solutions at a concentration of 0.1 per cent was induced by aging (minutes, hours, days, or years, according to the substance), heating, ultraviolet radiation (exposure for several minutes to a Hanovia Utility ultraviolet lamp) or by oxidants like ferric chloride. The decomposed solution was either directly applied to chromatograms and ionograms or extracted first in n-butyl alcohol. In some cases ether fractionation of the neutral and acid decomposi- tion products was performed. Chromatography and Electrophoresis Uni- and bi-dimensional ascending chromatography was per- formed with Whatman No. 1 paper and the following solvents: (a) water, (b) acetone and water (8:2), (c) 16 per cent NaCl and 2 per cent acetic acid (1:1), (d) 25 per cent acetic acid, and (e) isopropanol, 28 per cent ammonia and water (8:1:1). In the case of (a) and (b), the chromatogram was usually run in an atmosphere saturated with vapors of acetic acid. Addition of a few ml. of acetone on the walls of the tank improved some of the separations in the case of (a). In Fig. 3. A, lonochromatogram of ultraviolet decomposition products of indole-3- acetic acid. The limits of the fluorescent zones (a, c, e) and of the ultraviolet- absorbing zone f (lAA) in the ionogram were traced with a pencil (cf. Figure 4). The substances which remained at the origin (O) formed a reddish brown double line (zone b) due to chromatographing during the repeated application of the decomposed solution on the ionogram. The ionogram was next machine-sewed onto two filter paper strips. The sewing line on the upper strip is shown as a horizontal interrupted line on top of the ionogram at the bottom of the figure. Ascending chromatography with acetone and water 8:2. Spots II, III, and XII and its tail are double as they originate from the double line of zone b. Their slight displacement to the right of that line is jDrobably due to the holes of the sewing line which diverted the flow of the solvent during chromatography. B, Upper part of the chromatogram after exposure to ultraviolet radiation. Spots XIII and XIV faded considerably and new fluorescent spots (VIII, XV, XVI) ap- peared. After treatment of the chromatogram with A^ HCl, spots IX, X, XII and its tails, XIII, XV, and XVI exhibited different shades of red. VIII was slightly yellowish. Conventional hatching indicating approximate fluorescence colors as in Figure 7. 186 Bitancourt, Nogueira, and Schwarz order to avoid decomposition during chromatography, chromato- grams were always short (10 cm.) and were sometimes run in the cold (5 to 8° C.) or in an atmosphere of nitrogen. The inconveniences of this decomposition were put to a marked advantage in double chromatograms, i.e., two-dimensional chromatograms in which the same solvent is used in both directions (27). Multiple levels of pH were established in some chromatograms (6) by drawing longitudinal, evenly spaced, lines with a straight- edge and a capillary pipette filled with buffer solutions (Figure 2). Electrophoresis was initially carried out in an Elphor apparatus, with a voltage of 140 volts, and later in a Pheromatic II apparatus with 200 volts. Whatman No. 1 filter paper strips, 4 x 40 cm., re- ceived the solution on a 5 mm. wide transversal band in the mid- dle of the strij). The strip was moistened with a 0.1 M phosphate buffer solution and placed in the apparatus filled with the same buffer. The operation usually lasted 6 to 8 hrs. A combination of electrophoresis and chromatography was used in several instances. For this purpose after electrophoresis, the iono- gram, reduced to 3 cm. width, was machine sewed onto two strips of filter paper, one of them for dipping in the chromatographic solvent and the other one for running the chromatogram (Figure 3). Inspection of Chromatograms and lonograms and Color Reactions Chromatograms and ionograms were examined under long-wave (366 m^) and short-wave (254 m^^) ultraviolet light and fluorescent and ultraviolet-absorbing spots and zones were outlined with a pen- cil. The contrast between ultraviolet-absorbing spots and the sur- rounding paper was greatly enhanced by spraying the chromatogram with a 0.02 per cent alcoholic solution of safranin. Under short- wave ultraviolet light, absorbing spots appeared dark blue in contrast to the brilliant red of safranin. Significant changes of the color or intensity of fluorescence of some of the spots were produced by ex- posure of the chromatograms and ionograms to ultraviolet radiation for a few minutes. This also induced fluorescence in some of the nonfluorescent, short-wave, ultraviolet-absorbing spots. 14ie following reagents were ai)plied to the chromatograms and ionograms: FeCl.j reagents (acjueous solutions, Salkowski, Gordon- Weber); p-dimethylaminobenzaldehyde (Ehrlich) ; safranin; N HCl; 2,6-dichlorophenol-indophenol (DCPIP); 2,4-dinitrophenylhydrazine (DNPH); benzidine (Van Eck's); ammoniacal AgNO;,. In many cases more than one reagent was applied lo the same chromatogram or ionogram which for that purpose was cut into several longitudinal strips, one for each reagent (Figure 4). The composition of the rea- gents is given elsewhere (29). Pathways of Decomposition of Indole Derivatives 187 - O + Fig. 4. lonogram of the ultraviolet decomposition products of indole-3-acetic acid. Phosphate buffer 0.1 M, pH 7; 6 hrs., 140 volts. All the colored substances (reddish brown) of the decomposed solution applied at the origin O remained there (zone b). The lonogram was inspected under long-wave ultraviolet light which revealed fluorescent zones a, b', c, and e. Under short-wave ultraviolet light zone f (lAA) was seen as a dark zone due to the quenching of the blue fluorescence of the filter paper. The ionogram was next exposed to ultraviolet radiation through a stencil so that parallel longitudinal exposed bands alternated with unexposed bands. As a result of ultraviolet exposure the blue fluorescence of zone a was markedly intensified, that of zone c decreased and a bright turquoise green fluorescence appeared in zone £. A new zone, d, with strong yellowish fluorescence appeared, in part overlapping zone e. The ionogram was then cut longitudinally in four strips, each of which contained an exposed and an unexposed band. N HCl, Gordon-Weber reagent, and dinitrophenylhydrazine were applied to three of the strips. iV HCl produced a red color in zone d, greatly intensified in the irradiated portion and a pinkish buff coloration in the irradiated portion of zone f. Almost the same colorations were observed in the strip which received the Gordon-Weber reagent which in addition produced a purplish coloration in the unexposed portion of zone £. Dinitrophenylhydrazine produced a brown color in zones a, d, and the exposed portion of f. The color was intensified in the un- exposed portion of a and in the exposed portion of d. Conventional hatching in- dicating approximate fluorescence colors as in Figure 7. Sublimation Test The production of volatile indole derivatives like skatole and indole in solutions applied to a strip of filter paper and decomposed by exposure of the dry strip to ultraviolet radiation was detected by sublimation onto a clean strip of the same paper. The exposed and the clean strips, separated by a strip of Japanese lens paper, were placed between two glass plates and the whole was set on a hot plate at a temperature of 85° C. for 15 min. The clean sheet was then sprayed with Ehrlich's reagent. RESULTS Spontaneous and Induced Decomposition Judging from the discoloration of their aqueous solutions, several of the indole derivatives of this study decompose spontaneously in a few minutes, hours, days, or years. This was observed with TRPH, 188 Bitancourt, Nogueira, and Schivarz IPA, lAAL, lAA, N-HIAA, IGCA, lA, SK, and IN. This decomposi- tion is accelerated by heating and exposure to visible light and occurs in a matter of minutes with ultraviolet radiation. Results compa- rable to this radiation are obtained with minute quantities of an oxi- dant like ferric chloride. Salkowski and Ehrlich Reacting Spots In general only a small proportion of the spots and zones detected in our chromatograms and ionograms gave colored reactions with these reagents. IPA and lAA gave six spots, IGXA gave two, and TRPH and IGCA one each, besides their own spots. Three out of the six spots of IPA turned red with the application of N HCl which shows that the color reaction with Salkowski and Ehrlich's reagents is mostly due to the acid they contain. The same occmred with six spots of lAA and one of IGCA. A number of spots gave yellow or orange reactions with Ehrlich's reagent, in contrast wuth the purple or pink reactions of most of the indole derivatives. Fluorescent and Ultraviolet Absorbing Spots In contrast to the color reagents, ultraviolet light revealed many spots in our chromatograms and ionograms. Most of these spots are fluorescent and revealed by long-wave (366 m/^) ultraviolet light. Un- like many of their products of decomposition, only a few indole derivatives are fluorescent but they become so on chromatograms under several influences (aging, heating, ultraviolet radiation, alka- linization, application of oxidants). Nonfluorescent indole deriva- tives are usually short-wave ultraviolet-light absorbing stibstances and quench the blue fluorescence of the filter paper induced by that light. Most of the fluorescent spots and zones of our chromatograms and ionograms do not give any of the reactions of the preceding sec- tion. Dinitrophenylhydrazine Reacting Spots One spot or zone in the case of IGXA and lA and two in that of IPA, besides their own spots, gave a brown reaction with the car- bonyl reagent DHPH. TRPH and IGCA gave one spot each and lAA two (zones a and d, Figure 4). In the latter case, however, zone a showed a weaker reaction after ultraviolet exposure and zone d an increased reaction, showing that, as a result of exposure, the sub- stance of a was partially destroyed while that of zone d was trans- formed into a DNPH-reacting substance. Volatile Substances The sublimation test was positive in the case of SK and IN, as expected, and also in that of lAA and lAAL decomposed b) ultra- violet radiation on (he j:)apcr. PatJiways uf Decomposition of Indole Derivatives 189 Substances Separated by Electrophoresis Electropositive or basic substances and electronegative or acidic substances were detected in ionograms at pH 7 (Figure 4). Substances which remained at the starting line were mostly insoluble in the phosphate buffer at that pH. Some, however, may have been neutral or amphoteric. In chromatograms with multiple levels of pH (Figure 2) acidic, basic, and amphoteric substances exhibited a wavy pattern due to the different R( values of the forms of dissoci- -e- Fig. 5. Double chromatogram of indole-3-acetaldehyde. Ascending chromatogra- phy, with water, in an atmosphere saturated with vapors of acetic acid. A-B, distribution of spots after the first run; A, lAAL-sodium bisulfite addition com- pound, B, ether extract of a solution of the same compound. After the first run A was detached and sprayed with Ehrlich's reagent which revealed a purplish spot of lAAL or of the addition compound. In B lAAL was seen under short-wave ul- traviolet light as a dark elongated area. Three dots indicate the center of the spots of the presumed polymers. It is suggested that the ultraviolet absorbing spot that remained at the origin is also a polymer. Interrupted lines indicate a faint yellow fluorescent tail above and below the large spot of lAAL. C, same chromato- gram after the second run, sprayed with Ehrlich's reagent. lAAL now constitutes the large square spot near the center of the chromatogram. Left behind is another spot at the starting line, corresponding to the large spot of the first run in B but probably of the same polymer as at the origin. All three spots exhibited the same light blue color of lAAL in the Ehrlich reaction. In the square, large dots on the diagonal indicate the center of primary spots of the three presumed poly- mers; small dots indicate the secondary spots of the same polymers formed by polymerization and depolymerization during chromatography. 190 Bitancourt, Nogueira, and Scliwarz Fig. 6. Double chromatogratn of ultraviolet decomposition products of an alde- hyd;c indole derivative (lA, see footnote 1). Ascending chromatography. Acetone and water 8:2 in both directions in an atmosphere saturated with vapors of acetic acid. A, distribution of spots after the first run. B, after the second run. The diagonal of the chromatogram is indicated by an interrupted line. The center of the primary spots of the presumed polymers of lA is indicated by large dots, numbered I to VI; that of tlie secondary spots (of polymers formed by polymerization or depoly- merization during chromatography) by small dots. lA occupies the large spot 2 and its extension 2'. The outline of spot 2 was traced tiianks to its quenching of the blue fluorescence of tlie fdlcr j)aper under short-wave ultraviolet liglu. After spraying the chromatogram with Salkowski's reagent, the whole area of spot 2 and its exten- sion 2', inclusive of tlic part with a yellow fluorescence (spot 7'), was colored orange-yellow. No reaction was produced at the sites of the primary spots and some of the secondary spots of the presumable polymers showing that they were completely transformed by polymerization or dcpolymerization during the second chromatography. Conventional hatching indicating approximate fluorescence colors of the decomposition products (spots 1, 3, 4, 6 and 7) as in Figure 7. a lion. Neutral substances which are not affected by pH form a straight zone (Figure 2). Wavy patterns were also exhibited by com- pounds like lAAL and lA with no strong acidic or basic groups and lAA with water as the solvent, showed at least three different Rf val- ues. These two results are interpreted as possibly indicating forms of dissociation of the carbonyl and imino groups. Ether or ethyl acetate fractionation of the decomposed solutions Pathways of Decomposition of Indole Derivatives 191 and of the hydrazones of the DNPH reaction in the case of IPA has, on a few occasions, fmnished additional evidence of the acidic or neu- tral nature of some of the products of decomposition. Tautomerization and Polymerization The presence of two tautomers in chromatograms of IPA was demonstrated (26, 27, 29). It is possible that the presence of two forms of lA with different Rf values evidenced by the wavy pattern in ■ •.■.■.■.■.■.'i\'A\\.^ 1 RED ESgREEN E2] yellow m ^^^e'aS i^ ^S'S COLOR Fig. 7. Double chromatogiam of ultraviolet decomposition products of indole-3- acetic acid. Ascending chromatography. Acetone and water 8:2 in both directions, in an atmosphere saturated with vapors of acetic acid. A, distribution of spots after the first nm. B, after the second run. The solution was applied at the origin in the small circle (I) and the sheet was left overnight for equilibration in the chro- matographic tank. This resulted in diffusion up to the limit of the greater circle at the origin. The spots bet\\'een I and II and between III and IV seldom appeared in other chromatograms and were not numbered. The diagonal of the chromatogram in B is indicated as an interrupted line. The center of primary spots (substances present in the solution) is indicated as large dots, that of secondary spots (formed during chromatography from the substances whose primary spot is on the same horizontal line) as small dots. The isolated spot on the diagonal, in between the tails of spot V and IX is from a substance presumably present in the solution but entirely decomposed during the second chromatography. The secondary spots of IV, V and IX at the same horizontal level presumably originated from this substance. Fluorescence colors, ultraviolet absorption, and visible color indicated by con- ventional hatching. Heavier hatching indicates stronger intensity of fluorescence. o — ^— 1 T— ' ^— '- 01 CO — 0 1 1 I r- < X o l-H ■<1- M M (N rn r<^ ^- (N r^, .— (nj ^J sO < o o < O to-4 00 — - — — — . ° "^ C 3 > T3 t5 o p 3 £: o sx; « -3 i; S E U D ^ c> be 1 u > •^ ^ !- > i set d i2 10 3 00 "a 2 .2 -7? 0 CO u V E 3 c -1 T Z P u -U C 5 u -t; w 2 Q :^ 5 c V3 U C w Si en C o > V u J3 J2 RJ Vi -a c 3 o a S o u .0 00 u be R3 a c o o c o u u Pathways of Decomposition of Indole Derivatives 193 multiple pH chromatograms results from tautomerization at high pH values (22). The two aldehydic compounds investigated in this study, lAAL and lA, produce in chromatography unusually large spots (Figures 5 and 6). Thanks to the properties of double chromatograms (27) those spots can be interpreted in terms of different states of polymeri- zation of these compounds. Spontaneous precipitation occurring in aged solutions of lAAL and lA may also result from polymerization which probably occurs through the side-chain as in the corresponding aliphatic aldehydes. Solutions of lAA (0.1 per cent) in boiled (air- free) water exposed to ultraviolet radiation do not produce the brown discoloration of ordinary solutions but instead become milky. This also may be due to polymerization of which there is evidence in double chromatograms (Figure 7). It is seen that the spot in the upper right-hand corner is in reality quadruple and composed of two primary spots IX and X on the diagonal and two secondary spots, one of them a spot of IX produced from the substance of spot X and the other one, a spot of X, produced from the substance of spot XI. The best interpretation of this composite spot seems to be that IX is a polymer of X (I A A). In Table 1 is shown the number of spots and zones of decompo- sition products of indole derivatives in chromatograms and ionograms which exhibited the properties just described. The derivatives them- selves are included in the number whenever they possessed the cor- respondnig property. DISCUSSION The most significant result of these studies is perhaps the small number of spots and zones of our chromatograms and ionograms that give the Salkowski and the Gordon-Weber reactions. Only in the case of TRPH, IPA, and lAA was it possible to demonstrate the presence of the immediate product of degradation of the side-chain, IPA, lAAL and IGCA, respectively. The demonstration of IGXA in the case of IGCA and of lA in that of IGXA was doubtful. The prod- ucts of more advanced degradation of the side-chain are even more rare, and only lAA, among the decomposition products of TRPH and, with IGCA, among those of IPA, is detectable in some of our chromatograms. The identification of lA in the chromatograms of TRPH, IPA, lAAL and IGXA is doubtful in view of the possible confusion with o-aminobenzene derivatives with the same Rf. It seems, therefore, that degradation of the side-chain is not a major pathway in the spontaneous decomposition (aging) or the decomposi- tion induced by ultraviolet radiation- of the indole derivatives. 194 Bitancourt, Nogiieira, and Schwarz Other workers, however, have been more successful than we have in demonstrating the presence of indolic products of decomposition in chromatograms and ionograms of decomposed solutions of indole derivatives. Mayr (19), von Denffer and his collaborators (10), espe- cially Melchior who made the most extensive investigation in this field (20), and recently Kaper and Veldstra (15) were able to identify several indole derivatives or at least show the presence of several Salkowski reacting substances which we have failed to demonstrate. Except in the case of lAA and lAAL, we were not able to detect the presence of skatole, and much less of indole, which several of these authors have mentioned among the decomposition products of many of the indole derivatives of the currently accepted scheme of decomposition (Figure 1). With a much more refined sublimation method than the test we have devised, Behrens and Fischer (2) were able to demonstrate the presence of lA, skatole, and indole among the photolytic decomposition products of lAA. All these authors, however, have made little if any use of ultra- violet light to detect the presence of fluorescent substances among the decomposition products of indole derivatives and have overlooked those we find in our chromatograms and ionograms. Substances giving a yellow or orange-yellow color with Ehrlich's re- agent but not Salkowski and Gordon-Weber reactions were often found in our chromatograms and ionograms. Presumably they are primary amines, especially o-aminobenzene derivatives, which could result from cleavage of the pyrrole ring. Cleavage of that ring in tryptophan with the production of kynurenine, one of those deriv- atives, is well known and, at least in the case of enzymatic decompo- sition, has been shown to occur also with other indolic substances (18). Gordon and Weber (12) state that in excess of air inactivation of lAA by X-radiation appears chiefly due to ring opening. Another evidence of ring opening, with the production of an elec- tropositive amino group is given by the presence of zones on the negative side of ionograms (pH 7) of practically all the derivatives we studied. If the substances corresponding to those zones arc o-ami- nobenzene derivatives, the group in the ortho position must be neu- tral, which is suggested in the case of zone a of ionograms of lAA by its strong reaction with DNPH (Figure 4). One possibility is that zone a be o-amino])hcnylacetaldehyde or one of its hydroxyderiva- tives. The o-aminobcnzene derivative resulting from the opening of the pyrrole ring of indole in a reaction similar to that which pro- duces kynurenine from tryptophan is o-aminobenzaldehyde, a sub- stance which also gives a positive reaction with DNPH. None of the ^Enzymatic decomposition and decomposition induced by visil^Ic li,>;lit in liie |)icsence of fluorescent substances were beyond the scope of the present studies .nid will not be discussed here. Pathways of Decomposition of Indole Derivatives 195 spots and zones giving a yellow reaction with Ehrlich's reagent were identified as being anthranilic acid, an o-aminobenzene derivative which was found among the decomposition products of indole deriva- tives by other workers (20). In contrast with substances reacting with Salkowski, Gordon- Weber, and Ehrlich's reagents, fluorescent substances which do not give those reactions were numerous. It was rarely possible to estab- lish the correspondence of fluorescent spots of one indole derivative with those of another one. Spot II of the chromatograms of lAA (Figure 7) is an exception in that it was also apparently present in chromatograms of TRPH, IPA, and lAAL. It would seem, therefore, that most of the fluorescent substances originated separately from products resulting from degradation or changes in the nucleus of each derivative, rather than from degradation of the side-chain. In this connection, the possibility of an opening of the benzene ring, yielding a pyrrole derivative (5) which to our knowledge has not been considered to date, is worthy of attention. Whether degradation of indole derivatives starts by the side-chain, the pyrrole ring or the benzene ring, the final products must be ali- phatic compounds. Meyer and Pohl (21) claim that the end product of photolysis of lAA by riboflavin is anthranilic acid which, in ultra- violet light, decomposes further into glycine and formic acid with oxalic acid as an intermediate of the latter. It is possible that some of our fluorescent substances are indeed aliphatic compounds and represent final stages of decomposition of indole derivatives. The condensation of indole compounds with the formation of colored dimers like indigo and its inducement by ultraviolet radia- tion are well known (1). The possibility that dimerization occurs during the oxidation of lAA has been discussed by Ray and Thimann (25). Houff et al. (14) have shown that a colored dimer of lAA is an intermediate in the formation of N-HIAA, the main product of the Salkowski reaction. Evidence of polymerization in our chromato- grams might be due to such dimers. In Figure 7, spot IX might be the dimer of Houff et al. but we were not able to identify N-HIAA among the decomposition products of lAA because that substance diffuses extensively and quickly disappears in chromatograms. The case of lAAL and lA (Figures 5 and 6) is different in that polymerization probably occurs as a result of the condensation of the side-chains of the molecules involved, in a manner similar to that of the corresponding aliphatic aldehydes. Summing up, we may say that spontaneous decomposition, or the decomposition induced by ultraviolet radiation, of indole derivatives may follow four main courses and their combinations: (1) oxidation and other changes in the side-chain, (2) oxidation and opening of the 196 Bitancourt, Nogueira, and Schwarz pyrrole ring, (3) oxidation and possibly opening of the benzene ring, (4) polymerization. These possibilities have been represented in the diagram of Fig- ure 1. In such a diagram the concept of pathway is all but lost. In the interpretation of the facts this concept constitutes a serious drawback. We suggest that the intricate network of pathways be called a lattice and since it is a representation of all the possible ways in which indolic compounds may be decomposed, spontaneously or under the influence of pyhsical, chemical, or biochemical factors, it can be called the catabolic lattice of indole derivatives. SUMMARY Solutions of 14 indole derivatives decomposed by aging, heating, ultraviolet radiation, or oxidants like FeClg were studied chroma- tographically and electrophoretically. Fluorescence and ultraviolet absorption of spots and zones were detected under long-wave and short-wave ultraviolet light respectively. Significant changes in fluo- rescence and absorption were induced by exposure of the chromato- grams and ionograms to ultraviolet radiation. Salko^vski, Gordon- Weber, Ehrlich, and Van Eck reagents, safranin, N HCl, dinitro- phenylhydrazine, dichlorophenolindophenol, and ammoniacal AgNOg were applied to the chromatograms and ionograms, either to the whole or to longitudinal strips cut out from them so that more than one reagent could be used at a time. Only a small proportion of the decomposition products were in- dole derivatives reacting with Salkowski and Gordon-Weber reagents. A few of them were identified as the immediate product of degrada- tion of the side-chain. Of those which did not react, a number of spots and zones gave yellow or orange color reactions with Ehrlich's reagent and are probably o-aminobenzene derivatives but not anthra- nilic acid. Among them some moved to the negative side of the electrophoretic strips so that the side group in the ortho position must have been neutral. Double chromatograms produced large spots of lAA, lAAL, and lA which are interpreted as made up of from 2 to 6 polymers, present in the solution or formed during chromatography. The majority of spots were fluorescent and did not react \\ith any of the reagents. It is suggested that they might be pyrrole deriva- tives resulting from oxidation and cleavage of the benzene ring of the indole compounds, and aliphatic compounds, the last stages of de- composition of those substances. It is concluded that decomposition of indole derivatives may fol- low four main courses or a combination of them. Pathways of Decomposition of Indole Derivatives 197 LITERATURE CITED 1. Bacher, F. Chemische Reaktionen organischer Koipcr im ultravioletten Licht iind im Licht dcr Sonne. In: Abderhalden, Emil (ed.), Handbuch der Biolog- ischen Arbeitsmethoden. Abt I, Teil 2, 2 (1): 1339-1968. 1929. 2. Behiens, M., and Fischer, A. Zerlegnng von Stoffgemischen und Identifizier- ung von chemischen Substanzen durch Verdampfen und Kondensieren in einem Warmegradienten. Natunvis. 41: 13. 1954. 3. Bitancourt, A. A. Mecanismo genetico da tumoiiza^ao nos vegetais. (Abstr.) Programa da 2a. semana de genetica. p. 7. Piracicaba 8-12 de Fevereiro. 1949. 4. . Recherches physiologiques sur les auxines. Rev. Gen. Bot. 62: 498-591. 1955. 5. . Considera^oes bioquimicas sobre o cancer vegetal. Biologico, Sao Paulo. 23: 221-224. 1957. 6. , and Nogueira, A. P. Multiple pH levels in chromatograms. Science. 129: 99. 1959. 7. , Schwarz, K., and Nogueira, A. P. A decomposi^ao espontanea de alguns derivados indolicos. I. Metodos experimentais. Arq. Inst. Biol. Sao Paulo. 24: 169-182. 1957. 8. Bonner, J., and Wildman, S. G. Contributions to the study of auxin physi- ology. Symp. Soc. Stud. Evol. Growth. 6: 51-68. 1947. 9. Cohen, D., Ginzburg, B-Z., and Heitner-Wirguin, C. Metal-chelating properties of plant-growth substances. Nature. 181: 686,687. 1958. 10. Denffer, D. von, and Fischer, A. Papierchromatographischer Nachweis des ,8- indolaldehyds in photolytisch zersetzter lES-Losung. Naturwis. 39: 549,550. 1952. 11. Fischer, A. tJber die papierchromatographische und papierelektrophoretische Trennung von Indolderivaten. Planta. 43: 288-314. 1954. 12. Gordon, S. A., and Weber, R. P. The effect of X-radiation on indoleacetic acid and auxin levels in the plant. (Abstr.) Amer. Jour. Bot. 37: 678. 1950. 13. Henderson, J. H. M., and Bonner, J. Auxin metabolism in normal and crown gall tissue of sunflower. Amer. Jour. Bot. 39: 444-451. 1952. 14. Houff, W. H., Hinsvark, O. N., Weller, L. E., Wittwer, S. H., and Sell, H. M. The nature of an oxidation product of 3-indoleacetic acid. Jour. Amer. Chem. Soc. 76: 5654-5656. 1954. 15. Kaper, J. M., and Veldstra, H. On the metabolism of tryptophan by Agrobac- terium tumefaciens. Biochem. Biophys. Acta. 30: 401-420. 1958. 16. Kulcscha, Z., and Gautheret, R. Sur I'elaboration de substances de crois- sance par 3 types de culture de tissus de Scorsonere: cultures normales, cul- tures de Crown -Gall et cultures accoutumees a Thetero-auxine. Compt. Rend. Acad. Sci. Paris. 227: 292-294. 1948. 17. Link, G. K. K., and Eggers, V. Hyperauxiny in crown gall of tomato. Bot. Gaz. 103: 87-106. 1941. 18. Manning, D. T., and Galston, A. W. On the nature of the enzymatically cata- lyzed oxidation products of indoleacetic acid. Plant. Physiol. 30: 225-231. 1955. 19. Mayr, H. H. Zur Photolyse von Indol-3-Essigsaure bei papierchromatograph- ischen Arbeiten. Planta. 46: 512-515. 1956. 20. Melchior, G. H. tJber den Abbau von Indolderivaten I. Mitteilung. Photolyse durch ultraviolettes Licht. Planta. 50: 262-290. 1957. 21. Meyer, J., and Pohl, R. Neue Erkenntnisse zum Problem der phototropen Kriimmung bei hoheren Pflanzen. Naturwis. 43: 114, 115. 1956. 22. Morton, R. A., and Falimy, N. I. Indole-3-aldehyde from tissues. Nature. 182: 939. 1958. 198 Bitancourt, Nogiicira, and Schwarz 21. Ray, P. M., and Cuiiy, G. M. Intermediates and competing reactions in the photodestruction of indoleacetic acid. Nature. 181: 895,896. 1958. 25. , and Thimann, K. V. Steps in the oxidation of indoleacetic acid. Sci- ence. 122: 187, 188. 1955. 26. Schwarz, K. A decomposi^ao espontanea de alguns derivados indolicos. 111. Novos dados sobre a decomposi^ao do acido indolpiruvico. Arq. Inst. Biol. Sao Paulo. 26: 1-10. 1959. 27. , and Bitancourt, A. A. Paper chromatography of unstable substances. Science. 126: 607, 608. 1957. 2S. , and Bitancourt, A. A. A decomposi^ao espontanea de algims deri- vados ind61icos. II. Acido indolpiruvico. Arq. Inst. Biol. Sao Paulo. 24: 183- 197. 1957. 29. , and Bitancourt, \. A. Further evidence of tautomerism in chroma- tograms of indole-3-pyruvic acid. Biochem. Jour. 75: 182-187. 1960. 30. , Nogueira, \. P., and Bitancourt, A. A. Decomposigao enzimatica do triptofano e do acido indolacetico pelo extrato de ponteiros de abacaxi. (Abstr.) Ci. Cult. Sao Paulo. 8: 176. 1956. 23. Nogueira, A. P., and Bitancourt, A. A. A decomposi^ao espontanea de acido indolacetico. (Abstr.) Ci. Cult. Sao Paulo. 9: 163. 1957. PETER M. RAY University of Michigan The InterpretatLon of Rates of I n.dote-3 -acetic Acid Oxidation Although several fundamentally different mechanisms have been proposed to explain enzymatic oxidation of indole-3-acetic acid (lAA) by peroxidase-containing preparations, a feature common to all of these is that the oxidation is interpreted as a cyclical process involving several reactions. Goldacre (2) and Galston, Bonner, and Baker (1) suggested that HgOo is consumed in a peroxidase-catalyzed step, and reformed from Oo in an oxidase-catalyzed step. In the scheme pro- posed by Maclachlan and Waygood (3), lAA is supposed to be oxi- dized by Mn^3^ giving a radical which reacts with Oo; the resulting oxidant is used by peroxidase to oxidize a phenol, which in turn reoxidizes Mn+2 to Mn^^. Another possible mechanism, suggested recently by Yamazaki and Souzu (6), is that lAA is oxidized directly by peroxidase in a one-electron oxidation (V2 HoOo), giving a radical which reacts with oxygen to regenerate HoOo. I do not feel that any of the specific proposals about the mechanism of enzymatic lAA oxidation has been established unequivocally, and I think it is not yet possible to say whether some of the different enzyme preparations which have been studied actually operate by different reaction mechanisms. It may even be that some or all of the reactions of the formally distinct mechanisms actually go on together in a single reaction medium, and contribute to varying degrees to the over-all oxidation depending upon the conditions, the addition of phenols or of Mn, and so forth. In all of the suggested mechanisms, and very likely in any others which may be conceived, the reaction rates must depend upon concentrations of intermediates such as HoOo and radicals or other partially oxidized products derived from lAA. The purpose of this discussion is to explain the important bearing of this upon the meaning of lAA oxidation rates, and of progress [199] 200 P. M. Ray curves of lAA destruction, since such measurements are used widely not only as a basis for interpreting the mechanism of lAA oxidation, but also in physiological experiments in which lAA destruction is involved. The phenomena have been investigated in experiments performed with the lAA-oxidizing enzyme of Omphalia flavida (4, 5). When lAA is added to a peroxidase capable of oxidizing it, a characteristic lag or induction phase precedes attainment of a rapid reaction rate. This is illustrated with the solid line in Figure 1. It appears to be due to the fact that the reaction medium does not con- tain, initially, concentrations of the above-mentioned oxidation inter- mediates (such as H2O2) great enough to allow a substantial rate of oxidation. If H0O2 is added initially, no induction phase is ob- served; instead the reaction is occurring rapidly at the earliest pos- sible observation, as shown by the broken line in Figure 1. Evidently HoO., is either a reaction intermediate, or it can give rise to the actual intermediates rapidly. Under different conditions and with different enz\me preparations it is to be expected that the initial content of oxidation intermediates Avill vary. The initially observed rates of lAA destruction, as well as how pronounced the induction effect is, will vary accordingly, quite apart from variations in enzyme activity and effects of cofactors o o: W o < < / H202/ SATURATION c )ECONDARY^-'''EFFECTS / ^ STEADY/ "state : / STEADY/ '^TATE :/ '•1 /: ■ NO ADDITION /: 1 SATURATION y^ - H2O2 ADDED INITIALLY AND AT ARROW INDUCTIOTJ/PHASE 1 1 1 10 20 30 TIME MIN. Fig. 1. Illustration of the kinetic phases of enzymatic I.-\.\ oxidation observed with and without addition of H.Oo. In this example, the initial lAA concentration was 2 X 10-*M, and the top of the figure represents destruction of all the lAA. At zero time and at the arrow 5 x 10-"A/ H.O, was added to tiie sample shown l)v the broken line. The Interpretation of Indole-3-acetic Acid Oxidation 201 and inhibitors. This is an inescapable source of possible confusion in comparative experiments on lAA oxidation. The gradual increase in reaction rate which occurs during the induction phase must be ascribed to some process by which the total concentration of oxidation intermediates in the medium increases. The proposals mentioned concerning the lAA oxidation mechanism do not provide for such an increase in concentration, since every inter- mediate must be consumed and produced in equal amounts to main- tain the catalytic cycle and account for the reaction stoichiometry. Two principal types of processes can be considered in looking for an explanation of why the reaction rate increases during the induction phase: (aj a minor reaction occurs, separately from the main lAA oxidation process, which forms an lAA oxidation intermediate as a product, or (b) the lAA oxidation reaction itself forms a product which is or can give rise to one of the intermediates, so that as the cycle operates one of the intermediates is formed in excess of the amount consumed. We shall call processes such as (a) or (b) initiation reactions; they initiate a catalytic cycle dependent upon oxidation intermediates. Types (a) and (b) are fundamentally different kinetic- ally, for (b) is tied to the occurrence of the lAA oxidation reaction while (a) is not. With process (b) the reaction would appear to be autocatalytic, whereas with (a) it would not. With (a) the amount of lAA destroyed at time t after the start of reaction should be a linear function of t-; with (b) the log of the amount of lAA destroyed at time t should increase linearly with t once an appreciable rate of re- action is observed. We have concluded that the Omphalia enzyme reaction is in fact autocatalytic, that is, alternative (b). It should be noted that a positive distinction between initiation reactions (a) and (b) is not easy to make, as it must be based upon close study of the early part of the induction phase, because further complications in the kinetics, to be discussed below, appear as the reaction rate in- creases. By quantitative comparison between the kinetics of the induction phase and the rate effects of added HoOo, one can get some indication as to the concentrations of intermediates which must actually be formed dming induction. It appears that the amounts formed are much less than the amount of lAA which undergoes oxidation during this period, which suggests that the autocatalytic effect does not arise from a major product of lAA oxidation, but rather by a minor side reaction in the oxidation cycle. The lAA oxidation rate does not continue to increase indefinitely, but leads gradually into a steady maximum rate of oxidation which 202 P. M. Ray is maintained until exhaustion of substrate, or occurrence of secondary complications such as enzyme inactivation, cause it to fall. These phases are also illustrated in Figure 1 (solid line). The steady rate is, however, considerably lower than can be obtained by adding HoOo to the medium. The rate increases, with increasing concentrations of added HoOo, up to a maximum which we call the HoO-t saturation rate, and which we presume is due to saturation of the peroxidase with HoOo. This occurs at about 2 X lO^^M HoOo with the Omphalia enzyme, and is illustrated by the first part of the broken curve in Figure 1. It is apparent that in the absence of added H^Oo, the enzyme does not become saturated even when the rate has become steady, after induction. A further fact of significance is that when sat- urating amounts of H^Oo are added, it can be demonstrated that HoOo disappears gradually from the system, since after a time the rate falls to the same value as would be reached, after induction, if H2O2 had not been added, and at this point the HoOo saturation rate can be restored by adding more HoOo. These effects are shown in the broken curve of Figure 1. They indicate that processes are also oc- curring which lead to loss of oxidation intermediates from the sys- tem, processes which can be called termination reactions by analogy with organic auto-oxidation processes. A probable explanation of the steady rate of oxidation which is reached after induction is that it represents a steady state in which the rates of formation (initia- tion) and disappearance (termination) of oxidation intermediates have become equal — due, for example, to termination increasing more rapidly than initiation as the reaction rate rises (termination is evidently more rapid than initiation at the high rate attained by saturation with HoOo). Examples of possible types of termination re- actions are (a) a catalase-like reaction involving removal of HoOo, and (b) a reaction between free-radical intermediates such as P- -|- POo- -^ POoP. It will be evident that the lAA oxidation rate observed at steady state will be influenced not only by effects on enzyme activity and the main catalytic cycle, but also by any factors which influence the initiation or termination processes, and thereby the steady state con- centrations of intermediates. There is thus considerable danger in interpreting rate effects of experimental treatments as if the ob- served effects related only to enzyme activity or to the cyclic lAA oxidation mechanism. To proceed further with interpretation of rate effects on lAA oxidation it will be essential to develop a kinetic analysis of the proposed mechanism which includes assumptions about the nature of initiation and termination reactions. In our experience, the rates of lAA oxidation attainable by sat- The Interpretation of Indole-3 -acetic Acid Oxidatioji 203 uration with HoOo have proved to be a much more reliable and re- producible means of measuring enzyme activity than the steady state rates, presumably because the HoOo saturation rate is not af- fected by variations in the rates of initiation and termination. Finally, several examples exist in the literature of effects on lAA oxidation rates which involve secondary complications on the steady state rate, particularly effects on enzyme inactivation. Such effects must be distinguished carefully from true kinetic effects, because they cannot properly be used, in a kinetic sense, as evidence of the reaction mechanism, and they may also have quite different sig- nificance in physiology. It must be noticed that the early reaction phases illustrated in Figure 1 take place over amounts of lAA oxidation too small to be measured accurately by usual manometric methods, so that in manometry one is dealing usually with the steady state (whether or not HoOo is added) or with secondary rate complications. This lim- its the usefulness of manometry in investigation of lAA oxidation. LITERATURE CITED 1. Galston, A. W., Bonner, J., and Baker, R. S. Flavoprotein and peroxidase as components of the indoleacetic acid oxidase system of peas. Arch. Biochem. Biophys. 42: 456-470. 1953. 2. Goldacre, P. L. Hydrogen peroxide in the enzymic oxidation of heteroauxin. Austral. Jour. Sci. Res. B 4: 293-302. 1951. 3. Maclachlan, G. A., and Waygood, E. R. Kinetics of the enzymically-catalyzed oxidation of indoleacetic acid. Canad. Jour. Biochem. Physiol. 34: 1233-1250. 1956. 4. Ray, P. M. Destruction of indoleacetic acid. IV. Kinetics of the enzymatic oxidation. Arch. Biochem. Biophys. (In preparation). 5. , and Thimann, K. V. The destruction of indoleacetic acid. I. Action of an enzyme from Omphalia flavida. Arch. Biochem. Biophys. 64: 175-192. 1956. 6. Yamazaki, I., and Souzu, H. The mechanism of indoleacetic acid oxidase re- action catalyzed by turnip peroxidase. Arch. Biochem. Biophys. 86: 294-301. I960. R. L. HINMAN and P. FROST Union Carbide Research Institute A Model Chemical System for the Study of the Oxidation of I ndole-3 -acetic Acid by Peroxidase The oxidative degradation of indole-3-acetic acid (lAA) by plant ex- tracts was studied intensively during the 1950's (9). Despite the ad- vances made in understanding the enzymatic components, the re- quired cofactors, and the physiological significance of the reaction, elucidation of the complete process has remained elusive, in good part because the changes undergone by the organic substrate, lAA, are still unknown. It is the purpose of work under way in these lab- oratories to determine the identity of the products formed from lAA when acted upon by the lAA oxidase system. We have found that hydrogen peroxide in acidic media converts lAA to products the spectra of which resemble closely those of the products formed in the oxidation of lAA by peroxidase in the absence of added hydrogen peroxide. Since it is now almost certain that the effective component of the lAA oxidase of higher plants is a peroxidase (9), the HoOo/H^ system has considerable significance in relation to the biological ac- tivity of lAA. We are using the chemical system as a model to aid in investigating the chemistry of the oxidation of lAA by peroxidase. Previous attempts to identify the products of the oxidation of lAA by lAA oxidase have depended mainly on isolation of the products either in the free state or on paper chromatograms. Because of the multiplicity of products actually isolated (7, 10), and the possibility that the product formed initially may undergo decomposition under the conditions of isolation, we have chosen to study the nature of the product(s) by following the changes in the ultraviolet spectrum of lAA during its reaction with peroxidase and with the H0O2/H+ sys- tem. Following the course of these reactions by spectrophotometric techniques has one other advantage over techniques in which only residual lAA is determined. It is quite possible that lAA could be [205] 206 R. L. Hinman and P. Frost degraded by different pathways under different conditions, and tliat oxygen could be consumed and carbon dioxide evolved in each. Dif- ferences in these pathways could not be detected by assaying for residual I A A, but could be discerned by following the changes in the substrate as the reaction proceeds. For this purpose the spec- trophotometric technique is particularly well suited. This paper is a preliminary account of the chemistry of the model system, its relationship to the oxidations effected by peroxidase, and the spectrophotometric techniques employed in following the reactions. EXPERIMENTAL Changes in the ultraviolet absorption spectra were followed in quartz cuvettes of 1 cm. light path by means of a Beckman DK-2 re- cording spectrophotometer, equipped with the usual accessories for work in the ultraviolet region. The reference cell in each experiment contained the buffer system or solvent used in the sample cell. Correc- tions were made for the weak absorptions above 230 m^u, of peroxidase and hydrogen peroxide by subtraction of the absorbancies of blanks from those of the spectra being recorded. The reactions were carried out by pipetting solutions of hydro- gen peroxide or peroxidase in buffer into the cuvette, which con- tained a solution of lAA or some other substrate in the same buffer. The solution was stirred and the spectrum scanned. A typical en- zymatic reaction mixture contained lAA (final concn. 10 -*M) and peroxidase (10*' Af) in acetate buffer (O.IM sodium acetate -]- 0.04M acetic acid) at pH 5. A typical reaction in the peroxide system in- volved lAA (10-^M) and hydrogen peroxide (lO-^Tlf) in a buffer of 0.097M hydrochloric acid and 0.05M KCl (pH 1). Reactions were allowed to continue until changes in the spectra had ceased or were very slow, requiring from a few hours to several days depending on the reaction. Crystalline horseradish root peroxidase was obtained from Nutri- tional Biochemicals Corporation (Lot no. 7625). 2-Phenylindole-3- acetic acid was synthesized by the method of Bauer and Andersag (1), and 2-phenylskatole by the method of Kissman ct al. (5). Oxindole-3- acetic acid was a gift of Dr. Percy Julian, The Julian Laboratories, Chicago, Illinois. Other chemicals were pinified grades. THE PEROXIDASE SYSTEM The spectral changes which took place when lAA was treated with peroxidase under the conditions described above are shown in Figure 1. There was a very rapid increase in absorbancy in the 240 Chemical System for Oxidation of lAA by Peroxidase 207 > o z < m a: o (/) CD < X IN M^ Fig. 1. Changes in the ultraviolet spectrum during the oxidation of lAA (IQ-'M) b>° peroxidase (IQ-'A/). Solid line, in acetate buffer (pH 5); 1, 35 sec; 2, 10 min.; 3, 30 min.; 4, 4 hrs.; 5, 24 hrs.; broken line in phosphate buffer, pH 7.5, after 24 hrs. Continuation of curve 5 over range 0.8 to 1.8 absorbancy is given in reduced stale at lower left. to 260 m/i, region during the first 10 min., after which the rate of increase of absorbancy gradually fell off. The peaks at 248 and 254 mix could first be distinguished after about an hour, and reached their maximum in about 20 hrs. After that the peaks decreased slightly (0.06 unit) during the next 3 days. In the indole region (280 mfx) ab- sorption fell from 0.61 to 0.41 in the first 40 min., remained constant for the next 3 hrs., and then slowly fell as a new maximum formed at 285 m^ during the ensuing 3 days. The changes observed resemble closely those recorded by Ray (8) for the action on lAA of the lAA oxidase from Omphalia, and 208 R. L. Hinman and P. Frost of a crude peroxidase from horseradish root, both at pH 3.7. The principal differences are at 254 m^, where Ray observed only a shoul- der, and at 285 m^, where a new peak appeared in our experiments after 3 days. Since Ray's experiments were for periods under 2 hrs., it cannot be determined whether the maximum at 285 m^u, would have appeared. These minor differences may be due in part to differences in pH (at pH 7.5, Figure 1, the spectrum of the product(s) of lAA and peroxidase is quite different from that at pH 5), or to the presence of hydrogen peroxide in Ray's experiments. We found that the pres- ence of hydrogen peroxide in the mixture resulted in a general blur- ring of the spectrum, particularly in the 250 m/i, region, where the peak at 254 m^^ became a shoulder. Despite these minor differences the otherwise striking resemblance of the spectra from the peroxidase reaction and those from the lAA oxidase reaction confirm again the close relationship between the two, and the relevancy of using peroxidase as a model enzymatic sys- tem for studies of lAA oxidase. In addition to lAA, a number of related indoles were examined as possible substrates for peroxidase in the absence of added hydrogen peroxide. Under the conditions described for lAA in the experimental section, the spectra of (3-(indole-3-)propionic acid (IPA), ■y-(indole-3-)- n-butyric acid (IBA), a,a-dimethylindole-3-acetic acid, tryptophan, tryptamine, and skatole were unchanged during periods extending up to 90 hrs. THE MODEL CHEMICAL SYSTEM From other studies of the oxidation of indoles we have found that the rate of oxidation of some indoles by oxygen or hydrogen per- oxide is greatly accelerated by the presence of acid. The action of oxygen on lAA in aqueous solution at pH 1 produced red pigments, but there was little change in the ultraviolet portion of the spectrum. When lAA and hydrogen peroxide were mixed at pH 1, however, the spectrum of lAA underwent immediate changes, as shown in Figme 2. The peaks at 245 to 248 and 254 m/x are remarkably like those ob- served in the experiments with peroxidase. At pH 1 maximum ab- sorbancy was attained by the two peaks in the 250 m/x region in about 24 hrs., after which a slow decrease occurred. The indole peak at 279 m/x decreased very slowly while a new maximum appeared at 297 m^, complete in 72 to 90 hrs. In short, rapid changes took place at first in the 250 m^a region while the characteristic indole spectrum (278 to 288 m/x) underwent little change. Then, much more slowly, the indole portion of the spectrum ciianged completely, \\ith little disturbance of the peaks which had previously appeared in tlie 250 m/x region. Chemical System for Oxidation of lAA by Peroxidase 209 >- o z < m a: o en m < X IN Mfj. Fig. 2. Changes in the ultraviolet spectrum during the reaction of lAA (IQ-^M) and HoOa (10-^) at pH 1. Solid lines: curve 1, 30 sec; 2, 1 hr.; 3, 5.5 hrs.; 4, 28 hrs.; 5, 72 hrs. Broken line lAA (10-*A/). Continuation of curves 4 and 5 over range 0.8 to 1.8 absorbancy is given in reduced scale at lower left. The reaction of hydrogen peroxide with lAA is pH dependent. At pH 1 the initial reaction is very rapid; the peak at 254 m^ is present as soon as the spectrum is scanned (about 30 sec). From the accom- panying table it is clear that the reaction rate decreases markedly with increasing pH. In neutral or alkaline solution there is no change in the spectrum of mixtures of lAA and hydrogen peroxide. This agrees with Ray's (8) finding and confirms the earlier work of Siegel and Weintraub (12), who showed by other methods that lAA is not attacked appreciably by peroxides in neutral solution. When glacial acetic acid was used as the acidic medium, the peaks at 248 and 254 m^ appeared, but the spectral changes were much slower than in aqueous hydrochloric acid. However, in a 2:1 (v/v) 210 R. L. Hinman and P. Frost Table 1. Rates of oxidation of indole-3-acetic acid (lAA) and 7-(indole-3-)-«- butyric acid (IBA) in various media. Substrate* pH or Source of H + Time Required for First Appearance of Peak at 254 m^ Max. Absorbancy Attained at 254 m/i Time (hrs.) Required To Attain Max. Absorbancy at 254 m/i lAA 1 30 sec. 1.11 28 lAA 3 1 hr. 0.72 25 lAA 5 4 hrs. 0.67 46 lAA 6.6 73 hrs. 0.28 166 lAA 8 lAA glacial acetic acid 90 min. 0.79 23 lAA 67% acetic acid 15 min. 0.86 19 lAA 5t 30 min. 1.29 24 IBA 1 90 min. 0.90 48 * Initial concentration of substrate = \0~^M. Ratio of H2O2 to substrate = 10 in all experiments. t In the presence of \0~^M peroxidase; no H2O2 added. mixture of acetic acid and water the rate of increase of the peaks was about the same as in the mineral acid system, although the initial appearance of the peaks was still much slower (see Table 1). Decreasing the ratio of HoOo/lAA from 10:1 (which was the ratio used for most of the experiments) to 1:1 did not affect signifi- cantly the rate of growth of the peaks in the 250 m/x region, although the initial appearance of the peak at 254 m^u, required somewliat longer (5 min. when the ratio of 1:1). At a ratio of 1:2 the rate fell off much more, although the final height reached by the peaks was about the same as that of higher ratios, indicating that hydrogen peroxide was not limiting. At still lower ratios of oxidant to lAA hy- drogen peroxide was limiting, as shown by the fact that the maximum absorption was much less than that attained at higher ratios. It is not known whetiier the system also consumes oxygen, but from these results it seems unlikely. SPECIFICITY OF THE MODEL SYSTEM One of the most striking features of tlic H^Oo/H*^ system is its specificity. It appears to attack only those indoles which have a car- boxyl group (or a potential carboxyl group) in the side chain at the 3-position, and for greatest reactivity the carboxyl carbon must be Chemical System for Oxidation of lAA by Peroxidase 211 separated from the indole ring by one other carbon atom, as in lAA. This generalization is based on the following observations. (All ex- periments were carried out at pH 1 with a mole ratio of H2O2/IAA of 10:1.) (1) The spectrum of skatole remained unchanged for 25 hrs. (2) The spectrum of y-(indoIe-3-)-??-butyric acid (IBA) underwent rela- tively slow changes, but peaks at 248 and 254 m^u, were distin- guishable after 12 hrs. and reached maximum absorbancy in 48 hrs. (Figure 3). A shoulder with its center at 290 m/x appeared be- tween 24 and 48 hrs., corresponding to the maximum observed at 295 m^ when lAA was subjected to prolonged treatment with the H0O2/H+ system. (3) The spectrum of (3-(indole-3-)propionic acid (IPA) underwent a very slow increase in absorption in the 230 to 260 nifj^ region, while the indole peak at 280 rrifx slowly declined. No specific peaks appeared (Figure 3). (4) The spectrum of a,a-dimethylindole-3-acetic acid in which, as in lAA, the carboxyl group and the ring are separated by one carbon, changed more rapidly than did the spectrum of lAA, and was complete in 18 hrs. The new peaks were at 248, 259, and 293 m^ (Figure 3). In this case the peak at 293 m^ was formed about as rapidly as those at lower wave lengths, whereas with lAA the low^er peaks reached their maximum heights first, and the peak at 295 m^ required an additional 48 hrs. for complete formation (Figure 2). (5) The spectrum of 2-phenylindole-3-acetic acid changed rapidly and was complete in about 4 hrs. The spectral shifts are not directly comparable to those of the lAA reactions because the absorp- tion spectra of lAA and of 2-phenyl-IAA are quite different, as are those of their oxidation products. Nevertheless, it is signifi- cant that the spectrum of 2-phenylskatole, which lacks the car- boxyl group, was unchanged after 25 hrs. in the H2O2/H+ system. It is clear from these results that the Ho02/H^ system is selective in its attack of indoles. Any reaction which is limited to lAA and re- lated compounds would be of interest in considering the biological activity of lAA. The close relationship of the products of the chemical reaction and of the enzymatic reaction makes the chemical system considerably more significant. THE COURSE OF THE CHEMICAL REACTION AND THE NATURE OF THE PRODUCTS Although final answers have not been obtained to the questions of the detailed pathway of lAA oxidation in the H2O2/H+ system and 212 R. L. Hijiman and P. Frost >■ o z < CO q: o CO CD < X IN M/i Fi o q: o MOLES PER LITER Fig. 2. Growth effects upon Avena coleoptile cylinders (A), wheat roots (W), and flax roots (F) of (+)a-(4-methylphenoxy)propionic acid (4-MePOP) and (4-)a- (3, 5-dichlorophenoxy)propionic acid. Data presented as in Figure \. pionic and butyric acids are auxins of similar strength, the valeric acid shows intermediate character (at least in the oat cylinder test), while only antiauxin properties are revealed for the caproic acid. The presence of an auxin component in the activity of intermedi- ate substances like 2-naphthoxyacetic acid (4) or 4-BrPOA can hardly be disputed. In view of the gradual transition from such substances to those for which the restoration of oat coleoptile growth at higher concentrations remains well below the control level (e.g., 4-MePOA, Figure 3), the author does not hesitate to accept even a slight tend- ency to such a restoration as a sign of weak auxin activity. Such signs are often paralleled by indications of auxin activity in the flax root test. It shoidcl also be noted that the tendency to growth restor- ation at high concentrations of 4-MePOA is changed to a real stimu- lation for the nearly related substance (-|-)4-MePOP (Figures 2 and 3). The quantitative elucidation of the action of intermediate sub- stances along the lines used for the pure auxins and antiauxins meets with difficulties. It seems probable, however, that these can be obvi- 226 B. A berg MOLES PER LITER Fig. 3. Growth effects on Avena coleoptile of: A. Some pheiioxyacetic acids with different para-substituents (X), i.e. bromo (Br), methyl (Me), iodo (I), and ter- tiary butyl (tB). B. Some (+)phenoxyalkylcarboxylic acids, i.e. a-substituted n- butyric acid (B), n-valeric acid (V), and n-caproic acid (C). Data presented as in Figure 1. ated by the assumption of the cooperation of several similar, but not identical, auxin receptors. An alternative way would be to assume independent systems for auxin and antiauxin actions, but it seems that such an hypothesis generates more problems than it solves. If the initial inhibition of oat coleoptile cylinders is not related to the auxin system, it is diffi- cult to understand why it should be annihilated by the presence of [AA. The final stimulation, on the other hand, corresponds to an inhibition of flax root growth which is relieved by antiauxin applica- tion, and nothing is as yet known which makes it possible to differen- tiate this phase of the action from an ordinary auxin cllect. THE EFFECTS OF SUBSTITUENTS IN DIFFERENT POSITIONS The effect of pora-substitution has been discussed earlier (-1,6). 4-F- and 4-ClPOA show a considerably augmented auxin activity compared to ihc luisubstituted PGA. With increasing size of the jbara-substituent a change in the antiauxin direction takes place and is complete with 4-iP-POA and 4-NiPOA. In the mc'ia-substituted POA series a conspicuous antiauxin component of the activity is ap- parent already for .S-ClPO.\, but, on the othei- hand, a strong auxin Growth Regulating Effects of Pheyioxy Compounds 227 component is still present in 3-IPOA and 3-NiPOA. It is suggested that the meta compounds and, for example, 3,4-CloPOA are closely related to 2-NOA. The ori/io-substituted compounds generally show a fairly weak activity with both auxin and antiauxin components. In 2,3-Cl>POA the antiauxin component is fairly strong, and it is sug- gested that this substance is closely related to 1-NOA. The lack of stimulating effects on wheat root growth observed for 2-ClPOA re- appears for 2,6-Cl2POA, which may otherwise be characterized as an intermediate growth regulator. The presence of an auxin component in its activity is further indicated by the positive effects obtained in the pea curvature test (18). In 3,5-CloPOA the antiauxin component is wholly prevailing, but the appearance of a clear auxin component in (-f )3,5-Cl2POP Figure 2 shows that the presence of two meta sub- stituents is not wholly incompatible with auxin activity. The racemic 3,5-Cl2POP shows very weak activity in the pea curvature test accord- ing to Toothill et al. (18), but these authors hold that side-chain sub- stitution has a negligible effect on 3,5-derivatives. It seems to be an interesting task to study further how far 3,5-Cl2POA is related to phenoxyacetic acids with large /jrtra-substituents. The induction of auxin activity by further substitution in the ring of 3,5-Cl2POA (18), however, seems to indicate that simple dimensional factors can hardly be at work. Rather unexpectedly, 2,4,6-Cl3POA shows a purer antiauxin char- acter than does 2,6-CloPOA, and both a-propionic acids are stronger antiauxins than the acetic acid (Figure 4B). As the racemic 2,4,6- CI3POP is reported to show clear auxin activity in the pea curvature test (12, 18), this is apparently not true for all types of tests. The thymoxyacetic acid (2-iP, 4-Cl, 5-MePOA) exerts only antiauxin effects, presumably due to the large o>phcnoxy- acciic acid, growth, and ion absorption. Iti: R. L. ^Vain and F. Wightnian (eds.) The Chemistry and Mode of Action of Plant Growth Substances, pp. 253-259. Butterworth Sci. Pub!., London. 1956. Physiological Appruacli to Selective Action of 2,4-D 245 and Robertson-Cunninghame, R. C. Interrelationships between light intensity, temperature, and the physiological effects of 2:4-dichlorophenoxy- acetic acid on the growth of Lemna minor. Jour. Exper. Bot. 6: 156-176. 1955. 3. , and Sargent, J. A. The uptake of growth substances. II. The absorp- tion and accumulation of 2:3:5-triiodobenzoic acid by the root and frond of Lemna minor. Jour. Exper. Bot. 10: 480-503. 1959. 4. — . Sen, G., Birch, W. R., and Powell, R. G. The uptake of growth sub- stances. I. Factors controlling the uptake of phenoxyacetic acids by Lemna minor. Jour. Exper. Bot. 10: 33-54. 1959. 5. Johnson, M. P., and Bonner, J. The uptake of auxin by plant tissue. Physiol. Plant. 9: 102-118. 1956. 6. Sacher, J. A., and Glasziou, K. T. Effects of auxins on membrane permeability and pectic substances in bean endocarp. Nature. 183: 757,758. 1959. 7. Wedding, R. T., and Erickson, L. C. The role of pH in the permeability of Chlorella to 2,4-D. Plant Physiol. 32: 503-512. 1957. 8. Weintraub, R. L., Reinhart, J. H., and Scherff, R. A. Role of entry, transloca- tion, and metabolism in specificity of 2,4-D and related compounds. U. S. Atomic Energy Comm. TID-7512: 203-208. 1956. 9. Woodford, E. K., Holly, K., and McCready, C. C. Herbicides. Ann. Rev. Plant Physiol. 9: 311-358. 1958. DISCUSSION Dr. Henderson: Have you measured the lAA content of these plants after treatment? Professor Blackman: I haven't measured the lAA content. In fact, I don't know how I would measure the content of lAA which is physiologically active. Dr. Henderson: In a variety of plants, we found that 2,4-D reduced the rate of disappearance of I A A, while in others auxin disappear- ance was increased by 2,4-D. Professor Blackman: With TIBA, if you add lAA to the external solution at concentrations of 30 to 50 mg/1, you can prevent the loss of TIBA but not from roots. That's the only case I know where there is a clear-cut effect of averting the rate of loss by adding lAA. Dr. Burstrom: With the high addition of 2,4-D to your susceptible species, as well as in one of the cereals, you had the peak of accumula- tion after 8 to 12 hrs. In these species have you ever observed any cy- tological disturbances which may account for the inability of the tissues to retain the amounts of 2,4-D initially taken up in these concentrations? Professor Blackman: In the root uptake investigations we only used one mg/1, a very low concentration unlikely to cause cytological dis- turbances in a few hours. It is true that we have observed in Brassica alba that you can have injection of water into the intercellular spaces at high concentrations and still have 2,4-D being taken up by the stem segments. In this instance, this type of disturbance does not prevent accumulation of 2,4-D. For Triticiim I do not know whether there was 246 G. E. Blackman visible injury, but 1 would again stress that although the segments at high concentrations eventually lost water, they still took up 2,4-D. Dr. Bennet-Clark: In our laboratory we have extremely similar results which are to be published in the Journal of Experimental Botany. lAA accumulates in disks of tissue in essentially the same way that Dr. Blackman has described for 2,4-D. It rises to a maximum concentration and decreases again. The disappearance of I.\.\ from the tissue, which at one time we thought was due to the metabolism of lAA, the formation of conjugates, oxidation to COo, etc., is partly due to metabolism. Part is due to the output into the external solution. The point of interest in this connection is that the uptake process is oxygen-dependent, aerobic respiration-dependent, as Dr. Blackman shows for 2,4-D. The output is not dependent on aerobic respiration. The second feature of interest, as Dr. Blackman showed, is that the gross concentration of 2,4-D in the tissue was smaller than the gross concentration outside, and yet the material came out. This is defi- nitely also the case in our lAA experiments. If )ou consider that the lAA concentration inside is the weight of lAA per unit weight of tissue water, this gross concentration of lAA inside is smaller than the concentration of lAA outside during the phase at which the lAA is coming out. And yet this output of lAA is not an active secretion process; it is completely unaffected by cyanide, azide, anaerobic conditions, etc. And, therefore, I would judge that the gross lAA concentration inside and, by implication, the gross concentration of Dr. Blackman's 2,4-D inside, is not equal to the concentration of these substances in the effective site at which it is concentrated, and I would guess that it is concentrated in the cytoplasm rather than the vacuole. I would quite like to hear if that agrees with Professor Blackman's data. Professor Blackman: Yes, our ideas are very much like those of Bennet-Clark. ^\'e do not think that 2,4-D has got past the cytoplasm. We tlo, however, think that we have certainly obtained some very puzzling results with Lemna. Thus, if Lemna is first allowed to take up labeled 2,4-D and then transferred to culture solution, more, not less, 2,4-D comes out if the external solution contains cold 2,4-D (Blackman et al., Jour. Exper. Bot., 10: 'i'^, 1959). Egress is clearly not a simple exchange reaction. My feeling is that the 2,4-D accumulat- ing in the cytoplasm exerts a different effect from that located in the outer cytoplasm cell wall interface, and it is the concentration here which controls extension growth rather than what piles up behind the cytoplasm. Johnson and Bonner (Physiol. Plant. 9: 102, 1956) did Physiological Approach to Selective Action of 2,4-D 247 touch on this problem that the increased rate of extension growth of Avena coleoptiles is dependent on the external concentration of 2,4-D but that at each concentration the response is linear with time over several hours. If the rate of extension growth is dependent on the total internal concentration of 2,4-D, then the rate of extension growth should be curvilinear, not linear, since 2,4-D is steadily accumulating in the tissues. Dr. Andreae: In connection with Dr. Bennet-Clark's interesting ob- servations on lAA uptake and secretion, I should like to report our findings with lAA-treated tissues. Pea root tips accumulate lAA to ten times the external concentration. lAA, once inside the root tips, is metabolized and, according to our quantitative data, is not lost again to the external solution. Dr. Bonner: Prof. Blackman has shown us some most interesting results. We can conclude that the 2,4-D is actively accumulated by many kinds of cells and that from some kinds of cells it leaks out again. He has also shown us, beyond any doubt, that the uptake of one auxin can be inhibited by another. I think everyone that has investigated this subject would agree that the various auxins and anti-auxins are competitive with one another in their uptake into the cell. Now, I feel quite confident that someone is thinking that the way auxins interact with one another in controlling growth or the way anti- auxins inhibit the growth-promoting activities of the auxins, is merely by preventing the uptake of the active material into the cell. Well, I wish to state that this isn't so. The effect of anti-auxins in inhibiting growth is due to some different phenomenon than the effect of anti- auxins on inhibiting the uptake of active auxins. That this is so can be shown by simple quantitative relationships. The concentration of lAA which is required to half-inhibit the growth promotion caused by 2,4-D in Avena coleoptile section, is somewhat lower than the 2,4-D concentration used. The concentration of lAA required to in- hibit by half the rate of 2,4-D uptake by the Avena coleoptile section is of the order of one thousand times the concentration of the 2,4-D used. The 2,4-D uptake is less sensitive to the presence of lAA than is the promotion of growth of the section by 2,4-D. So apparently there are two entirely different processes. ROBERT M. MUIR University of Iowa CORWIN HANSCH Pomona College Chemical Structure and Growth- Activity of Substituted Benzoic Acids Since the discovery by Zimmerman and Hitchcock in 1942 (12) that substituted benzoic acids would induce growth responses in plants, many studies have been made of the effects of these compounds in a wide variety of plant responses. In these investigations the usual find- ing is that unless the benzoic acid has a halogen substituent in one or both ortho positions it is inactive except in the case of a few com- pounds with methyl substituents in the ortho position (7, 8, 9, 11). The activating effect of an electronegative substituent in the ortho posi- tion may be explained by the electronic theory of organic reactions in that the low electron density of the benzene ring would promote dis- placement of the electronegative radical in reaction with a nucleo- philic substance. This reaction may be represented for 2,8,6-trichloro- benzoic acid which Bentley (1) found to be very active in promoting cell elongation as follows: COOH COOH Ck 1 /CI CK J\ /X + ci- ci ^/ ^ci In this interpretation the activity of the benzoic acid structure is dependent upon a suitable electron density at the ortho position. Re- cently Fukui et al. (2) have calculated the pi-electron distribution for various benzoic acid derivatives by the molecular-orbital method. The frontier electron density in the lowest vacant orbital of the ground state is calculated as the approximate superdelocalizability (S/[^']) for a reaction with a nucleophilic reagent. Some of these values [249] 250 R. M. Muir and C. Hansch Table 1 . Chemical reactivity and auxin activity indices of substituted benzoic acids. Substituent on Benzoic Acid S/(N)* Auxin Activity Index t 2,5-Dichloro- 0.7771 0.7186 0.7108 0.7093 0.7074 0.6918 0.6825 0.6810 0.6693 0.6501 0.6199 0.6096 0.6079 0.5564 1 0 2,4-Dichloro- 0 0 2,3,6-Trichloro- Ca. 10.0 2-Chloro- 0 05 2,3,5-Triiodo- 2,4,6-Trichioro- . ... 50.0 0 0 2-Bronio- 0.1 2-Iodo- 0 0 2,6-Dichloro- 2,6-Dibronio-. 0.1 0 0 4-Chloro . . 0 0 2-Fluoro- . 0 0 3,4,5-Triiodo- 0 0 4-Fluoro- 0 0 * Approximate superdelocalizability at ortho position for reaction with a nucleo- philic reagent [After Fukui et al. (2)]. t Molar concn. of indole-3-acetic acid inducing an elongation of 0.15 mm. Molar concn. of growth regulator inducing an elongation of 0.15 mm. are given in Table 1 together with oin- vahies for the activity of the various benzoic acids in promoting elongation at low concentrations compared with indole-3-acetic acid at 100. Compoimds with S/*-"^'^ values less than 0.6693 at the ortJio posi- tion do not promote elongation while those with a value of 0.6693 or greater do except for some such as those substituted in the para position. The inactivating effect of substitution in the para position has been recognized for a long time. Veldstra (10) has suggested that since 2,3,6-trichloro-4-fluorobenzoic acid is active, the inactivation by substitution at the para position is due to the size of the substituent and the small size of fluorine permits activity. Since the S/<^'' values for 2,4-dichloro- and 2,4,6-trichloro- benzoic acids are high but the compounds are inactive, some steric hindrance of the chlorine atom at the para position must exist. However, fluorine in the para posi- tion may also result in an inactive compound, for we have found 2-chloro-4-fluorobenzoic acid (IX) to be completely inactive in pro- moting elongation. In general the introduction of fluorine into the benzoic acid struc- ture has an inactivating effect as far as the promotion of elongation is concerned. We have examined a number of fluorine-substituted ben- zoic adds and all except 2-fluoro-6-chlorobenzoic acid have been in- active. Of ]:)nrti( ular significance is the effect of replacing the chlorine Substituted Benzoic Acids 251 COOH /F /\: COOH COOH n COOH .F COOH .F NH2' NO; IV V vn vni COOH .F VI COOH .CI F IX at the 2 position in 2,5-dichlorobenzoic acid with fluorine. Ahhough the 2,5-dichloro compound has the highest S/'^'> value yet calculated for the series and is one of the more active compounds, the 2-fluoro-5- chlorobenzoic acid (VIII) is completely inactive. Professor Fukui has kindly sent us his calculations of S/<^^ values for the 2-chloro-4-fluoro- and 2-fluoro-6-chloro- compounds and these are given in Figure 1. It is readily apparent that the effect of fluorine is not a consequence of its size but of its contribution to the electron density at the ortlio position. Fluorine in the para position reduces the S/*^' value at the ortJw position of the chlorine atom to 0.6614, below the value associated with activity in elongation, and the com- pound is completely inactive. With fluorine at the ortho position the S/'^' value for the other ortho position is increased to 0.7152. Thus 2-fluoro-6-chlorobenzoic acid has an auxin activity index of 0.1, which is the same as the value for 2,6-dichlorobenzoic acid. The S/'N' calculations are of interest also in connection with an apparent effect of the size of the iodine atom on activity. Although 252 R. M. Muir and C. Hansch the Sr'<^') value for ortho iodobenzoic acid is higher than 0.6693, the compound is inactive, presumably because of the large size of iodine and the consequent steric hindrance in displacement. If, however, the Sj.'<'N') value is increased by additional iodine substitution in the 3 and 5 positions giving 2,3,5-triiodobenzoic acid, then the compound is very active. Fukui et al. (2) have calculated approximate superdelocalizability at the ortho positions for reaction of the benzoic acids with an electro- philic reagent and with a radical reagent. No relation is found be- tween these values and the auxin activity of the molecules. Thus our 0 2693 02893 0 2 899 0 2879 HO, ^0 0.5096 HO^^O 0.0 4 4 4 00005 0.00 07 0.1429 0.6 9 45 Fig. 1. Approximate superdelocalizability values (Sr"^""^) as calculated by Fukui for 2-chloro-4-fluorobenzoic acid (left) and 2-fluoro-6-chlorobenzoic acid (right). suggestion of the nucleophilic character of the substrate with which the growth regulators react (3, 6) has been substantiated by the the- oretical chemistry of molecular orbitals. In the course of examining substituted benzoic acids for auxin activity, a few have been found to inhibit the elongation of cells of Avena coleoptiles at low concentrations. Of these, 4-ethyl-3-mercapto- benzoic acid (EMBA) (4) has been the most interesting. It was found to inhibit elongation by 40 per cent at lO'^M and to repress the effects of growth regulators promoting elongation at concentrations as low as lO'^M. The interaction of EMBA and these growth regulators is shown in Table 2. The growth induced by the synthetic regulators appears to be much more sensitive to the effect of EMBA than the growth induced by the natural hormone, indole-3-acetic acid. In gen- eral, the degree of the inhibitory action of EMBA appears to be in- versely related to the relative activity of the regulators in promoting elongation. Similar and lesser inhibition is demonstrated for the Substituted Benzoic Acids 253 Table 2. Percentage inhibition of growth of Avena coleoptiles in- duced by growth regulators in the presence of 2 X 10"^/ 4-ethyl-3- mercaptobenzoic acid (5 X XO'^M with lAA). Growth Regulator Molar Concn. Per Cent Growth Inhibition Tndnlp-'^-arptic acid 2 X 10-7 5 X 10-7 2 X 10-6 5 X 10-7 5 X 10-6 2 X 10-6 5 X 10-7 5 X 10-6 5 X 10-7 5 X 10-6 5 X 10-7 5 X 10-6 2 X 10-7 5 X 10-7 5 X 10-6 1 X 10-5 2 X 10-5 5 X 10-5 1 X 10-4 2 X 10-4 65 2,4-Dichlorophenoxyacetic acid 2,4,5-Trichlorophenoxyacetic acid Tndolp-'^-butvric acid 45 35 80 55 30 85 45 85 1 -TVanhthaleneacetic acid 85 45 1 -Nanhthaleneacetonitrile 40 50 2,6-Dimethyl-3-bromobenzoic acid 2,6-Dichlorobenzoic acid 50 50 25 10 100 65 30 elongation induced by indole-3-acetic acid, 1-naphthaleneacetic acid and 1-naphthaleneacetonitrile, the compounds most active in promot- ing elongation. Greater inhibition occurs in the case of 2,4-dichloro- phenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and indole-3- butyric acid, which are less active. The greatest inhibitory effect is found with 2,6-dichloro- and 2,6-dimethyl-3-bromo- benzoic acids, which have the lowest activity as auxins. EMBA, at one-tenth of the concentration of 2,6-dichlorobenzoic acid, results in an inhibition of 30 per cent of the elongation effect of the latter. These results suggest a competitive type of inhibition, and Line- weaver-Burk plots of the reciprocals of elongation and regulator con- centration (5) conform to expectation for the interaction of EMBA and indole-3-acetic acid. The interaction with 2,4-dichlorophenoxy- acetic acid (Figure 2) and 2,6-dimethyl-3-bromobenzoic acid appears to be competitive at high concentrations of the auxin, but at low con- centrations the inhibition tends to be complete. The inhibition by EMBA also appeared to be reversible in an experiment in which in- 254 R. M. Mitir (mil C. Hanscli 10 X h- $ o ir o 10 15 20 25 30 X 10^ MOLAR CONCN. 2,4-D Fig. 2. Inhibiting effect of 4-ethyl-3-mercaptobenzoic acid at 1 x 10"^M and 2 X lO-'M on tlie growth of Avena coleoptile sections induced by 2,4-dichlorophenoxv- acetic acid. dole-3-acetic acid at 2 X lO'^M and EMBA at 5 X 10-^M were re- placed after 5 hrs. by EMBA at the same concentration and indole-3- acetic acid at 4 X 10-^M. The higher concentration of indole-3-acetic acid resulted in a growth rate nearly the same as that of the tissue in the absence of EMBA. To evaluate the chemical grouping responsible for the inhibition brought about by EMBA, the effects of several related compounds were examined and the data are given in Table 3. The S-meihyl eilier Table 3. Percentage inhibition of elongation of Avena coleoptiles produced by indole-3-acetic acid in presence of substituted benzoic acids. Substituted Benzoic Acid 3-Mercapto-4-ethyl- 3-Methyimcrcapto-4-ethyl-.. . . 2-Mercapto- 3-Mercapto- 4-Mercapto- 4-Ethyl- 4-Chloro- 2,4-Dichioro- .... Molar Concn. 5 X 10-6 5 X 10-6 2 X 10-^ 2 X 10-'' 2 X 10-4 2 X 10-4 1 X 10-" 1 X lO-* Molar Concn. of lAA 2 X 10-7 65 20 10 15 50 35 35 15 5 X 10-7 45 20 5 10 40 30 30 20 2 X 10-6 35 10 5 10 40 20 30 20 Substituted Benzoic Acids 255 derivative is not as effective in inhibiting the elongation induced by indole-3-acetic acid, even though being less polar it should penetrate more rapidly to the reaction site. Thus the reaction at the thiol group must be the primary element in the inhibition effect. The aromatic thiol group itself, however, even in the meta position, gives rise to only slight inhibitory effect for the benzoic acid structure. The in- hibition by jbara-substituted benzoic acids is, in general, of the same order for thiol, chlorine, and ethyl groups and only about one-fourth or less of the inhibition brought about by EMBA. Thus the action of EMBA appears to be quite specific and to depend on the effect of the ethyl substituent on the thiol group. This effect is probably elec- tronic in character, with the ethyl substituent releasing electrons to the ring and rendering the thiol group S- with a greater potential for reaction. On the basis of this study of the inhibitory action of 4-ethyl-3- mercaptobenzoic acid on elongation, the following inferences may be formed: (1) Since the degree of inhibition in interaction with the various auxins is related to the activity of the auxins, the EMBA mole- cule reacts at the substrate site where the auxins react and induce growth. (2) Since the reaction involves the thiol group, it is possible that the substrate site is a sulfhydryl, as has been suggested on the basis of considerations involving only the auxin structure. SUMMARY The growth activity of substituted benzoic acids appears to be de- pendent primarily upon electronic characteristics determined by the substituents, but these electronic characteristics may be counteracted in part by steric factors. ACKNOWLEDGMENT The authors express their profound gratitude to Professor Fukui of the University of Tokyo for making available his calculations of pi-electron distribtitions and thus making possible for the first time the quantitative appraisal of the electronic factors in the activity of growth regulators. LITERATURE CITED 1. Bentley, J. A. Growth-regiilating effect of certain organic compounds. Nature. 165: 449. 1950. 2. Fukui, K., Nagata, C, and Yonezawa, T. Electronic structure and auxin ac- tivity of benzoic acid derivatives. Jour. Amer. Cliem. Soc. 80: 2267-2270. 1958. 3. Hansch, C, Muir, R. M., and Metzenberg, R. L., Jr. Further evidence for a chemical reaction between plant growth-regulators and a plant substrate. Plant Physiol. 26: 812-821. 1951. 256 R. M. Miiir and C. HanscJi 4. Hansch, C, Schmidhaltcr, B., Reiter, F., and Saltonstall, W. Catalytic synthesis of heterocycles. VIII. Deiiydrocyclization of o-ethylbenzenethiols to thiana- phenes. Jour. Org. Chem. 21: 265-270. 1956. 5. McRae, D. H., and Bonner, J. Diortho substituted phenoxyacetic acids as anti- auxins. Plant Physiol. 27: 834-838. 1952. 6. Muir, R. M., and Hansch, C. The relationship of structure and plant-growth activity of substituted benzoic and phenoxyacetic acids. Plant Physiol. 26: 369-374. 1951. 7. , and Hansch, C. On the mechanism of action of growth regulators. Plant Physiol. 28: 218-232. 1953. 8. , and Hansch, C. Chemical constitution as related to growtli regulator action. Ann. Rev. Plant Physiol. 6: 157-176. 1955. 9. Pybus, M. B., Smith, M. S., Wain, R. L., and Wightman, F. Studies on plant growth-regulating substances. XIII. Chloro- and methyl-substituted phenoxy- acetic and benzoic acids. Ann. Appl. Biol. 47: 173-181. 1959. 10. Veldstra, H. On form and function of plant growth substances. In: R. L. Wain and F. Wightman (eds.). The Chemistry and Mode of Action of Plant Growth Substances, pp. 117-133. Butterworth Sci. Publ., London. 1956. 11. , and Westeringh, C. van de. Growth-substance activity of substituted benzoic acids. Rec. Trav. Chim. Pays-Bas. 71: 318-320. 1952. 12. Zimmerman, P. W., and Hitchcock, A. E. Substituted phenoxy and benzoic acid growth substances and the relation of structure to physiological activity. Contr. Boyce Thompson Inst. 12: 321-343. 1942. DISCUSSION Dr. Smith: We have studied the effect of dichloro- and dimethyl- substitution of benzoic acids, and I would like to show you some of our results in relation to those obtained by Drs. Muir and Hansch. These assays utilized three test systems (Ann. Appl. Biol. 47: 173. 1959), but the data from the pea test are representative of our results. As seen in Table 1, and as stated by Dr. Hansch, the monochloro acids are inactive. However, not only is the 2,6-dichloro- active, but marked activity is also shown by the 2,3-, 2,5- and 2,3,6- derivatives. Similar, but less marked results, were obtaind with mono- and dimethylbenzoic acids. Here again, mono-substitution renders the compound inactive, but some activity is shown by the 2,3-, 2,5-, and 2,6- acids. Dr. Wain: Dr. Smith has raised an interesting point. In structure activity relationships it is important to study whole series. One finding that must be noted is the quite high activity of the 2,3- compounds which to date has been overlooked. In view of this activity, one has to ask whether a removal of the 2- or 6- substituent is essential in the growth reaction. 1 think a very ingenious hypothesis has been put forward here, but 1 am rather dubious about it myself, especially when one has to accept the elimination of a methyl group from the 2 or 6 position, in order to explain the activity of the 2,6-dimethyl- and 2,3,6-trimethylbenzoic acids. Substituted Benzoic Acids 257 Table 1. Activities of chloro- and methyl-substituted benzoic acids in the pea curvature test. * Substituted Benzoic Acid 2-Chloro-. . . . 3-Chloro- 4-Chloro- 2,3-Dichloro-. 2,4-Dichloro- 2,5-Dichloro-. . 2,6-Dichloro-. . 3,4-Dichloro- . . 3,5-Dichloro-. . 2,3,6-Trichloro- 2-Methyl- 3-Methyl- 4-Methyl- 2,3-Dimetiiyl- . 2,4-Dimethyl- . 2,5-Dimethyl- . 2,6-Dimethyl- . 3,4-Dimethyl- . 3,5-Dimethyl- . Molar Concentrations 10-7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 io-« 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 10-5 0 0 0 0 0 3 0 0 0 4 0 0 0 0 0 0 0 0 0 10-4 0 0 0 2 0 5 1 0 0 5 0 0 0 1 0 0 0 0 0 10-' 0 0 0 4 0 5 3 0 0 6 0 0 0 4 0 2 1 0 0 * Activity on an arbitrary scale: 0 = inactive; 6 = highly active. Dr. Muir: I would like to make it clear that in the first place we were looking at a series of compounds which we were trying to ex- plain. We found that in the fluorine series we had a rather unusual situation as far as halogen substituents were concerned, and our dis- course was primarily concerned with them. I don't see, except for a few compounds which Dr. Smith studied, any real difference from our position other than in the methyl-substituted compounds. These may yield wholly different results, and until we have further electronic data to analyze this picture, I don't believe they constitute too serious an objection. Our primary purpose was to explain the relationship of structure as it is shown in the effects of these compounds on Avena coleoptile tissue. Dr. Wain: I would just like to say again what Dr. Smith has said. All these results have been obtained in three different tests by Dr. Wightman so that the results are of general applicability as far as we can see in these different tests, using different types of tissue. Dr. Fawcett: In his discussion of the highly active 2,3,6-trichloro- benzoic acid. Dr. Muir postulates a nucleophilic reaction involving a replacement of the chlorine atom in the 6 position. In 2,3,5,6-tetra- chlorobenzoic acid, which has similar activity, a nucleophilic replace- ment of either of the ortho chlorine atoms would be subject to con- 258 R. M. Muir and C. Hansch siderable steric hindrance. Usually, in these circumstances the re- placement rate becomes very small. Dr. Muir: The selection of the ortho position for the attack as shown in the reaction of 2,3,6-trichlorobenzoic acid is determined by the S/<^'> values. AVhere there is only one halogen-substituted position, that one is it. In the cases where there are two ortho substituents, as Fukui, Nagata, and Yonezawa have indicated, the one with the greater S/^N> would be most likely to be displaced in the reaction. Their cal- culations of superdelocalizability show that one may be of a different value from the other, depending upon the substituents in the ring. Dr. Wain: We are not really concerned necessarily with reaction at the set 2 or 6 position. We were under the impression from all of your papers that it is the 2 or 6 position which was involved. But I now take it that any other position will do, providing that it is suit- ably activated. Is that right? Dr. Muir: Partially. Where there is steric hindrance, even though the electronic value or density is favorable, the steric hindrance T\ill make the molecule inactive. Dr. Ray: In relation to the idea that 4-ethyl-3-mercaptobenzoic acid is reacting with the same site as do the auxins, I wonder if there is any w^ay that you can explain why you get reversal of this inhibition by some auxins but not by others? It seems that, from the data you show, in the case of some auxins the inhibition was quite independent of the auxin concentration. Dr. Muir: My explanation would be that in not all cases did we have the appropriate range in which to demonstrate competition. In some instances this was outside of the ranges in which the reversi- bility is most easily shown. Dr. Wain: I would like to ask whether 4-ethyl-3-mercaptobenzoic acid reacts chemically with any of these auxins. For instance, have melting point curves been constructed to show whether you get com- pound formation or a simple eutectic? Dr. Hansch: The answer is no. Dr. Wain: One further point. The pH is very important in rela- tion to all thiol compounds which are readily oxidized to disulfides under alkaline condition. The pH here was on the acid side, wasn't it? Dr. Muir: It was controlled at 5.6. Dr. Hansch: The values for superdelocalizability which vou have just seen and which Professor Fukui was so kind to fiunish us repre- sent only a small number of those he has made. A more extensive list has been published (Jour. Amer. Chem. Soc. 80: 2267. 1958). F. G. TEUBNER^ S. H. WITTWER JANE Y. SHEN- Michigan State University Relationship of Molecular Structure to Biological Activity In the H-Arylphthalatnlc Adds' The activity of a number of substituted iV-phenylphthalamic acids in stimulating fruit set of tomato was reported by Hoffmann and Smith in 1949 (8). Since then, these chemicals have received only infrequent attention as fruit setting agents (3), although the closely related iV-1- naphthylphthalamic acid has shown considerable promise as a se- lective herbicide (4). The action of iV-m-tolylphthalamic acid in caus- ing increased flower formation in the tomato (23) has led to the prac- tical use of this chemical to obtain increased flower and fruit num- bers and, thereby, higher yields of greenhouse grown tomatoes (33). It was subsequently found that a number of the chloro- and methyl- substituted iV-phenylphthalamic acids possessed flower forming activ- ity and that the structural requirements of these compounds for alter- ing flower formation apparently differed from those for auxin ac- tivity in the substituted benzoic and phenoxyacetic acids (24). At present, only two other synthetic growth regulators, a-(2- naphthoxy)phenylacetic acid (17) and the 2,3,5-triiodobenzoic acid (6, 31, 34, 35), have been clearly demonstrated to increase flower forma- tion in the tomato. Although 2,3,5-triiodobenzoic acid has been con- sidered an auxin antagonist (5), or auxin synergist (26), it also has auxin properties (1, 13). Similarly, a-(2-naphthoxy)phenylacetic acid is highly active in stimulating parthenocarpic fruit set (18) but has not, to our knowledge, been evaluated for auxin activity in any of the more classical assays. This report deals with the molecular structure of the 1 Subsequently: Department of Horticulture, University of Nebraska, Lincoln, = Subsequently: Division of Biological and Medical Research, Argonne National Laboratory, Lemont, Illinois. ^Journal article No. 2485 from the Michigan Agricultural Experiment Station. [259] 260 Teubner, Wittwer, and Shen substituted AT-arylphthalamic acids as related to their biological ac- tivity in tomato flower formation, and as auxins in the stimulation of tomato parthenocarpy and elongation of Avena coleoptile sections. MATERIALS AND METHODS Chemical^ Most of the substituted AT-arylphthalamic acids used in these in- vestigations were prepared by reacting equal molar amounts of phthalic anhydride and the appropriate aromatic amine in benzene at 30 to 80° C. The products were dissolved in dilute base, reprecipi- tated with hydrochloric acid, washed thoroughly with water, and dried. The sterically hindered 2,6- and 2,4,6-substituted phenyl de- rivatives were prepared by heating the amine and phthalic anhydride together without solvent at 150 to 200° C. to form the imide, then hydrolyzing to the phthalamic acid with one equivalent of sodium hydroxide. Purity of the preparation was checked by titration to ob- tain the equivalent weight, and good agreement with theoretical val- ues was found. Neutral equivalents were a better criterion of purity than melting points since most of the N^-arylphthalamic acids decom- posed as they melted, liberating water and forming the imide. Tomato flower formation. The procedure was essentially that de- veloped by Teubner and Wittwer (24) and was based on the observa- tion that the first inflorescence of the tomato (Lycopersicon esculen- tum, 'Michigan-Ohio Hybrid') is initiated 9 days after cotyledon expansion (21). The inflorescence is most susceptible to modification by chemical treatment when the first flower primordium is differen- tiating, which, in the test variety, corresponded to plants having two plumule leaves and a third leaf one-half to one inch in length (24, 33) . Plants treated with the N-arylphthalamic acids at this stage developed branched flower clusters with five to seven flowers on each branch. Over a limited concentration range, which varied for each active chemical, the flowering response was directly proportional to concen- tration. The maximum responses obtained with this method have been clusters consisting of three to four branches and 28 flowers. Higher concentrations either reduced branch and flower numbers or produced severe formative effects. Solutions of the various N-arylphthalamic acids were prepared just prior to treatment by dissolving weighed amounts of each chemical in a small quantity of acetone and diluting to the appropriate volume with water. No deleterious effects were observed for concentrations of acetone up to 5 per cent, the maximum employed in the studies. The * Personal communication from Dr. A. E. Smith, Agricultural Chemicals, Nauga- luck Clhemical Division, United States Rubber Company. TJw N-Arylphthalamic Acids 261 solutions were applied with a compressed air sprayer and plants were sprayed to the drip point. In other studies, responses equivalent to those from spraying have been obtained by applying 0.01 ml. of each concentration to the apex of the plant (21). Flower numbers were determined after 6 weeks or when the final flower bud was clearly visible. Tomato parthenocarpy. Luckwill's test (11) was employed, and 0.01 ml. of each solution was applied directly to ovaries of tomato flowers emasculated just prior to anthesis. Six ovaries were treated with each concentration of a chemical. Solutions were prepared as described above, and ovaries treated with 5 per cent acetone did not differ from nontreated ovaries. Ovary diameters were measured to the nearest 0.1 mm. with a vernier caliper 5 days after treatment. Avena straight grozath. The procedure used for growing and sec- tioning Avena coleoptiles was that described by Leopold (10) using 'Brighton' oats. Coleoptiles 2 to 3 cm. in length were selected and a single 5 mm. section was cut 3 mm. below the tip. The sections were floated, ten to a dish, without removing the leaf, in 10 ml. of the test solutions which contained a phosphate-citrate buffer (pH 5.0) and 3 per cent sucrose (15). Since most of the substituted A''-aryl- phthalamic acids dissolve only with difficulty below pH 6.0, the so- lutions were prepared by dissolving appropriate amounts of each chemical in the K0HPO4 buffer component (pH 8.5) and then adding the citric acid and sucrose. No visible precipitation occurred in 10"^ M solutions prepared in this manner. Lengths of the Avena sections were measured after 20 hrs. to obtain maximum differences between the treated and the control sections (16), and to minimize hydrolysis of the phthalamic acids at pH 5.0 (3). Measurements were made to the nearest 0.5 mm. Elongation was expressed as per cent of the final length of control sections grown in the phosphate-citrate-sucrose mixture. Statistical The statistical significance of the differences between the means of control and treated samples was based on the "t" test as outlined by Goulden for nonpaired variates (7) . RESULTS Activity in Tomato Flowering In preliminary studies tomato plants were treated with a number of phenyl-substituted N-phenylphthalamic acids at concentrations ranging from 5 to 500 p.p.m. in order to establish an effective range for each. It was found that substitution of chloro- or methyl- groups 262 Teubner, Wittwer, and Shen at the ortho or meta positions enhanced flower forming activity while hydroxyl- or nitro- groups at either the ortho, meta, or para positions in the phenyl ring rendered the weakly active N-phenylphthalamic acid inactive. Substitution of chloro- or methyl- at the para position did not abolish the flower-forming activity of iV-phenylphthalamic acid, although these derivatives had a high degree of phytotoxicity which often masked their weak flower-forming activity. On the other hand, substitution of bromo-, carboxyl-, amino-, dimethylamino-, or acetyl- gioups at the para position resulted in derivatives which were inactive at concentrations up to 500 p. p.m. The iV-2,5-dimcthoxy- and iV-2-methyl-5-isopropylphenyIphthalamic acids were also mactive in the preliminary tests. Replacement of the phenyl ring with a 1-naphthyl group (xV-1- naphthylphthalamic acid) resulted in high phytotoxicity and forma- tive effects, but no increase in flower numbers. On the other hand, N- 2-naphthylphthalamic acid exhibited no phytotoxic or formative ef- fects and did give a slight increase in flower numbers.^ On the basis of these preliminary tests, all of the monochloro- and dichloro- together with four of the six possible trichloro- derivatives were evaluated in the tomato flowering test over their most effective concentration ranges. In the same test A^-phenylphthalamic acid was included together with its monomethyl- and several dimethyl- deriva- tives. The results obtained are presented in Table 1 together with their statistical significance. It is apparent that mono-ortho and mono- meta substitutions on the phenyl ring are far more effective in en- hancing the tomato flower forming activity of N-phenylphthalamic acid than is para substitution. Furthermore, monomethyl substitution is as effective, or nearly so, as monochloro substitution. The positional effects of the mono-substituted derivatives are even more apparent if the relative activities of the disubstituted derivatives are examined. Thus, A''-2,3-dichlorophenylphthalamic acid reflects the activating ef- fect of both ortho and meta substitution and is the most active deriva- tive examined to date. In contrast to the effect of monomethyl substitution, the 2,3-di- methyl derivative has surprisingly low activity. Similarly, 2,5-disubsti- tution results in a slight depression of activity relative to the mono- substituted derivatives. The relatively low activity of the parach\oro and para-methyl derivatives is enhanced considerably by ortho substi- tution and less so by a meta substituent. In these cases, however, the relative activity of the disubstituted derivative is somewhat less than "Only a single lest of tlie napluliyl derivatives has been conducicd so thai these results should be considered as tentative. «; .2 s u V o a: T3 V en t« OJ u u .s be c o a 3 CO .■a C *^ «! Is bI I Si O U _3 T3 V ■w CO U h CO C O c u o a "o X CM ■ O 00 ■ O O cO 00 CM ■ r-- CO • 00 r-~ ^o r- •^ IT) CV CO ^ c<^ o COS COQ • CNl CN -^ X :eO0 CO O O ' 00 T— c CO 00 CM ^ ■ Lor-ot--oo-*ooocMr^oooocNoo-Ocoio-OLnr-CNOc^iOLor~~ X CM I o I o X I o X CM ovit=~r--co .ooiJ-icocooocMcoo<:>r-oooooor~-ro vjj'frJ^V- .oot--or-ooooooo ■-o\OLnr--sO'^OLn\ooor~-oo-0'0\o ■ lO CO CO O ■^ CO • r~~ r~~ \o o r-- \o CM ■^ CO CO r~- 00 vO 00 ■ o o ■ -co o r-' ■ • o CO • CM CO o to _2 u a, • • ■ CO '.'.'.'.'. • • ■ • t~- '.'.'.'.'.'.'.'.'.'■ I* l' l' l" • I* I I 1 • ■ • ■'iiAAAOOOO ■ • • "SL'SL'SL'SL ■ • • -OOOOOOu'-hUUi 1 ii"S"S"2^ O O O o O O O O 0-y--5-5-= >->->'*-' •!r;ir!-i^, ■ u u u — — — -3.^ — _cjn_C-Cj::jrj3 w u w u «^:s:s2 y y y y a .y -c 'c -c 'c ii jii uU -S •= .s .s IZicMcon-cO'^insO'Tt-u-iLn io^^^lo^cm co ^ co^io^ \o^^_^ CM^CM^CMCm'^co'cO CO^-*^-^ "*^ CM cm CM CO cm' cm" CM co" c o 1) a o C3 c u u -4-* -M 2 C to W h CI. ^ IT) 2 o ^ s o o CO C w ~0 u > u c c C ^ 264 Teubner, Wittwer, and Shen the corresponding mono-o)-^/?o or mono-meta compound. These rela- tionships can also be applied to the trisubstituted iV-phenylphthalamic acids which, in general, exhibit somewhat lower activity than the most active of the related disubstituted forms. It is of particular interest that di-orara-chlorophenoxyacetic acid, a typical auxin, had diameters of 5.4 and 8.3 mm. at 10-^ and IQ-^M, respectively. This activity com- pared favorably with that of N-S-chlorophenylphthalamic acid, but was far less than that of the 2,3-dichloro- and 2,3,5-trichlorophenyl- phthalamic acids. Indole-3-acetic and 2-naphthoxyacetic acids were only one-tenth as active as para-chlorophenoxyacetic acid, and showed an appreciable growth rate only at the highest concentration (lO'^AI) tested. In tomato ovary growth as with flower formation di-or///o substi- tution abolished activity. The low activity of the mono-para deriva- tives in flower formation was also reflected in their failure to promote parthenocarpic fruit development. In the latter case, there was a stimulation of abscission of the young ovaries after 2 days which may have masked any growth promoting effects. Non-treated ovaries did not abscise during the test period of 5 days. The abscission caused by mono-para substitution did not occur with the di-ortho derivatives, although the 3,4-disubstituted chloro- and methyl-compounds showed similar but less pronounced effects than tiie n\ono-para derivatives. Activity in Avena Section Straight Growth The high activity of some of the substituted A'^-phenylphthalamic acids in stimulating fruit development prompted an evaluation of their activities in the Avena section test. Earlier studies conducted primarily with N-phenylphthalamic acid had given variable results s O c V S o V > V 13 u o a V a be "3 u CIS cQ > :S o x; X ■^ s ^ 6 3 a en c S^ C J^ tfl 13 1 in !- . -2 H ■ge U-i ~ ' O t, ^ u . rt CM > 'C > o T3 u U C O c u c o u I o X I o X I o X X I o X CM .=! u Si a c Oh _2 ■S ."2 c ^ • • -00 ■ ■ lOcN • ■ to • • ■•'^ • Thi^ vO . ■CM ■ CMt^ CM CM CMiri ■* ■ 00 CM CM O r- CM 00 OC» + + ■^ • CN Lo r~- LT) CM ■,>.>,>, o J- b o b b -t^-f: -^-c j= J3 -c O — ^ ^^ — ^ ^ >x ?^ >^ +J 4-* ■*-» -♦-> p o o o c _ _ ... K7 I I I I I I I I I I I I I I I I I I I I ^CMcn-^cn-^ino-^inLninoincMcn-^mino-* cM'cNrcM'cM'm'cn en -^[^rf^-^" cM^cM^cvfrn CM cm" cm" c u s- O C o G W 00 t^ T3 - ^ C o u 1; a "o 1 ^ s W -n ^1 C ^ o £; '^ c o en ^ in *" OT! _ C O P3 c rs o c o bo Si - 4-< -^^ s e 266 Teubner, Wittwer, and Shen because of solubility difficulties, loss of activity of solutions on stand- ing, and the possible hydrolysis of the amide liberating the aromatic amine and phthalic acid (3). The measures taken to avoid these diffi- culties are given under Methods. The results presented in Table 3 are those of a single test, but have been confirmed for the chloro-substituted derivatives in at least two other tests. The per cent elongation relative to controls shows essentially the same structure-activity relationships that Avere apparent in both flower formation and fruit parthenocarpy. Mono-para and di- ortlio substitutions either abolish or have no effect on the activity of N-phenylphthalamic acid. The most active compounds were the N- 2,3-dichloro-, A''-2,3,5-trichloro-, and N-3,5-dichlorophenylphthalamic acids. The high activity of the latter was surprising in view of its lower activity in tomato flower formation and ovary development. In addition to the derivatives listed in Table 3, the three (ortho, meta, and para) mono-hydroxy and mono-nitro derivatives were tested and were inactive. The closely related N-1-naphthylphthalamic acid was almost as active as A^-2-chlorophenylphthalamic acid at 10"^ and 10"^ M, giving 115 and 117 per cent elongation, respectively. xV-1-naphthyl- ph thalamic acid did not, however, stimulate the growth of Avena sec- tions at concentrations lower than 10-^Af and retardation (88 per cent of controls) occurred at lO-^M. On the other hand, N^-2-naphthyl- phthalamic acid was one of the more active derivatives tested with 133, 125, and 120 per cent elongation at 10 3, lO*, and 10-^M concen- tration, respectively. It should be noted (Table 3) that the maximum elongation obtained with any of these derivatives was only 129 per cent, while comparable results with indole-3-acetic acid at \Q''M were 147 per cent of the final length of control sections. DISCUSSION The results clearly indicate that modifications of structure, through substitution of chloro- and methyl- groups on the phenyl ring, have precisely the same effects on auxin activity (Tables 2 and 3) as on ac- tivity in tomato flower formation (Table 1). It is not possible, at present, to resolve the question of whether these chemicals function as auxins, or in some other manner, in the control of tomato flower formation. However, it is evident that only those derivatives which have activity in stimulating Avena straight growth and tomato par- thenocarpy are able to increase flower numbers in the tomato. Consideration of the structural relationships of the substituted N- phenylphthalamic acids on the basis of their activity in Avena straight growth (Table 3) indicates requirements for activity distinctly dif- c '-t-> > c u x: ex 3 4-1 u e c rt * o o > to ■Zi c <:: c « e J2 U w ++ 1 CO -orounr^oO'^ — i o o ooocncsocv T-H ^H,— (^^ ,— . 1 "*^" F^>^ fiOa : •*-*- ++ ^/>r r~-cNi-^ooo^^ocNooLnuo-*sD(Nir-~r~-Tt-r-~oo o ooj^o — cocno^oocso^t-o^ooo ^— t -t— XI V LO — _++ ++ ,^ „ „ A (• =M>5 z^fjor^fjon fjCn^^^ CO 1 cNoooOr-irimr^CNOo]-^'— CNfNjoooor^oooOLn u o O^OO — — OOOeN-- — i^^OOOOOOO H w C _o P3 '•^ -!-> C V u c o u 1 ■* so CM rO cTm (N o ^ _^-H--l-+ III! in ^ ri c^ (NsO-OfNOvOOtNl o OOOOtNOOOOC^lOl^O^OOOOOOO u (fl O S 1 Lo o ro m o'f^i cd Subs phth 4iA4 4 6ESSS . 1 1 1 1 pppoooooo-2^-2^>->. >,— t: •=; -5 Phenyl -phenyl 0, »d M — 1 *-^ — ^ ^^ ^^ ^^ *— « _r!'^_Ci-^r*r-'f-'aJUiDflJ h7 I I < Ill II ^ CgCNCNricTirOcO-^-^rf CMCNOim (N (N (N r<^ 1 C 4J , ] O i) u > 111 V D. ■!-» r-- 2 n- 1) o ^ u. < V 0 < ^ o lO ca o = •M c ( 1 c o -t-i CL Ki bn ^-^ C -4-1 O (fl W «>3 c «) lU bf) u c o V V nj ■I-' JZ c n> 1) o < > < q: 0.50 0.25 Mr. 2. The relative distribution of radioactivity in 10 nnn. bean stem sections as a function of duration of exposure to l-C"-2,4-D. Curve 1, 1-hr. exposure. Curve •2. 2 hrs. Curve 3, 4 hrs. Curve 4, 8 hrs. Curve 5, 24 hrs. Cnr\c 6, 72 Ins. [280] -1 < O CO ID u. en \- z O o o o o > O < o o < a: MM Fig. 3. The effect of various treatments on the localization of radioactivity in bean stem sections following exposure to l-C"-2,4-D. Curve 1, 1-hr. exposure. Curve 2, 2-hr. exposure. Curve 3, 4-hr. exposure. Curve 4, 2-hr. exposure to unlabeled 2,4-D followed by 2-hr. exposure to radioactive 2,4-D- Curve 5, 2-hr. exposure to radio- active 2,4-D followed by 2 hrs., with no additions. Curve 6, 2-hr. exposure to radio- active 2,4-D followed by 2-hr. exposure to unlabeled 2,4-D. [281 ] 282 M. K. Bach and J. Fellig > > O < UJ > < UJ HUU i 1 1 1 ; 1 1 1 300 - - r -1 - 200 - - .100 - - n - J. 1 1 - 6.5 HOURS Fig. 4. The eflctt of polarity on the locaHzation of radioaclivily in bean stems fol- lowing exposure to l-C"-2,4-D. The relative radioactivities 5 mm. from the point of application are compared. Open bars, planted basal end down. Closed bars, planted apical end down. to 3 mm. from the point of application, even in the presence of the unlabeled 2,4-D- This suggests that the dilution of label in this area was very gradual, perhaps because the radioactive material was bound in some way to the cellular material. Figure 4 shows the effect of polarity on the transport of radioactive material through the stem sections by comparing the relative radioactivities 5 mm. from the point of application of l-Ci'*-2,4-D. The results were factored as in Figiue 2. In contrast to the absence of polarity effects on the total up- take of 2,4-D, the translocation of radioactivity in the sections was clearly faster in the apex to base direction. uptake and Fate of C'-Iabeled 2,4-D in Bean Stems 283 The Fate of Radioactive 2,4-D in the Stem Sections As has been reported by Fang et al. (11), 2,4-D is relatively im- mune to oxidative attack by plant tissues. Accordingly, less than 1 per cent of the radioactivity which was taken up by the stems could be recovered as COo in a KOH trap. Thus the activity which is lost from the stems must be presumed to be liberated into the agar me- dium at the basal end of the stems. However, as would be expected from the results shown in Figures 2 and 3, it proved very difficult to demonstrate a diffusion of radioactivity from the stems into the agar. The gradient established in the stems results in very little radioac- tivity near their basal ends. Undoubtedly the concentration in the agar immediately adjacent to the stems cannot exceed the concen- tration in the stems at that point. For technical reasons it has not proved possible to concentrate the radioactivity from sufficiently large volumes of agar to get significant counts, or to identify the com- pounds involved. Experiments which are designed to establish a com- plete balance sheet for the radioactivity applied to the stems are now in progress. In agreement with the results of Jaworski et al. (18) , and of Butts and Fang (9) , who used whole plants, we found that the stem sections converted 2,4-D to at least three compounds resolvable by paper chro- matography. One of these, having an Rf of 0.5 in the solvent of Ja- worski et al. (18), was formed in the largest amount, and, after 3 days' incubation, comprised up to 60 per cent of the total radio- activity in the extracts. Figure 5 shows the distribution of radioactivity .45 .50 .60 .70 .80 Rj UNITS Fig. 5. The resolution of the radioactive products formed from l-C"-2,4-D in beans by paper chromatography. 284 M. K. Bach and J. Fcllig on a typical chromatogram. The compound having an Rf of 0.5 was eluted and rechromatographed. A single radioactive peak with the same Rf was found. As was reported by Jaworski and Butts (17), refluxing in 2N HCl for 2 hrs. liberated a compound having an Rj similar to that of 2,4-D in the phenol-water and butanol (17) solvent systems. Addition of l-Ci»-2,4-D during the isolation procedure did not give rise to any radioactive peaks on the chromatograms other than that of 2,4-D itself. It must be concluded, therefore, that this material is a compound formed from 2,4-D by the plants. The sug- gestions that this compound is a glycoside (17) or a peptide (9) of 2,4-D must be viewed with some doubt, particularly since the band of Rf 0.5 on the chromatograms does contain carbohydrate as well as ninhydrin positive material. These ninhydrin and carbohydrate con- taining bands do not, however, coincide with the radioactive band on rechromatography. The Biological Activity of the Product Formed From 2,4-D In order to define the time during which the initiation of pro- liferation takes place, it was important to establish whether the compound of Rf 0.5 which is formed from 2,4-D by the bean stem sections has the capacity to induce proliferation, or, indeed, possesses any other biological activity. Table 2 summarizes the results obtained when lO^M concentrations of this material (as calculated from ra- dioactivity) were applied to bean stem sections. The material used had been obtained by mass isolation using the paper chromatography technique described above, and thus contained several nonradio- active contaminants of unknown concentration. Upon rechroma- tography it showed only one radioactive spot (Rf 0.5). It will be seen that this material was completely inactive in causing the proliferation of the bean stems, even though it was applied at ten times the equiva- lent concentration which would be necessary for the maximum ef- Table 2. The activity of a crude prepara- tion of the main radioactive product formed by beans from carboxyl-C'''-2,4-D in the bean stem proliferation assay. Sample Gain in Fresh Weight, Per Cent Control 24 ± 6 lO-" A/ 2,4-D 166 ± 14 10-'A/Rr0.5 band 14 ± 7 uptake and Fate of C^'>-laheled 2,4-D in Bean Stems 285 fectiveness of 2,4-D. Furthermore, the material appeared to be slightly inhibitory both in the bean stem sections and in the Avena internode test (23). In bean stems no evidence of callus formation at the apical cut surface could be found in the sections which had been exposed to this preparation, while the control stems always show some swelling at this point. In the Avena test a concentration of 3 X lO'^M of this material was slightly inhibitory to growth, while lower concentrations were totally inactive. The maximal activity of 2,4-D in the Avena assay is found between lO*' and 10-^M. It appears likely, therefore, that this material is a detoxification product of 2,4-D and does not have the biological properties of the auxin itself. This conclusion is also supported by the findings of Fang et ah (10) that 3-(/7-chloro- phenyl)-l,l-dimethylurea (CMU) is converted to a similar inactive product in beans and by those of Hay and Thimann (13) on the fate of 2,4-D in vivo. The findings of Andreae and Good (1) that 2,4-D is not detoxified by the formation of the aspartyl derivative nearly as readily as are other herbicides, seem to be contradictory to these find- ings. However, in the absence of any reliable information on the na- ture of this material, and in view of the large differences of experi- mental conditions employed, the two findings may not be incom- patible. SUMMARY Using an in vitro incubation system it was shown that exposure to 10-'*M 2,4-D for 4 hrs. is sufficient to cause the initiation of ex- tensive callus formation in bean stem sections. The uptake of 2,4-D during this period is dependent on the concentration of 2,4-D sup- plied and on the area exposed. Further, no clear-cut difference be- tween the various internodes of young bean plants could be demon- strated, nor could a difference in uptake from the apical or basal end of the stems be shown. After apical application of l-Ci^-2,4-D to the stems, the peak of radioactivity was a few mm. below the point of application and traveled down with time. The width of the peak increased markedly at longer exposures. Apical application of unlabeled 2,4-D accelerated the disappearance of radioactivity from stems which had previously been exposed to Ci^-2,4-D, although the location of the peak of radio- activity remained essentially unchanged. Decarboxylation and release of C"Oo appear to be a minor path in the metabolism of 2,4-D by the bean stems. Most of the radioactivity was recovered in the form of a complex of unknown structure which yielded 2,4-D upon acid hydrolysis. 286 M. K. Bach and J. Fellig ACKNOWLEDGMENTS The authors wish to thank Mr. Waker J. Skraba and Mr. Donald A. Salisbury for the preparation of the radioactive 2,4-D, and Dr. Donald G. Crosby and Mr. Robert V. Berthold for the Avena inter- node assays. The devoted and competent assistance of Miss Mary Jane Persohn is gratefully acknowledged. LITERATURE CITED 1. Andreae, W. A., and Good, N. E. Studies on 3-indoleacetic acid metabolism. IV. Conjugation with aspartic acid and ammonia as processes in the metabo- lism of carboxylic acids. Plant Physiol. 32: 566-572. 1957. 2. Bcal, J. M. Effect of indoleacetic acid on thin sections and detached segments of the second inteinode of the bean. Bot. Gaz. 102: 366-372. 1940. 3. . Histological responses to growth-regulating substances. In: F. Skoog, (ed.) Plant Growth Substances, pp. 155-166. Univ. \Vis. Press, Madison, Wis. 1951. 4. Berger, J., Smith, P., and Avery, G. S., Jr. The influence of auxin on respira- tion of the Avena colcoptile. Amer. Jour. Bot. 33: 601-604. 1946. 5. Bergmann, F. H., Towne, J. C.. and Burris, R. H. Assimilation of carbon dioxide by hydrogen bacteria. Jour. Biol. Chem. 230: 13-24. 1958. 6. Bloch, R. Abnormal plant growth. Brookhaven Symp. Biol. 6: 41-54. Brook- haven Nat. Lab., New York. 1954. 7. Bonner, J. Plant Biochemistry. 537 pp. Academic Press, Inc., New Voik. 1950. 8. Bryson, J. L., and Mitchell, T. J. Improved spraying reagents for the detection of sugars on paper chromatograms. Nature. 167: 864. 1951. 9. Butts, J. S., and Fang, S. C. Tracer studies on the mechanism of action of hormonal herbicides. U. S. Atomic Energy Comm. TID-7512: 209-214. 1956. 10. Fang, S. C., Freed, V. H., Johnson, R. H., and Coffee, D. R. Absorption, trans- location, and metabolism of radioactive 3-(p-chlorophenyl)-l,l-dimethylurea (CMU) by bean plants. Jour. Agi". Food Chem. 3: 400-402. 1955. 11. , Jaworski, E. G., Logan, A. V., Freed. V. H., and Butts, J. S. The ab- sorption of radioactive 2,4-dichlorophenoxyacetic acid and the translocation of C» by bean plants. Arch. Biochem. Biophys. 32: 249-255. 1951. 12. Gall, H. J. F. Some effects of 2,4-dichlorophenoxyacetic acid on starch diges- tion and reducing activity in bean tissue cultures. Bot. Gaz. 110: 319-323. 1948. 13. Hay, J. R., and Thimann, K. V. Fate of 2,4-dichlorophenoxyacctic acid in bean seedlings. II. Translocation. Plant Physiol. 31: 446-451. 1950. 14. Hoagland, D. R., and Snyder, W. C. Nutrition of strawberry plant under con- trolled conditions: (a) effects of deficiencies of boron and certain other ele- ments: (b) susceptibiHty to injury from sodium salts. Proc. Amer. Soc. Hon. Sci. 30: 288-294. 1933. 15. Humphreys, T. E., and Duggcr, W . M.. Jr. The effect of 2,4-dichlorophenoxy. acetic acid on the respiration of etiolated pea seedlings. Plant Phvsiol. 32: 530-536. 1957. 16. , and Dugger, \V. M., Jr. Effect of 2,4-dichloropiicnoxyatctic acid and 2,4- dinitroplicnol on the uptake and metabolism of exogenous substrates by corn roots. Plant Physiol. 34: 112-116. 1959. 17. Jaworski, E. G., and Butts, J. S. Studies in plant metabolism. II. The metabo- lism of C'^-labcled 2,4-dithlorophenoxyacetic acid in bean plants. Arch. Biochem. Biophys. 38: 207-218. 1952. uptake and Fate of C"<-laheled 2,4-D in Bean Stems 287 18. , Fang, S. C, and Freed, V. H. Studies in plant metabolism. V. The metabolism of radioactive 2,4-D in etiolated bean plants. Plant Physiol. 30: 272-275. 1955. 19. Klein, R. M., and Manos, G. The use of metal chelates for plant tissue cul- ture. Ann. N. Y. Acad. Sci. 88: 416-125. 1960. 20. Kraus, E. J., Brown, N. A., and Hamner, K. C. Histological reactions of bean plants to indoleacetic acid. Bot. Gaz. 98: 370-420. 1936. 21. Laibach, F., and Fischnich, O. Ober eine Testmethode zur Priifung der kal- lusbildenden Wirkung von Wuchsstoffpasten. Ber. Deutsch Bot. Ges. 53: 469-477. 1935. 22. Muzik, T. J., and Cruzado, H. J. Differentiation of bean internode segments in tissue culture with added indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and maleic hydrazide. Bot. Gaz. 120: 57-59. 1958. 23. Xitsch, J. P. Methods for the investigation of natural auxins and growth in- hibitors. In: R. L. Wain and F. Wightman (eds.). The Chemistry and Mode of Action of Plant Growth Substances, pp. 3-31. Butterworth Sci. Publ. Lon- don. 1956. 24. Reinhold, L. Release of ammonia by plant tissues treated with indole-3-acetic acid. Nature. 182: 1022, 1023. 1958. 25. Vlitos, A. J., and Meudt, W. The role of auxin in plant flowering. 1. A quan- titative method based on paper chromatography for the determination of in- dole compounds and of 3-indoleacetic acid in j^lant tissues. Contr. Boyce Thompson Inst. 17: 197-202. 1953. 26. Weintraub, R. L., Reinhart, J. H., ScherfE, R. A., and Schisler, L. C. Metabo- lism of 2,4-dichlorophenoxyacetic acid. III. Metabolism and persistence in dormant plant tissue. Plant Physiol. 29: 303, 304. 1954. 27. White, P. R. The Cultivation of Animal and Plant Cells. 239 pp. Ronald Press, Inc., New York. 1954. V. H. FREED Oregon State College F. J. REITHEL University of Oregon L. F. REMMERT Oregon State College Some Physical-Chemical Aspects of Synthetic Auxins With Respect to Their Mode of Action It has become very apparent that the mechanism of action of synthetic plant growth regulators and the relation of their chemical structure to this activity is inextricably interwoven. Many attempts have been made to relate structure of the compound to activity, or to afford an explanation of the mechanism of action. Either case involved the necessity of considering the other facet of these interrelated prob- lems. Thus, in attempting to explain why the varying of the organic structure of a synthetic plant growth regulator modifies its activity, it became necessary to invoke a mechanism of action compatible with the observations. Most of the work attempting to relate structure of synthetic growth substances to their plant growth regulating activity has been from the standpoint of the organic chemistry of these molecules (1, 9, 13, 17, 26) . It has been found that the number and kind of atoms in the molecule as well as their arrangement in relation to each other are of prime importance in determining whether or not the molecular spe- cies will be active. However, it must be remembered that the physical properties and geometry of the molecule are related to activity also. This paper is concerned with an attempt to relate some physical chemical aspects of synthetic plant growth substances to the biological action of these chemicals. * Taken in part from a doctoral dissertation submitted to the Graduate School, University of Oregon, by senior author. Also Technical Paper No. 1263, Oregon Agricultural Experiment Station. [289] 290 Freed, Reithel, and Remmert Shortly after the isolation and identification of indole-3-acetic acid, it was discovered that certain synthetic acids possessed the ability to produce responses from plants similar to that of lAA. Koepfli et al. (17) examined the activity of a number of indole derivatives, and this was followed in turn by the discovery of the activity of 1-naphthalene- acetic acid (36) and the phenoxyacetic acids (37, 38) as well as the biological activity of a number of related substances (33, 38). It was only natural that in the course of such studies attempts would be made to relate the structure of these molecules to their activity and to speculate as to their possible mode of action. It is nearly a requirement that any proposed theory of the relationship of structure to growth regulating activity must be premised on a mechanism of action conso- nant with the molecular structures involved (14). A number of interesting theories concerning the mode of action of these substances and the significance of molecular structure relation- ships to activity have been proposed (3,5,9,11,18,19,22.23). Most of the theories, wherein the organic structure of molecules was con- sidered, propose a mechanism of action involving a chemical reaction between an enzyme molecule and the growth regulator (10,11,18). It would probably be naive to assume that the action of these chemi- cals could come about in any way than through their effect on the enzymes of the organism. On the other hand it becomes a more diffi- cult task to obtain conclusive evidence as to the mechanism by which a growth regulator interacts with an enzyme. Various mechanisms have been proposed, ranging from the suggestion that a growth regu- lator serves as a prosthetic group involving a two-point attachment to the enzyme surface (10, 11), and that the growth regulators are chelat- ing agents for essential metals (15). The interaction of growth regulating chemicals with the enzymes of the protoplasm by physical rather than chemical forces has been considered in the theory of Veldstra (30) and the work of Brian and Rideal (8). Some support for this theory comes from the detailed con- sideration of dosage-response curves obtained with growth regulating chemicals. Northern's observation of a reduced viscosity of the cyto- plasm upon treatment with a growth regulator was explained on tlie basis of disassociation of the proteins of the cytoplasm (24). This ob- servation would be consonant with the theory of adsorption on the surface of select proteins, weakening the intermolecular bonds and resulting in a loss of viscosity of the gel of the cytoplasm. The in- creased streaming of cytoplasm noted upon treatment witli plant growth regulators would also lend to bear this out (33). Consideration of possible physical interaction of the growth regu- lating phenoxyacetic acid with proteins of the organism came about Physical-Chemical Aspects of Synthetic Auxins 291 through the reports indicating laikue to demonstrate the competitive effects ot 2,4-D on enzymes (27, 28). While many enzymes have been shown to be affected by the presence of this chemical, there seems to be a lack of evidence of a clear-cut competitive effect of sufficient order of magnitude to account for the rather magnificent biological re- sponses induced by the chemical. BIOLOGICAL ACTIVITY OF CHLOROPHENOXYACETIC ACIDS In order to evaluate any physical differences that may be signifi- cant in the relation of structure to activity and mode of action of the chlorophenoxyacetic acids, it was necessary to assess and rate the biological activity of members of this series. Such rating was done by means of the root growth test using corn and lupine. The results of this rating are to be found in Table 1. The values given in Table 1 are the molar activity in relation to 2,4-D which was taken as 100. These values are in general accord with many published values given in the literature. It is noted in this table that there is a very low order of activity for those compounds substituted in the 2 and 6 position, which ob- servation was reported by Muir and Hansch (22), Leaper and Bishop (18), and Osborne et al. (26). It was suggested by Muir et al. (23) that the 2,6-disubstituted compounds were relatively inactive because of the requirement for free ortlw position with which to react with a sub- strate molecule. Although it has been suggested several times that the phenoxyacetic acids probably reacted with the protein to form a new chemical species, the failure to find such a species by means of C^^- labeled 2,4-D or a specific effect with any of the intermediate Krebs cycle substrates (27) would appear to argue against specific chemical Table 1. The percentage molar activity of some phenoxyacetic acids as measured in root- growth tests with corn and lupine. Substituted Phenoxyacetic Acid Molar Activity, Per Cent of 2,4-D Parent Compound .... 2-Chloro- 0.05 9.4 3-Chloro- 15.0 4-Chloro- 53.0 2,4-Dichloro- 2,6-Dichloro- 3 4-Dichloro- 100.0 11.0 78.8 3,5-Dichloro- 2,4,5-Trichloro- 2,4,6-Trichloro- 2,3,5-Trichloro- 32.7 98.5 35.0 21.2 292 Freed, Reithel, and Remmert reactions. The suggestion of Veldstra (30) that the compounds may be acting at a protein-lipoid surface leads to speculation that the action of these compounds may be physical in nature. If the action of these compounds is physical, then there should be some measurement which ^^•ould show obvious differences in the physical properties of the biologically active and the biologically inactive members of the series. The work of \\'right (35) in correlating the biological activity of insect repellents to their infrared absorption spectra suggested that the infrared spectra of the chlorophenoxyacetic acids may reveal some relation to activity. INFRARED AND ULTRAVIOLET SPECTRA OF CHLOROPHENOXYACETIC ACIDS A series of chlorinated phenoxyacetic acids was examined quali- tatively for infrared absorption, using 1 mg. of chemical to 300 mg. of potassium bromide in a pellet form. The spectra ^vere run from about 2 fx out to 14 /x (Figure 1). The most obvious difference noted between an active (2,4-D) and a relatively inactive compound (2,6-D) was to be found in the absorption in the region of 12 ju, (825 cm.-^). The major difference noted in this region is that the biologically active compound had an intense absorption band at 794 cm.-^, whereas the inactive compound had only a moderately strong absorption in this region, characterized by a missing Q branch. This fact is illustrated by 1 r 2,4-Dichlorophenoxyocetic ocid 2,6-Dichlorophenoxyocetic ocid J L 14 12 10 8 6 4 2 MICRONS Fig. 1. Infrared absorption spectra of 2,4- and '^.G-dicliloioplicnDwacclic aciil Physical-Chemical Aspects of Synthetic Auxins 293 Table 2. Infrared absorption of some phenoxyacetic acids and other growth sub- stances. Acid Relative Absorption in 850 to 789 Cm.-' Region * Presence or Absence of Q Branch M W M S s M M S w s w M M S 3-Chlorophenoxyacetic 4-Chlorophenoxyacetic 2,4-Dichlorophenoxyacetic 2,5-Dichlorophenoxyacetic 2,6-Dichlorophenoxyacetic 3,4-Dichlorophenoxyacetic 3,5-Dichlorophenoxyacetic 9 4 5-Dirhinrnnhenoxvacetic + + + + + -f 9 4 6-Dirhloronhenoxvacetic 9 4-DihrniTionhenoxvacetic + TnHolp-3-acetic + 1 -TVanhthaleneacetic + * S = strong; M = medium; W = weak absorption. the absorption spectra for 2,4-D and 2,6-D. A series of such compounds was examined for infrared absorption in this region. The spectra were assigned a code number and were then examined and sorted into two groups, one being those thought to have appreciable biological activity and those having moderate or low biological activity. Follow- ing the sorting, the spectra were decoded and the absorption spectra and biological activity were found to give a positive correlation of 0.80. A summary of the absorption characteristics of several of the chlorophenoxyacetic acids, 1 -naphthalene acetic acid, and indole-3- acetic acid is given in Table 2. The infrared absorption of substituted aromatic compounds in the 800 cm.-i region has been suggested by Bellamy (2) as being due to the in-plane, out-of-plane vibrations of the nuclear substituents. If this is indeed the case, the absorption spectra noted here might sug- gest that the biologically active compounds have a uniformly coupled plus-minus movement of the nuclear substituents, where the com- pounds of lesser biological activity have an alternate vibration pat- tern. In the former case, this behavior would make for a greater pos- sibility of interaction of the ring with a hydrocarbon chain such as forms the skeleton of a protein molecule. The apparent success in relating biological activity to infrared ab- sorption spectra of the chlorophenoxyacetic acids raised a question as to whether there may be a similar relation between the electronic configuration of these molecules as indicated by their ultraviolet ab- 294 Freed, Reithel, and Remmert Table 3. Ultraviolet absorption and water solubility of some phenoxyacetic acids. Substituted Phenoxyacetic Acid m/i Em X 10-3 H2O Solubility Moles L Parent Compound 2-Chloro- 269 273 273 279 283 279 278 282 284 289 287 1.4 1.7 1.5 1.3 1.9 2.2 0.31 1.6 1.4 1.2 0.67 7.2 X 10-3 3-Chloro- 11.6 X 10-3 4-Chloro- 4.3 X 10-3 2,4-Dichloro- 2.7 X 10-3 2,5-Dichloro- 2.4 X 10-3 2,6-Dichloro- 7.2 X 10-3 3,4-Dichloro- 2.3 X 10-3 3,5-Dichloro- 5.5 X 10-3 2,4,5-Trichloro- 9.9 X 10-4 2,4,6-Trichloro- 6.2 X 10-4 sorption and their activity. Accordingly, the ultraviolet absorption spectra of a number of these compounds were determined with a Gary recording spectrophotometer and the molar extinction coefficient in the neighborhood of 280 fx calculated. The results of these determina- tions are given in Table 3. The results in Table 3 are not as unequivo- cal as was the case with the infrared absorption. It will be noted in the monosubstituted compounds that the molar extinction coefficient is actually less for the compound of greatest activity. However, the relationship between absorption spectra and biological activity holds very well for the di- and tri- substituted isomers studied. However, when these ultraviolet absorption data are studied in relation to the solubility of the compounds, a slightly different picture emerges. Thus, the molar solubility of the 2- and 3-chlorophenoxyacetic acids is ap- proximately twice and three times that of the p-chlorophenoxyacetic acid, being 7.2 X 10-=^Af, 11.6 X lO-^M, and 4.3 X lO-^M, respectively, for these compounds. In light of the relationship of water solubility to biological activity, it is reasonable to suppose that a higher molar dosage of the more soluble compounds would be required to achieve the same degree of biological activity, all other factors being equal (12) . Table 4 represents an attempt to summarize the relation of the physical properties to biological activity. It will be noted that a negative value for any of the properties is associated with a compound of low activity. Thus, the importance of these properties to activity is clearly demonstrated. ACTION OF 2,4-D ON ROOTS AND MITOCHONDRIA If, as previously suggested, the primary event in ihc action of these synthetic growth regulators is adsorption, it should be possible to demonstrate a ready reversibility of the action of these substances (9). Tims, it would seem that removal of a plant or an organism from a short exposure to the chemical should result in a recovery from Physical-Clieniical Aspects of Synthetic Auxins 295 Table 4. The relationship of biological activity to absorption spectra and water solubility of phenoxyacetic acids. HoO Solubility Relative >6 X lO-iand IR Absorption Substituted Biological <5 X 10-3 With UV Em 280 Phenoxyacetic Acid Activity Moles/L Q Present 1.2 X 10'^ Parent Compound. . .05 — — + 2-Chloro- 9.4 — + 3-Chloro- 15.0 — + + 4-Chloro- 53.0 + + + 2,4-DichIoro- 100.0 + + + 2,5-Dichloro- 89.0 + + + 2,6-Dichloro- 11.0 — — — 3,4-Dichloro- 78.8 + + + 3,5-Dichloro- 32.7 — — + 2,4,5-Trichloro- .... 98.5 + + + 2,4,6-Trichloro- .... 21.2 " the effects of tfie chemical. The exposure period would have to be of sufficiently long duration to assure that adsorption was not a limiting factor; however, this was demonstrated by Blackman (7) and Osborne (25) to be a relatively short period. Such an experiment was per- formed with corn seedlings, exposing them to a concentration of 6.7 X lO-'^M of chemical per seedling for varying lengths of time up to 48 hrs. Removal of the seedling from an exposure up to 24 hrs. resulted in a complete recovery, indicating that up to this period of time the action of the chemical was reversible. Measurement of the response was made 48 hrs. after the beginning of the exposure. A more definitive experiment of this type would be an exposure of subcellular particles from the plant to a chemical such as 2,4-D, removal from exposiue, washing of the particles, and a measurement of enzymatic activity. Mitochondria would seem to be the fraction of choice since isolation of these particulate fractions from plants and measurement of their activity is now a well-established practice. Mi- tochondria were isolated from cabbage in the usual manner, one ali- quot of the preparation being held as a control; another incubated with 2,4-D, followed by washing with the suspending media to remove excess 2,4-D; a third aliquot was washed with the suspending medium to serve as the washed control. Previous experience had demonstrated that a 10 min. exposure to 2,4-D resulted in equally good inhibition as longer exposures. In order to assme complete saturation of the mitochondria with 2,4-D, they were incubated for 30 min. before re- moving by centrifugation and washing. Samples of the control, the washed control, and the 2,4-D treated mitochondria were tested for enzymatic activity in a Warburg apparatus. To aliquots of the latter 296 Freed, Reithel, and Remmert Table 5. Washing as a factor in the reversal of 2,4-D inhibi- tion of oxygen uptake by mitochondria. O, Uptake, Ail/Hr Per Cent Treatment No 2,4-D + 2,4-D Inhibition Control 533 2,4-D washed 515 188 64 Washed control 468 192 58 two 2,4-D was added to assure that the activity was still 2,4-D sensi- tive. The results of this study are to be found in Table 5. It will be noted that exposure of the mitochondria to 2,4-D fol- lowed by washing to remove the chemical, resulted in a complete re- covery of the activity as measured by oxygen uptake. However, addi- tion of 2,4-D to the Warburg vessel demonstrated that the mechanism responsible for oxygen uptake was still 2,4-D sensitive. A similar situa- tion was to be found with the washed control. It should be noted in passing, however, that long exposure of the mitochondria to 2,4-D, 40 min. or more, resulted in an irreversible loss of their oxidative capacity. The behavior noted with these mitochondria strongly supports the theory that the primary event in the action of these chemicals is adsorption. It is obvious that had this chemical reacted with con- stituents of the mitochondria to form a chemical complex or com- pound, simple washing would not have removed a sufficient amount of the chemical to restore the full oxidative capacity of the mitochon- dria. On the other hand, if this chemical were simply adsorbed by physical forces on the surface of the enzyme, one would expect that washing would remove substantially all of the 2,4-D, thus permitting recovery of the oxidative ability. This apparent adsorption on the surface of protein also affords a possible explanation of why 2,4-D gives a rather general nonspecific inhibition of oxygen uptake of mitochondria using different members of the Krebs cycle intermedi- ates. In seeking further evidence in support of the theory that 2,4-D is adsorbed by proteins, a study of 2,4-D adsorption by proteins was made by equilibrium dialysis. This study clearly demonstrated the adsorption involving four molecules of chemical per molecide of protein. One of the characteristics of an adsorbing system is tiiat less of the solute species will be adsorbed by a surface as temperature in- creases (9). Therefore, it should be possible to ascertain whether or Physical-Chemical Aspects of Synthetic Auxins Table 6. Effect of temperature on the 2,4-D inhibition of oxygen uptake by mitochondria. 297 O, Uptake, fil/Hr 25° C. 30° C. VTitnrhondria 1068 ± 45 285 ± 6 1078 ± 72 \4itnrhnndria. + 2,4-D, 4.24 X lO-Mf 394 ± 5 Per rent inhibition 73 63.4 not the chemical was being adsorbed by the protein of the mitochon- dria by measuring the oxygen uptake at two different temperatures. If the 2,4-D is adsorbed, one would expect less inhibition of oxygen up- take at a higher temperature. This proposition was tested by using a mitochondrial preparation from cabbage, one aliquot of which was run at 25^^ C, the other at 30° C. In each case a control was com- pared with 2,4-D treated samples. The results of this study are re- ported in Table 6. The data in Table 6, which represents an average of triplicate runs, clearly demonstrate that the rate of inhibition decreases with temperature. It is interesting to note that the decrease in activity, which is 13.7 per cent, is in good agreement with the increase in water solubility of 2,4-D over this same temperature range (18.5 per cent). The increase in the solubility again would make for decreased adsorption by virtue of the change of chemical potential of the solute species. These findings afford further support of the theory that the primary event involved in the action of growth regulators is one of adsorption. ACTION OF 2,4-D ON CRYSTALLINE ENZYMES Since it would appear from the foregoing data that physical ad- sorption of the chemical by an enzyme surface is involved in the biological action of these chemicals, the question arises as to the consequence to the protein of this adsorption. While many of the more common cases of enzyme inhibition come about by the chemi- cal reacting with a particular functional group of the protein or competing with the substrate for a specific site, cases of inhibition by adsorption are less well known. While the adsorption probably occurs at sites adaptive to the structure of the compound and is therefore specific, these sites are not those occupied by the substrate. This is borne out by the noncompetitive nature of the inhibition of 298 Freed, Reitlicl, and Rcjninert peroxidase found in this laboratory. It would appear rather that there may be one of two alternative consequences of this adsorption: (a) the adsorption restricts energy transfer to the protein molecule, thus modi- fying the rate of reaction, or (b) the adsorption of the solute species results in a modification of the structure of the protein such that the kinetics of the reaction it catalyzes is changed (29, 32). It has been suggested that inasmuch as many proteins contain flu- orescent centers, the fluorescence intensity of such a species might cor- relate with enzymatic activity. Indeed such has been shown to be the case (21, 31). It has been suggested by Karreman et al. (16) that the fluorescent emission is the mechanism by which energy may be trans- ferred from an enzyme to its substrate molecule. It would appear, therefore, that if 2,4-D reduced the fluorescence intensity of the en- zyme, this would indicate interference with energy transfer. Also, if the enzymatic activity of the protein were affected by the concentra- tion of 2,4-D, which reduces fluorescence intensity, this would indi- cate that adsorption interferes with energy transfer. Upon testing this theory with glyceraldehyde phosphate dehydrogenase and a-amylase, no discernible effect on enzymatic activity was found at a concentra- tion of 2,4-D at which fluorescence intensity was markedly reduced. This measurement was made using the Aminco-Bowman spectrophoto- fluorometer. Inasmuch as reduction in fluorescence intensity did not correlate with the change in enzymatic activity, it was felt that the effect of the chemical in modifying the enzymatic activity of the protein Avas not due to restriction or modification of energy transfer within the pro- tein. From these considerations the assumption was made that the chemical exerts its influence by modification of the structure of the enzyme molecule. It is a well-known phenomenon that the modifica- tion of a catalyst's surface materially changes the property of the sm- face as a catalyst. In order to study this problem, attention was then turned to finding an enzyme, the activity of which was known to be a function of the structinal integrity of the protein molecule. Peroxi- dase (horse-radish) has been cited as an example of a protein capable of undergoing reversible denaturation with heat. Thus, the degree of reversible denaturation of peroxidase may be followed by measure- ment of its enzymatic activity as a function of temperature. It was reasoned that if the adsorbed 2,4-D brought about a change in the structure of the molecule, this should facilitate the heat denaturation of peroxidase. Accordingly a study of the rate of change of horse- radish peroxidase activity with increasing temj>crature witli and with- out 2,4-D was followed. The results of this study are shown in Figure 2. Physical-Chemical Aspects of Synthetic Auxins 299 0.25 H 0.05- O < 25 35 45 TEMPERATURE "0 55 Fig. 2. The effect of 2,4-D on the enzymatic activity of peroxidase as a function of temperature. It is immediately apparent from this graph that not only has 2,4-D accelerated the loss of activity of the peroxidase, but also the optimal activity of the peroxidase has been shifted from 45° C. down to 35° C. in the presence of 2,4-D. These data lend support to the assumption that the mechanism of action of 2,4-D in modifying en- zymatic activity of a protein is a result of the change of structure of the enzyme molecule. Consideration of these data raises the speculation as to how 2,4-D and other synthetic auxins may at one concentration cause growth stimulation and at higher concentrations inhibition. Reports in the literature, indicating that low concentrations of 2,4-D or other syn- thetic growth regulators may stimulate the activity of a certain en- zyme and at higher concentrations cause inhibition of this enzyme, reinforce interest in this speculation. It is postulated that the en- zymatic stimulation associated with low concentrations of 2,4-D could come about through a slight reversible modification of the enzyme structure which would make the enzyme a more efficient catalyst, but that as additional molecules of 2,4-D are added to this surface the modification of the structure becomes increasingly severe with a con- sequent loss of catalytic property. In order to ascertain whether or not 2,4-D is capable of both stimulation and inhibition of an enzyme by variation in concentra- 300 Freed, Reithel, and Remmert tion, a number of crystalline enzymes were tested for activity in vary- ing concentrations of 2,4-D. In the case of glyceraldehyde-3-phosphate dehydrogenase, a marked stimulation in activity was obtained with a concentration of about 100 p. p.m. of 2,4-D, and a very marked inhi- bition of enzymatic activity was noted at 1,000 p. p.m. of 2,4-D. In the case of glucose-6-phosphate dehydrogenase, a 40 p.p.m. 2,4-D solu- tion resulted in a 22 per cent increase in activity, whereas a 1,000 p.p.m. concentration resulted in an appreciable loss of enzymatic ac- tivity. A similar situation was found to prevail with isocitric dehy- drogenase. Little stimulation was found with the peroxidase, but in- hibition by 2,4-D was appreciable above 500 p.p.m. \Vhile these con- centrations of chemical appear quite high in considering the small amounts of 2,4-D required to bring about an auxin-like effect, it should be remembered that the media in which the enzymatic assays are made are not those of physiological conditions. Thus, in general the pH tends to be higher than would be found in the cell, reducing the efficiency of the 2,4-D because of increased ionization. SUMMARY The mechanism of action of a synthetic growth substance and the structural relations to activity appear to be two very closelv inter- related problems. Seemingly, the solution to one affords at least some indication to the solution of the other. The data presented in this pa- per would appear to indicate that the primary event in the mechanism of action of tiie chlorophenoxyacetic acids is that of adsorption on a protein surface, and that as a consequence of this adsorption, the structure of the protein is modified with a consequent change in its enzymatic activity. This theory of the molecular level mechanism of action of the synthetic growth substances affords an explanation of how the same molecule may both stimulate growth at a lo^v concen- tration and bring about inhibition at a higher concentration. The lines of evidence on which this postulate is based are as follows: (1) The finding that the growth regulators can reduce the vis- cosity of cytoplasm and can stimulate streaming of the cyto- plasm. (2) The surface activity and behavior of growth substances as shown by Veldstra (30), Brian and Rideal (8), and Linser (19). (3) The finding of Marinos (20) that exposure at high concentra- tions of growth substances results in the shrinkage of the cy- toplasm and a leakage of the cell constituents. (4) Examination of the ultraviolet and infrared absorption spectra suggests that the geometry of the molecule is important and that interpretation of these data suggests a plane surface per- Physical-Chemical Aspects of Synthetic Auxins 301 mitting the establishment of dipole or Van der Waal forces where adsorption may be involved. (5) Water solubility appears to provide a natural limit to the behavior of these compounds. The demonstration of the ad- sorption of these compounds by proteins and by mitochondria. (6) The change in structtire of the peroxidase molecule. These observations suggest that adsorption and consequent modi- fication of protein structure may be the basic action of plant growth regulating phenoxyacetic acids. It may be inferred that not all en- zymes or proteins are going to adsorb the chemical with equal facility and, therefore, there will be a marked difference among enzymes as to the response to the chemical. Likewise, the same enzyme derived from different sources would quite likely have different affinities for the chemical and, therefore, would show a different response in terms of change of enzymatic activity. This in part may help to account for the selectivity of these chemicals. ACKNOWLEDGMENTS The authors wish to acknowledge their indebtedness to F. J. Wit- mer for assistance in obtaining the infrared spectra and to R. E. Hughes for assistance on certain of the enzyme assays. LITERATURE CITED 1. Abeig, B. On the effects of j!?ara-substitiition in some plant growth regulators with phenyl nuclei. I7i: R. L. Wain and F. Wightman (eds.), The Chemistry and Mode of .Auction of Plant Growth Substances, pp. 93-116. Butterworth Sci. Pub!., London. 1956. 2. Bellamy, L. J. The Infrared Spectra of Complex Molecules. 2nd ed. 425 pp. John Wiley and Sons, Inc., New York. 1958. 3. Bennet-Clark, T. A. The kinetics of auxin-induced growth. In: R. L. Wain and F. Wightman (eds.), The Chemistry and Mode of Action of Plant Growth Substances, pp. 310-312. Butterworth Sci. Publ., London. 1956. 4. , and Kefford, N. P. The extension growth-time relationship for Avena coleoptile sections. Jour. Exper. Bot. 5: 293-304. 1954. 5. Bentley, J. A. The naturally-occurring auxins and inhibitors. Ann. Rev. Plant Physiol. 9: 47-73. 1958. 6. Berger, J., and Avery, G. S., Jr. The mechanism of auxin action. Science. 98: 454, 455. 1943. 7. Blackman, G. E. Interrelationships between the uptake of 2:4-dichlorophenoxy- acetic acid, growth, and ion absorption. In: R. L. Wain and F. Wightman (eds.). The Chemistry and Mode of Action of Plant Growth Substances, pp. 253- 259. Butterworth Sci. Publ., London. 1956. 8. Brian, R. C, and Rideal, E. K. On the action of plant growth regulators. Bio- chim. Biophys. Acta. 9: 1. 1952. 9. Cassidy, H. G. Adsorption and chromatography. In: A. Weissberger (ed.), Technique of Organic Chemistry, v. 5. 360 pp. Intersci. Publ., Inc., New York. 1951. 302 Freed, Reithel, and Remmert 10. Foster, R. J., McRae, D. H., and Bonner, J. Auxin-induced growth inhibition a natural consequence of two-point attachment. Proc. Xat. Acad. Sci. U. S. 38: 1014-1022. 1952. 11. , McRac, D. H., and Bonner, J. Auxin-antiauxin interaction at high auxin concentrations. Plant Physiol. 30: 323-327. 1955. 12. Freed, V. H., and Burschel, P. The relationship of water solubility to dosage of herbicides. Zeitschr. Pfl.-krankii. Pfl.-schutz. 64: 477-479. 1957. 13. Irvine, V. C. Studies in growth-promoting substances as related to X-radiation and photoperiodism. Univ. Colo. Stud. 26: 69, 70. 1938. 14. Ishizaki, H. Studies on the mechanism of fungicidal action. V. The theoreti- cal measurement of the activity of a haptophoric group. Bui. Fac. .\gr. Mich. Univ. 9: 48. 1955. (English translation.) 15. Johnson, E. J., and Colmer, A. R. Tiie relationship of raagnesiiun ion and molecular structure of 2,4-dichlorophenoxyacetic acid and some related com- pounds to the inhibition of respiration of Azotobacter vinelandii. Plant Physiol. 33: 99-101. 1958. 1'). Karreman, G., and Steele, R. H. On the possibility of long distance energy transfer by resonance in biology. Biochim. Biophys. Acta. 25: 280-291. 1957. 17. Koepfli, J. B., Thimann, K. V., and Went, F. W. Phytohormones: structure and physiological activity. 1. Jour. Biol. Chem. 122: 763-780. 1938. 18. Leaper, J. M. F., and Bishop, J. R. Relation of halogen position to physio- logical properties in the mono-, di-, and trichlorophenoxyacetic acids. Bot. Gaz. 112: 250-258. 1951. 19. Linser, H. Zur Wirkungsweise von Wuchs- und Hemmstoffen. I. Wachstums wirkungen von Indol-3-Essigsaure und Eosin sowie pflanzlicher Wuclis- inui Hemmstoffe im Gemisch an der .^wena-KoIeoptile. Biochim. Bioph\s. Acta. 6: 384-394. 1951. 20. Marinos, N. G. Responses of Axiena coleoptile sections to high concentrations of auxin. Austral. Jour. Biol. Sci. 10: 147-163. 1957. 21. .Maxwell, E. S., de Robichon-Szulmajster, H., and Kalckar, H. M. Yeast uridine diphosphogalactose-4-epimerase, correlation between activity and fluorescence. Arch. Biochem. Biophys. 78: 407-415. 1958. 22. Muir, R. M., and Hansch, C. The relationship of structure and plant-growth activity and substituted benzoic and phenoxyacetic acids. Plant Physiol. 26: 369-374. 1951. 23. , Hansch, C. H., and Gallup, A. H. Growth regulation by organic com- pounds. Plant Physiol. 24: 359-366. 1949. 24. Northen, H. T. Relationship of dissociation of cellular proteins In auxins to growth. Bot. Gaz. 103: 668-683. 1942. 25. Osborne, D. J. Growth of etiolated sections of pea intcrnode following ex- posures to indole-3-acetic acid, 2,4-dichlorophenoxyacctic acid and 12.5 die liloio- benzoic acid. Plant Physiol. 33: 46-57. 1958. 26. , Blackman, G. E., Powell, R. G., Sudzuki, F., and Xovoa, S. Growth regulating activity of certain 2:6-substitutcd phenoxyacetic acids. Nature. 174: 742. 1954. 27. Switzer, C. M. Effects of herbicides and related chemicals on oxidation and phosphorylation by isolated soybean mitochondria. Plant Phvsiol. 32: 42-44. 1957. 28. , Smith, F. G., and Loomis, W. E. Factors affecting oxidation and phos- phorylation of soybean mitochondria. Plant Physiol. 30 (suppl.): xxxi. 1955. 29. Ungar, G., and Kadis, S. Effect of insulin on sulphydryl groups in muscle. Nature. 183: 49, 50. 1959. Physical-Chonical Aspects uf Synthetic Auxins 303 30. Veldstia, H. The relation of chemical structure to biological activity in growth substances. Ann. Rev. Plant Physiol. 4: 151-198. 1953. 31. Velick, S. F. Fluorescence spectra and polarization of glyceraldehyde-3-phos- phate and lactic dehydrogenase coenzyme complexes. Jour. Biol. Chem. 233: 1455-1467. 1958. 32. Westphal, U. Steroid-protein interactions. III. Spectrophotometric demon- stration of interaction between proteins and progesterone deoxycorticosterone and Cortisol. Arch. Biochem. Biophys. 66: 71-90. 1957. 33. ^Voodford, E. K., Holly, K., and McCready, C. C. Herbicides. Ann. Rev. Plant Physiol. 9: 311-358. 1958. 34. Wort, D. J. Influence of 2,4-D on enzyme systems. Weeds. 3: 131-135. 1954. 35. Wright, R. H. Physical basis of insect repellency. Nature. 178: 638. 1956. 36. Zimmerman, P. W., and Hitchcock, A. E. The response of roots to "root- forming" substances. Contr. Boyce Thompson Inst. 7: 439-445. 1935. 37. , and Hitchcock, A. E. Formative effects induced with /3-naphthoxyacetic acid. Contr. Boyce Thompson Inst. 12: 1-14. 1941. 33. , and Hitchcock, A. E. Substituted phenoxy and benzoic acid growth substances and the relation of structure to physiological activity. Contr. Boyce Thompson Inst. 12: 321-343. 1942. DISCUSSION Dr. Wain: I would like to point out the clanger of assuming that the reason phenoxyacetic acid is held to a protein and is unable to pass through a membrane in dialysis, is due to the fact that the ring system has been associated with the protein. There may be an asso- ciation of poles of opposite charge. There could be, for instance, a protein with a free NHo group that could very readily associate with the carboxyl group of the phenoxyacetic acid to give an association of the molecule with the protein which has nothing to do with the ring system. Nevertheless, I fully agree that the ring system is all im- portant, and what is more, the ring system must be an unsaturated ring system. The unsaturated benzene ring is flat, and it is well known that flat surfaces can readily attach themselves to other surfaces by Van der Waal's forces. Benzene itself is capable of forming molecular compotmds with mercuric cyanide and platinum chloride, for ex- ample, and presumably is providing one coordinate valency. It may well be, therefore, that there is something of this kind taking place with the benzene ring to give: a free pair of electrons. I'm not sug- gesting that that known pair is donated to form a rigid coordinate bond, but the electron density outer surface may very well be slightly modified by this kind of tendency so as to give an association with the benzene ring, which after all is perhaps significant in view of the fact that there must be unsaturation in the ring. Many people work- ing in this field of auxinology are closing their eyes and are ignoring the fact that ethylene, carbon monoxide, propylene, etc., are active as plant growth regulators. Dr. van Overbeek: I agree with Dr. Freed on the emphasis he has 304 Freed, Reitlicl, and Remmert placed on a physical mode of primary action. I can imagine that the effect of 2,4-D on mitochondria could be due to swelling of the mito- chondrial membranes. I would like to ask how 2,6-D and the other homologues behave. Dr. Freed: We have measured the effect of 2,6-D, 2,4,6-T, and a nimiber of other compounds on the oxygen uptake of mitochondria. We do find that many of these compounds have an effect. This is as would be postulated since most of them are capable of being adsorbed to a greater or lesser degree. This has a parallel in that as a homolo- gous series is examined for biological activity or activity on mitochon- dria, a few members may be found that are innocuous at quite a range of concentrations, but most of them will have measurable ac- tivity with a few having outstanding activity. This suggests that the structural requirement for activity is not absolute, which is consonant with an adsorption phenomena. Hence the moderate activity of 2,4,6-T is not unexpected. Dr. AVain, we are fully aware of the possible implication of the carboxyl group in the absorption phenomenon. "When I say absorp- tion I'm thinking in rather broad terms. The ultraviolet spectra, of course, also implicate electron density because of the electronic con- figuration of the ring that has been modified both by the carboxyl group and the chlorine substituents. Dr. van Overbeek: That's why I asked the question, because ab- sorption is necessary but not the timely point. Dr. Freed: It appears from these studies that adsorption is the first event in the action of these compounds, but the subsequent effect may well be the crucial mechanism in their action. Dr. Bonner: Well, I know it's very attractive to think about how auxin molecules can be adsorbed, presumably by 2-point attachment, to enzyme molecules and cause them to be changed and to do some- thing different than they otherwise would do. It's attractive to think that auxins might do their work by affecting, for example, the mito- chondria which we know are very important, and the powerhouses of the cell, and I yield to no one in my admiration for mitochondria. One of the most fascinating and indeed useful aspects of auxins is, however, that they don't work on all kinds of organisms. We saw, for example, that auxins bind to bovine serum albumen, but it is a well- known fact that auxins do not work on cows. Now, it is a very remark- able fact, I think, that auxins work on sudi a small part of the spec- trum of living organisms. They work on higher plants and a few algae but they don't work on all organisms. It seems to me quite clear, therefore, that when we try to find out how auxin does its work we have to think about processes that go on in and arc unique to plants Physical-Chemical Aspects of Synthetic Auxins 305 and to algae and a few other organisms and not about processes that are common to all living things. For example, one of the lessons of comparative biochemistry is that mitochondria are very much the same in all living creatures. We all have the same kind of mitochon- dria, and I would think that, for example, if 2,4-D or lAA did its work on mitochondria, it ought then to work on all kinds of organisms. And it seems to me that the hypothesis that auxins exert their effects by general and nonspecific adsorption to enzymes breaks down in the face of the facts of comparative biochemistry. Dr. Freed: Your points are well taken, Dr. Bonner. Now I call your attention to the following: (A) It is known that a given enzyme isolated from different tissues will have slightly different substrate specificities yet catalyze the same essential reaction. This indicates that we are dealing with a popula- tion of molecules, some of which may give a remarkable response to a given chemical and still others of the same population give only a slight response. Thus the problem is one of degree rather than an all or none effect. This appears to arise from the differences in the struc- ture of the protein. Such variation at the molecular level may account in part for the variation among different organisms in their response to lAA. However, one of the consequences of this postulate is that many organisms may be responding to lAA but either so slightly or in such a manner that we have not yet observed the response. (B) In support of the foregoing it must be remarked that the first demonstration of the ability of 2,4-D to inhibit oxygen uptake by mitochondria was performed with mitochondria isolated from animal tissue rather than from plants. (C) Finally, it should be noted that lAA has recently been found to have a marked effect on certain functions of mammalian metabo- lism. Thus, Mirsky and his coworkers (Endocrinol. 59: 369. 1956) have found lAA to function as an antidiabetic agent in mammals. It thus appears that compounds may affect a wider range of or- ganisms than previously suspected. This would seem to be compatible with the theory advanced. Dr. Leopold: It is very exciting to think that adsorptive features of auxins might be relevant to their activities in biological systems. We have, like Dr. Freed, been very much taken by this possibility and have done some measurements of the influences of molecular structure on adsorption of auxins onto charcoal. This system suffers from its lack of biological specificity, to be sure, but it might tell us something about the influence of molecular structure on a simple adsorptive function. We found that compounds showing the great- est activity as auxins also showed the greatest adsorption onto char- 306 Freed, Reithel, and Remjnert coal. lAA was very good, 2,4-D and 2,4,5-T were excellent, but when we examined a whole series of chlorinated phenoxy acids the corre- lation with growth activity was not good because the more you chlorin- ate the more adsorption you get regardless of where the chlorine is placed in the ring. Thus, 2,4-D and 3,5-D are of equal activity in this adsorption system, 2,6-D shows a little less adsorption, but 2,6-D is very much in the same class as 2,4-D. 2,4,6-T, and 2,3,6-T are less adsorbed than the other trichlorinated ones but they were better than 2,4-D. I think that it may have implications in some aspects of auxin function in cells, but there's not a good correlation between auxin activity and adsorption, in our system. Dr. Wain: I think it's a very important point that Dr. Leopold raised. The compound with a ring system with more chlorine atoms can in fact be absorbed and can act as a competitive antagonist. But it's not necessarily a growth substance because other features of the molecule are important. JAMES BONNER California Institute of Technology On the Mechanics of Auxin-Induced Growth' It is a cornerstone of growth-substance lore that auxins cause an in- creased rate of plant cell elongation. The question of how auxins bring about such auxin-induced cell elongation constitutes a classical problem of auxinology. We shall here consider the state of knowledge on this subject. The present discussion is restricted to information concerning but a single plant tissue, the Avena coleoptile section, about which much is known. The elongation of the Avena coleoptile is normally controlled by auxin produced in the apex of the organ. The native hormone, which appears to be chemically identical with indole-3-acetic acid (I A A) (29), moves by polar transport to the lower regions of the coleoptile and there promotes elongation. Excised sec- tions of the coleoptile, floated in solution, respond to added lAA by increased growth rate, and such sections constitute a convenient sys- tem for the study of auxin-induced growth. Two characteristics of the response of Avena coleoptile sections to lAA deserve particular note. The first, as illustrated in Figure 1, is that the response is large. The rate of elongation of the section in the presence of added lAA (at optimal concentration) is seven- to tenfold greater than the rate of elongation in the absence of the growth sub- stance. The second noteworthy characteristic is that elongation in the absence of added lAA is not controlled by endogenous growth sub- stance. This conclusion is based upon the fact that the rate of elonga- tion of sections in the absence of added lAA is not slowed by added competitive inhibitors of auxin action. In the response of the Avena ^ Much of the work here summarized has been made possible by the continued and generous support of the Herman Frasch Foundation. The present discussion is primarily a summary of work done with this support and is not intended as a general revie\v of the entire field. [ 307 ] 308 ]. Bonner O cr o 0 6 12 TIME IN HOURS 18 Fig. 1. Progress curves for growth of Avena coleoptile sections. Basal medium of sucrose, 0.09A1 and K-malcate buffer, 0.0025Af, pH 4.5. IA.\ as indicated. Temp. 25° C. Initial length of sections 5.0 mm. coleoptile section to added lAA we have, then, not only a large re- sponse but also one that is pure — uncontaminated by residual native growth substance. THE DRIVING FORCE OF CELL ELONGATION We will first ask. What powers the elongation of plant tissue? This is a general question and one relevant to all cell extension whether auxin-controlled or not. In the case of the Avena coleoptile section, the driving force of cell elongation is the osmotic pressure of the vacuolar contents of the individual cells. This is clear from the information in Figure 2. Sections placed in solutions isotonic (0.42Af) with themselves essentially fail to elongate, either in the presence or absence of lAA. Sections placed in solutions containing an osmotically active solute (mannitol) in concentrations lower than isotonic grow at reduced rates. The rate of section elongation is, in fact, essentially a hyperbolic function of concentration of external solute. Classical osmotic lore tells us that: Net DPDT„,„e = O.P.T,,sue - W.P. - O.P.Kx.ornal where DPD designates diffusion pressure deficit, O.P., osmotic pres- sure, and W.P., wall pressure. Mechanics of Auxin-induced Groivth 309 X »- o (T. 0.5 - 0.6 EXTERNAL CONCN., M Fio-. 2. Initial growth rate of Avena coleoptile sections as a function of external os- motic concentration. Growth in mm/section/4 hrs. Osmotic solutes made up of sucrose, Q.09M plus varied concentrations of mannitol. After Ordin, Applewhite, and Bonner (20). The DPD of Avena coleoptile sections growing in solutions over the concentration range of Figure 2 has been shown to be null, within the precision of measurement (Ordin et ah, 20) . The grow- ing sections are therefore in osmotic equilibrium with the solution in all cases. In addition O.P.Tissue is identical, or nearly so, for sections in solutions of different O.P.Externai- We may conclude therefore that it is the tension to which the cell walls of the tissue are subject which is altered by changing O.P.Externai W.P. = O-P-Tissue — O.P.Externai and that rate of section growth as a function of O.P.Externai is in fact a measure of the rate of cell wall deformation as a function of cell wall tension. Our first conclusion is therefore that the rate of cell extension depends on the tension to which the wall is subjected by the osmotic pressure of the cell contents. One further conclusion can be drawn from the information in Figure 2. This concerns the difference in growth rate between lAA treated and control sections. Since tissue osmotic concentration is not 310 /. Bonner altered by lAA treatment, the wall tension developed by lAA treated sections must be identical with that developed in control sections. The more rapid growth of lAA treated sections must therefore be due to more rapid yielding of the wall. Our second conclusion is therefore that lAA treatment increases the deformability of the cell wall. ROLE OF OSMOTIC CONCENTRATION The factors which immediately control the rate of Avena coleop- tile section growth are then the osmotically induced load or tension on the cell wall and the resistance of the wall to deformation under load. If grow^th rate of the section is to remain constant with time, as is often desirable in growth studies, it is evidently essential to ar- range circumstances under which both of these factors remain con- stant. Thus the growth rate of sections which elongate in water (con- taining lAA) steadily decreases with time as shown in Figure 3. This is due to the fact that as the section takes up water and elongates, its osmotically active solutes are progressively diluted, as is indicated in Figure 3. The tension to which the cell wall is subjected therefore O o I I FINAL OSMOTIC CONCN. 0.49 M 0.32 M 12 18 TIME IN HOURS 24 Fig. 3. Progress curves for growth of Avena coleoptile sections in lAA, 2 X 10 ''.ll, in presence or absence of sucrose, 0.08.\/. Initial osmotic concentration, 0.42M. Mechanics of Auxin-induced Growth 311 decreases correspondingly. The maintenance of constant growth rate requires the addition to the external medium of an absorbable solute which is then taken up by the tissue and contributes to tissue osmotic concentration. By appropriate choice of solute and concentration, con- ditions may be arranged so that the uptake of solute balances the growth-induced dilution of cell contents to maintain constant tissue osmotic concentration. In the experiment of Figure 3 this has been achieved by the addition to the medium of OMM sucrose, which in fact constitutes the standard medium for the growth of Avena coleop- tile sections. A wide range of absorbable solutes can, however, replace sucrose in this function (Ordin et al, 20) . It is important that the role of tissue osmotic concentration in determination of section growth rate be generally understood since lack of such understanding in the past has led to acrimonious dispute (Bennet-CIark and Kefford, 2; Bonner and Foster, 7; Marinos, 18) . I ROLE OF CELL WALL RIGIDITY The effect of lAA in increasing the rate of coleoptile section elongation is due, in last analysis, to the effect of lAA in decreasing cell wall resistance to deformation under load as has been demon- strated above. There are, however, additional reagents which may be used to experimentally alter cell wall deformability. The chief of these is the calcium ion. The lAA-induced growth rate of sections is decreased in the presence of calcium ions (Thimann and Schneider, 26) as is illustrated in Figure 4. The relation of steady state growth rate to calcium ion concentration is a hyperbolic one, and the Kg or calcium ion concentration required to elicit half-maximal inhibition is ca. 3 X 10"^ equiv. That the effect of calcium ion is upon cell wall resistance to deformation is demonstrated by the fact that sections in solutions of varied calcium ion concentration are all in osmotic equilibrium with the solution (DPD = O) and that the initial osmotic concentration of the tissue is unaffected by calcium. Wall pressure therefore equals osmotic pressure in all cases. Since the force exerted on the wall is then independent of calcium ion concentration, it fol- lows that the reduced rate of extension of sections in calcium-contain- ing solutions is due to reduced rate of deformation of the walls in response to this constant force. The effect of the calcium ion in increasing cell wall resistance to load is shared by the magnesium ion which is, however, less effective. Monovalent cations such as Na+ and K+ are essentially without effect on growth rate, at least in concentrations of 1 to 10 mequiv/1. Potas- sium ions do, however, act as an antidote against the inhibitory effect 312 J. Bonner CALCIUM CONCN., MEQ./L. Fig. 4. Inhibition of lAA-induced growth of Avena coleoptile sections by CaCL. Based on steady state rates over a 6 hr. growth period. Basal medium contained su- crose, 0.09i\f. After Cooil and Bonner (12). of calcium ions on section growth rate. The experiment of Figure 5 summarizes the growth interrelations of Ca+^ and K+ ions. Sections growing in the absence of either ion, when transferred to calcium- containmg solution, quickly assume a new and slower steady state growth rate. If such sections are transferred to water, their growth continues at the rate characteristic of the calcium-containing solution and only slowly increases. Transfer of the sections to K^ ion-containing solution results in immediate reversal of the calcium inhibition. We may summarize the information of Figure 5 by saying that growth inhibition by calcium ions behaves as thougii it were mediated by ex- changeably bound ions. MECHANICAL PROPERTIES OF COLEOPTILES UNDER EXTERNAL LOAD The elfects of lAA and of inorganic ions on the cell walls of coleoptile sections can be readily and rigorously demonstrated by methods which measure their deformability under artificially im- Mechanics of Auxin-induced Growth 313 O o TIME IN HOURS Fig. 5. Progress curves of lAA-induced Avena coleoptile section — growth as influ- enced by addition of calcium ions (10 mequiv/1) as well as by subsequent with- drawal of calcium. Basal medium contained sucrose 0.09M and lAA 2 X lO'W. A. Sections in basal medium throughout. B. Sections transferred to basal medium plus calcium at first arrow and thence to KCl (1 mequiv/1) at second arrow. C. Sections transferred to basal medium plus calcium at first arrow and thence to basal medium minus salt at second arrow. D. Sections transferred to basal me- dium plus calcium at first arrow and left in this solution. E. Sections in basal medium plus calcium throughout. Modified after Cooil and Bonner (12). posed external load. The results of such measurements confirm and extend the conclusions reached above on the basis of growth rate studies, namely, that lAA increases cell wall deformability while cal- cium ions decrease it. A convenient measurement is that of the rate of bending of a coleoptile which is rigidly supported at one end and loaded with a weight on the other. The mechanical analysis of this method, which was first used by Heyn (15), is as follows: The turgid cylindrical section of plant tissue, composed of cells, may be thought of as a structure composed of bags of water, surrounded by intercon- nected walls. When the section is subjected to a force normal to its long axis, one side of the structure is placed in compression, the other in tension. The side which is under compression cannot, as a first approximation, compress. It is composed of incompressible liquid. The 314 ]. Bonner O U- UJ O 0 10 20 30 LlI se 40 50 WEIGHT APPLIED WEIGHT REMOVED SLOPE = RATE OF PLASTIC BENDING 0 10 15 20 25 30 TIME IN MINUTES Fig. 6. Progress curve of deformation of a 2 cm. Avena coleoptile section in re- sponse to an applied force. The section is held rigidly at one end, a constant bending force applied to the other. Angle of deformation is plotted as a func- tion of time. After Tagawa and Bonner (24). side which is in tension can, however, yield lo this tension since the supporting cell walls can stretch. The property measured in such a bending experiment is therefore the stretchability of the cell walls of the section. The principal complication is the possibility of pressure-induced water flow through the tissue from the compression to the tension side. Such flow may be expected to occur but to become significant only over time periods longer than those required {ca. 5 min.) for the measurement of rate of deformation of the wall. Figure 6 presents data on the time course of deformation under load of an Avena coleoptile section. The initial rapid elastic deforma- tion is followed by a period of steady plastic deformation. The rate of such plastic deformation under load is influenced both by lAA and by inorganic ions. Figure 7 presents data on rate of plastic de- formation, at constant external load for sections equilibrated with varying concentrations of lAA. It is evident that the lAA concentra- tion dependence for cell wall deformability closely resembles that for lAA-induced section growth and that, in fact, the concentrations Mechanics of Auxin-induced Groiuth 315 en (T O X CX) o CD lAA CONCENTRATION, M. rig. 7. lAA concentration dependence of (left) plastic deformation and of (right) growth of Avena coleoptile sections. In plastic deformation experiment, sections were equilibrated with lAA for 60 min. and plastic deformation then measured for 4 min. In growth experiment, sections were incubated in sucrose 0.09M, K-maleate buffer 0.0025M, pH 4.5, and lAA for 18 hrs. of lAA which ehcit half maximal response are identical within the errors of measurement. That calcium ions increase the resistance of section cell walls to deformation under external load is shown in Figure 8. The calcium ions responsible for this effect appear to be exchangeably bound, as is shown by the fact that the cell wall stiffening effect persists when the tissue is transferred to water but is discharged when the tissue is transferred to K+ ion-containing solution. The effects of calcium and of potassium on cell wall deformability as measured by rate of de- formation under external load are then similar to the effects of the same ions upon cell wall deformability as measured by rate of cell extension in response to internal (osmotically induced) load. CHEMICAL BASIS OF CELL WALL PROPERTIES The facts adduced above lead to the conclusion that lAA increases cell wall deformability and that cell wall deformability is decreased in the presence of calcium ions. lAA increases cell wall deformability even in cell walls made stiff by the presence of calcium ions. In such 316 ]. Bonner O Q LU OQ O H C/) < 8* 40 _ 0* 120 TIME IN MINUTES Fig. 8. Time course of inhibition of plastic deformability of Avena coleoptile sections by added calcium ions (20 mequiv/1) and of reversal of the inhibition by potassium ions (20 mequiv/1). Sections equilibrated in solutions for varied times as indicated and plastic deformation then measured over a 4 min. period. walls the material which interacts with calcium limits deformability. That lAA does its work upon the same material which interacts with calcium is therefore implied although not rigorously demonstrated. It will now be shown by independent, chemical, methods that lAA and calcium ions exert their effects upon the same cell wall constitu- ent, namely, the pectic material. The nature of the material within the coleoptile which interacts with calcium ions to cause cell wall stiffening can be attacked in a straightforward manner. It has been shown above that the responsible ions behave as though exchangeably bound to the tissue. Living Avena coleoptile sections possess a readily measurable cation exchange ca- pacity. The characteristics of such binding closely resemble those of the binding which causes cell wall stiffening, as is summarized in Table 1. The data of Table 2 establish that the capacity of such sections to bind calcium ions exchangeably is entirely due to the free Mechanics of Auxin-induced Growth 317 Table 1 . Comparison of parameters which characterize interaction of calcium ions and Arena coleoptile sections with respect to cell wall stiffening and exchangeable binding. Parameter Cell Wall Stiffening Exchangeable Binding Ca"'"''" concn. required to produce 0.5 max. effect 3 X 10-3 ^• 2-3 X 10-3 JV Time reouired for 0.5 eauilibration Ca. 10 min. Ca. 10 min. (nonesterified) pectic carboxyl groups of the cell wall. It may be con- cluded therefore that, in the presence of calcium ions at least, it is the cell wall pectic material which limits cell wall deformability. Whether this is also true in the absence of added calcium ions cannot be deduced. It is of interest, however, that the cell walls of sections taken from Avena seedlings germinated in distilled water contain measurable calcium and, in fact, a quantity sufficient to bind with approximately one-fifth of the free pectic carboxyl groups of the cell wall. The pectic material of the Avena coleoptile makes up but 5 per cent of the total weight of cell wall substance (Jansen et a\., 16). Cellu- lose constitutes another 25 per cent (Bishop et aL, 3), while polysac- charides which yield on hydrolysis xylose, arabinose, glucose, and galactose make up the bulk of the remainder. It appears remarkable that a minor constituent such as pectic material should play a major role in determining cell wall mechanical properties. It is indeed clear that we have inadequate knowledge of the structural arrangements of cell wall components other than cellulose, and such knowledge will be needed before detailed discussions of cell wall properties can be fruitfully undertaken. INFLUENCE OF lAA ON PECTIC METABOLISM Since lAA increases rate of cell extension by causing increased cell wall deformability, it is evident that lAA in some way alters cell Table 2. Demonstration that the exchangeably bound calcium oi Avena coleoptile sections is held principally by free (nonesterified) pectic carboxyl groups of the cell wall. After Jansen rf a/. (16). Cation exchange capacity of living coleoptile sections Cation exchange capacity of cell walls Cation exchange capacity of cell walls calculated on basis of free pectic carboxyl groups Cation exchange capacity of living sections pre- dicted on basis of 2 Cation exchange capacity of living sections predicted on basis of 3 3.1 X 10-3 Mequiv/g fresh wt. 0.18 Mequiv/g dry wt. 0.165 Mequiv/g dry wt. 3.6 X 10"3 Mequiv/g fresh wt. 3.3 X 10-3 Mequiv/g fresh wt. 318 /. Bonner wall chemistry. Such alterations have been sought since the beginnings of auxinology. Only in recent years, however, and after the advent of appropriate methodology have they been foiuid. "With such methodology it has been shown that the application of lAA to Avena coleoptile sections results in an increased rate of pectic synthesis by the tissue. That lAA influences pectic metabolism was first detected by the use of C^^ inethyl-labeled methionine. The methyl gioup of methionine serves as donor of the methyl ester groups of pectic substances (Ordin et al., 21; Sato, 23). The application of lAA to coleoptile sections in the presence of C^^ methyl-labeled me- thionine (or of other methyl donors, as formaldehyde), increases the rate of appearance of labeled pectic methyl ester groups (Ordin et al., 21, 22; Jansen et al., 16). It was subsequently found that the in- creased rate of pectic methyl ester formation in the presence of lAA is paralleled by increased rate of polygalacturonic acid formation (Al- bersheim and Bonner, 1). lAA therefore increases the rate of forma- tion of pectic material. The effect of lAA upon pectic synthesis pos- sesses all of the earmarks of an authentic auxin-controlled reaction, with characteristics similar to those of lAA-induced gro^v•th. Thus the effect of lAA upon rate of pectic synthesis is a rapid one, manifest within 15 to 30 min. after application of the auxin. It is inhibited by antiauxins such as 2,4,6-trichlorophenoxyacetic acid. It takes place only under aerobic conditions (Cleland, 10). It is inhibited, as is growth, by ethionine. It occurs in sections which are restrained from growing by the presence of isotonic mannitol solution, a condition under which auxin-induced cell wall plasticization continues to oc- cur. It woidd appear therefore that lAA-induced cell wall plasticiza- tion and lAA-induced alteration in pectin synthesis are similar and possibly identical reactions, although there is as yet no rigorous proof that this is so. Further analysis of the effects of lAA on pectic metabolism re- quires a knowledge that the pectic material of Avena coleoptile sec- tions includes three principal forms (Jansen et ah, 16). The first is the so-called hot water soluble pectin (extracted from the cell wall by hot water). This fraction is, as summarized in Table 3, highly esteri- fied. A second pectic fraction is the cold water soluble, 70 per cent al- cohol precipitatable material. This fraction, which constitutes ap- proximately 5 per cent of the total pectic substance, is presumably removed from the cell wall during the preparation of the latter but can be recovered from the cold water washings by alcohol precipita- tion. The third pectic fraction — that which remains in tiie wall after cold and hot water washing— is known as the residual pectin. Mechanics of Auxin-induced Grozuth 319 Table 3. Characteristics of the pectic materials of the cell walls of Avena coleoptile sections. After Jansen f/ a/. (16). Fraction Whole cell walls Cold H2O soluble, 70 per cent EtOH insol Hot HP soluble Residual Per Cent AUA* in Fraction 5.3 4.0 23 43 Per Cent of Total AUA 91 5 14 78 Per Cent Methyl Esterified 40 ca 100 90 31 * AUA = Anhydrouronic (galacturonic) acid. It makes up some 80 per cent of the total, is roughly 30 per cent ester- ified, and contains essentially all of the free pectic carboxyl groups which contribute to the cation exchange capacity of the wall. This residual pectin may be solubilized by hot water in the presence of the chelating agent ethylenediaminetetraacetic acid (EDTA). The effects of lAA, so far as pectic material is concerned, are con- centrated in the hot and cold water soluble pectic fractions. Informa- tion bearing on this point is summarized in Table 4 for two types of experiments. In the one type of experiment, sections were merely in- cubated for 15 hrs. in the presence of glucose and in the presence or Table 4. Cell wall pectic synthesis during the growth of Avena coleoptile sections. Summarized after Albersheim and Bonner (1). Analytical Increase in AUA* Radioactivity of AUA** Mg/100 Mg Ratio CPM/100 Ratio Cell Wall lAA/ Mg lAA/ Pectic Fraction Per 15 Hrs. Control Cell Wall Control Control 0.32 11,850 Total wall < lAA 1,5 1,2 0.49 14,300 [Control 0.055 650 Cold H.,0 Soluble lAA 3,8 2,3 0.145 1.500 Control 0.102 2,500 Hot H.,0 Soluble lAA 1.6 1 .6 0.166 4,030 Control 0.17 8,700 Residual lAA 1.1 1 .0 0.18 8,800 * AUA = Anhydrouronic (galacturonic) acid. ** Sections incubated 5 hrs. in uniformly labeled C"-glucose, 2.5 X 10~*M, 1.9 mc/millimole. 320 ]. Bonner absence of lAA, and analytical determinations were made by an ap- propriate, sensitive, and specific method (Albersheim and Bonner, 1) of the initial and final amount of pectic material in each cell wall fraction. In the second type of experiment, sections were incubated for 5 hrs. in C^^-labeled glucose, and the amount and specific radio- activity of the pectic galacturonic acid of each fraction were deter- mined. It is clear that in the presence of lAA the rate of formation of cold water soluble pectin is increased twofold or more and that of hot water soluble pectin 60 per cent, while the rate of synthesis of residual pectin is but little influenced. The effect of lAA on pectic metabolism is, then, to increase rate of production of the more soluble pectic molecules. One can feel in- tuitively that this should in some way increase cell wall plasticity but as yet no rigorous demonstration of how it does so has been achieved. EFFECT OF AUXIN ON INTERACTION OF CELL WALL COMPONENTS The walls of the parenchymatous cells of the Avena coleoptile possess, in common with the primary walls of other cylindrical plant cells, a pattern of arrangement of cellulose microfibrils known as tube structure. The cellulose microfibrils are disposed in a manner pre- dominantly normal to the long axis of the cell and thus in a 'barrel- hoop" fashion. That this is so may be readily observed by electron microscopy (Miihlethaler, 19), although it was first deduced on the basis of birefringence measurements (Bonner, 5). Such measmements enable one to draw conclusions as to the orientation of cellulose micro- fibrils within the wall since the larger index of refraction of cellulose lies parallel to the long axis of the microfibril. It is characteristic of the microfibrillar network that, when stretched, the microfibrils tend to align themselves in the direction of stretch. The amount of stretch- ing required to elicit a given degree of reorientation is a measiue of the interaction between the units of the network as has been shown for model systems (Bonner, 5). It is of interest that lAA treatment ex- erts a profound effect on the tendency of the cell wall microfibrils of Avena coleoptiles to reorient in response to mechanical shear. This has been demonstrated by the following general technique: coleoptile sec- tions (from which the epidermis had been stripped) were plasmolyzed in glycerine, clamped at the two ends, and stretched longitudinally under a polarizing microscope. As the tissue is stretched, the initial negative anisotropy (microfibrils statistically at right angles to the shear axis) diminishes, becomes null (statistical isotropy), and finally becomes positive (microfibrils statistically parallel to the shear axis). The course of micronbrillar reorientation whh increasing stretch is Mechanics of Auxin-induced Growth 321 + 60 (BREAK) 10 20 30 (BREAK) 40 50 STRETCH, 7o OF LENGTH Fig. 9. Reorientation of cellulose microfibrils of Avena coleoptiles (one-half coleop- tiles, epidermis removed) during longitudinal stretching as a function of prior (2 hrs.) lAA treatment. Path difference under polarized light measured with Senarmont compensator. After Bonner (5). shown in Figure 9, which also contrasts the behavior of lAA-treated and nontreated sections. It is clear that the reorientation of the wall microfibrils in response to shear is dramatically decreased by the pres- ence of lAA. This effect is not an artifact of lAA-induced growth since it occurs equally strikingly in lAA-treated but nongrowing sections. How are we to understand the effect of lAA treatment upon the response of the wall microfibrillar network to shear? Evidently the microfibrils reorient in the direction of shear because they interact, stick to one another here and there. In lAA-treated tissue this inter- action is decreased and ability of the microfibrils to slide past one an- other correspondingly increased. lAA treatment of Avena coleoptile sections results in but slight effects on cellulose (Boroughs and Bon- ner, 9; Ordin et al, 21, 22) . However, the facts available suggest that the pectic material of the wall may constitute the glue through which the microfibrils interact. 322 J. Bonner ACTIVE AND PASSIVE ASPECTS OF lAA-INDUCED GROWTH It has already been shown that cell extension in final analysis is stretching of the cell wall clue to osmotically controlled water up- take. In this sense cell extension itself is a passive process. Cell ex- tension in the Avena coleoptile section, however, is accompanied and in fact controlled by at least three distinguishable, metabolically powered and hence active processes. The first is the active accumula- tion of solute molecules or ions which, by maintaining the osmotic concentration of the cell contents, maintain the turgor-induced load on the cell wall. The second is the deposition of new cell wall ma- terial which normally keeps pace with cell extension. The third is the auxin-induced plasticization of the wall. That the plasticization process may be experimentally separated in time from actual cell extension has been shown over the years by Heyn (15) , Thimann (25), and by Cleland and Bonner (11). In this type of experiment, sections are supplied with lAA but restrained from growing by the presence of a suitably high external concentration of nonabsorbable solute. The sections are then allowed to expand in water under conditions in which the action of auxin is blocked by the presence of a suitable in- hibitor. An effect of the auxin pretreatment is manifested by growth of the auxin-treated sections greater than that of nonauxin-treated control sections. Alternatively, the cell wall deformability under ex- ternal load of auxin-treated (but nongrowing) sections may be measured directly (Cleland, 10). 0.3 1-0.2 H O z u ^ 0.1 o I 0.0 X o 0.1 lAA AIR No I AA No AIR Mannitol , _L + IAA^L_ I AA I 2 3 TIME. HOURS 0.2 - .0.1 - 0 20 40 60 80 I AA PRE- TREATMENT, MIN Fig. 10. Experimental separation of lAA-induced wall softening from tlic act of turgor-controlled cell extension. Left, Avena coleoptile sections placed in 0.3Af mannitol with or without IA.\ for an initial pretreatment inider aerobic conditions and then transferred (with an intermediate treatment) to water under anaerobic conditions. Right, residual auxin effect showing increase in length directly propor- tional to length of treatment, .\fter Cleland and Bonner (11). Mechanics of Auxin-induced Groxvth 323 Experiments of this type have shown that lAA-induced cell wall plasticization takes place even though the section is not elongating. This plasticization is, however, of a special kind in that it is made and stored in an amount which is proportional to the length of time dur- ing which the tissue is treated with auxin. This relation is shown in Figure 10. A brief auxin pretreatment makes it possible for a speci- fied amount of auxin-induced residual growth to take place in the second phase of the experiment. Twice as long an auxin pretreatment permits twice as much residual growth. Clearly, the action of lAA is not to bring about a general decrease in wall rigidity but rather to change the wall in such a manner that it can yield by a specified amount. Further characteristics of auxin-induced wall softening include the facts that the process is aerobic, inhibited by varied respiratory inhib- itors, and suppressed by ethionine. The act of elongation itself, on the contrary, is not inhibited by anaerobiosis and is relatively less sensitive to metabolic inhibitors. ROLE OF RESPIRATION It has been known for many years that the application of auxins, including lAA to plant tissue, including Avena coleoptile sections, results in rapid and considerable increases in respiratory rate (Bon- ner, 4, 6). It has been natural, therefore, to seek an understanding of auxin-induced growth in terms of the respiratory response. It ap- pears, however, that such search is fruitless. lAA-induced increase in respiratory rate, in the Avena coleoptile at least, does not accompany lAA-induced cell wall softening in nonelongating sections (0.3M mannitol) (Ordin et aL, 20). The respiratory increase which accom- panies lAA-induced growth is therefore an artifact of extension rather than a direct effect of lAA. It is also clear, however, that lAA-induced cell wall softening requires respiratory energy, supplied perhaps in the form of ATP since the phosphorylative uncoupling agent, 2,4-dini- trophenol, blocks lAA action. INTERACTION OF CELL WALL AND CYTOPLASM Since the ultimate effect of lAA treatment is upon the cell wall and since lAA-induced cell wall softening requires the participation of mitochondrial respiration, it is perhaps obvious that wall and cytoplasm interact in auxin-induced growth. It is nonetheless of in- terest that auxin-induced cell wall softening does not occur in plas- molyzed sections in which cytoplasm and wall are in only tenuous contact (Cleland, 10). Such sections are not injured by the treatment since they respond to lAA upon deplasmolysis. 324 ]. Bonner ROLE OF PECTIN ESTERASE There has been extensive discussion of the possible role of pectin esterase in auxin-induced growth. Glasziou (13) and Glasziou and In- glis (14) in particular have suggested, on the basis of experiments with tobacco pith and Jerusalem artichoke tuber tissue, that auxins function by binding and thus inactivating pectin esterase. Avena coleoptile sections do indeed contain readily detectable amounts of pectin esterase (Jansen et ah, 17), essentially all of which is bound to cell wall. lAA is, however, without direct effect either on this binding or on the activity of the enzyme in Avena coleoptile sections (17). It has already been shown above that the role of lAA in pectic metabo- lism lies in an earlier step than that mediated by pectin esterase. SUMMARY There appear to be two general approaches to the study of auxin action. The first is to determine with what material added auxin in- teracts within the cell, find out what the interaction product does, and so step by step, trace the sequence by which cell wall softening is id- timately brought about. The second approach is to start at the op- posite end of the chain, namely with the final result of wall softening, discover what chemical changes bring about this effect, and step by step trace the sequence forward to the initial interaction of auxin with plant. Both approaches have been used but we are as yet far from linking them. On the one hand, it appears that auxin does in- teract within the plant with a specific receptor entity and that this interaction involves two point-combination of auxin and receptor (Bonner and Foster, 8). Identification of the auxin-receptor complex by the use of labeled auxin has so far failed, and we conclude only that the complex is present in the plant tissue at very low concentra- tion, of the order of 1 part in 100,000,000 or less. The approach from analysis of cell extension itself has, however, been, as shown above, appreciably fruitful. It is clear that auxin-induced growth is the re- sult of auxin-induced cell wall softening and that this is in turn as- sociated with auxin-induced alteration in the synthesis of cell wall pectic material. Elucidation of the way in which auxin influences pec- tin synthesis requires that the enzymology of pectic synthesis first be understood, which it is not. This then is the present state of the study of the mechanism of auxin action in the Aveyia coleoptile section. The facts presented above are numerous and complex. It may be j)crhaps of passing value to summarize them in terms of a model which, although it may very well be incorrect, will nonetheless serve to help us remember some of the facts. This model, which concerns the parenchymatous cells of coleoptile section and disregards the epi- Mechanics of Auxin-induced Growth 325 dermal cells (which serve merely to slow the growth of the section) (Bonner, 5), starts with the observations of Miihlethaler (19) and of Wardrop (27, 28) that dining elongation of coleoptile parenchyma cell walls the constituent cellulose microfibrils are steadily separated from one another and dispersed from their initially transverse orien- tation. In addition, however, new, transversely oriented microfibrils are steadily added on the inner surface of the wall. Evidently as the wall is stretched during elongation, the microfibrils are pulled apart into a more disperse network and reoriented in the direction of stretch just as in inanimate model systems. We may imagine then that as the wall is stretched, as junction after junction yields and breaks under tension, it becomes progressively weaker. The mechanical strength of the wall is maintained only because of the constant addi- tion of new material to its inner surface. The present model then assumes that the mechanical strength of the cell wall is primarily determined by the most recently deposited material. Further questions with which the model concerns itself are: What properties of the wall determine how tightly the cellulose microfibrils are linked together? What determines how readily they may be pulled apart? The model nominates the pectic molecules for this function. The long random coils of pectic material intertwine the microfibrils as fungal hyphae intertwine the clay particles in a soil, binding the whole into an interconnected network. And in this function long pectic chains will evidently be more effective than short ones. The addition of auxin to the tissue encourages the production of short pectic chains. This model has many attractive features since, as con- sideration will reveal, many aspects of cell wall softening can be in- terpreted within this one framework. The particularly unattractive feature of the model lies, however, in the fact that it would seem to be most difficult to discover whether it corresponds to reality. LITERATURE CITED 1. Albersheim, P., and Bonner, J. Metabolism and hormonal control of pectic substances. Jour. Biol. Chem. 234: 3105-3108. 1959. 2. Bennet-Clark, T. A., and KefEord, N. P. The extension growth-time relationship for Avena coleoptile sections. Jour. Exper. Bot. 5: 293-304. 1954. 3. Bishop, C. T., Bailey, S. T., and Setterfield, G. Chemical constitution of the primary cell walls of Avena coleoptiles. Plant Physiol. 33: 283-289. 1958. 4. Bonner, J. The action of the plant growth hormone. Jour. Gen. Physiol. 17: 63-76. 1933. 5. . Zum Mechanismus der Zellstreckung auf Grund der Micellarlehre. Jahrb. Wiss. Bot. 82: 377-412. 1935. 6. . The growth and respiration of the Avena coleoptile. Jour. Gen. Physiol. 20: 1-11. 1936. 7. , and Foster, R. J. The growth-time relationships of the auxin-induced growth in Avena coleoptile sections. Jour. Exper. Bot. 6: 293-302. 1955. 326 J. Bonner 8. Bonner, J., and Foster, R. J. The kinetics of auxin-induced growth. In: R. L. Wain and F. Wightman (eds.), The Chemistry and Mode of Action of Plant Growth Substances, jip. 295-309. Butterworth Sci. Publ., London. 1956. 9. Boroughs, H., and Bonner, J. Effects of indoleacetic acid on metabolic path- ways. Arch. Biochem. Biophys. 46: 279-290. 1953. 10. Cleland. R. E. The hormonal control of cell wall properties. Ph.D. Thesis, Calif. Inst. Tech. 1957. 11. , and Bonner, j. The residual effect of auxin on the cell wall. Plant Physiol. 31: 350-351. 1956. 12. Cooil, B., and Bonner, J. The nature of growth inhibition by calcium in the Avena coleoptile. Planta. 48: 696-723. 1957. 13. Glasziou. K. T. The effect of auxins on the binding of pectinmethylesterase to cell wall preparations. Austral. Jour. Biol. Sci. 10: 426-434. 1957. 14. , and Inglis, S. The effect of auxins on the binding of pectin methyl- esterase to cell walls. Austral. Jour. Biol. Sci. 11: 127-141. 1958. 15. Heyn, .\_ N. J. Der Mechanismus der Zellstreckung. Rec. Trav. Bot. Neerl. 28: 113-244. 1931. 16. Jansen, E. F., Jang, R., Albersheim, P., and Bonner, J. Pcctic metahrlism of growing cell walls. Plant Physiol. 35: 87-97. 1960. 17. , Jang, R., and Bonner, J. Binding of enzymes to Avena coleoptile cell walls. Plant Physiol. 35: 567-574. 1960. 18. Marinos, N. G. Responses of Avena coleoptile sections to high concentrations of auxin. Austral. Jour. Biol. Sci. 10: 147-163. 1957. 19. Miihlethaler, K. Elektronenmikroskopische Untersuchungen iiber den Fein- bau und das Wachstum der Zellmembranen in Mais- und Haferkoleoptilen. Ber. Schweiz. Bot. Ges. 60: 614-628. 1950. 20. Ordin, L., Applewhite, T. H., and Bonner, J. Auxin-induced water uptake .by Avena coleoptile sections. Plant Physiol. 31: 44-53. 1956. 21. , Cleland, R., and Boimer, J. Influence of auxin on cell-wall metabolism. Proc. Nat. Acad. Sci. U.S. 41: 1023-1029. 1955. 22. , Cleland, R., and Bonner, J. Methyl esterification of cell wall constituents under the influence of auxin. Plant Physiol. 32: 216-220. 1957. 23. Sato, C. Methyl group synthesis in plant metabolism. Ph.D. Thesis, Univ. Mich. 1955. 24. Tagawa, T., and Bonner, J. Mechanical piopcrtics of the Avena coleoptile as related to auxin and to ionic interactions. Plant Physiol. 32: 207-212. 1957. 25. Thimann, K. V. Studies on the physiology of cell enlargement. Tenth Sym- posium on Development and Growth. Growth Suppl. 15: 5-22. 1951. 26. , and Schneider, C. L. The role of salts, hydrogen-ion concentration and agar in the response of the Avena coleoptile to auxin. Amer. Jour. Bot. 25: 270-280. 1938. 27. Wardrop, A. B. The mechanism of surface growth in parenchyma of Avena coleoptilcs. Austral. Jour. Bot. 3: 137-148. 1955. 28. . The nature of surface growth in plant cells, .\usiral. Jour. Bot. 4: 193-199. 1956. 29. Wildman, S. G., and Bonner, J. Observations on the chemical naiiuc and formation of auxin in the Avena coleoptile. Amer. Jour. Bot. 35: 710-746. 1948. Mechanics of Auxin-induced Growth 327 DISCUSSION Dr. Bennet-Clark: The suggestion that has just been made that growth is only enlargement and that division in two is not growth seems to me quite incorrect. Cell division involves complex differen- tiation. Cell enlargement can be an osmotic intake of water involving no differentiation, as for example, recovery from wilting. The growth processes that we have been discussing are those promoted by so-called auxins or growth hormones, the very existence of which was first revealed by studies of tropisms. I would like to re- fer to the behavior of two geotropic mechanisms which seem to be remarkably different. The first is that of the first internode or meso- cotyl of Zca mays which is a desirable research object as on geotropic stimulation it bends very sharply with small radius of curvature, which means that the length and volume of cells on the convex side are markedly larger (some 30 to 40 per cent) than those on the con- cave side and consequently analysis of the differences between them is facilitated. This bending is caused by different growth rates of the t\\'o sides of the mesocotyl. The other quite distinct behavior is that of a grass node. When placed on its side, the lower side expands and the node becomes sharply bent upwards because these lower-side cells expand about 200 to 300 per cent in length and volume. Is this to be called growth? They do not have any normal capability of growth like mesocotyl cells, and expand only in response to the gravitational stimulus. Some of the earlier workers in the last decade of the nineteenth century showed that a geotropic stimulation of stems (inflorescence axes) resulted in increase of extensibility of the tissue on the lower side. A given bending moment caused greater deflection away from the lower side than that away from the upper side. With a grass node this situation is reversed: it bends more readily towards the upper than to the lower side. This is associated with the fact that cells on the lower side are more turgid than those of the upper side, and they are consequently more rigid. The increase in their osmotic pressure "blows them up" and causes the expansion and the rigidity. Structurally these cells are very remarkable: The tissue has a concertina-like appearance and the cells have relatively thin walls at right angles to the long axis of the node and very thick walls oblique to this long axis. On expansion, which is sometimes as much as a fourfold extension, these thick oblique walls pull out like the oblique folds of a concertina. This can hardly be described as growth. It does, however, involve factors important in growth; the first is the increase 328 y. Bonner in the plasticity of the thick oblique walls, and the second is genera- tion of the necessary osmotic pressure to maintain or increase turgor. Both these processes only occur after the geotropic stimulus. I would not like to state that we have "hormones," one producing plas- ticity and another generating high osmotic pressure until a better and more extensive survey of growth substances separated by chroma- tography has been completed. (I remember my first chief's advice: "Beware of the devil because the devil always sends positive results first!") The point I want to make is that in almost all of our discussion about auxin action and about the activity of substituted phenoxy- acetic acids, indoles, and so forth, we assume that because our test process is an expansion in length, it is always the same molecular process that is involved in bringing about the expansion in length. In the node it is quite clear that there are at least two (possibly more) completely separate processes: one is wall softening and the other is build-up of osmotic pressure. In the node this osmotic pressure is quite certainly due to entry of or production of sugars in the lo^ver-side cells. The data will be published soon. In corn mesocotyls, however, we find that the sugar concentration decreases, whereas that of potassium ions increases very markedly on the lower side. Growth initiated by so-called hormones involves this complex of processes and may thus really require a complex of hormones for its completion. The process initiated by a synthetic substance like 2,4-D may be a different process from any of those initiated by the natural gravitational hormone or by lAA, though each of these possibly dif- ferent processes provides the same end result — namely cell extension. I would like to refer to the views put forward by me earlier. Indole- 3-acetic acid does affect wall extensibility, and I thought cross link- ages between polysaccharide molecules, especially pectins, the most likely point of attack, and that the mechanism was through control of the methylation of pectin. This view was based on preliminary analyses of the methoxyl content, and we thought also that we had demonstrated a transmethylation accelerated by lAA. Later work in our laboratory has shown that there is no difference in the degree of methylation of wall pectin in stems and mesocotyls as a consequence of increased extensibility caused by lAA. We had, of course, thought that increased methylation and consequent de- creased calcium in the wall was the cause of increased extensibility. W^e still have to find a molecular mechanism to explain this in- creased wall extensibility, and I now no longer favor the pectin- methylation hypothesis. DAPHNE J. OSBORNE Department of Agriculture, Oxford MARY HALLAWAY Department of Botany, Oxford The Rote of Auxins In the Control of Leaf Senescence. Some Effects of Local Applications of 2 A-Oichlorophenoxy acetic Acid on Carbon and Nitrogen Metabolism. It has long been recognized that senescence in detached leaves is re- tarded when root initials are formed along the petiole. The green color is then retained, and net protein syntheses and the accumula- tion of metabolites occur as long as the blade remains attached to living roots. If the roots are removed, the processes of senescence again take place. It has now been shown that the aging processes in detached leaves can be arrested by treatment with certain chemicals and that the presence of roots is therefore not essential for preventing senescence of the blade. For example, Richmond and Lang (5) found that chlorophyll and protein breakdown in detached Xanthium leaves was retarded by treatment with kinetin. Person, Samborski, and For- syth (3) showed similar effects in detached wheat leaves following treatments with benzimidazole, and Brian, Petty, and Richmond (1) reported that both autumnal yellowing and subsequent leaf fall could be retarded in a number of deciduous species by spraying gibberellic acid on the leafy branches. More recent work (2) has shown that suit- able applications of both 2,4-dichloro- and 2,4,5-trichlorophenoxy- acetic acids (2,4-D and 2,4,5-T) are also effective in delaying certain degradative processes of leaf aging, and it is clear that the role of auxins in the control of leaf senescence must also be considered. The surface treatment of both attached and detached autumn leaves of Prunus serrulata (2) revealed that droplet applications of the butyl ester of 2,4-D (10 to 100 ^ag.) in ethanol, would result in a retention of green and photosynthetically active chlorophyll below the spot, while the rest of the blade became yellow and senescent. Under these conditions, no roots were formed and there was no apparent cell enlargement or cell division. Autoradiograms of leaves treated [329] 330 D. J. Osborne and M. Hallawny with a radioactive butyl ester of 2,4-D labeled with C^* in the car- boxyl group showed that radioactivity was initially concentrated be- low the treated area with only traces of activity in the remainder of the blade. In attached leaves, activity was eventually confined to the areas below the applied drops and corresponded to the areas of tis- sue which remained green. It had therefore been possible to arrest senescence in a specific group of cells by a relatively high dose of 2,4-D and to maintain these cells within an area of similar cells containing little or none of the acid or related C^^-containing com- pounds. This method of applying 2,4-D offers a means of studying the effects of relatively high auxin concentrations upon the general metabolism of groups of cells in situ, and for studying differential senescence within a single leaf blade. The present communication is concerned with some of the changes which occur in the carbon and nitrogen fractions in the leaf blades of Euonymus japonica following such local applications of the butyl ester of 2,4-D. MATERIAL AND METHODS The experiments were carried out during the summer months of 1959 on well-developed Euonymus bushes growing out of doors. Only leaves from the second year wood were used. One spot of 2,4-D butyl ester in ethanol (26 /i.g//xl) was applied by micropipette to the lamina on one side of the main vein of the adax- ial surface of the blade, so that each leaf received 50 /xg. of the ester. Each drop spreads over an area approximately 1 to 1.5 cm. in diameter, and the outer margins of the spread were finely marked round Avith a nonwater soluble white ink. Control leaves receiving ethanol only were marked in a similar way. Within six days the leaf tissue bordering on a treated spot became visibly lighter green and later became progressively more yellow imtil the twelfth day from treatment, when the 2,4-D-treated area appeared as a fresh green spot in an otherwise yellowing blade. At intervals of 1, 3.5, 6, 12, and 13 days from the initial treatment, the respiration of different portions of the blade was measured and the distribution of nitrogen within these parts was determined. The estimations were carried out on 1 cm. leaf discs cut from the blade, antl each sample comprised six discs. From each 2,4-D-treated leaf, one disc was cut from within the area delimited by the spread of the spot, and a sec- ond from the untreated and opposite half of the leaf. One disc was cut from each control leaf. Role of Auxins in Control of Leaf Senescence 331 Respiration Measurements Oxygen consumption was measured by the Warburg teclinique. In the samples from the 2,4-D-treated areas it was essential that residual ester remaining on the surface of the discs should not penetrate the tissues through the cut edges. To avoid this, the six leaf discs were balanced on edge in an upright position on a thin layer of 2 per cent agar which covered the bottom of the flask. Water was added to the side arm and potassium hydroxide to the center well. Each flask was wrapped in a black bag and equilibrated at 20° C. for one hr.; measurements of oxygen consumption were made at half-hour in- tervals up to six hrs. The discs were removed from the flasks and stored in 80 per cent ethanol and later used for the estimation of the nitro- gen fractions. Nitrogen Measurements Total nitrogen, alcohol-insoluble and alcohol-soluble nitrogen Avere estimated separately. Each sample of six discs was macerated in 80 per cent (v/v) ethanol in a VirTis homogenizer and the suspen- sion made up to volume. After removal of an aliquot of the suspen- sion for the estimation of total nitrogen, the remaining suspension was centrifuged and an aliquot of the supernatant liquid used for the determination of alcohol-soluble nitrogen and the whole of the remaining precipitate used for the determination of the alcohol- insoluble fraction. Each sample was digested with selenium catalyst, made alkaline with alkaline metaborate, and the ammonia distilled off in vacuo into borate buffer which was titrated against HCl in the usual way. RESULTS There is a measurable effect of 2,4-D on attached Euonymits leaves as shown, within 24 hrs., by an increase in the rate of oxygen con- sumption in discs of leaf blade cut from the treated areas (Figure 1). The values for the rate of oxygen uptake of subsequent samples con- tinue to rise until the thirteenth day after treatment, when the 2,4-D- treated tissues are green and the remainder of the leaf is yellowing. Discs from the treated spots are then respiring oxygen over three times faster than discs from the control leaves. The rate of oxygen consumption of discs from the untreated halves of 2,4-D-treated leaves shows an initial, but less marked stimulation, but this rate falls off again after the sixth day and then remains 30 per cent higher than that of the controls. 332 D. J. Osborne and M. Hallaway J 300 O LJ ? I cn LlJ (T li. 200 C3 tr o X 100 "I r "1 r T r 2,4-D treated areos of leaf 2,4-D untreoled areas of leaf Control leoves 8 DAYS AFTER TREATMENT 10 14 Fig. 1. Oxygen consumption by leaf discs cut from 2,4-D-treated Euonymus leaves and from control leaves at various time intervals after treatment. Values indi- cated by triangles at I and 3.5 days are for ethanol-treated control leaves. Dupli- cate values are indicated bv vertical lines. The nitrogen values given in Figure 2 show that after 3.5 days no measurable changes had occurred in any of the nitrogen fractions; but by the sixth day a significant fall in both total and alcohol- insoluble nitrogen had taken place in the untreated areas of the 2,4-D-treated leaves. This coincided with the first visible signs of yellowing in the untreated parts of the blade. At no time was there a net loss of total nitrogen or of protein in the 2,4-D-treated areas. The evidence suggests that there is an ac- tual increase in both these fractions and it seemed likely that some of the nitrogen lost by the untreated parts of the leaf might be ac- cumulated within the 2,4-D-treated tissues. This supposition is sup- ported by the fact that yellowing occms first in the tissues immediately surrounding the treated spot; the premature senescence of these sur- rounding tissues might well be due to a migration of readily respir- able substrates from the areas of low auxin concentration towards those of high auxin concentration in which there is a high rate of oxygen consumption. Evidence for the acciunulation of nitrogen compounds and possibly also for carbon comjjoiuuls by the 2,4-D- treatcd tissues was obtained from the following experiments. Evidence for the Accumulation of Carbon Compounds The leaves of a small branch of an outdoor Euonymus bush were labeled with C^^ in the following way: the branch was enclosed in a Role of Auxins iti Control of Leaf Senescence 333 4 - X in I X (/> iij a: li- 0 e> TOTAL NITROGEN • 2:4-D treated areas of leaf 3 2:4-0 untreated areas of leaf O Control leaves INSOLUBLE NITROGEN ^'^-^ SOLUBLE NITROGEN 6 8 10 DAYS AFTER TREATMENT 14 Fig. 2. Nitrogen content of discs cut from 2,4-D-treated Euonymus leaves and from control leaves at various time intervals after treatment. Values which are significantly different (P = 0.65) from the respective control at any one time- point are marked with an asterisk. polyethylene bag attached to a flask containing BaCi403. Carbon di- oxide was liberated by injecting an excess of perchloric acid through a vial closure to give an internal concentration of approximately 2 per cent COo and a total of 50 microcuries of C^^ as Ci^Oa- Four hours later the bag was removed, and drops of 2,4-D ester in ethanol, or ethanol only, were applied to the leaves in the manner previously described. At intervals of 2.5, 6.5, and 12 days from the application of 2,4-D, 2i DAYS > 6i DAYS 12 DAYS CONTROL TREATED 2:4-D Fig. 3. Autoiadiogiams of Euoriyniiis leaves exposed lo CO, for 4 liouis. Left. Leaves suhscfiiieiitly treated willi drop of elhaiiol. Right. Leaves sul)sequently treated with a drop of ethanol conlaiiiiiig the l)iii\l csici of LM 1). Leaves sam- pled 21/^, 6i/[,, and 12 davs after tu\iiiiuiii. Role of Auxins in Control of Leaf Senescence 335 sample leaves were removed from the branch and subjected to the usual processes of autoradiography. Reference to the autoradiogram photographs reproduced in Fig- ure 3 indicate that there was an appreciable fixation of Ci^02 by all the leaves. Two and one-half days after treatment with 2,4-D some of the leaves showed considerable darkening near the boundary of the treated area indicating either an accumulation or a greater retention -^ of C^"* material. This is clearly visible (Figme 3, top) in several of the leaf veins leading to the treated spot. The center of the spot ap- pears paler than the surrounding leaf after 2.5 days, possibly as a re- sult of the more rapid loss of C^* as C^^Os due to the relatively higher rate of respiration in these areas. By 6.5 days and later, there is a marked concentration of C^^ in all the 2,4-D-treated spots. Evidence for the Accumulation of Nitrogen Compounds It should be noted that control nitrogen values for attached leaves (Figure 2) show daily variation. This variability from occasion to occasion in outdoor control plants is not surprising and has been discussed by Steward et al. (6) in considerable detail. He has shown that a wide variety of environmental conditions can produce marked effects upon the nitrogen composition of plants. Although the data in Figure 2 show an increase in both total and protein nitrogen in the 2,4-D-treated spots, a more critical investigation was made using de- tached leaves so that the majority of the original nitrogen should remain within the leaf and none could be transported away from the blade to other parts of the plant. The detached Euonymus leaves were kept in a relatively damp atmosphere in the following way. The cut end of each petiole was placed in a small tube containing 0.5 ml. of distilled water. To prevent drying out the base of each tube was embedded in 3 per cent agar in the bottom of a larger specimen tube which was plugged with cot- ton wool. The leaves were treated with a spot of ethanol containing 50 |Ltg. 2,4-D or with ethanol only, and were stored out of doors away from direct sunlight. Nineteen days later, the 2,4-D-treated spots were still green, but the remainder of the blade was yellowing. Respiration and nitrogen determinations were made on discs cut from the leaves, and the results are listed in Table 1. It is seen that there is again a / higher rate of oxygen consumption in the 2,4-D-treated tissues than ' in the untreated parts of the leaf, although both tissues are respiring faster than the controls. There is a loss of both total and alcohol- insoluble nitrogen in the untreated and yellowing areas of the 2,4-D- treated leaves and a statistically significant increase in the total nitro- ly' 336 D. J. Osborne and M. Hallaway Table 1 . Values for the respiration rate and for the nitrogen fractions of detached Euonymus leaves 19 days after treatment with 2,4-D or ethanol. Respiration ;ul. OJUv/G Fresh Wt. Nitrogen Fractions as Mg. N G Fresh Wt. Treatment Total Alcohol insoluble Alcohol soluble 0 days Control leaves 3.72 3.21 0 43 Detached for 19 days 108 195 164 3.81 4.38 2.83 3.22 3.33 2.11 Ethanol-treated area of control leaves 0 73 2,4-D-treated area of leaves (green) 2,4-D-untreated area of leaves (yellow) 0.99 0.49 Sign. diff. of nitrogen values P = 0.05 0.29 0.38 0.20 gen oi the treated spots. There is no evidence of a net hydrolysis of protein in the 2,4-D-treated spots, and the increase in total nitrogen in these areas can be accounted for by an increase in soluble nitrogen which must have been accumulated from the untreated parts of the blade. This confirms previous results obtained with our detached cherry leaves (2). DISCUSSION It is tempting to speculate if the maintenance of differential rates of metabolism within a leaf by local variations in the auxin con- centration could be a controlling factor in determining the move- ment of metabolites within the blade and thereby determining the tUiferential states of senescence of the cells. The roles of kinetin, ben- zimidazole, and gibberellin in controlling leaf senescence might also be due, in part, to an effect upon the accumulation of metabolites in treated parts of the blade. The mechanism for the stimulation of oxygen uptake in the 2,4-D- treated tissues remains to be investigated more fully. Since these tis- sues retain a photosynthetically active chlorophyll (2) and lose neither total nor protein nitrogen, in spite of the considerable changes which occur in these constituents in the surrounding cells, it might be that 2,4-D is acting in a manner analogous to that suggested for the thyroid hormone in animal tissues (4) by a stimulation of the basic metabolic rate of the cells. Role of Auxins in Control of Leaf Senescence 337 These experiments are still in their early stages. The method of applying 2,4-D to a leaf is providing us with a very valuable way of studying the effects of 2,4-D on groups of cells in situ, where they are surrounded by tissues in which the applied auxin is either absent or in a relatively very low concentration. SUMMARY There seems little doubt that the presence of a relatively high con- centration of 2,4-D in isolated groups of cells in Euonymus leaf tissue can maintain within these cells an abnormally high rate of respiration. The evidence from autoradiograms of Ci^-labeled leaves has shown that carbon compounds either move into or are preferentially re- tained in the areas of high oxygen consumption below the spot of 2,4-D. The values from the nitrogen determinations indicate that nitrogenous compoimds are accumulated within the 2,4-D-treated areas. These treated cells therefore comprise a specialized part of the leaf in which there is a high rate of metabolism and no net protein breakdown; they seem to act as metabolic sinks to which nitrogen and possibly carbon materials are drawn from the surrounding cells, with the result that there is a premature senescence in the untreated parts of the leaf which contain only a relatively low concentration of the applied auxin. The maintenance of differential metabolic rates within a leaf by local variations in the auxin concentration could be a controlling factor in determining the movement of metabolites within the blade and thereby determining the differential states of senescence of the cells. The roles of kinetin, benzimidazole, and gibberellin in controlling leaf senescence might also be due in part to an effect upon the accumulation of metabolites in treated parts of the blade. ACKNOWLEDGMENTS We are indebted to Mr. R. G. Powell of the Agricultural Research Council Unit of Experimental Agronomy, Oxford, for devising the simple method for supplying the attached Euonymus leaves with C^^Oo and to Dr. D. C. Smith for his advice with nitrogen determinations. We also wish to thank Prof. G. E. Blackman for his constant interest and encouragement. LITERATURE CITED 1. Brian, P. W., Petty, J. H. P., and Richmond, P. T. Effects of gibberellic acid on development of autumn colour and leaf -fall of deciduous woody plants. Nature. 183: 58-59. 1959. ^2. Osborne, D. J. Control of leaf senescence by auxins. Nature. 183: 1459, 1460. 1959. 338 D. J. Osborne and M. Hallaway 3. Person, C, Samborski, D. J., and Forsyth, F. R. Effect of benzimidazole on de- tached wheat leaves. Nature. 180: 1294, 1295. 1957. 4. Pitt-Rivers, R., and Tata, J. R. Thyroid Hormones. 247 pp. Pergamon Press, Inc., New York. 1959. 5. Richmond, A. E., and Lang, A. Effect of kinetin on protein content and sur- vival of detached Xanthium leaves. Science. 125: 650, 651. 1957. 6. Steward, F. C, Crane, F., Millar, K., Zacharius, R. M., Rabson, R., and Mar- golis, D. Nutritional and environmental effects on the nitrogen metabolism of plants. Symp. Soc. Exper. Biol. 13: 148-176. 1959. DISCUSSION Dr. Galston: I remember some recent papers by Dr. Reinhokl who found that the application of auxin to sunflower hypocotyl sections causes a rapid egress of ninhydrin-positive nitrogenous materials, largely ammonia, I believe. Yet, in your experiments, there was, if anything, a competitive advantage conferred by the feeding of auxin to the treated cells. I wonder if you can resolve this apparent dis- crepancy? Dr. Osborne: I can only give you a suggestion. In Dr. Reinhold's tissue there was no available surplus of substrates upon which the rapidly metabolizing tissues could draw, whereas in these leaf tissues we have substrates in the surrounding area of untreated leaf as a source of supply for the 2,4-D-treated cells. The auxin-treated area does not then have to break down nitrogenous materials inside its own piece of tissue to keep pace with the high metabolic rate. In Dr. Reinhold's experiments the loss of nitrogenous materials to the ex- ternal solution was depressed by the addition of sucrose or succinate. Dr. Galston: Do you imply that there is an increased senescence of the neighboring cells caused by a local application of 2,4-D? Dr. Osborne: Yes, and may I add just a few comments on some work I did in the tropics on leaves of Combretiiui (Jour. Trop. Agric. 35: 145. 1958). Spot applications of 2,4-D were made on leaves and within 7 days the blade had yellowed, leaving the 2,4-D-treated area as a green spot on a yellow background. The leaves then ab- scised. II one collected leaves on the first two da)s after treatment and diffused the petioles into blocks of agar and tested the blocks in an abscission test, the petiolar diflusate was abscission-retarding. This might be expected following applic:ition of a stibstance such as 2,4-D. After about 3 days, when yellowing of the leaves was ap- parent, the petiolar diffusate had little or no activity in the abscission test. After 4, 5, and 6 days, the leaves became increasingly yellow and petiolar diffusate became increasingly abscission-accelerating. Since petiolar diffusates from naturally senescing leaves are abscission-ac- celerating, I suggest that these results give a further indication that Role of Auxins in Control of Leaf Senescence 339 one is, in fact, getting increased senescence in the sm-rounding tis- sues following a local 2,4-D treatment. Dr. Wareing: ^Ve have been doing some experiments which bear on Dr. Osborne's results. We are primarily interested in the possible effect of auxin on translocation, and have been feeding labeled sugar to older leaves of bean plants in the basal region of the plant and tracing the movement of the sugar without any applied lAA. As many people have found, we got movement from the applied mature leaf toward the young growing leaf. There is no appreciable movement into already mature leaves between the applied leaf and the shoot apex. If lAA is applied to one of these mature leaves, then sucrose moves into that leaf instead of into the young growing leaves. So, here again, ^ve have movement of labeled nutrients toward a region where hormone is applied. Dr. Crafts: We have found, using labeled urea, that we get an ex- tremely rapid splitting of the urea and synthesis of the labeled car- bon dioxide into sugar. The sugar moved in a perfectly normal fash- ion, indicating that this labeled urea may be a much handier tool than the sugar — cheaper, much more readily absorbed by the plant, and apparently perfectly normal in its distribution. Dr. Bach: A minor technical point: Are you sure that the labeling you observed after the application of radioactive 2,4-D is actually in the tissue, not on top of it, or perhaps dissolved in the waxy layers? Dr. Osborne: As Dr. Crafts has just said, perhaps the tissues would not respond unless some of the 2,4-D entered the leaf. The green areas correspond closely to the areas of radioactivity, but I have not extracted the leaves to determine the amount of activity actually within the tissue. Dr. Freed: In Dr. Osborne's discussion here I was reminded of a famous name in auxinology, that of Dr. Ezra J. Kraus. He retired to Corvallis, Oregon, and shortly after his retirement suggested an ex- periment which consisted of treating a plant with lAA and 2,4-D and then measuring the amount of sugar and phosphorus accumulating in the various areas of the leaf. Now this was not with exogenously applied sugar or phosphate but with only the normal metabolic su- gars, etc. Results showed an accumulation in the hypocotyl of both the sugar and phosphate in the plant. Radioautograms and chiomato- graphic studies showed that the 2,4-D accumulated in the bean hypo- cotyl, bearing out Dr. Osborne's findings. Dr. Osborne: We have done a few experiments in which we fed the plants labeled phosphate and then put on the 2,4-D spots after- wards, but we found no evidence of an accumulation of phosphate in 340 D. ]. Osborne and M. HaUaway the 2,4-D-treated areas after 1 1 days. The leaf seems to be uniformly labeled. Dr. Freed: ^Vell, 2,4-D applied to a plant apparently inhibits phos- phate uptake, but the endogenous phosphate accumulates in the areas with 2,4-D largely because of the stimulated respiration. Dr. Wittwer: A number of years ago, we studied the effect of di- ethyl ether on the movement of calcium in the bean plant, then fol- lowed this with work on the effects of growth substances that sus- pended polarity. We found that in line with the results just men- tioned by Dr. Osborne, there are a number of mineral nutrients that are altered in their transport and distribution in the plant following the application of such growth substances as 2,3,5-triiodobenzoic acid, 1-naphthaleneacetic acid, and maleic hydrazide. Samish (Plant Analy- sis and Fertilizer Problems, pp. 156-165 [Institute de Recherches pour les Huiles et Oleagineaux, Paris, France, 263 pp. ], 1954) and Kessler and Moscicki (Plant Physiol., 33: 70. 1958) both reported a greening up of chlorotic foliage following treatment with growth substances. Maleic hydrazide, 1-naphthaleneacetic acid, and 2,3,5-triiodobenzoic acid have promoted the movement and transport of iron and calcium in tissues where otherwise it would not occur. S. M. SIEGEL and F. PORTO Union Carbide Research Institute Oxidants, Antioxidants, and Growth Regulation The re-establishment of interest in oxygen toxicity (6,8,9, 10, 11,25) has provided a new orientation in oxygen biochemistry. Recognition of the dual role of oxygen in life process opens the way to a more fundamental understanding of interrelationships involving the phys- ical organic chemistry of oxidation, the metabolic machinery of the cell, and processes of growth and differentiation. Interest in oxygen poisoning has led to the discovery that many radiation protectant-reducing compounds often active in catalytic quantities also provide a biological defense against oxygen (8, 11, 25). Such an overlap of protective agents is a logical consequence of the well-established interdependence between oxygen and ionizing radia- tion in inflicting cellular damage (8, 16). It has been suggested that damage produced by these agents is no more than an intensification of activity along already-existing radical-mediated (that is, one- electron) pathways of oxidation-reduction (25, 28), and that deterior- ative changes so effected, even by oxygen alone, differ little from the terminal processes of senescence as they ordinarily take place. Thus, it has been concluded (29) that "The life-shortening action of radia- tion involves the induction of ... . degenerative changes and stimu- lates in many respects acceleration of the natural aging process." The demonstration in the early 1950's of oxygen and antioxidant effects in heat damage led similarly to a concept linking tissue damage and accelerated aging processes (2). These and other considerations, together with the recognition of antioxidant activity as a function of electron mobility (26, 27), have led to the formulation of the Regulator-Antioxidant Hypothesis, which seeks to relate regulation of growth, development, and aging [341] 342 S. M. Siegel and F. Porto to the balance of oxidants (electron acceptors) and antioxidants (electron donors) in the cell tissue or organism: A. Definition and properties of antioxidants. 1. Any electron donor which inhibits the oxidation of suitable labile substrates with high stoichiometric efficiency is an anti- oxidant. 2. Antioxidants may operate by: a. Reaction with an intermediate which is the electron-de- ficient member of an oxidation-reduction equilibrium. b. Trapping or deactivation of radicals or ions. c. Reaction wuth an oxidant which would otherwise attack a labile substrate. d. Reaction or interaction with the electron-deficient form of an oxidation (electron-transporting) catalyst. Both electron- transfer and TT-complex formation are included. 3. Antioxidant efficiency derives from the following mechanisms: a. Participation in a cycle wherein a suitable electron source can regenerate the antioxidant from its oxidized form. b. Trapping of radical or ionic initiators of chain reactions. c. Reducing the efficiency of an electron-transport catalyst with which it interacts. B. Biochemical activities of antioxidants derive from the following: 1. Antioxidants protect labile cellular components against attack by primary oxidants such as O2 or derivative oxidants including H0O2 and other peroxides, oxidizing radicals, ions, or such moieties when bound in macromolecules. a. Non-specific protection entails a general shift of oxidation- reduction equilibria toward the reduced conditions. b. Specific protection may be conferred by thermodynamic and geometric restrictions which fix the antioxidant in electron transfer chains or localize it on selectively adsorb- ing surface sites of macromolecules or membranes. 2. Antioxidants may influence or regulate specific metabolic path- ways: a. By reductive activation of the inactive oxidized forms of enzymes (e.g., Enz - SS - R -[- e -f- H^^ ^ Enz - SH -[- ' ^ ' ^)- b. By blocking electron-transport catalysts, antioxiciants may "shut down" specific metabolic pathways (e.g., inhibition of flavoproteins by formation of a 7r-complex between anti- oxidant and riboflavin). c. As mobile electronic systems by serving themselves as electron carriers or carrier moieties of oxidizing enzymes. Oxidants, Antioxidants, and Growth Regulation 343 Oxidant toxicity - the physiological consequences of antioxidant action. 1. Their essential role in metabolism notwithstanding, oxygen and other oxidants attack essential cellular components, and bring about a cumulative loss in essential functions. 2. Ontogeny (the developmental sequence of the individual) be- gins with an excess of antioxidants, a relatively low oxygen tension, and frequently, a low oxygen demand. 3. As development proceeds, oxidized substances accumulate. The rate of oxidant accumulation will vary with the environmental oxygen supply. Increase in environmental oxygen may be gradual, but frequently involves a discontinuity such as hatch- ing or birth. 4. As development proceeds further in continuous contact with oxygen, oxidized cellular components accumulate, increasing the ratio oxidant/antioxidant (or electron acceptor /electron donor). As the acceptor-donor balance shifts toward the oxi- dized state, cellular activities are altered: a. A loss in proliferative capacity - decreased mitotic activity - stabilized cell populations in tissues and organs. Differ- ential oxidant tolerance results in unequal cessation of suppression of cell division. b. Certain cellular growth activities may persist in the more oxidized state, or may be favored at a higher acceptor-donor ratio than cell division. Cell enlargement, especially in plants, is indicated here. c. Further shifts in the acceptor-donor balance terminate all growth processes, but allow maintenance of the individual cell. 5. Maintenance of the nongrowing cell, tissue, or organism char- acterizes the prematuration state. a. Prematurity is an unstable transition state. The acceptor- donor balance either attains a plateau or lies within range of values analogous to a range of maximum buffering capacity. b. Further maturation processes may entail chemical changes indicative of the progressive domination of the cell by oxi- dants and oxidation. Examples are melanization and lignifi- cation. c. Initiation of major secondary chemical changes may shorten the transition state leading to rapid loss of function. 6. Beyond the efficient "buffer range," accumulation of oxidative 344 5. M. Siegel and F. Porto "faults" is an accelerated process leading to deterioration of structures and functions in nucleus and crytoplasm. Senescent degeneration is then under way. 7. Attainment of some large acceptor/donor ratio corresponds to major losses in maintenance and synthetic capacities (e.g. fail- ure in protein synthesis) limiting survival of the cell, etc., to the lifetime of residual enzymes and other already formed species. The terminal phase of senescence is in progress. 8. Antioxidants which promote growth may do so by maintenance of a relatively youthful, reduced state. They enhance cellular abilities to resist oxidant damage. 9. Increased oxidant stress shifts the role of antioxidants from functions in quantitative growth control to functions in survival on an all-or-none basis. 10. Under reduced oxidant stress hormonal requirements will be lowered. Growth can be enhanced by increase in antioxidant or decrease in oxidant as long as their ratio lies within suitable limits. Further, maturation processes will be retarded. 11. Some low value for the acceptor/donor ratio should correspond to the chemically reduced state which favors proliferating cells. Electron mobility is characteristic of most estrogens, androgens, and carcinogens, and addition of such donors must lower the ratio into this critical region. ANTIOXIDANT ACTIVITY AND GROWTH General Considerations The existence of a relationship between reducing agents and growth is established. In animal systems, the thiol-disulfide equilibrium has been implicated in cellular multiplication (1, 19). Thyroxine, a powerful antioxidant (23), is implicated directly in growth processes, possibly by way of its ability to shift the thiol-disulfide equilibrium toward the reduced state (13). Other substances which stimulate tissue growth or regeneration include phenols, polynuclear hydrocarbons, arylamines (10,11), carbazoles, stilbenes, and phenothiazine, and tri- phenylmethane dyes (1, 17, 19). Among these structures a common fea- ture is their high level of electron availability. All of these molecular types contain mobile tt- orbital electrons or coupled ??(nonbonding)- TT - systems if atoms such as N, S, or O are on or in the aromatic system. Antioxidant or electron-donor properties may also be found among plant regulators, including indoles, aryloxy compounds, unsaturated hydrocarbon derivatives, coumarins, etc. (19, 23, 26, 27). Among the conventional reducing agents involved in growth processes are ascorbic acid and the thiols. In contrast with the growth-promoting inhibitors Oxidants, Antioxidants, and Growth Regulation 345 of oxidation, more oxidized substances may be physiologically inactive or even inhibitory. Oxindoles are representative of the inactive state; quinones as thiol reagents and H-abstractors serve as harmful oxidants. Quinonoid substances have been implicated in normal cessation of growth and onset of differentiation in plants (18, 30). Quinones are known experimentally for their antimitotic and radiomimetic activities (19). Experimental Studies Methods employed in evaluation of antioxidants may be found in published or forthcoming accounts. The experimental oxidation systems used include iodide-HoOo (26,27); eugenol, peroxidase- HoOo (22); eugenol, celery vascular tissue-HoOo (21,24); and to a lesser degree iodide, peroxidase-HoOo and pyrogallol-Oo (23). Measured oxidation products are, respectively, iodine; dimerization product; lignin; and in the last two systems iodine, once again, and a phenol polymer-polyquinone mixture. The growth responses studied included germination of onion {Allium cepa, 'Yellow Globe'); elongation of turnip radicle {Brassica rapa, 'Purple Top White Globe'); elongation of cucumber hypocotyl sections (Cuciimis sativus, 'Improved Long Green'); and elongation of flower stalk sections from Taraxacum officinale. All test materials were cultured in solutions buffered at pH 6.65 (0.066Af phosphate) under 50 footcandles constant illumination (22, 27). The chemical test systems have been used to demonstrate anti- oxidant activity in the following compounds: Indoles, including in- dole, indoline, methyl- and phenylindoles, lAA; other indolealkanoic acids, 5-hydroxy-IAA, tryptamine, tryptophane, serotonin, and car- bazole; Pyrroles, including pyrrole, methyl- and phenylpyrroles, the bile pigments, and pyrrolidine; Hydrazines, including hydrazine and its salicylyl-, malonyl-, maleyl-, isonicotinyl-, aryl-, and alkyl- deri- vatives; Diazines and miscellaneous heterocyclic compounds, including purine, mercapto-purines, benzimidazole, pteridines, and benzthio- phene; Amities, including alkylamines, phenylethylamines, and aryl- amines; Aryloxy compounds, including diphenylether, benzofuran, anisole, thyronine, and iodothyronines (thyroxine, for example); and various thiols, phenols, ascorbic acid, and Co^^ salts. Activity-constitution relations have emerged in some instances (23, 27). Thus antioxidant efficiency among the indoles is reduced by electron-withdrawing groups, as in oxindole, oxindone, or indole aldehyde. Less marked reduction results from 3-substitution. Both deactivating effects are illustrated in the series indole > skatole>> indoleformic acid. Deactivation may also be effected by decreased 346 5. M. Sicgcl and F. Porto electron availability at the heteroatom, as in indole > benzthiophene> benzof uran > indene. Other examples of chemically meaningful effects are deactivation of phenylhydrazine by the p-nitro group and enhancement of the activity of pyrrole by methylation at positions 2 and 5. Hydrogen- ation of pyrrole (yielding pyrrolidine) reduces its efficiency to the level of aliphatic amines which usually are weak reducing agents, whereas indole, after hydrogenation to indoline, retains its funda- mental aromatic character (as an A'^-substituted aniline), i.e. remains highly active. Of approximately 80 compounds studied, only one-fourth have been tested as growth regulators, although most are known to have some form of biological activity. Growth of the turnip radicle in IQ-^M test solutions was stimulated by indole, methylindoles; isoni- Table 1 . Growth promoting activity of antioxidants. Concn. M X 106 Elongation of 0.9 Mm. Cucumis Sections in 12 Hrs. Relative to: Compound Endogenous as 100 lAA as 100 Pyrrole Skatole Serotonin 2.0 5.0 0.1 1.0 2.0 2.0 1 .0 1.0 1.0 1.0 1.0 164 130 133 175 160 138 128 128 170 142 150 100 100 100 120 94 Thyroxine Hydrazines Methyl- 1,1 -Dimethyl- 1,1-Diphenyl- 1-Naphthyl- Isonicotinyl- 61 45 45 105 63 75 Elongation of 1 .2 Mm. Taraxacum Sections in 24 Hrs. Relative to: Endogenous as 100 lAA as 100 Pyrrole Skatole 1.0 10.0 1.0 2.0 1.0 1 n 1 .0 1.0 1.0 1.0 135 147 177 138 122 122 133 133 144 153 30 31 63 Thyroxine Hydrazines Methyl- .... 1,1 -Dimethyl- 1.1-Diphcnyl- 1-Naphthyl- /)-Bromophenyl- Isonicotinyl- 32 18 18 27 27 36 43 Oxidants, Antioxidants, and Groioth Regulation 347 cotinyl hydrazine (isoniazid) and other hydrazines; and by tyraminc and mescaline. The phenylethylamines promoted growth only 20 to 25 per cent, but the other compounds, all more efficient anti- oxidants, increased growth 90 to 150 per cent. In the two section tests, extensive comparisons were made between lAA and a number of other antioxidants as elongation promoters (Table 1). Cucumis elongation is promoted to approximately the same degree by lAA, pyrrole, skatole, serotonin, and diphenylhy- drazine. The other compounds listed show one-half to three-quarters of lAA activity. Taraxacwn elongation is more specific for lAA, but even the weakest growth promoters were one-fifth as active as lAA. When chemical and biological activities are considered jointly, it is apparent that substances of different constitution but high anti- oxidant efficiency, show qualitatively similar biological effects (Table 2). Percentage deviations from appropriate controls have been tabu- lated to show both the direction and magnitude of the effects. Although some correlation may be found in comparing specific chemical and biological tests, experience has shown that this is not generally true. We must recognize that, ideally, antioxidant activity is a measure of electronic behavior whereas over-all biological effec- tiveness also depends upon thermodynamic and geometric properties which are the determinants of transport and localization. Lignification is one of the processes which characterize maturation and cellular senescence in the green plant (21,23). The experimental biosynthesis of lignin proceeds through a radical initiated chain mechanism and offers a model for the natural process. Experimentally, quinones accelerate lignin synthesis, a role consistent with their ap- pearance in differentiating tissues. In contrast, various metabolites, especially DPN, are powerful inhibitors of lignin formation. Exten- sive lignification is thus indicative of cellular oxidation-reduction imbalance. It can be "forced" upon young cells experimentally but is most pronounced in xylem differentiation where it accompanies or is accompanied by protoplastic degeneration. lAA and other anti- oxidants both inhibit lignin synthesis and promote elongation. The growth-promoting activities of some biological agents acting as anti- oxidants in their proposed protective and metabolic roles may be reflected in their ability to delay the onset of lignification and at- tendant deteriorative oxidative processes. OXIDANT-ANTIOXIDANT INTERACTIONS IN GROWTH General Considerations Application of reductants accelerates regeneration whereas callus formation is inhibited by oxygen and other oxidants (28). Cysteine, Co+2 salts, propyl gallate, and sympathomimetic amines have been 348 5. M. Siegel and F. Porto used successfully to protect ciliates, rodents, and vascular plants against oxygen poisoning. The culture of Clostridium tetani, an obligate anaerobe, in air can be accomplished in the presence of C0+2 salts (3). In this case, the antioxidant protects the organism against ordinary atmospheric levels of oxidant. Both growth and enzyme levels in embryos of Phaseolus vulgaris ('Red Kidney') are reduced by incubation at elevated oxygen ten- sions (20, 23). Other signs of oxygen damage in a variety of plants sug- gest disturbance of the protoplast membrane and accelerated aging of leaf tissue (6). Pea root tissues damaged by incubation in pure Oo show increased peroxide production and increased lAA destruction (7). The presence of C0+2 suppresses excessive oxidations and reduces visible injury. Mitochondria can be protected against harmful levels of oxygen by thyroxine (15). Enzymes whose activity is thiol-dependent are sometimes readily inactivated by oxygen, even at atmospheric level (14). Tyrosyl groups may be reasonably put forth as an addi- tional oxidation-sensitive member of the peptide chain. Peroxides participate in the depolymerization of deoxyribonucleic acid and even more drastic chemical changes occur at elevated oxygen ten- sions (4, 12). Organic peroxides formed from cellular components can in turn attack other constituents such as thiols (5). Experimental Studies The study of oxygen and oxidant toxicity consists of simple germination and growth tests as previously described. Experimentally, responses of plant systems both to increased oxidant and to temporary removal of oxidant stress will be considered. The recognition of peroxides as probable intermediates in oxygen poisoning has led to their use in experiments. Damage to lettuce seed {Lactuca sativa, 'Iceberg') incubated in hy- drogen peroxide was reduced markedly in the presence of hydrazine, although the latter alone at the concentration used was also toxic (Table 3). A reciprocal relationship is indicated in the mutual can- cellation of toxic effects of oxidant and antioxidant. A similar re- sponse is the removal of inhibitory effects of high levels of lAA by elevated oxygen tension. Organic peroxides, which are not decom- posed by catalase, are generally more toxic than HoOo. Nevertheless, it is possible to obtain an appreciable amount of protection against their effects by the use of suitable antioxidants. For example, indole has been employed as a partial protectant against p-menthane hydro- peroxide. o u u O O 00 CM o CO in n- I SO CO + + + o o C .2 -a 'x o -o c o bo c o c -g c -a o 1/ V _> *4-» a. c a -a .2 '■*-> a < -a V o 3 -d o i< V be C O be ■*-' C V u lU Ph N 2 'S o c o >- o o C7^ O o lO o so CO CO o o CO o o u-i in 1 + + + CO in 00 00 o (N CO to 1 + + + o -a c O + "o in in in (M in + + + < < CO •^ in t~-- I I CO so ^ + + + U 2 ^ c 3< XXX I o X X X X o o CM 6 c _o '■*-» -a O -o "y. p C a o O ffi -i -^ V -o ■■3 o o G V bO 3 o c bD 3 C^ 0 C 0 u 0 s -a a, Si be a 3 0 3 s 'S ^ ^J ! 0 h G "(3 o 350 S. M. Siegel and F. Porto Table 3. Interaction of peroxides and antioxidants in seed germination. Oxidant and Concentration Turnip Seed Germination (Per Cent) With Indole at: /?-Menthane hydroperoxide, .\/ X 10-^ 0 0. 05 0.50 1.0 X \Q-^M 0 79 60 50 50 2 44 52 55 58 5 12 16 20 30 50 0 6 6 10 Lettuce Seed Germination (P( Hydrazine at :r Cent) With HP2, M X 10-3 0 0. 2 1.0 X lO-^M 0 96 80 60 10 60 66 82 20 8 14 36 50 0 0 0 Growth promotion under ordinary atmospheric conditions is com- mon to a number of protectants against oxygen stress. Thus lAA and indole reduce damage by oxygen, ozone, and organic peroxide; mesca- line and isoniazid protect against ozone; C0+2 protects against oxy- gen, hydrogen peroxide, and organic peroxides. One example will illustrate the effects of simultaneous treatment with several protectants: lettuce seed in 5 X 10"^ Af p-menthane hydro- peroxide failed to germinate even after 100 hrs. at 25°C. With lO-^M ascorbic acid, 4 seeds germinated in a population of 200; with 10-^M lAA or CoCU, 10 germinated. Addition of ascorbic acid to lAA had no further effect, but mixtures of CoClo either with ascorbic acid or lAA permitted germination of 14 seeds. Finally, all three protectants combined enabled 26 seeds to germinate. If the antioxidant properties of hormones are functionally im- portant, hormonal recjuirements should be sensitive to environmental oxidant level. This proposition was tested with elevated levels of oxygen and peroxides. Reduction in oxidant level should lower the hormone requirement. Elimination of the aerobic Co^- requirement for C. tctani by removal of oxygen provides a partial test of the pro- posed relationship. AVhen 2-week-old cucumber seedlings are held under anaerobic condition (argon) for a limited period, the subse- quent growth of hypocotyl sections is enhanced, and scnsiti\ity to Oxidants, Antioxidants, and GroxvUi Regulation 351 Table 4. The effect of anaerobic pre-conditioning on growth of cucumoer hypo- cotyls. Treatment Elongation in 12 Hrs., Mm. In Buffer In 1.5 X 10-«MIAA lAA Buffer (per cent) Air control 1.0 2.1 210 Argon: 1.5 hrs. Air: 4.5 hrs. 1.0 2.0 200 3.0 hrs. 3.0 hrs. 1.4 2.2 154 6.0 hrs. 0.0 hrs. 2.8 2.0 71 Submerged in water 6.0 hrs. 2.1 2.1 100 lAA diminished (Table 4). The data show that a period of anaerobic pre-conditioning can replace the external auxin requirement, and even render moderately inhibitory an ordinarily optimal lAA level. Anaerobic pre-conditioning may be equivalent to addition of an anti- oxidant such as Co+^ which increases growth by preventing lAA destruction, or may effect a more general increase in the reduction potential of the cell, delaying oxidation of many labile components. SUMMARY This paper calls attention to a new interrelationship between oxi- dation and growth. The antioxidant properties of known hormonal entities provide the link between these two processes. Geometrically varied growth promoters share in common the ability to inhibit lignin synthesis and other oxidations diagnostic of unbalanced oxidation- reduction states in maturing (aging) cells. The toxic effects of oxygen constitute a major contribution to degenerative changes in cell senes- cence. Accordingly hormonal substances with antioxidant properties have been assigned a protective role by buffering the cell against cumu- lative damage by oxidants. The dynamic and continuous relation be- tween oxidants and antioxidants is illustrated by substances which promote growth under ordinary aerobic conditions, become survival factors at elevated oxidant levels, and yet lose their effectiveness, or inhibit, when the oxidant level is reduced. Three general functions are proposed for antioxidants as bioregulators. First, antioxidants serve as protectants for specific structures or through their general effects on oxidation-reduction balance. Second, antioxidants may serve as metabolic regulators by selectively blocking specific pathways. Finally, as mobile electronic systems, antioxidants may act as cofactors in electron transport. 352 5. M. Siegel and F. Porto LITERATURE CITED 1. Brachet, J. Cliemical Embryology. 533 pp. Interscience Publishers, Inc., New York. 1950. 2. Commoner, B., Lippincott, B., and Passonneau, J. V. Electron-spin resonance studies of free-radical intermediates in oxidation-reduction enzvme systems. Proc. Nat. Acad. Sci. U. S. 44: 1099-1110. 1958. 3. Dedic, G. A., and Koch, O. G. Aerobic cultivation of Clostridium tetani in the presence of cobalt. Jour. Bact. 71: 126. 1956. 4. Demyanovskaya, N. S., and Znamenskaya, M. P. Effect of oxvgen on deoxy- ribonucleic acid of mycelium of actinomycetes. Dokl. Akad. Nauk SSSR. 114: 856-858. 1957. 5. Dubouloz, P., and Fondarai, J. Sur le metabolisme des peroxydes lipidiques. Action des peroxydes lipidiques sur les groupements thiols proteiqucs. Bui. Soc. Chim. Biol. 35: 819-826. 1953. 6. Eliasson, L. The inhibitory effect of oxygen on the growth of wheat roots. Physiol. Plant. 11: 572-584. 1958. 7. Galston, A. W., and Siegel, S. M. Antiperoxidative action of cobaltous ion and its consequences for plant growth. Science. 120: 1070, 1071. 1951. 8. Gerschman, R., Gilbert, D. L., Nye, S. W., Dwyer, P., and Fenn. W. O. Oxygen poisoning and x-irradiation: a mechanism in common. Science. 119: 623-626. 1954. 9. , Gilbert, D. L., Nye, S. \V., Nadig, P. W., and Fenn, \V. O. Role of adrenalectomy and adrenal-cortical honnones in oxygen poisoning. .\mer Jour. Physiol. 178: 346-350. 1954. 10. Gilbert, D. L., Nye, S. W., Price, W. E., Jr., and Fenn. AV. O. Effects of autonomic drugs and of adrenal glands on oxygen poisoning. Proc. Soc. Exper. Biol. Med. 88: 617-621. 1955. 11. , Nye, S. W., Gilbert, D. L., Dwyer, P., and Fenn, W. O. Studies on oxvgen poisoning: protective effect of /3-mercaptoethylamine. Proc. Soc. Exper. Bioi. Med. 85: 75-77. 1951. 12. Gilbert, D. L., Gerschman, R., Cohen, J., and Sherwood, \V. The influence of high oxygen pressures on the viscosity of solutions of sodiiun desoxyribonucleic acid and sodium alginate. Jour. Amer. Chem. Soc. 79: 5677-5680. 1957. 13. Goldstein, B., and Gotortseva, E. The effect of the thyroid hormone on SH groups. Biokhimiya. 22: 994-998. 1958. 14. ITaugaard, N. Oxygen poisoning. XI. The relation between inactivation of enzymes by oxygen and essential sulfhydryl groups. Jour. Biol. Chem. 164: 265-270. 1946. ' 15. Kripke, B. J., and Bevcr, A. T. Thyroxine and succinate oxidation. Arch. Biochem. Biophys. 60: 320-328. 1956. 16. Kronstad, W. E., Nilan, R. A., and Konzak, C. F. Mutagenic effect of oxygen on barley seeds. Science. 129: 1618. 1959. 17. Levy, B. M. New method for the rapid determination of lathvrogenic agents. Science. 129: 720. 1959. 18. Reeve, R. M. Histological and histochemical changes in developing and ripen- ing peaches. 1. The catechol tannins. .Amer. Jour. Bot. 46: 210-217. 1939. 19. Sexton, W. A. Chemical Constitution and Biological Activity. 2nd ed. 124 pp. D. Van Nostrand, Inc., New York. 1953. 20. Siegel, S. M. Effects of exposures of seeds to various physical agents. II. Phy- siological and chemical aspects of heat injury in the red kidney iiean embryo. Bot. Gaz. 114: 297-312. 1953. Oxidants, Antioxidants, and Growth Regulation 353 21. . Structural factors in polymerization: the matrix in aromatic hio- polvmer formation, pp. 37-63. In: T. Hayashi (cd.) Subcellular Particles. Amer. Physiol. Soc. Washington. 1959. 22. . Elongation-promoting activities of antioxidants. Amer. Jour. Bot. (In preparation.) 23. , and Frost, P. Inhibition of iodide oxidation by thyroxine and other antioxidants. Proc. Nat. Acad. Sci. U. S. 45: 1379-1382. 1959. 24. , Frost, P., and Porto, F. Effects of indoleacetic acid and other oxidation regulators on in vitro peroxidation and experimental conversion of eugenol to lignin. Plant Physiol. 35: 163-167. 1960. 25. , and Gerschman, R. A study of the toxic effects of elevated oxygen tension on plants. Physiol. Plant. 12: 314-323. 1959. 26. , Porto, F., and Frost, P. Inhibition of pyrogallol oxidation by 3-indole- acetic acid. Arch. Biochem. Biophys. 82: 330-334. 1959. 27. , Porto, F., and Frost, P. Bio-regulatory activity and nitrogen function in organic compounds. Antioxidant properties and their physiological signi- ficance. Physiol. Plant. 12: 727-741. 1959. 28. Sinjukhin, A. The role of redox potential changes in plant regeneration pro- cesses. Biophysika. 3: 284-288. 1958. 29. Upton, A. Ionizing radiation and the aging process. Jour. Geront. 12: 306-311. 1957. 30. Van Fleet, D. S. Analysis of the histochemical localization of peroxidase related to the differentiation of plant tissues. Canad. Jour. Bot. 37: 449-458. 1959. A. W. GALSTON and RAVINDAR KAUR Yale University The Intracellular Locale of Auxin Action: An Effect of Auxin on the Physical State of Cytoplasmic Proteins' Modern theories of the mechanism of auxin action emphasize the demonstrable effects of auxin in increasing the plastic extensibility of the cell wall (4). These effects, first noted by Heyn more than 25 years ago, are attractive in explaining the cell elongational aspects of auxin action, since it is clear that the wall must be plasticized if elongation is to occur. This type of theory is not completely satis- factory in explaining auxin action, however, in that auxin is also known to produce marked changes in the cytoplasm (5, 8), and to initiate mitotic activity in certain cells (7). These latter effects are difficult to account for in terms of changes in cell walls mediated by auxin. In the course of investigations on the intracellular location of Ci^-carboxyl-labeled 2,4-dichlorophenoxyacetic acid (2,4-D) fed to green and etiolated pea stem segments, we discovered fortuitously a marked effect of this compound and of other auxins on the physical state of the proteins in a centrifugal supernatant devoid of all cellular particulates. The effect noted was a marked decrease in the heat coagulability of the proteins in the auxin-treated tissues as compared with the control tissues. The magnitude of the effect is as great as the often cited eflEects of auxin on the cell wall, and is therefore to be considered as of possible significance in the growth reaction initiated by auxin. We wish to emphasize that the altered heat coagulability of the cytoplasmic proteins is probably not, per se, the important physical property possibly related to growth; it is, however, a possible •Aided by grants from the National Science Foundation and the U.S. Public Health Service. We are indebted to Mary Lyons and Drs. S. Maheshwari and N. Maheshwari for assistance with the experiments on heat coagulability of the proteins. [355] 356 A. W. Galston and R. Kaur indicator of some fundamental change in the configuration of the protein molecules, the exact nature of which remains to be delineated. MATERIALS AND METHODS 'Alaska' peas were used throughout these investigations, being grown under conditions already described in another article in this volume (2). Etiolated and light-grown sub-apical stem sections were grown overnight in the presence or absence of an auxin. After meas- urement of growth, the sections were homogenized in ice-cold 0.25M sucrose -\- .00 IM ethylenediaminetetraacetic acid (EDTA) in a pre- chilled mortar and pestle to yield a suspension of 12.5 mg. fresh wt/ ml. This suspension was repeatedly centrifuged at various speeds in an International Refrigerated Centrifuge (Model PR-2) to yield, suc- cessively, cell wall fragments and unbroken cells, chloroplasts (when present), mitochondria, microsomes, and a final centrifugal superna- tant fraction containing the soluble proteins of the cell. The fate of the nuclei in such a fractionation is unknown, but they or their fragments are presumed to sediment along with the plastids or possibly mi- tochondria. In certain of the experiments, C^^-carboxyl-labeled 2,4-D ^vas used as the auxin. This was purchased from Nuclear Chicago Com- pany, and had an activity of 950 microcuries per millimole. Aliquots from the fractionation were pipetted on to l^^" X %2" stainless steel planchets, and were counted in a stream of Q-gas with a Nuclear Chi- cago model D-47 gas flow counter equipped with a micromil window, mounted in a model M-5 sample changer, and attached to a model 186 decade Scaler. RESULTS The fractionation scheme employed for tracing the intracellular localization of exogenously applied labeled 2,4-D is shown in Figure 1. W'ith green sections, the 2,4-D concentration applied was the 3 X 10-^M, which is approximately optimal for growth in the light and in the presence of 1 per cent sucrose. Etiolated sections were given this same concentration of 2,4-D, although it is approximately 100 times too high for their optimal growth in length, but about optimal for increase in fresh weight. The distribution of radioactivity in the various fractions of green and etiolated cells is seen in Table 1. It should be noted that the final supernatant contains a major part of the activity, as does the first precipitate, P,, consisting of unbroken cells and cell walls. ^Vhen the ?! fractions were reground and recentrifuged, considerable ac- ti\ity was lost from the precipitate to the wash medium, two such 50 to 200 5 mm. long sub-apical pea stem sections Crushed in pre-chillcd mortar and honiosrcnizcd with 8 to 16 ml. of 0.25M sucrose + O.OOlAf EDTA. Clentrifuged 30 min. at 600 x gravity. ^ CELL \\ ALLS and TISSUE FRAGMENTS Si CHLOROPLASTS P.. Centrifuged 15 min. at 1,500 X gravity MITOCHONDRIA P3 S3 Centrifuged 15 min. at 12,000 X gravity Centrifuged 60 min. at 24,000 X gravity MICROSOMES P4 1 S4 FINAL SUPERNATANT Aliquots for: PROTEIN PRECIPITATION DIALYSIS Fig. 1. Fractionation scheme for green pea stem sections. Table 1. Localization of radioactivity in cell fractions of pea stem sections incu- bated overnight with 3 X 10"^^/ 2,4-D (950 juc/millimole, carboxyl-labelled). Description Av. Corrected C.P.M. Per Cent of Total Activity Fraction Green Etiolated Green Etiolated Pi Cell walls and tissue fragments 2,480 2,510 34 39 Si First supernatant 4,800 3,969 66 61 P2 Chloroplasts 498 7 P3 Mitochondria 407 403 6 6 P4 Microsomes 268 211 4 3 S4 Final supernatant 3,640 2,175 50 34 Table 2. Effect of regrinding and washing on C" content of cell wall fraction. Description Av. Corrected C.P.M. Per Cent of Original Counts Retained Fraction Green Etiolated Green Etiolated Pi Cell walls and tissue fragments 2,480 2,510 Pla Pj ground, washed and centrifuged 500 740 20 29 Pl» Pla ground, washed, centrifuged 270 360 11 14 Table 3. Eflfect of washing in sucrose — EDTA on C'^ content of various precipitate fractions of green sections. Fraction Description Av. Corrected C.P.M. Per Cent of Original Counts Retained P. Chloroplasts 169 P^a Washed cliloroplasts 10 6 Pa Mitochondria 156 P.K. Washed mitochondria 13 8 P4 Microsomes 107 P4a Washed microsomes 10 9 Intracellular Locale of Auxin Action 359 operations sufficing to reduce the radioactivity in the precipitate to 11 to 14 per cent of the original level (Table 2). We thus consider that the wall fractions themselves contain little or no firmly bound 2,4-D. The same appears to be true of the other particulate fractions, in which a single washing in sucrose-EDTA reduces the radioactivity to under 10 per cent of the original value (Table 3). It is thus clear that the great bulk of the applied 2,4-D is not bound to any visible particulate in the cell, and must be assumed to be in the soluble phase. Our next experiments were designed to test the possibility that the 2,4-D was bound to some macromolecule in the final supernatant (S4) fraction, since several previous investigations had reported the presence or formation of auxin-protein complexes in plant cells (3, 6, 10). Dialysis of the final supernatant against the sucrose-EDTA medium used in preparing the homogenate revealed a ready outward passage of the label (Table 4). Two successive 16-hr. dialyses at 2° C. against 100 volumes of medium reduced the residual counts to about 3 per cent of the original value. Since this did not differ significantly from the behavior of 2,4-D added in vitro to control supernatant pro- tein (from sections not treated with auxin during the overnight growth test), this experiment offers no evidence for the binding of ap- plied 2,4-D to any non-dialyzable component of the final supernatant. Attempts were next made to precipitate proteins by 0.5M tri- chloroacetic acid, by saturated (NH4)oS04 (pH 6.1), and by boiling (100° C. for 8 min.), and to test the precipitates for C^^ content. The first two methods yielded only very weak activity in the washed pre- cipitates; however, the boiling experiment, while providing no direct evidence for protein-bound 2,4-D, led to an observation of great in- terest. We noted that those homogenates derived from auxin-treated cells yielded little or no precipitate, the solution merely turning opal- escent after immersion for 8 min. in a boiling water bath. The con- trols, on the other hand, invariably yielded a copious bulky-white precipitate. This experiment could be rendered quantitative by cen- Table 4. Dialysis of final supernatant of green sections. Average Corrected Fraction Description C.P.M. S4 Final supernatant 1,390 Sir Residue of S4 dialysis 435 S4rl Residue of S4r dialysis 45 360 A. W. Galston and R. Kaur Table 5. The effect of various concentrations of 2,4-D on the growth and protein coagulability of green and etiolated pea stem sections. Green Sections Etiolated Sections 2,4-D Molarity Increase in fresh wt., per cent Turbidity reading Mg. dry wt. heat- coagulated protein Increase in fresh wt., per cent Mg. dry wt. heat- coagulated protein 0 (initial control) 0 (final control) . . 10-6 10-5 33 67 93 86 113 136 122 87 56 37.7 27.5 28.7 20.1 17.6 55 114 130 124 65.3 53.6 40.3 21.9 1 o-< 18.4 triiuging down the precipitate, transferring it quantitatively to a tared weighing pan, drying overnight at 90° C, cooling in a desic- cator, and weighing to the nearest 0.1 mg. Alternatively, in those in- stances where flocculent precipitates failed to form, the quantity of suspended material could be estimated turbidimetrically in a Klett- Summerson photoelectric colorimeter equipped with a #42 blue filter. Sample data from such measurements are presented in Table 5. It is clear that increasing concentrations of 2,4-D sharply reduce the quantity of heat coagulable proteins, whether measured gravi- metrically or turbidimetrically. Other experiments (1) have revealed that indole-3-acetic acid (lAA) is approximately as active as 2,4-D in decreasing the heat co- agulability of the proteins; the weak auxins phenylacetic acid and 2,3,5-triiodobenzoic acid are slightly active, and the antiauxin p- chlorophenoxyisobutyric acid is completely inactive. The effect ap- pears not to be produced in vitro by a mixing of control protein with the auxin. There also appears to be no effect of auxin on total pro- tein, since the precipitates deposited in 0.5Af trichloroacetic acid are equal in control and auxin-treated fractions. The effect is thus prob- ably to be interpreted as an auxin-induced alteration of the physical state of the cytoplasmic proteins. DISCUSSION While the auxin effect we have described in this paper is new to plant physiology, there are certain previous references in the liter- ature which appear to be closely related to it. For example, Thimann and Sweeney (8), in an elegant series of papers, showed that lAA ap- plied to Avcna coleoptile cells markedly increased the rate of cyto- plasmic streaming. The effect was apparent within minutes, and the dose response relationships and the inhibition by various agents par- Intracellular Locale of Auxin Action 361 alleled closely the results obtained in growth experiments. In a some- what similar series of experiments, Northen (5) applied auxins in lanolin paste to one side of 'Navy' bean petioles, and after a suitable incubation period, centrifuged the petioles at right angles to their long axis. Cells on the auxin-treated side showed predom- inantly displaced contents, while those on the control side did not. Northen interpreted these results as indicating an auxin-mediated decrease in cytoplasmic viscosity. This interpretation would har- monize well with the Thimann-Sweeney experiments, since the rate of cytoplasmic streaming must be assumed to be inversely propor- tional to the viscosity of the cell contents. Similarly, our results can best be interpreted as an alteration of the physical state of the cyto- plasmic proteins, which could be reflected in altered heat coagulabil- ity patterns, as well as in viscosity and other physical properties. Various workers, on the basis of structure-activity studies with various auxin analogues, have proposed attachment of auxin to a sur- face of some colloidal or polyphasic system, with a resultant swelling (hydrophily) of this system (9). Our results are also consistent with these observations. The significance of these findings is as yet difficult to assess. At most, they may provide an explanation of how auxin acts in promot- ing cell growth; at the least, they provide an alternative to the cur- rent cell-wall theories of auxin action which, it seems to us, are in- herently incapable of accounting for all aspects of auxin action. SUMMARY C"-carboxyl-labeled 2,4-D was applied to etiolated and green pea epicotyl sections at concentrations promoting growth. The sections were subsequently homogenized in 0.25M sucrose + .00 IM EDTA and the various particulates separated by centrifugation and counted for C^*. No evidence could be found for the attachment of the 2,4-D to anything in the cell, the great preponderance of it remaining in the final centrifugal supernatant fraction. Attempts at heat coagu- lation of the proteins of this fraction revealed that 2,4-D greatly de- creased the amount of protein so deposited without affecting total protein content. This effect of auxin on the physical state of the cytoplasmic proteins appears to be correlated with auxin-induced growth. LITERATURE CITED 1. Galston, A. W., and Kaur, R. An effect of auxins on the heat coagulability of the protein of growing plant cells. Proc. Nat. Acad. Sci. U. S. 45: 1587-1590. 1959. 2. , and McCune, D. C. An analysis of gibberellin-auxin interaction and its possible metabolic basis. This volume, pp. 611-625. 362 A. W. Galston and R. Kaiir 3. Gordon, S. A. Auxin-protein complexes of the \sheat grain. Amer. Jour. Bot. 33: 160-169. 1916. 4. Heyn, A. N. J. On the relation between growth and extensibility of the cell wall. Proc. Akad. Wet. Amsterdam. 33: 1045-1058. 1930. 5. Northen, H. T. Relationship of dissociation of cellular proteins by auxin to growth. Bot. Gaz. 103: 668-683. 1942. 6. Siegel, S. M., and Galston, A. W. Experimental coupling of indoleacetic acid to pea root protein /// z'/i'o and in vitro. Proc. Nat. Acad. Sci. I'.S. 39: 1111- 1118. 1953. 7. Snow, R. Activation of cambial growth by pure hormones. New Phytol. 34: 347-360. 1935. 8. Thimann, K. V., and Sweeney, B. M. The effect of auxins on protoplasmic streaming. Jour. Gen. Physiol. 21: 123-135. 1937; 21: 439-461. 1938; 25: 841-854. 1942. 9. Veldstra, H. The relation of chemical structure to biological activity in growth substances. Ann. Rev. Plant Physiol. 4: 151-198. 1953. 10. Wildman, S. G., and Gordon, S. A. The release of auxin from isolated leaf ]:)roteins of spinach by enzymes. Proc. Nat. Acad. Sci. U.S. 28: 217-228. 1942. KENNETH V. THIMANN and NORIKO TAKAHASHI' Harvard University Inter relationships Between Metallic Ions and Auxin Action, and the Growth Promoting Action of Chelating Agents The part played by metallic ions in growth processes has gradually become more prominent in recent years. It is now clear that the ions of calcium, potassium, manganese, iron, and cobalt greatly modify the growth of plant sections. In addition it has been reported by Heath and Clark (10) that ethylenediaminetetraacetic acid (EDTA), 8-hydroxyquinoline, and other chelating agents have a small but definite growth promoting effect on wheat (Triticum) coleoptile sections and a growth inhibiting effect on wheat roots. The parallel- ism between the action of indole-3-acetic acid (lAA) and EDTA suggested to them that the two substances might act in a similar way, though it was stressed that their actions could not be identical. Since subsequent workers could find no real effect of EDTA on roots, all of what follows is restricted to shoot tissue. As an explanation of the effect of chelating agents on growth, it was proposed (10) that growth is normally restrained in some way by a metal, and that growth promoting substances in general act by chelating this metal. In the case of lAA, a comparison was drawn between the nitrogen atom adjacent to the 6-membered ring and that in the chelating agent 8-hydroxyquinoline. However, this ex- planation is evidently most improbable, for several auxins, such as 1-naphthaleneacetic and 2,3,6-trichlorobenzoic acids, could have only very weak chelating ability, yet their growth promoting action is very strong and on some plant material stronger than that of lAA; while on the other hand EDTA and 8-hydroxyquinoline are extremely powerful chelators, yet their growth promoting activities are rela- tively small. In Heath and Clark's 10 mm. wheat coleoptile sections ^Subsequently: Japan Women's University, Bunkyo-Ku, Tokyo, Japan. [ 363 ] 364 K. V. Thimann and N. Takahashi the growth promotion caused by lAA was also very small and no larger than that caused by EDTA. But with lupine (Lttpinus) hypo- cotyls, Weinstein et nl. (25) found grow^th increments of 30 to 50 per cent with lAA, and EDTA was just about as effective. Under appropri- ate conditions elongation of 100 per cent is easily produced in oat (Avena) coleoptile sections, while it will be shown below that EDTA causes in them only a relatively small elongation. It is true that Cohen et al. (5) have demonstrated some chelation of both lAA and NAA with Cu+2 jons, but this took place in 50 per cent ethanol and in 0.02 M solution, and they found no chelation with Ca^^ q^ Mg*^. AVhen Recaldin and Heath (17) examined the reaction with ferric ions, what they observed was a slow development of color accom- panied by Oo uptake and lAA breakdown, in other w^ords, a chem- ical reaction akin to the Salkowski reaction. Nakazawa (13) could find no evidence that lAA chelates with iron in pea seedlings. Thus, evidence that appreciable real chelation takes place under physiological conditions is lacking, and even if it did take place, there are as yet no indications of a causal relationship to growth. However, in favor of some relationship between growth and chela- tion is the increasing evidence mentioned above that metallic ions strongly influence growth. Recently it has been suggested, indeed, that calcium specifically controls growth through its linkage to pectic acid groups in the cell wall (1), and it has been shown that calcium raises the osmotic content of Avena coleoptile cells w^hile at the same time lowering tlie suction pressure — an effect interpreted as due to "tightening" of the cell walls (6). Experiments relating auxin action to pectin meihylesterase (3, 7, 8, 16) are interconnected with this concept, though the effect does not appear to be a simple one. In any event, chelating agents might indeed promote growth by re- moving some of the hypothetical wall-bound calcium. The fact that the ferric-EDTA complex does not promote growth, under conditions where free EDTA does, supports this idea (25). The whole phenomenon seemed to warrant restudy of the action both of chelating agents and of some metal ions on growth. MATERIALS AND METHODS The EDTA used was a three times recrystallized sample of the sodium salt sold as Sequestrene AAA, and kindly supplied by the Alrose Company of Providence, R.I. The stock solution was atljusted to pH 5.6. The 8-hydroxyquinolinc (8-HOQ), and other chelating agents were CP chemicals. Avena coleoptile sections 10 mm. long were cut in the usual manner about 3 mm. below the tip from seedlings of 'Victory' oats 74 hrs. old, grown in water at 25° C. after receiving Metallic lotis, Auxin Action, and Chelating Agents 365 5 hrs. of red light administered from the third hour after soaking. The first internodes (mesocotyls) from simihir plants grown in com- plete darkness were sectioned in weak green light (15, 18), 4 mm. sections cut 2 mm. below the coleoptilar node being used. Both types of sections were allowed to grow in the solutions in complete dark- ness. All section elongation was measured after 24 hrs. at 25° C. unless otherwise indicated. Potato discs used had a 9 mm. diameter and were 1 mm. thick. They were cut from 'Katahdin' tubers of selected large size and washed 24 hrs. in water before use (9). The growth media contained 2 per cent sucrose, unless its absence is specified, together with either 5 X lO'^M KCl, CaCla at 5 X 10'^^^ or lower, or both KCl and CaCL, or more generally potassium phos- phate buffer, 0.0 IM, pH 5.5. The Action of Some Metallic Ions on Growth In the first place the powerful growth inhibiting action of calcium must be emphasized. Since this inhibition was first reported on coleoptile sections in 1938 (23) it has been confirmed by many work- ers; it was shown very strikingly in pea stem sections, which show no similar inhibition by magnesium (24). Unlike other ions, calcium does not promote growth at low concentrations (24). On roots the effect is one of growth promotion, as has long been known, and in recent years studied especially by Burstrom. Other ions, however, notably potassium (23), manganese (2), fer- rous iron (19), and cobalt (12), promote growth. Some of the effects of buffers are largely due to their potassium content rather than to their anions — a fact often lost sight of. The action of Fe+2 has been ascribed to two different effects (19) (see Discussion). Cobalt is particu- larly powerful, being nearly 100 times as effective as manganese on coleoptile sections. It also strongly promotes growth of pea stem sections which show very little response to manganese (21). Two observations suggest that cobalt operates by way of the organic acid metabolism: (a) when acetate is added growth is no longer pro- moted, but is strongly inhibited; (b) in presence of cobalt the normal excretion of hydrogen ions is prevented (21). On the other hand, Busse (4), from the facts that cobalt causes the growth of coleoptile sections to continue for a longer time than normal and that their respiration is not promoted but slightly inhibited, has concluded that cobalt inhibits a process of cell-wall deposition which otherwise would have made growth slow down. Recently Dr. Kenneth Wright in my laboratory has extended the cobalt effect to potato tuber slices (Figure 1). Preliminary experi- ments with this tissue showed that the optimum concentration, about 366 K. V. Thimann and N. Takahashi Fig. 1. Increase in fresh weight of potato tuber disks in water, in l-naphtha- Icneacetic acid, 1 mg/1, or in 1-naphthaiencacctic acid, 1 mg/1 with 10-^\/ CoCL. Data courtesy Kenneth Wright. \0-^'M, is the same as with Avena. The promotive eliect, though not quite as large as witli Avoia and Pisinn, is clear enough. It will be noted that promotion of growth by cobalt appears as soon as promo- tion by auxin does, and that its effect is exerted dining the period of most rapid growth. Thus, there is no indication here of a delayed effect like that just mentioned. Also, in our experiments with galac- tose, cobalt was found not to interfere with the conversion of this sugar to wall material (22). Even with the Avena coleoptile our results do not indicate a delay of more than 12 hours in tiie onset of the cobalt effect (Figure 2). o o a: o 120- H 100 z O >? 80 60 m 40 _i o 20 O o 10 2 o (T 0 U. UJ o -10 z UJ or UJ -20 -30 0 10 15 20 25 30 TIME IN HOURS Fig. 2. A. Time course of the effect of cobalt chloricie on the elongation of 10 mm. Avena coleoptile sections. All media contain sucrose 10 g/1, KCl O.OOIM and lAA I mg/1 (=6.10-''i\/); all concentrations of cobalt indicated on the curves are x 10-°M; mean of 2 complete experiments, each including all 4 cobalt concentrations and the control, and of 3 experiments at the 18i4 hour point. B. Time course of the positive or negative increments due to cobalt, derived from the above. 368 K. V. Thimann and N. Takahashi The inhibiting action of 3 X 10-^M Co*^ jj indeed detectable at 9 hrs. (Figure 2B), and the promotion by lO-^M and 3 X 10-^M Co^^ at almost the same time. However, it is true that the increments only become really large after 12 hours' growth. Another difficulty in regard to Busse's interpretation is that if the promotion of giowth by low concentrations of Co"- is due to inhibition of the deposit of cell-wall, no explanation is offered for the inhibition of growth by higher concentrations. That 3 X 10"^Af Co+2 should strongly promote a process which lO'^M Co^^^ equally strongly inhibits seems most unlikely. For these reasons the conclu- sion of Busse can only be accepted with reserve at present. The Action of Chelating Agents The essential results of Heath and Clark (10) could readily be confirmed. For Avena coleoptile sections the optimum concentration of EDTA was found to fall somewhat below 10-^M, and lO'^M was generally inhibitory. The increment caused by EDTA occurred in all growth media tested; in CaCU its value was not significantly different from that in KCl, buffer, or water. In the absence of sucrose it aver- aged about 10 per cent, in 2 per cent sucrose about 5 per cent. Since these increments are small differences between two fairly large num- bers, they are subject to fairly wide variation; nevertheless the data agree rather well. Other chelating agents have been studied less thoroughly. AVith 8-hydroxyquinoline (8-HOQ) the optimum concentration lay near 3 X 10-^i\i^ i.e., about one-third of that with EDTA. A representa- tive group of three experiments is shown in Table 1; the increments (in 2 per cent sucrose) are comparable to those obtained with EDTA. A few trials with 9,10-phenanthroline, on the other hand, did not show significant growth promotion. In the hope of improving the magnitude of the effect, sections Table 1. Effect of 8-hyclroxyquinoline on elongation oi Avena coleoptile sections. Elongation in Mean Concn. Per Cent Increase of 8-HOQ Over Controls, X in-->.i/ I II III Average Per Cent u 42 44 40 42.0 1 46 49 48 47.7 5.7 3 49 45 46 46.7 4.7 10 40 46 43 43.0 1.0 30 44 36 34 38.0 -4.0 100 24 24 Medium consisted of 2 per cent sucrose plus O.OLU phosphate buffer at pH 5.0. Metallic Ions, Auxin Action, and Chelating Agents 369 of Avena first internodes were used, since these have been found more responsive to very low auxin concentrations (15, 18). However, to our surprise, first internode sections showed no marked response, either to EDTA or 8-HOQ, over the whole range of concentrations up to lO-^M. They showed excellent response to lAA, however, at a concentration which showed only a small growth promotion in coleoptiles. I- < (3 O _J Ixl 120 no 100 T T T Su 2% + EDTA .001 .01 0.1 1.0 10 CONCN. OF lAA (MG/L) Fig. 3. Effect of EDTA on promoting the elongation of coleoptile sections, as a function of lAA concentration and presence of sucrose (Su). All experiments in O.OIM phosphate, pH 5.5. 370 K. V. Thiynann and N. Takahashi The failure of the first internode to respond suggested that the chelating agents are not acting as weak auxins but in some other way. This was tested by studying their action in presence of a strong auxin, namely lAA. It was then found that in presence of lAA, EDTA at 2.5 X lO-^M could increase growth by up to 50 per cent (Figure 3). The action could be exerted in all growth solutions and in presence or absence of sucrose, though without sucrose the in- crement is only 5 to 10 per cent. At optimum sucrose, the increment caused by EDTA is largest when the lAA concentration is from 0.1 to 1 p. p.m., but at low sucrose it is best at from 1 to 10 p. p.m. It is clear, therefore, that the action is not simply that of a weak auxin. Comparable results were obtained with 8-HOQ, though the increment was smaller; for example, in 3 experiments with lAA 0.1 p. p.m. 8-HOQ at the optimum level (10-^M) gave an average increment of 13.0 per cent. With this knowledge a return was made to the first internode sections and it was found that in presence of lAA these sections did respond to EDTA. In Table 2 representative data are compared ^vith those from coleoptile sections in the same experiment. Though more sensitive to low lAA concentrations than the coleoptile, the first internode gives a response to EDTA only one-quarter as great. For further comparison, internode sections from etiolated 'Alaska' peas were used. Again the effect of EDTA was best in presence of auxin, but even at the optimal lAA level, namely 1 mg/1, the incre- ment due to EDTA was only an additional 11 per cent. Addition of sucrose could increase the EDTA effect somewhat, but the response of these sections is still considerably smaller than that of coleoptiles. The effectiveness of EDTA, therefore, is a function of the tissue used, as well as of the amount of auxin present. Although the action of EDTA is thus exerted mainly in combin- ation with lAA, this is not a general interaction with auxins since. Table 2. Effects of EDTA on sections from Avena coleoptile and first internode under the same conditions. All solutions contain sucrose 2 per cent and KCl 0.005A/; 24 hours. Solution Elongation in Per Cent of the Initial Length lAA X 10-M/ 0 6 6 EDTA X lO-^Af 0 0 6 Coleoptile 28.8 46.5 90.5 First internode 21.5 57.5 67.9 Increment due to EDTA 44.0 10.4 Metallic Ions, Auxin Action, and Chelating Agents 371 surprisingly enough, it is not shown in combination with NAA or with 2,4-D. Table 3 shows representative figures, the mean of three complete experiments with each of these auxins. lAA at one or two concentrations was included for comparison in each series; the con- trast between the clear, positive effect of EDTA with lAA and its slight negative effect with the other auxins is evident. Evidently, therefore, the action of the chelator is specifically con- cerned with that of lAA. It is reasonable to suggest that its very small but repeatable effect in the absence of added auxin is exerted by potentiating the endogenous lAA. The Interaction Between EDTA and Calcium As stated at the outset the promotion of growth by chelators might be due to their removing an endogenous growth-restraining concentration of calcium. It follows that the chelator should also be able to remove a growth restraint caused by externally added calcium. In other words, growth inhibition caused by added calcium should be antagonized by EDTA. Experiments to test this can be carried out in two ways: the calcium can be added directly to the growth solution or it can be taken up by the plants beforehand. The former method has the obvious disadvantage that chelation of the calcium can occur in the solution so that the effect on the plant is masked. The second is in some respects more problematical but it is freer from objection. While both methods have been used, and lead to the same conclusion, only the second will therefore be described here. Ave7ia seeds were soaked in CaCL solutions of various strengths from 0.02M to 0.00 IM, and after 3 hrs. were laid out on filter paper in the usual way. Sections were cut from the coleoptiles 72 hrs. later and their growth compared with that of control sections (from seeds that had been soaked in water), both being tested on the same day. With low CaClo concentrations the differences were not always sta- tistically signficant, but with 0.02M they were real. As a mean of 10 experiments in sucrose-KCl the control sections grew 38.9 ± 2.3 per cent, the sections from Ca-soaked seeds 30.5 ± 1-5 per cent. In order to be sure that this decrease in growth was due to calcium taken up into the coleoptile, the procedure was repeated using Ca'*^. After 76 hours' growth the plants were dissected and the radioactivity counted. The activity expressed as percentage of the total was distributed as follows (mean of 10 plants individually sectioned and counted): roots 24, seed 39, coleoptile 32, leaf 5. Thus one-third of the Ca^s taken up was present in the coleoptile. V en O o 3 £ o c c o V c C _o "o o K <^ V CO Q cm' u o O) < < ^ ^ 4^ ^ ^ < O H <-) P lO tij i/S ^M O ffi " a o u c 4) V in .> 3 JD ,w 9; 4-< a en O -C CO a W ■M c u u u ex CO CO o -^ CO * O m IT) r~- rO CO o m ^- 1—1 ^— ' ^— d ^- *— ■ ^— ' ^- 1 1 < oo' CO in o o" S" < \0 ■<*• r-~ in 00 CO ^^ o CM CN Cv ^- eg o ■ + + bo ^ ~~^ ^ — ^ ^■"^ ^ ^"^ "" •-■ eg o T— < 1—" ^- CO G .2 <— ■^ ■^ 1 1 '*j 03 4^ C «J ,^ — ^ H — 0 < 00 in r-^ o < O u-i r- 1— ' ,-M (N t— • "x o_ ^ + 3 y ^^ ■ — ■ < •4-) «! a .2 CO CO o CN <3 CO '- r- un eg \0 O o ^ o CN Cv Cn CN be 1 1 c _o u V bo CO CO o (N ^ ,- n! ,_^ -M o ^H ^^ o r- r~- o C <5 sO o o U o 1 u, V Pl. ^^ T— " 00 rO o o 00 lO O o 1 lO r- CN CO ■t ,r— o CN o ■^ •* in ^ LTl ^ + \0 vO + < < h h c c Q s < < 2 ir c I. 0 1 eg" 1 c ir t due to E < 1— 1 < > ^ c ' B ) cu 1 t c 3 t- t - ^ r ) a, 1 t c j 4 a o XI V u V V u c o o o V -a 3 u C 4J < bo" c 0.2 O eg 4— u C V u c o u en C bo •- C in c cn O V U o c c o w o 5 o « = -4-4 L- *^ cn ^ U D tn j- bcS O ■? -C t) m c u ;- !5 « eg rt u o _ - V J5 C O Metallic Ions, Auxin Action, and Chelating Agents 373 Table 4. Effect of soaking Avena seeds in calcium chloride on subsequent sensi- tivity of the coleoptile sections to EDTA. All figures are elongation in per cent of the initial length. Mean of four separate experiments. Soaking Treatment Elongation of Sections Control Plus EDTA Increment Due to EDTA Growth in Sucrose-KCl Water 40.8 36.3 43.9 39.5 3.1 CaCU 0 02M 3.2 Growth in Sucrose-KCl Plus lAA Water 66.5 59.7 102.7 95.3 36.2 CaCIo 0.02M 35.6 Sections from the plants of calcium-soaked seeds were now tested for their reaction to EDTA, in parallel and on the same day with sections from control plants. The results of four such experiments (all done in sucrose plus KCl plus lAA 0.1 mg/1) are averaged in Table 4. Without auxin the increments are very small and show no difference. In presence of lAA the increment due to EDTA in the control sections averaged 36.2 per cent of the length. In sections made from plants pretreated with Ca+2 the increment averaged 35.6 per cent of the length. The agreement is good and the conclusion clear; the growth promotion caused by EDTA in the sections enriched in Ca+2 is no greater than in the controls. In these experiments the growth inhibition is known to be due to Ca^^ which has been proved to be taken up by the tissue, yet EDTA does not overcome it in the least. The growth promoting effect of EDTA cannot therefore be due to chelation with calcium. In two papers which appeared after this work was done, Ng and Carr (14) have shown that EDTA does not remove as much calcium from intact or homogenized coleoptiles, or from filter paper, as does citrate-phosphate buffer, and although their data show very small and unsatisfactory growth promotion by EDTA (of the order of 5 per cent greater than controls) their conclusion that EDTA could hardly act by chelating wall-bound calcium agrees with the above. The complementary approach to the same problem would be to measure the actual loss of calcium from the sections during growth. For this purpose seeds were soaked in Ca^^ CU as before and sections cut for growth experiments from the resulting coleoptiles. After 24 hours' elongation the radioactivity both of the solution and of the dried and powdered sections was determined. Unfortunately these 374 K. V. Thiynann and N. Takahashi experiments, though extensive, have yielded no conclusive result. It was at first found that while EDTA alone had no effect, yet in the presence of both lAA and EDTA a significant amount of Ca^s was lost from the sections and recovered in the growth solution. Subsequent more extensive tests have revealed a degiee of variation between experiments which makes this result doubtful. More exten- sive washing of the sections has not greatly reduced the variation. It must be concluded that if EDTA does remove any calcium it does not do so when added alone, and even the combination of EDTA with lAA removes so little, if any, that it is at the borderline of significance even with the highly sensitive radioactivity method. The chemical and photometric calcium determinations of Ng and Carr (14) lead to the same conclusion. DISCUSSION One possible explanation of the large growth promotion which EDTA causes in presence of auxin might be merely that its action is proportional to the amount of elongation occurring. This is nega- ted not only in Figure 3, but also by the data of Table 2 (and of numerous similar experiments) which establish that first internode sections in low auxin concentrations elongate more than coleoptile sections, yet show a smaller EDTA effect. The failure of EDTA to promote growth appreciably in presence of NAA or 2,4-D suggests that EDTA in some way protects lAA from destruction within the tissues. While this is not impossible it is not wholly satisfying for the following reasons: (a) destruction of lAA in coleoptile sections in vivo is not very great, as witness the high recovery of C" from carboxyl-labeled lAA in transport experi- ments; (b) Figure 3 shows that at the lowest lAA level, where pre- sumably destruction has its largest effect, EDTA is inactive; (c) in Pisum sections, where lAA destruction is known to occur actively, the effect of EDTA is relatively small. On the other hand, the strong promoting effect of manganese on the lAA oxidizing system would certainly give a basis for inhibition by a chelating agent. It seems more probable, however, that it is some other reaction of lAA, perhaps its conjugation, that is metal-promoted and there- fore sensitive to EDTA. It would be of interest to investigate the several side reactions which remove lAA in vivo. At this point we return to the actions of specific metal ions men- tioned at the outset. Recently, Shibaoka and Yamaki (19) have found that ferrous ions promote growtii of Ax'cua coleoptile sections in I.\A but not in XAA. Metallic Ions, Auxin Action, and Chelating Agents 375 and conclude that the action is due to inhibition of lAA destruction. However, they also showed that the translocation of botJi lAA and NAA was promoted, and conclude that Fe^^ has two different effects — to inhibit destruction and to promote transport. The parallelism with the action of EDTA is suggestive. The several parallels with the action of cobalt are also notable, for cobalt, like EDTA, shows little or no effect in the absence of auxin, but causes up to 50 per cent increment in presence of optimal auxin; both cobalt and EDTA are somewhat more effective on coleoptiles than on pea stems, and both are considerably more effective in presence of sucrose than in its absence (21). Cobalt differs from EDTA in that it promotes growth in solution equally well with NAA and lAA, but again there is a suggestion of similarity, since when acetate is present cobalt becomes strongly inhibitory but this occurs only with NAA and not with lAA. It is worth noting that it is not impossible for a metal ion and a chelating agent to act in the same way, for, if a particular catalyst owes its activity to a metal complex, then an exogenous metallic ion could compete at its site of action, while the chelating agent, by form- ing a chelate in situ, could prevent the metal and its site of action from coming together. Such an action need not be in the wall and indeed the possibility should be considered that it occurs in the mitochondria. It is known that the activity of these bodies is strongly influenced by their Ca+2 (11) and since the energy for growth of coleoptiles, pea stems, and freshly-cut potato sections is supplied via cytochrome oxidase, which is in the mitochondria, a close relation between metal ions and growth could be suspected here. Further, EDTA, as well as manganese, is known to prevent the swelling of mitochondria which occurs in isotonic solutions (20).2 It seems a safe prediction that the mode of action of auxin will not be fully understood until the role of the several metal-dependent reactions has been elucidated. ACKNOWLEDGMENTS This work was supported by a grant from the National Science Foundation. The experiments on potato disks were carried out by Dr. Kenneth Wright while on a leave of absence from Smith College, Northampton, Mass. For skillful technical assistance in the later work the writer is indebted to Mrs. C. ^Vinkler Kurland. ''The subsequent finding of Whitehouse, Staple, and Kritchevsky (Arch. Biochem. Biophys. 87: 193. 1960) that cobalt activates the oxidation of cholesterol, pyruvate, and octanoate by rat liver mitochondria supports this line of reasoning. 376 K. V. Thimann and N. Takahashi LITERATURE CITED 1. Bennet-Clark, T. A. Salt accumulation and mode of action of auxin. A pre- liminary hypothesis. In: R. L. Wain and F. Wightman (eds.), The Chemistry and Mode of Action of Plant Growth Substances, pp. 28^-291. Butterworth Sci. Publ., London. 1956. 2. Bonner, J. Limiting factors and growth inhibitors in the growth of the Avena coleoptile. Amer. Jour. Bot. 36- 323-332. 1949. 3. Bryan, W. H.. and Newcomb, E. H. Stimulation of pectin methylesterase activity of cultured tobacco pith by indoleacetic acid. Physiol. Plant. 7: 290-297. 1954. 4. Busse, M. Cber die Wirkungen von Kobalt auf Streckung, Atmung, und Substanzeinbau in die Zellwand bei Avenakoleoptilen. Planta. 53: 25-44. 1959. 5. Cohen, D., Ginzburg, B.-Z., and Heitner-Wirguin, C. Metal-chelating proper ties of plant-growth substances. Nature. 181: 686,687. 1958. 6. Cooil, B. J., and Bonner, J. The nature of growth inhibition by calcium in the Avena coleoptile. Planta. 48: 696-723. 1957. 7. Glasziou, K. T. The effect of 3-indolylacetic acid on the binding of pectin methylesterase to the cell walls of tobacco pith. Austral. Jour. Biol. Sci. 10: 337-341. 1957. 8. The effect of auxins on the binding of pectin methylesterase to cell wall preparations. Austral. Jour. Biol. Sci. 10: 426-434. 1957. 9. Hackett, D. P., and Thimann, K. V. The nature of the auxin-induced water uptake by potato tissue. Amer. Jour. Bot. 39: 553-560. 1952. 10. Heath, O. V. S., and Clark, J. E. Chelating agents as plant growth sub- stances. A possible clue to the mode of action of auxin. Nature. 177: 1118- 1121. 1956; 178: 600, 601. 1957. 11. Kunz, W., Friedel, W., Miiller, F., So, P. van, and Strack, E. Uber die Wirkung von Athylendiamin-tetraessigsaure auf isolierte Lebermitochondrien. Zeitschr. Physiol. Chem. 310: 265-274. 1958. 12. Miller, C. O. Promoting effect of cobaltous and nickelous ions on expansion of etiolated bean leaf disks. Arch. Biochem. 32: 216-218. 1951. 13. Nakazawa, K. On the chelation of lAA in roots. Me. Fac. Lib. Arts Educ. Yamanashi Univ. 81: 156-161. 1957. 14. Ng, E. K., and Carr, D. J. Effect of pH on the activity of chelating agents and auxins in cell extension. Physiol. Plant. 12: 275-287. 1959. 15. Nitsch, J. P., and Nitsch, C. Studies on the growth of coleoptile and first internode sections. A new, sensitive, straight-growth test for auxins. Plant. Physiol. 31: 94-111. 1956. 16. Ordin, L., Cleland, R., and Bonner, |. Influence of auxin on cell-wall metabolism. Proc. Nat. Acad. Sci. U.S. 11: 1023-1029. 1955. 17. Recaldin, D. A., and Heath, O. V. S. Chelation or complex-formation by indoleacetic acid in vitro. Nature. 182: 539,540. 1958. 18. Schneider, C. L. The effect of red light on growth of the Avena seedling with special reference to the first internode. Amer. Jour. Bot. 28: 878-886. 1941. 19. Shibaoka, H., and Yamaki, T. Mechanism of growth promotion by ferrous sulfate. Bot. Mag. Tokyo. 72: 203-214. 1959. 20. Slater, E. C, and Cleland, K. W. The effect of calcium on the respiratory and phosphorylative activities of heart-muscle sarcosomes. Biochem. Jour. 55: 566-580. 1953. Metallic Ions, Auxin Action, and Chelating Agents 377 21. Thimann, K. V. Sludles on the growth and inhibition of isolated plant parts. V. The effect of cobalt and other metals. Amer. Jour. Bot. 43: 241-250. 1956. 22. , Craigie, J., Krotkov, G., and Cowie, L. Utilization of uniformly labeled C"-galactose by etiolated Avena coleoptiles. Amer. Jour. Bot. 45: 295-297. 1958. 23. , and Schneider, C. L. The role of salts, hydrogen-ion concentration, and agar in the response of the Avena coleoptile to auxins. Amer. Jour. Bot. 25: 270-280. 1938. 2!. , Slater, R. R., and Christiansen, G. S. The metabolism of stem tissue during growth and its inhibition. IV. Growth inhibition without enzyme poisoning. Arch. Biochem. 28: 130-137. 1950. 25. Weinstein, L. H., Meiss, A. N., Uhler, R. L., and Purvis, E. R. Growth-pro- moting effects of ethylenediamine tetra-acetic acid. Nature. 178: 1188. 1956. DISCUSSION Dr. Nitsch: At the time Dr. Thimann reported the very large effects he obtained with cobalt as an lAA synergist on Avena coleop- tiles (Amer. Jour. Bot. 43: 241. 1956), we tried the effect of cobalt on oat first internodes. To our great surprise, we got little, if any, stimulation (J. P. Nitsch and C. Nitsch. Plant Physiol. 31: 94. 1956). We generally got a small cobalt effect with coleoptiles. We repeated these experiments several times but never obtained the large cobalt promotion which has been reported, although manganese gave results which agreed with those of other workers. The explanation for the discrepancy between Dr. Thimann's and our results came only this year when Busse's article appeared (Planta. 53: 25. 1959). Busse has shown that the cobalt effect is large under two conditions: (1) that the sections be long, and (2) that the test last for more than 24 hrs. We used 4 mm. sections, taken 3 mm. below the tip of the coleoptile, or 2 mm. below the node (in the case of first internodes), whereas Dr. Thimann used 10 mm. sections. In addition, we always measured elongation 20 to 24 hrs. after the start of the treatment whereas Dr. Thimann measured elongation after 48 hrs. These differences in technique, differences in the oat variety (we used 'Brighton'), and the fact that we added a citrate-phosphate buffer to the sucrose solution, explain the discrepancies observed in the results. They indicate that cobalt may retard a sort of aging process which allows lAA to act longer on older parts of the coleoptile. Dr. Thimann: I have never done any large number of experi- ments with cobalt using 4 mm. sections. We have found perfectly good cobalt effects in 24 hrs. with both Avena coleoptiles and either 'Alaska' or 'Laxton's Progress' pea stem sections, although it is true that we have observed greater responses in 48 hrs. Busse's data on coleoptile sections show cobalt promotions from about 6 hrs. on. His conclusions are essentially that the effect of cobalt appears when the 378 K. V. Thimann and N. Takaliashi growth of the controls is slowing down and that this slowing down is due to the deposition of cell wall material which cobalt prevents. According to Busse, the main effect of cobalt is to allow stretching for a longer period of time. I am not personally convinced that this is the whole explanation, although I have not made extensive time studies. As you saw, cobalt exerts its effect on potato slices while the control slices are still growing well. It is true that if you measure it over a very short time, 3 to 4 hrs., there is only a very small cobalt effect, so that while perhaps the effective times may vary with the experimental conditions, there is an increasing effect of cobalt with time. I found two effects of cobalt linked to organic acid metabolism; namely, that in the presence of acetate, the cobalt exerts only an in- hibition and that cobalt prevents the normal excretion of organic acids by coleoptiles and pea sections. I think that the cobalt effect is probably quite complex. Dr. Bonner: It might be appropriate to mention some further work which bears on the same final question. It is not otherwise similar to Prof. Thimann's work. We know that calcium is inhibitory to growth of isolated tissue sections. The question is. How does the calcium do it? Wliat does it combine with to bring about this drastic inhibitory effect? Experiments that have been published for some years show that one characteristic of calcium-induced growth inhibition of Avena section growth is that the calcium ions that exert the inhibition are bound exchangeably. The same is true of the bound calcium ions which decrease the mechanical deformability of coleoptile tissue. Apparently the calcium ions that make the tissue stiff and not grow rapidly or not bend rapidly are ions that go into the tissue and combine with something; then they stay there and don't come out in water but have to be encouraged out by some other kind of ion such as sodium or potassium. One can measme things about these exchangeably bound ions, the concentration needed to obtain half maximal inhibition of bendability, and the time constants for ecpiilibration of the tissue with the ion. We can also find out, by classical chemical methods, whether the tissue can bind calcium ions exchangeably. We can take tissue and put it in some labeled calcium solution, find out the amount of ion bound by the tissue in such a form that it will not leak out in water but can be exchanged by unlabeled calcium, potassium, or some other cation. In this way it is possible to determine that Avena coleoptile sections, for example, do bind calcium ions exchangeably and they have a certain cation binding capacity that we can calculate in milliequivalents per gram of sections just as if one were a soil scientist. We can also determine Metallic Ions, Auxin Action, and Chelating Agents 379 the kinetic constants for this binding. We can determine what con- centration of calcium is needed to get half maximal binding of the exchangeable bound calcium, and we can measure the time constant for the binding. Now, the happy coincidence is that both the con- centration of calcium needed for half-maximal effect and the time constant for the binding is about the same for the exchangeable binding of calcium as it is for the binding of the calcium which causes inhibition of growth rate or inhibition of bendability. There- fore, I conclude that it really is exchangeably-bound calcium that goes into the tissue and gets bound somewhere that exerts these effects. The only remaining question then is to discover what is in the tissue that binds the calcium. This is not so hard to do as it might seem. If we drop our tissue in boiling water and kill it, it still retains its cation binding capacity. If we take tissue and make a cell wall preparation from it, we find that this portion has essentially all of the cation-binding capacity of the intact tissue. So, I think the calcium ions that inhibit the growth rate and are exchangeably bound are bound in the cell wall. The final question is, What is it in the cell wall that binds the calcium? There are free carboxyl groups in the cell wall and, as has been shown by Peter Ray and by our own group during the past two years, there are free pectic carboxyl groups in the cell wall and these account quantitatively for the cation-binding capacity of the wall. It seems to us that it is these free pectic carboxyl groups that bind the calcium in the cell wall which are then the calcium ions that cause inhibition of growth rate and inhibition of cell wall bendability. Just one last comment. In these same experiments it has been shown that if we grow Avena seedlings in distilled water and then harvest the coleoptile sections and make cell wall preparations from them, then these cell wall preparations contain calcium to such an extent that there is enough calcium to occupy about one-fifth of all the free caiboxyl groups in the cell wall. We propose, therefore, that perhaps one way that EDTA works in causing increases in growth rate is to pry out some of this calcium which is endogenously present in the cell wall, calcium which is contributed to the cell wall from calcium which the seed contains. Perhaps here again we're dealing with rigidity of cell wall induced by calcium. Dr. Fawcett: The discovery by Cohen, Ginzburg, and Heitner- Wirguin (Nature. 181: 686. 1958) that the ultraviolet absorption spec- tra of lAA and of NAA are profoundly altered by the presence of cupric, but not calcium or magnesium ions, led them to postulate that the cupric ion reacted with these acids to form chelate complexes. 380 K. V. Thimann and N. Takahashi However, it is not necessary to use a chelation mechanism to interpret these absorption spectra. By measuring the pH, Cohen et al. also confirmed that cupric nitrate reacted differently from calcium and magnesium nitrates when added to solutions of lAA and NAA. From a study of several closely related acids comprising active and inactive growth regulators, I found that the pH changes, observed upon addition of cupric ions, do not correlate with growth regulating activity and, moreover, are explicable by mechanisms not involving chelation (Nature. 194: 796. 1959). We have recently compared a range of chelating agents with lAA, 2,4-D, and NAA in four different biological tests. The lAA, 2,4-D, and NAA were highly active in all tests. Of the forty chelating agents tested most were inactive, though weak activity was shown by nine of them, namely, ethylenediaminetetraacetic, diethylene- triaminepentaacetic, l,2-diaminoc);c/ohexanetetraacetic, citric, tar- taric, gluconic, nicotinic, succinic, and nitrilotriacetic acids. This slight but significant activity was shown only in the wheat coleoptile test. The remaining chelating agents were inactive in the wheat coleoptile, the pea segment, the slit pea curvature, and tomato epinasty tests. Thus, the results obtained in these experiments provide strong evidence against the idea that plant growth regulating activity is explicable in terms of chelation. PETER M. RAY University of Michigan Problems in the Biophysics of Cell Growth It has become accepted that growth of plant cells by enlargement must depend upon phenomena occurring in the primary cell wall which result in stretching under the force imposed upon it by tur- gor pressure, and consequently, that the stimulation of growth by auxin must involve an effect on the properties of the cell wall. It is usual to refer to this effect as a softening or plasticizing of the cell wall and to think of growth as a plastic stretching of the cell. It must be remembered that this need not be a direct effect; it may be exerted via metabolic processes in the protoplasm. The present remarks are made to help clarify our thinking about cell wall growth by pointing out that at least two fundamentally dif- ferent mechanisms of growth may be confused under the idea of plas- tic stretching. It is convenient to illustrate these mechanisms with the pectate theory of cell wall growth proposed by Bennet-Clark (1) and Ordin, Cleland, and Bonner (5, 6). This does not imply a belief on the author's part that the pectate theory is necessarily a factual de- scription of the important events involved in cell wall growth; it serves well, however, to illustrate some molecular principles which appear to be generally applicable whatever the actual chemistry of the growth process. It must be assumed that the resistance of the cell wall to stretch- ing by the force of turgor is due to some type of bonding between the molecular units of the cell wall, the polysaccharides composing the microfibrillar material and the matrix in which this is embedded. Growth, then, must involve the breaking of certain of these bonds in some manner. According to the pectate theory, the critical bond- ing forces are visualized as being electrostatic cross links between polyuronide chains, whose free carboxyl groups have formed salts with Ca+2 (Figure lA). [381] 382 P. M. Ray I © I Polyuronide choin # Carboxyl group O Methyl esterified carboxyl © Calcium ion Me Methyl group ©. I I ©« Me © Me ©■ ^1 Fig. 1. Illusliation of the plastic and molecular mosaic mechanisms of growth based on the pectate theory. A shows cross link in unstrained wall. B shows elastic expansion under turgor. In C the cross link is being broken by forcible separation; the polyuronide chains now assume relaxed configurations (D), and the carboxyl groups enter into salt linkage with other adjacent carboxvls (E). B' sliows again a bond under turgor stress; in C, bond is broken by methylation of the carlioxyls. After the released chains reach relaxed positions (D'), methyl ester groups arc remo\ed and carboxyls enter into salt linkage with other adjacent carboxyls (E'). Under the force of turgor these critical bonds are put under stress, as shown in Figure IB. This will involve an increase in the bond length from the rest length, as well as elastic strain elsewhere in the cell wall structure. One way in which the structure could expand irreversibly would be that at some point the strain on the electro- static bond becomes great enough to break the bond — i.e., further increase in bond length does not increase the restoring force between the bonded groups. The two polyinonide chains will then spring apart, taking up more relaxed configinations, and the carboxyls will enter into salt linkages with other carboxyls to which they now find themselves adjacent. This process will be repeated after similar bond breaking elsewhere in the structure has reimposed strain on the re- gions in which the two original carboxyl groups are located. This process amounts to a passive distortion of the cell wall struc- Problems in Biophysics of Cell Growth 383 ture under force and can be called a plastic extension. The rate of extension will be greater, the greater the turgor pressure, since more bonds will be under a strain great enough to break them. To make the structure more plastic and to stimulate growth at constant turgor pressure (as auxin does), it is necessary to reduce the total number of bonds present. In terms of the pectate theory this requires an in- crease in the methyl ester content, so that fewer carboxyl groups are left unesterified and capable of forming salt linkages. Another way in which the cell wall structure under strain might grow may be recognized by supposing that, in fact, turgor force is not great enough to break critical bonds in the structure — salt linkages in the pectate theory. Through metabolic machinery the cell, however, may be capable of breaking these bonds by insertion of a methyl ester group on one or both carboxyl groups (Figure IC). When this hap- pens, the no-longer linked polyuronide chains will move apart to relaxed configurations, just as in the previous example, and subse- quently they may enter into bonding with adjacent carboxyl groups after metabolic removal of the methyl groups (Figure IE'). The cell wall will extend as these events take place successively in all parts of the structure. This kind of growth is not a passive process as every unit of en- largement depends upon an act of metabolism; it cannot be consid- ered a passive plastic stretching. Nevertheless, it is dependent upon turgor, because in the absence of strain in the structure, the bonded uronide chains will not separate when the bond between them is broken by methylation, but instead they will merely resume bonding subsequently. Also, note that the amount of growth achieved per active growth event will be greater, the greater the strain on the units acted upon, so the growth rate will increase with increasing turgor pressure, as was the case with pure plastic stretching. So an increase in the growth rate at constant turgor pressure is not to be looked for in a change in the number of bonds present but in an increased rate of bond breaking - of methylation in the pectate theory. One can see that an increased growth rate could be obtained in the presence of auxin, without any change in methyl ester content, provided there is an increased rate of metabolic turnover of uronide methyl ester groups. This second picture of how the cell wall might grow is really an extension, to molecular dimensions, of the concept of mosaic growth advanced by Frey-Wyssling and co-workers (3,4). This conceives of extension as resulting from localized transformations of elastic strain into fixed deformation through some loosening process carried on by the protoplasm. The areas of loosening (as interpreted from electron micrographs) were much larger than the bond distances 384 P. M. Ray which would be involved in the mechanism discussed here. We shall call the active process of bond breaking pictured here the molecular mosaic growth mechanism in contrast to the passive plastic type of deformation discussed earlier. The basic difference between them is, in sum, whether it is the force of turgor or an act of the cell which breaks the bonds as the cell grows. These contrasting mechanisms provide some interesting problems. Many of the characteristics of the growth process could result from operation of either mechanism — this was illustrated above by the dependence upon tmgor pressure. It is important to note that in the measurement of cell wall plasticity (7) one may be dealing ^vith either mechanism of enlargement and therefore not necessarily ^\•ith strict plasticity at all. On the other hand, a clearer view of the prob- lem may suggest experimental approaches useful in revealing the actual mechanics of cell enlargement. As indicated above, one dis- tinction can be made between the number of bonds present and the rate at which bonds are broken. It should be emphasized again that these bonds may not in fact be calcium salt bridges between uronide chains; the distinction will apply to whatever critical bonds are actu- ally involved in cell enlargement. It is not, of course, necessarily the case that cell growth can be described by any single type of molecular event, and it seems possible that both plastic and molecular mosaic types of extension may con- tribute to over-all growth. This does not diminish the importance of the distinction. It also can serve as a starting point for considering more complex forms of bonding which may be involved in cell growth. One example will be given: Suppose that the basis for rigid- ity of the cell wall matrix is interlocking of polysaccharide chains which run randomly and occasionally happen to be caught together in loops, as in a knitted fabric. Extension of such a structure can be brought about only by severing one or both chains in the vicinity of a loop (a molecular mosaic, since splitting of the covalent bond pre- sumably would occur by a metabolic process). Expansion of the structure would soon result in other chains, which were previously not in contact, becoming entangled, and thus assuming the stress on the wall and restoring rigidity. In this model, new forces tending to stillen the wall arise automatically upon extension, and auxin treat- ment of cells under no turgor could result in a subsequent limited extension of the wall, as has been observed in some experiments (2, 8). SUMMARY There are a number of problems in the biophysics of cell wall growth concealed under the vague concej)t of plasticity. The possible Problems in Biophysics of Cell Growth 385 mechanics of growth range from a viscosity problem to questions of macromolecular metabolism. It will be important to obtain further experimental evidence as to what types of bonding are important in the cell wall's mechanical properties and in its ability to expand irre- versibh. LITERATURE CITED 1. Bennet-Clark, T. A. Salt accumulation and mode of action of auxin. A prelim- inary hypothesis. In: R. L. Wain and F. Wightman (eds.), The Chemistry and Mode of Action of Plant Growth Substances, pp. 284-291. Butterworth Sci. Publ., London. 1956. 2. Cleland, R., and Bonner, J. The residual effect of auxin on the cell wall. Plant Physiol. 31: 350-354. 1956. 3. Frey-Wyssling, A. Macromolecules in Cell Structure. 112 pp. Harvard Univ. Press, Cambridge, Mass. 1957. 4. , Miihlethaler, K., and Wyckoff, R. Microfibrillenbau der pflanzlichen Zellwande. Experientia. 4: 475,476. 1948. 5. Ordin, L., Cleland, R., and Bonner, J. Influence of auxin on cell-wall meta- bolism. Proc. Nnt. Acad. Sci. U.S. 41: 1023-1029. 1955. 6. , Cleland, R., and Bonner, J. Methyl esterification of cell wall con- stituents under the influence of auxin. Plant Physiol. 32: 216-220. 1957. 7. Tagawa, T., and Bonner, J. Mechanical properties of the Avena coleoptile as related to auxin and to ionic interactions. Plant Physiol. 32: 207-212. 1957. 8. Thimann, K. V. The physiology of growth in plant tissues. Amer. Sci. 42: 589-606. 1954. B. KESSLER Z. W. MOSCICKI R. BAK National and University Institute of Agriculture Rehovot, Israel The Effects of Decapitation and Growth Regulators on the Movement of Calcium. in Apricot Trees' The movement of calcium in plants is very limited or negligible (1,4) once it has reached certain organs, particularly leaves and fruits. Calcium is not withdrawn from leaves prior to leaf fall in the autumn, and is regarded as being immobile in the phloem. Bledsoe et al. (2) showed that Csl*'^, supplied to the root environment of peanut plants, moved directly to the stems and foliage, but could not move through the phloem to reach the developing fruits in any significant quantity. The latter findings have been confirmed by several authors (8), based on the immobility of radiocalcium applied as a foliar spray (4, 12, 19). Bukovac et al. (4) reported that there was little movement of radiocalcium in the plants, but that after the plants had been anes- thetized with diethyl ether, considerable quantities of Ca^^ moved from the site of application to other parts of the plants. A similar effect upon the movement of Ca^'^ was obtained with triiodobenzoic V acid (TIBA) (12), which interferes with auxin transport (14, 15, 16). The effect of TIBA upon the movement of metal ions, in addition to being a problem of translocation, became of interest in the light of suggestions that the activity of plant growth regulators might, in part, be due to their metal-chelating ability (6). This paper describes the effect upon downward translocation of radiocalcium of several growth regulators. MATERIALS AND METHODS One-year-old apricot seedlings and 6-year-old apricot trees were used for these experiments. Trees of similar performance were se- lected and each experiment was carried out in quadruplicate. The •Publication of the Agricultural Research Station, Rehovot, Israel. Series No. 216-E. [387] 388 Kessler, Moscicki, and Bak following growth inhibitors were tested: triiodobenzoic acid (TIBA), maleic hydrazide (MH), coumarin (CM), dichloroanisole (DCA), and m-tolylphthalamic acid (MTPA) (18). Decapitation of the terminal end of the stem was introduced as an additional treatment and was carried out just before the start of the experiments. Each tree was sprayed with solutions containing 100 p.p.m. of one or another of the above substances. One terminal leaf of each plant was then painted with 0.1 ml. of Ca^^ (0.8 ^c/ml). The same amount of radiocalcium was applied to control plants which had been sprayed with water as pretrcatment. For analysis the fifth leaf below the treated one was collected, dried, and weighed. This leaf was washed and the radioactivity counted. Both a thin end-window Geiger-Mueller tube and a gas flow counter fitted with a micromil window (Nuclear-Chi- cago) in an atmosphere of Q-gas were used. All counts are corrected for background and self absorption to infinite thinness. The results are expressed as total counts per minute (c.p.m.) per g. dry matter. For measurements of the time effect, six similar branches on each tree were treated as above, and at intervals after the application of Ca^s, samples were collected from these six branches. Thus, any change in the movement of Ca could be detected. In another series of experiments the effect of different concentra- tions of TIBA was tested. Concentrations of from 0 to 100 p.p.m. of DAYS AFTER TREATMENT Fig. 1. The movement of foliar applied Ca" in l-year-old apricot trees and the effect of MH and dichloroanisole upon this transport. Movement of Calcium 389 TIBA were applied prior to the application of Ca"*^. Leaves were collected for analysis 9 days after treatment. All treatments and repli- cates were completely randomized. For all tests with the young trees the same control experiment (shown in Figure 1) was used. RESULTS It can be seen from Figure 1 that in young apricot trees there exists a certain movement of foliarly-applied calcium. The radioac- tivity reached 241 c.p.m. 7 days after treatment, in contrast to that obtained with one-year-old apple trees (12). The effect of DCA was only noted after 3 days, after which a rise in the counts became ap- parent. MH, on the other hand, inhibited movement of Ca'*^ during the first 3 days and later caused it to increase. TIBA was particularly effective (Figure 2). This substance completely inhibited the move- ment of Ca^^ in the first 4 days after treatment, but later caused the movement of Ca to increase to give 28,000 c.p.m. Decapitation, coumarin, and MTPA produced rather unexpected results (Figure 3). Decapitation and MTPA increased calcium move- ments during the first 4 days while coumarin was ineffective. But in all three cases the radioactivity dropped again after the initial in- crease, reaching a low of about 50 c.p.m. on the 7th day, as against to b UJ < >- tr a d Z cc lli a. tn o u DAYS AFTER TREATMENT Fig. 2. The effect of TIBA upon the basipetal transport of Ca*^ in 1 -year-old apricot trees. 390 Kessler, Moscicki, and Bak 300 kiJ < >- tr. o 200 - DC Hi 0. in Z o o 100- DAYS AFTER TREATMENT Fig. 3. The effect of decapitation, coumarin, and MTPA upon the basipetal transport of Ca*' in 1 -year-old apricot trees. 241 c.p.m. for the control (Figure 1). This drop in radioactivity must be interpreted as being caused by a stop in the supply of Ca^^ from the treated terminal leaf, while Ca^^ continued to move out from the counted leaves. If we compare the effect of these treatments upon the movement of Ca^^ in one-year-old trees to that in 6-year-old trees, similar trends are obtained. From Table 1 it can be seen that in 6-year-old apricot trees, there was a considerably greater movement of foliar applied calcium than in younger trees. In the old control trees the radioactiv- ity reached 15,560 c.p.m. on the 7th day after the treatment, as against 241 c.p.m. in the young controls (Figure 1). TIBA again showed a considerable promoting effect, while the radioactivity in MTPA- treated leaves dropped as before, after an initial stimulative effect. It is of particular interest that in this case decapitation strongly inhib- ited the downward movement of Ca^^^ As previously stated, we must also assume that in older trees MTPA stops the movement of Ca-*-"^ from the terminal treated leaf after a time while Ca^^ continues to move out from the counted leaf. Preliminary experiments in which the radioactivity of various leaves along the shoots was counted showed this assumption to be apparently true (Table 2). Movement of Calcium 391 Table 1 . The effect of various growth inhibitors and decap- itation upon the translocation of foliarly-appHed Ca''^ from the terminal to the fifth leaf in 6-year-old apricot trees. Treatment Control TIBA MTPA Decapitation Counts Per Minute, 2, 4, and 7 Days After Treatment 5,055 20,307 7,191 799 12,345 27.683 21,888 848 15,560 48,775 3,830 695 Table 2. Time course of the distribution of radioactivity from^Ca''^ along MTPA-treated branches of 6-year-old apricot trees. No. of Leaf Below Treated Terminal Counts Per Minute, 2, 4, and 7 Days After Treatment 2 4 7 3 45,529 30,740 31,078 5 7,191 21,888 3,830 7 133 4.533 15,683 9 158 585 6,346 In view of the activity of TIBA on the movement of calcium, the effect of different concentrations of this substance was tested. From Figure 4 it can be seen that the optimal concentration of TIBA was about 50 p.p.m. while higher concentrations were less effective. DISCUSSION The experimental results reported in this paper suggest that cal- cium is able to move in a downward direction in apricot trees, to some extent. The translocation of calcium in white pine trees has already been demonstrated by Ferrell and Johnson (7). But this movement is very different in various tree species and in trees of dif- ferent ages of the same variety. It was previously found that there is no downward movement of calcium in one-year-old apple trees (12), a finding which is in conformity with the generally held opinion that little or no basipetal movement of calcium occurs within the plant. Comparing the movements of calcium in apple and apricot trees, it 392 Kessler, Moscicki, and Bak a. UJ I- I- < >- a. o LJ 0. < 200 100 60 01 O < < 40 CO Q UJ h- cc o a. en 20 TIP < X < Fig. 3. Relation between auxin transport and mean cell length in 8-day-old bean hypocotyl. Data from Jacobs (8). data derived from Avery and Burkholder (1) indicates that in the Avena coleoptile a similar relation exists to that in the bean hypo- cotyl - more cells correlate with more transport (Figure 4). The development of auxin transport through the tip of young hypocotyls 2 to 8 days old was then studied. No auxin was transported acropetally in any stage. Basipetal transport first appeared in the 5-day tip and steadily increased in amount between the 5- and 8-day stages (Figure 5). But does this gradual development of auxin transport, found in excised sections, have any significance in the normal development of the plant? We found evidence that it does (9). There are two regions of maximum elongation in the hypocotyl: the first occurs in the tip in the very early stages (2 to 3 days after germination); the second develops just below, and becomes the major zone of giowth by 6 to 7 days. Studies on excised sections show that growth in this second zone is limited primarily by auxin, and yet this zone does not produce any significant amount of auxin itself — it gets auxin from the upper parts of the plant. But it cannot get the auxin it needs for growth until the tip region, standing between it and the auxin Auxin Collected - 0.3 22 ,».V,i.i.w.u.u.^;.7.;.;.;.:.;.;,;,v; H DISTANCE FROM TIP OF AVENA COLEOPTILE.M Fig. 4. Relation between auxin transport and mean cell length in 30 to 33 mm. long Avena coleoptiles. Transport data are from Went and White (25), cell-length data from Avery and Biirkholder (1). AGE OF SEEDLINGS, DAYS Fig. 5. The gradual development of basipctal auxin iraii-sport in the tip of young bean hypocotyls. The solid triangles represent native auxin; open circles represent native auxins plus added lAA. Solid lines represent auxin in base to apex collec- tions; dashed lines represent auxins in apex to base collections. Data from Jacobs (8). Polar Movement of Auxin in Slioots 403 supply, tlifferentiates a system for transporting auxin. Confirming this interpretation is the high and statistically significant correlation between the growth of the hypocotyl and the amount of added auxin transported basipetally through the tip. In other words, the ability of the tip to transport auxin apparently controls the growth pattern of the 3- to 8-day hypocotyl (9). The rate of auxin transport in the sections from Avena coleoptile was shown to be 9 to 12 mm/hr in the critical paper by Went and White (25). The old and unresolved question as to whether auxin moved faster through the vascular strands of the coleoptile cylinders than through the other cells was answered by them in the negative — although their published figures seem to show a slight increase in transport ^vhen the added auxin was over the vascular strand. Since the coleoptile is famous for being nonmeristematic, I thought it would be interesting to study the transport properties of a meristem. Accordingly, the intercalary meristem of the gynophore of Arachis was investigated (7, 10). Auxin transport was strictly and basipetally polar, and the rate of transport was 10 mm/hr (Figure 1). In view of the results mentioned below, it should be noted that vascu- lar tissues are maintained intact across the elongating meristem (7). Other determinations of rate are few. Guttenberg and Zetsche (5), using a somewhat different method, provided evidence that the rate of auxin transport in sections cut from the hypocotyls of Helian- thus annuus was also 10 to 12 mm/hr. Using a third method, Gregory and Hancock (4) estimated the rate at 5 mm/hr in sections from apple stems; however, a critical examination of their paper makes clear that this is a minimum rate — the maximum is 10 mm/hr, the same as for the other two organs studied. This uniformity is quite remarkable, considering the differences in the material used. Gregory and Hancock had also been impressed by the flimsy basis of van der Weij's conclusions about temperature effects on auxin transport, so they examined the point with apple stems. Contrary to van der AVeij's conclusions, though not his data, they found that increasing temperature resulted in increasing rate of transport. Although the earlier work on coleoptiles left the impression that strictly polar transport was a characteristic of all cells, we had reason to think otherwise. During the period that auxin transport was gradually appearing in the tip of the bean hypocotyl, the only obvious histological change was the differentiation of vascular tissue. Similarly, the course of xylem regeneration in Coleiis would be easier to under- stand if auxin were normally moving in the vascular tissue rather than through all the stem tissues. Accordingly, transport tests were run in which various areas of the stem were excised. No transport 404 W. P. Jacobs was found in the pith alone, but as much transport as in the com- plete controls was found once the vascular area was included in the transport section (13). The most obvious conclusion is that the normal, high-speed transport of auxin goes on in the vascular tissues of stems. Added auxin moves only basipetally in sections from the Avena coleoptile so long as surface leakage is forestalled and so long as more or less physiological concentrations of lAA (indole-3-acetic acid) are added — this was shown by Went and White in 1939 (25) and con- firmed by Soding and Raadts in 1952 (19). If one doubted the uni- versality of strictly basipetal auxin movement, where should one look for acropetal transport? Certainly not in seedlings, and probably not in determinate organs. A portion of stem from an adult plant, histologically much more complex than the coleoptile and so located that one would expect upward movement of metabolites, would be a likely guess. Internode 2 of adult Coleus satisfies these requirements (11). In addition, Coleus is from a large family (Labiatae) hitherto never critically examined in its auxin relations. When the auxin relations of a rigorously standardized clone of Coleus blumei Benth. were investigated, substantial acropetal trans- port of added auxin was found in internode 2 in addition to the expected basipetal movement (Figure 6). Exhaustive refinements of technique to prevent surface leakage — including lowered humidity No. Apex — ^ Base AA Curvature Relation of blocks to sections Base Apex Curvature lAA No. 10 157 8 7.5 i 1.2 0.9to.4 5.5 I 1.0 0.6 I 0.3 49 10 8 149 16.6 Auxin transported 4.9 44 Fig. 6. Determination of acropetal and basipetal movement of auxin in sections cut from young inlernodes of Coleus wlien 2 mg/1 of indole-3-acetic acid in agar is added (shaded blocks). The apical end of the section is designated A, lAA represents the collected auxin calculated as though it were all indole-3-acetic acid. From Jacobs (11). Polar Movement of Auxin in Shoots 405 and vaseline rings around all sections — did not affect the acropetal movement of auxin (11, 12). With 2 mg/1 of lAA in agar added at each end, the ratio of basipetal to acropetal auxin movement was 3:1. What reason was there to think that this acropetal transport in isolated stem sections was of significance in the more intact plant? Evidence was obtained from parallel studies of xylem regeneration in this same standardized internode. The leaves are the major source of auxin in Coleus, and excising the leaves causes a decrease in the number of xylem cells regenerated, the decrease being exactly pro- portional to the amount of diffusible auxin produced by the leaf. When synthetic lAA is substituted for the leaves so as to give exactly the same amount of auxin as they produce, the lAA exactly replaces the xylogenic effect of the leaves (11, 14). Thus we have detailed evi- dence that auxin from the leaves is the factor normally limiting xylem regeneration. Now, when regeneration occurs in plants with all their leaves left on, their course of regeneration parallels the ob- served 3:1 ratio of auxin transport — i.e., regeneration is mostly basipetal, but with a significant amount of acropetal regeneration occurring, too. And Figure 7 shows more quantitative data: if we 0 AMOUNT OF AUXIN TRANSPORTED 20 65 1 AUXIN FROM ABOVE ■BII^^^^^H^H AUXIN FROM RFI n\A/ ^^^H 1 I 5.6 NUMBER OF XYLEM STRANDS I Fig. 7. Relation bet^veen amount of auxin transported through excised sections of Coleus and the number of xylem strands regenerated in the plant when leaves above and below are excised. From Jacobs (12). 406 VV. F. Jacobs compare regeneration in plants in which only distal sources of auxin are lett on (i.e., leaves 1, 2, and the apical bud) with plants with only proximal sources of auxin (leaves 3 to 8), we see that the number of xylem cells regenerated is nicely proportional to the observed auxin transport (12). Our conclusion is that the acropetal auxin transport is of real physiological significance.^ Another idea is suggested by musing over the results with the bean hypocotyl and Coleiis internode 2. In both cases lAA was added at a concentration of 2 mg/1 of agar. And in both cases, very exact relations could be observed between the amount of auxin transported under these circumstances and the amount of growth or of xylem regeneration. This suggests that, contrary to what one might expect from the reports of van der Weij (20) or Went (23), there is 7iot a steady increase in auxin transported as one increases the amount added, but rather a plateau - and that 2 mg/1 is on the plateau. We are currently checking the hypothesis in two ways: by direct assay with the Ave7ia curvature test of transport through isolated sections ringed with vaseline (Figure 8), and by indirect test using xylem regeneration in Coleiis again as a measure of how much auxin is getting into the more intact plant. Xylem regenerated under the influence of the shoot tip could be completely replaced by 1 per cent lAA in lanolin. And as evidence of saturation was the fact that 10 per cent lAA in lanolin gave no further increase in the number of xylem strands which regenerated [Figure 5 in Jacobs ct al. (15)]. The Avena curvature results are not definitive, since they do not yet include dilutions of the collected auxin. (This means that there is a possibility that the Avena curvatures for the transport of 1.25+ mg/1 are above the proportionality range of the Avena bioassay. We are, of course, checking this possible artifact now.) But, so far as they are valid, the two tests confirm the hypothesis and each other. Auxin transport in Coleus seems to show a saturation effect at concentra- tions of \-{- mg/1 of agar. Experiments already described, plus others ilescribed b) Jacobs in 1954 (12), provided evidence that the transport capacity at satur- ation normally limits how much auxin is available in the plant. Al- though the leaves normally produce auxin at a level matching the saturation capacity of the stem (12), their level of production is sub- ject to fluctuation as the environment changes. The potential im- ^The appearance, wiili flowering, of acropetal auxin transport in stems ot Coleus has been reported (16), the report criticized on grounds of inadccjuate technique (12), and found unconfinnable by Haupt (6). Professor Leopold has told me that his attempts to repeat the experiments have given inconsistent results. Polar Movement of Auxin in Shoots 407 en UJ Ld OC O LJ Q UJ cr Z) I- cr UJ > < CONCENTRATION OF lAA ADDED, P.RM. Fig. 8. Evidence of saturation in auxin transport in young internode sections of Coleus. The sections were prediffused for 2 hrs., ringed with vaseline (to obviate surface leakage), then 5 mm. sections were cut from the slightly longer pre- diffused sections. Transport time was 25 hrs. portance of the saturation capacity in the physiology ol the plant is thus obvious: it could act as a controlling valve which prevents excess amounts of auxin from being transported around the plant and thereby disturbing the balanced coordination of normal development. That such a saturation capacity is not limited to Coleus is indi- cated by the results in bean hypocotyl mentioned above and by trans- port tests on Avena coleoptiles (Goldsmith, Ph. D. thesis, Harvard). From the earlier view of normal auxin transport as something unique, strictly one-way, and unchangeable by various environmental factors, a new view has thus developed since 1939. Auxin transport, in these later researches, appears as a function which varies with the physiological and histological state of the tissue, which is responsive to changes in the environment and which is 7iot always strictly polar. In addition, it has been shown not only that auxin transport controls the relative polarity of various developmental phenomena, but also that organs have a maximum capacity in the physiological range of concentrations for transporting auxin, and that this set transport capacity serves an important regulatory function by buffering the plant from sudden increases in auxin level. What of the future? We need to know how general are the results now known in detail for seedling organs and a few vegetative stems. We need critical direct experiments on auxin transport in roots, 408 W. P. Jacobs with the resuks meaningfully related to the normal physiology. To better understand the role of the plateau capacity of auxin trans- port, we need to know how stable is this plateau level. From the current view of auxin transport it would be reasonable to expect that continued exposure to higher than normal auxin levels would cause gradual regulation in the plateau capacity. Knocking out auxin movement by the addition of chemicals (2, 3, 17, 18) may lead to an understanding of the biochemical basis of auxin transport. It would be particularly intriguing if a chemical could be found which would actually knock out the polarity — i.e., allow equally fast move- ment of auxin in both directions in a normally strictly polar organ like the coleoptile. ACKNOWLEDGMENT Grateful acknowledgment is made to the National Science Founda- tion and to the American Cancer Society for grants in support of the research reported here. LITERATURE CITED I. Avery, G. S., Jr., and Burkholder, P. R. Polarized growth and cell studies on the Avena coleoptile, phytohormone test object. Bui. Torrey Chil) 63: 1-15. 1936. 2. Clark, W. G. Electrical polarity and auxin transport. Plant Physiol. 13: 529-552. 1938. 3. du Buy, H. G., and Olson, R. A. The relation between respiration, proto- plasmic streaming and auxin transport in the Avena coleoptile, using a polarographic microrespirometer. Amer. Jour. Bot. 27: 401-413. 1940. 4. Gregory, F. G., and Hancock, C. R. The rate of transport of natural auxin in woody shoots. Ann. Bot. II. 19: 451^65. 1955. 5. Guttenburg, H. v., and Zetsche, K. Der Einfluss des Lichtes auf die Auxin- bildung und den Auxintransport. Planta. 48: 99-134. 1956. 6. Haupt, W. Gibt es Beziehungen zwischen Polaritiit und Bliitenbildung? Ber. Deutsch. Bot. Ges. 69: 61-66. 1956. 7. Jacobs, W. P. The development of the gynophore of the pcaiuit plant, Aracliis hypogaca L. 1. The distribution of mitoses, the region of greatest elongation, and the maintenance of vascular continuity in the intercalary meristem. Amer. Jour. Bot. 34: 361-370. 1947. 8. . Auxintransport in the hypocotyl of Phaseolns vulgaris L. Amer. Jour. Bot. 37: 248-254. 1950. 9. . Control of elongation in the bean hypocotyl by tiie ability of the hypocotyl tip to transport auxin. Amer. Jour. Bot. 37: 551-555. 1950. 10. . Auxin relationships in an intercalary meristem: further studies on the gynophore of Aracliis hypogaea L. Amer. Jour. Bot. 38: 307-310. 1951. 11 . The role of auxin in differentiation of xylem around a wound. Amer. Jour. Bot. 39: 301-309. 1952. 12. . Acropetal auxin transport and xylem regeneration — a quantitative study. Amer. Nat. 88: 327-337. 1954. Studies on abscission: the physiological basis of the abscission-speed- ing effect of intact leaves. Amer. Jour. Bot. 42: 594-604. 1955. Polar Movement of Aiixm in Shoots 409 14_ Internal factors controlling cell differentiation in the flowering plants. Amer. Nat. 90: 163-169. 1956. 15 ^ Danielson, J., Hurst, V., and Adams, P. What substance normally controls a o-iven biological process? II. The relation of auxin to apical dom- inance. Developmental Biol. I: 534-554. 1959. Hi. Leopold, A. C, and Guernsey, F. S. Auxin polarity in the Coleus plant. Bot. Gaz. 115: 147-154. 1953. 17. Niedergang-Kamien, E., and Leopold, A. C. Inhibitors of polar auxin trans- port. Physiol. Plant. 10: 29-38. 1957. 18. , and Skoog, F. Studies on polarity and auxin transport in plants. 1. Modification of polarity and auxin transport by triiodobenzoic acid. Physiol. Plant. 9: 60-73. 1956. 19. Soding, H., and Raadts, E. tJber die Leitung des Heteroauxins in der Hafer- koleoptile. Ber. Deutsch. Bot. Ges. 65: 93-96. 1952. 20. Weij, H. G. van der. Der Mechanismus des Wuchsstofftransportes. Rec. Trav. Bot. Neerl. 29: 379-496. 1932. 21 _. Der Mechanismus des Wuchsstofftransportes. II. Rec. Trav. Bot. Neerl. 31: 810-857. 1934. 22. Went, F. W. Wuchsstoff und Wachstum. Rec. Trav. Bot. Neerl. 25: 1-116. 1928. 23. . Salt accumulation and polar transport of plant hormones. Science. 86: 127,128. 1937. 24. , and Thimann, K. V. Phytohormones. 294 pp. Macmillan, New York. 1937. 25. , and White, R. Experiments on the transport of auxin. Bot. Gaz. 100: 465-484. 1939. A. C. LEOPOLD and S. L. LAM Purdue University Polar Transport of Three Auxins' In understanding the polar system which transports auxin through plants, it is particularly relevant to know whether the transport sys- tem is specific to indole-3-acetic acid (I A A). There is evidence in the earlier literature that some other auxins may be carried in the trans- port system (10), but it is not clear whether these compounds may all be moved with the same polarity and the same velocity as indole-3- acetic acid, nor is it clear how effectively the transport system can distinguish between auxins of different molecular structure. Experi- ments on these questions were undertaken in an effort to characterize further the polar transport system in plants. METHODS The auxins were assayed in every case by means of the Avena cur- vature test, following the procedure described by Leopold (4). The auxins were transported through stem sections taken from the epicotyl of sunflower seedlings grown in the greenhouse between December and April. The method is identical to that described by Niedergang-Kamien and Leopold (6). A donor block was placed at the apical end of a 5 mm. stem segment, and the auxin transported into a receptor block at the basal end was assayed after 120 min. of transport at 25° C. Unless otherwise indicated, all transport tests with indole-3-n-butyric acid continued for 180 min. The experiments with transport inhibitors were carried out by the method of Niedergang-Kamien and Leopold (7). The inhibitor was applied in an agar block at the basal end of the sunflower stem sec- tion, and the auxin was simultaneously applied as a 5 /^l. droplet at 1 Journal Paper No. 1493, Purdue University, Agricultural Experiment Sta- tion, Lafayette, Indiana, U.S.A. [411] 412 A. C. Leopold and S. L. La m the apical end. After 30 min. the stem section was blotted and trans- ferred to a fresh receptor block in which the transported auxin was collected for an additional 90 min. In these experiments, the con- trols were transferred from a plain agar block to a fresh receptor block at the same time that the inhibitor treatments were transferred. This represents the only modification of the earlier technique. RESULTS Preparatory to a comparison of the transport of several different auxins, five auxins were tested for activity in the Avena curvature test. Included in this test were indole-3-acetic acid (lAA), 1-naphtha- leneacetic acid (NAA), indole-3-n-butyric acid (IBA), indole-3-propi- onic acid (IPA), and 2-naphthoxyacetic acid (NOA). Characteristic Avena test data for these five auxins are shown in Figure 1. Of course the greatest curvature response was obtained with lAA. Normal cur- vature responses were also obtained with NAA and IBA, though less curvature was obtained with these per unit of auxin. IPA and NOA yielded essentially no curvatures. The Avena curvature test is, therefore, a valid assay for the three auxins: lAA, NAA, and IBA. The transport of these three auxins in both basipetal and acrope- 10 10"' 10'=" MOLAR CONCENTRATION OF AUXIN 10" Fig. 1. Response curves for five auxins in the Avena curvature test: indole-3- acctic acid (IaA), indolc-3-n-butyric acid (IBA), 1-naphthaleneacctic acid (NAA), indole-3-propionic acid (IPA), and 2-naplulioxyacctic acid (NO.^). Polar Transport of Three Auxins 413 Fig. 2. flower time 1 BLOCK The acropetal and basipetal transport of three auxins applied to sun- stem sections in various concentrations. Symbols as in Figure 1. Transport 20 min. for lAA and NAA, 180 min. for IBA. tal directions was also measured. Each was presented in agar donor blocks to sunflower stem sections. Concentrations of the auxins were varied between 3 X lO-'^M and lO^Af. Assays of the receptor blocks provide data such as those shown in Figure 2. Looking first at the basipetal transport, it can be seen that each of these three auxins is transported downward in the manner usual for lAA. The amount of auxin transported is presented simply as the Avena curvature without correction for difl;erences in activities in the Avena curvature test or for length of transport period. It can be readily seen, however, that downward transport was obtained, and that NAA and IBA were ap- parently transported in somewhat smaller quantities than lAA. The tests of upward transport show that within the range of concentra- tions of donor blocks tested (10-^ to IQ-^AI) essentially no transport was obtained. We can, therefore, conclude that each of these three auxins is transported in a polar manner. A further comparison of the transport of the three auxins can be made by measurements of the velocities of transport. Velocity of transport was measured in the manner originally used by van der Weij (9). Sunflower stem sections were provided with donor blocks 414 A. C. Leopold and S. L. Lam at the apical end for different periods of time. The donor blocks and the stem sections were removed from the receptor blocks which were then assayed for auxin. The concentration of lAA in the donor block was IQ-^M, and the concentration of the NAA and IB A was 10-^M in each case. The results of velocity measurements are shown in Figure 3. It can be seen that 40 minutes of transport time were necessary before lAA could be detected in the receptor block. The stem section was 5 mm. long, and so the velocity can be estimated at 7.5 mm/hr. The first NAA detectable in the receptor block appeared after about 45 min., indicating a velocity of 6.7 mm/hr. The first IB.\ detectable appeared after about 95 min. indicating a transport velocity of 3.2 mm/hr. Each of these values was determined several times with separate experiments and the variation was not in excess of 1 mm/hr. Another means of asking whether the three auxins are transported by similar or dissimilar physiological processes is to measure the sen- sitivity of each of them to inhibitors of transport. For these experi- ments the following inhibitors were selected: 2,4-dichlorophenoxy- acetic acid (2,4-D), a-methoxyphenylacetic acid (MOPA), 2,3,5-triiodo- 160 DONOR TIME, MINUTES Fig. 3. Determinations of the velocity of transport of three different auxins. Sun- (loucr sections transporting the auxins are applied to receptor blocics for in- ci easing periods of time and the time of initial response in subsequent Avena test is indicated l)y tlie arrow. Symbols as in Figure 1. d <; __ ^ ,^ oZ 2? a V c O i; in o ■ u lO u V M o ^ CL -^ a a ■%^ ^ r- SO +- -w Tf tN O -, 1 iJo 03 '" .2< 4-» -*-» •4-* i^ <^ G c CO u s^ or so CJ (N CU > Z2 a '- D. o. so in 00 -^ — ' ■■ — ^ t.O O '^ ■-s _ c C C C ^ 2 <^ 4J 00 <-> V o (J CQ >= ■ t^ • u • i_ " J-, CM V ^ V •^ V h2 a a a ^*- r^ Ti- 00 CO O o 00 00 C^ — o *■ — ' "xl a c c libito BA, <5: §2 V 00 V o u • u .1 u a; CO o -Ci 1— ( a o a 00 ir> m sO !-■ . _ ^ go 4-t C c c Q^ U u i; «'- u 00 «J Tj- o §< 1 -f "^ u u u '^ k • t/ ■^ aj CO (J ■s< ^"2 cs a ^ a a 1 >z ■* LO LD ;2;^ CJ 1^ >-T3 ^-^ •^ s c^ CO .S< o §< ti t- r-- Tf fli o £§ r~- in CO Jjci -C C T— ( T-H ^-^ Si . c J3 C HI 4-> .S «« s o "^ ■mS ti cs ©■r- to S. 5 B CQ •'3 ti +^ ♦^ « vm s-> O 0 S c .2 « *^ r'- c 3H ■ •-4 3 < C c c _0 ■*- +-» .IE ,;-^ IS V . a V's. (U .- : V IS JiK . 5^ : !c = >g Tab gle ex dIBA 3 (. ^ 3^ J 3 " 1 ^ s- .S c < 4 2 fC 3 416 A. C. Leopold and S. L. Lam benzoic acid (TIBA), 2-naphthoxyacetic acid (NOA), and indole-3- propionic acid (IPA). Each of these except the MOPA and NOA has been reported as being an inhibitor of lAA transport, and these last two had been found to be inhibitors in experiments in this laboratory. Results of typical inhibition experiments are presented in Table 1. The tests were conducted with each transported auxin in one test, so that the relative effectiveness of the various inhibitors as they in- fluence one auxin is a more reliable comparison than as they influ- ence the several auxins. The data show clearly that each of the in- hibitors is effective against the transport of any of the three auxins. Within this array of transport inhibitors, then, there is no evident distinction between the auxins being transported. It is clear that TIBA is by far the most potent inhibitor of transport of each of the three auxins, causing more than 80 per cent inhibition of transport in each instance at a concentration of only \{)-^M. The inhibitions obtained with the other inhibitors appear to be comparable to one another. It might be mentioned that the naphthalene containing inhibitor NOA was not apparently more effective against the trans- port of NAA than the other inhibitors. Similarly, IPA was not more effective against the transport of the indole auxins than the other in- hibitors tested. DISCUSSION The experiments reported here provide a comparison of the trans- port characteristics of three different auxins: lAA, NAA, and IBA. It is clear that all three are polar in their movement, and they vary somewhat in velocities. They are all sensitive to the same transport inhibitors, and there does not appear to be a selectivity by transport inhibitors for the different auxins. The transport of the three auxins therefore appears to be very similar indeed, except for the diilerences in velocity. While lAA and NAA appear to have very closely similar transport velocities, IBA is markedly slower. It has been established by Fawcett et al. (1) that IBA is metabolized in plant tissues giving rise to lAA, and while it is not certain that this conversion must occin- before growth activity is obtained, it is reasonable to assinne that there would be a gradual production of lAA in the tissues during transport tests. The delay in conmiencement of cmvatine responses in the time curves with IB.\ may well be due to the time required for the con- version of the IBA into lAA, which then would, of course, have the transport characteristics of that auxin. A comparison of the transport velocities of these three auxins plus anthraceneacetic acid was made by Went and ^Vhile (10) with some- Polar Transport of Three Auxins 417 what different conclusions. They interpreted their experiments as indicating that IBA transport was 25 per cent slower than lAA. Our experiments indicate IBA to be about 50 per cent slower. They found NAA to be transported 58 per cent more slowly than lAA, and oin- experiments indicate the rate to be almost the same as for lAA. Ex- amination of the method of determination of velocity used by Went and White suggests that precise calculations would be very difficult to make by their method. It involves the determination by photokymo- graph of the moment of initial curvature of Avena coleoptiles when the transporting coleoptile sections are placed unilaterally upon the tip and a donor agar block on top of that. The determination of the moment of initial curvature is complicated by nutational movements of the coleoptile, by initial negative curvatures of the coleoptile, and by uncertain rounded sections of the time curve which make defini- tion of the unresponding and responding sections rather uncertain. Using the more precise method of van der Weij (9), we feel that the velocities reported here are quite exact within 1 mm. per hour vari- ability at most. The possibility exists, of course, that the velocity of NAA transport in Avena coleoptiles may be slower than in sunflower stems. The strictness of polarity of transport of lAA reported by the earlier workers is confirmed here, and in addition the same is found for IBA and NAA. The report by Went and White (10) of lack of transport for indole-3-propionic acid is also confirmed here. In view of the close similarities of the characteristics of transport of lAA, NAA, and IBA, it can be deduced that the transporting tissue cannot distinguish between these three auxins, except for the velocity difference for IBA as discussed above. Molecules with closely similar structures can be effectively differentiated against. Thus there is ap- parently no transport of indene-3-acetic acid (8), of indole-3-propi- onic acid, cis-cinnamic acid (10), the phenoxyacetic acids (5), or of 2- naphthoxyacetic acid. Indirect evidence may permit the inclusion of phenylacetic acid in this list (2). It appears, then, that the polar transport system is not specific for the indole ring, but it may be spe- cific for a two-cyclic ring since none of the phenyl and phenoxy auxins seem to be transported in this system. The inhibition of apparent transport by two benzoic acid auxins (3) indicates that these mono- cyclic auxins may not be transported either. It has been suggested by Niedergang-Kamien and Leopold (7) that an adsorptive phase may be involved in the polar transport system. Since the most likely site of adsorptive attachment of an auxin would be on the aromatic ring, it is reasonable to suppose that the speci- ficity of the transport system for certain ring structures may be related to the adsorptive characteristics produced by the ring. 418 A. C. Leopold arid S. L. Lam SUMMARY The transport of indole-3-acetic, indole-3-«-butyric, and 1-naphtha- leneacetic acid through sunflower stem sections has been examined. Evidence is provided that the transport of each of these auxins is polar in nature. The velocities of transport of indole-3-acetic acid and of 1-naphthaleneacetic acid are closely similar, while a slower apparent rate obtains for indole-3-n-butyric acid. The sensitivities of transport of the three auxins to transport inhibitors were also meas- ured, and no striking differences between the three auxins were found. It is suggested that the three auxins move in the same trans- port system, and the system is not exclusively specific for the com- mon plant growth hormone, indole-3-acetic acid. LITERATURE CITED 1. Fawcett, C. H., Wain, R. L., and Wightman, F. Beta-o\idation of omega- (3-indolyl) alkanecarboxylic acids in plant tissues. Nature. 181: 1387-1389. 1958. 2. Gustafson, F. G. Lack of inhibition of lateral buds by the growth-promoting substance, phenylacetic acid. Plant Physiol. 16: 203-206. 1941. 3. Keitt, G. W., Jr., and Skoog, F. Effect of some substituted benzoic acids and re- lated compounds on the distribution of callus growth in tobacco stem explants. Plant Physiol. 34: 117-122. 1959. 4. Leopold, A. C. Auxins and Plant Growth. 354 pp. Lniv. Calif. Press, Berkeley. 1955. 5. Niedergang, E. Chemical modification of polarity and auxin transport in plants. Ph.D. Thesis, Univ. of Wis. 1954. 6. Niedergang-Kamien, E., and Leopold, A. C. Inhibitors of polar auxin trans- port. Physiol. Plant. 10: 29-38. 1957. 7. , and Leopold, A. C. The inhibition of transport of indolcacctic acid by phenoxyacetic acids. Physiol. Plant. 12: 776-785. 1959. 8. Thimann, K. V. On an analysis of the activity of two growth-promoting sub- stances on plant tissues. Proc. Kon. Akad. Wet. Amsterdam. 38: 896-912. 1935. 9. Weij, H. G. van der. Der Mechanismus des W^uchsstofftransportes. Rec. Trav. Bot. \terl. 29: 379-196. 1932. 10. Went, F. W., and White, R. Experiments on the transport of auxin. Bot. Gaz. 100: 465-484. 1939. BRUCE B. STOWE^ Harvard University The StLmutatlon of Auxin Action by Llpldes Studies with the gibberellins have exposed a major paradox: intact dwarf peas respond markedly to treatment with gibberellic acid, whereas epicotyl sections cut for bioassay from similar plants respond hardly at all (1,20). Thus a conventional straight growth section bioassay for growth substances is almost useless for the estimation of gibberellins. More importantly, this observation exposes a hiatus in our understanding of the mechanism of growth in these sections, and moreover, this gap seems particularly amenable to experimental analysis. Our own measurements of the growth, under optimum conditions, of dwarf peas and of the sections obtained from them are summarized in Table 1 (18). It is apparent that, as has been reported by others, gibberellic acid (GA3) itself has only a marginal influence on the sections; more significant is its further promotion of the effect of indole-3-acetic acid (I A A). Even so, the sections under these optimal conditions are not even two-thirds of the length they would have reached on the intact plant, and only one-third of the length they would have achieved with GA3 treatment. A likely explanation of this growth deficiency is that some factor required for gibberellin action is normally supplied to the section by the rest of the plant. In previous work (16) we found that a fat fraction could be isolated from peas which markedly promoted sec- tion growth, and it was natural to test this for its ability to restore part of this missing growth. This fat fraction was not a specific sub- stance, however, as a wide variety of fatty acid esters could also in- crease the growth obtained. 1 Subsequently: J. W. Gibbs Laboratory, Department of Botany, Yale Uni- versity, New Haven, Conn. [419] 420 B. B. Stoxue Table 1 . Comparison of percentage increase in length in 24 hrs. of sections from, and of intact zones on, dwarf 'Lax- ton's Progress' peas. Plants and sections continuously exposed to medium by rotating on shaker within petri dishes. Data of two experiments (18), with standard deviations. Percentage Increase in Length Medium II 10 Mm. Sections Basal medium* 44.9 ± 5.6 39.9 ± 3.5 + GA3 (0.3 mM) 54.6 ± 5.3 39.9 ± 7.9 + lAA (1.7 nM) 67.5 ± 7.0 65.1 ± 10.5 + GA3 + lAA 84.3 ± 6.4 70.1 ± 12.0 10 Mm. Zone on Intact Plant Basal medium* + GA3 + lAA 132.4 ± 15.3 201.1 ± 14.6 125.0 ± 16.8 234.0 ± 24.1 * Sucrose, 1.25 per cent + 50 fxM C0CI2 + 5 mM KH2PO4 (pH 5.5). Figure 1 shows the data obtained both with the dwarf 'Laxton's Progress' pea and the 'Alaska' pea. In each case, section elongation can nearly be doubled by an extract of pea glycerides or with pure methyl esters of certain fatty acids. The Tween detergents, which are also fatty acid esters, also showed growth-promoting activity. It is apparent that the differences between the dwarf and the 'Alaska' pea sections are small. In fact, although dwarf pea sections have been used predominantly in this work, each time results have been checked on 'Alaska' pea sections the response has been very similar. When the new lipide factor is added to the tests of Table 1, using one of the most active compounds, methyl linoleate at 68 |.u\/, the section growth is increased to 113.8 ± 7.3 in the first test and 107.4 ±: 9.7 in the other. Even so, these increments still bring growth up to only four-fifths of that of untreated plants and to not quite half the growth anticipated in the presence of GA;;. In the hope that some other li})ide might futhcr stimulate the growth response of the sections, some thirty-odd pure substances have been examined. Table 2 summarizes these results, which indi- cate that many alkyl compounds with a chain length greater than twelve carbon atoms are capable of accentuating the growth of pea ALASKA LAXTONS PROGRESS 140 100 - 60 20 ^^ r -II- / I u l^^[ H ■Hh A Pea Glycerides j::! 10 30 100 0 ■ih 10 30 100 LJ O cr UJ Q_ 1- < CD O _l UJ 140 100 - 60 20 ^^ r -H yj /^-o' T=± I I + Tween 20 0 Tween 80 rx - ''«=y -i-// — \ — I — ^ — 0 I 3 10 30 100 B Tweens T r / + J L I 3 10 30 100 20 0 -//- T T-VA-r X Methyl linoleate • Methyl oleate 1 — r k-4t=^.— /7 C Fatty Acid Esters I -ih 4 10 20 40 100 0 ■■ i 2 4 10 20 40 100 CONCENTRATION, MG./L. Fig. 1. Percentage elongation of 10 mm. pea epicotyl sections after 24 hrs. in 1.25 per cent sucrose + 50 ^M CoCL + 5 niM KH,PO, (pH 5.5) + 1.7 ^Af lAA -i-O.SAiMGAa plotted against a logarithmic scale for various lipides in mg/1. In (A) bars represent standard deviations; crosses and circles are different ex- periments. Reproduced from (16). 422 B. B. Stoiue Table 2. Comparison of effectiveness of different classes of simple alkyl lipides in enhancing auxin-induced growth of 'Laxton's Progress' pea epicotyl sections. Full details are published elsewhere (18); the most active compounds are cited in text. Alkyl Lipide Class Compounds less than C12. Fatty acid methyl esters. . Triglycerides Fatty alcohols Monoglycerides Free fatty acids Efficacy Many esters, glycerides, alcohols tested; none active. All 9 tested (in C12 to C20 range) found active. All 4 tested (tri-palmitin, -stearin, -olein, and -linolein) active. 4 (in C18 to C22 range) active, 2 (Cu and Cie) inactive. 2 (C16 and Cig) active, 4 (Cu, Cig, C20, and C22) inactive. All 7 tested (in C12 to C22 range) found inactive. sections. The free fatty acids are ineffective, despite the fact that their esters are among the most active compounds. The most active simple alkyl substances discovered to date are triolein, trilinolein, methyl mynstate, and selachyl alcohol. All are of comparable ac- tivity, but none can bring section growth entirely back to normal — values like those reported above for methyl linoleate being typical of the group as a whole. Thus it is likely that yet other factors are required for optimal growth, and in particular for the growth promotion caused by gib- berellic acid. Lockhart's (13) and Galston and Warburg's (9) results may also be interpreted as indicating that the rest of the plant pro- duces a factor which enhances gibberellin action on the sections. Nonetheless, attempts to extract such an additional factor have not been successful. It seems likely that the inissing substance could be the caulocaline postulated some years ago by \Vent (24). It was de- duced that this was synthesized in the roots and could only travel through living tissue. Went showed that it recjuired pea tip auxin, but pea tips are now known also to produce gibberellin (13). Alternatively, it may be that gibberellic acid and dihydrogib- berellic acid are not the active form of gibberellin but are converted to it in some other part of the plant. This would fit in ^\'ith the results of Phinney (15) reported at this conference, which indicate a biochemical pathway of gibberellin synthesis in maize. Since in this study a technique had been adopted Avhich made it possible to prepare stable aqueous emulsions of nearly any fat-soluble substance (18), it was thought desirable to investigate the effect of the hitherto inaccessible fat-soluble vitamins on the section bioassay. Tests of vitamins A, Do, E, K^, and (3-carotene revealed that of these E and K, were as effective as the simple alkyl lipide comj)ounds cited Stimulation of Auxin Action by Lip ides 423 earlier, and were active at even slightly lower concentrations (19). The absolute growth, however, remained no greater than before. The other substances were inactive. Vitamins E and K^ possess a bicyclic ring with an isoprenoitl side chain. In animals the side chain is not essential for vitamin K activity and, accordingly, the side-chainless vitamin K5 and menadione analogues were tested on pea sections (19). No growth promotion was noted, but when vitamin K/s side chain, phytol, was tested by itself, growth promotion was found. A less exact analogue of the vitamin E side chain, farnesol, was ineffective. However, the activity of phytol makes it seem likely that the potency of both vitamin E and K^ is due to their possession of a long chain hydrocarbon substituent. Some common property of a wide variety of long-chain hydro- carbon compounds is thus responsible for the greater elongation of the pea stem sections. Tests of a number of components of lipide metabolism, such as coenzyme A, thioctic acid, acetate, pyruvate, cytidine, choline, etc., and of compounds involved in isoprenoid synthesis, such as mevalonic acid and its lactone, as well as adenine, niacinamide, and ribose, have not revealed any connection of this phenomenon with well-known biochemical pathways. Nor have physical factors such as surface action of these compounds or in- creased uptake of auxin been found to be implicated in any tests yet carried out. The details of these largely negative findings have therefore been left to other publications (18, 19). In this connection, one of the most significant features of the lipide effect is the fact that the optimal amounts required are much too small to meet any major nutritive requirement of the sections. Table 3 shows that a 4 //if concentration of vitamin Ki - scarcely more than twice the concentration of lAA used - suffices for a Table 3. The dependence on auxin of the response to Upides of 10 mm. 'Laxton's Progress' pea epicotyl sections. Percentage increase in length with standard devia- tions after 24 hrs. (17). Treatment No Lipide Plus Methyl Linoleate (50 ijlAI) Plus Vitamin Ki (4 mM) Plus Vitamin E {\0 ixM) Basal medium* 39.1 ± 3.6 38.4 ± 3.2 40.6 ± 3.4 35.7 ± 5.9 + GA3 (0.3 txM) 41.8 ± 15.0 46.9 ± 5.8 51.2 ± 8.2 43.6 ± 9.4 + lAA (1.7 juM) 55.7 ± 6.2 68.7 ± 6.5 62.9 ± 7.6 65.9 ± 4.6 -f GA3 + lAA 67.4 ± 5.6 99.7 ± 5.0 104.3 ± 10.2 94.0 =t 6.2 ■ Sucrose, 1.25 per cent + 50 fiM C0CI2 + 5 mM KH2PO4 (pH 5.5) + 0.004 per cent Pluronic F-68. 424 B. B. Stowe considerable accentuation of section growth over that achieved with lAA plus GA3 alone. The concentrations of vitamin E and methyl linoleate employed, although higher by 6 to 30 times that of lAA, still may be considered to lie within a hormonal range. Further examination of the data of Table 3 reveals that the lipides are completely ineffective in the absence of an auxin (other tests have shown that 1-naphthaleneacetic acid and 2,4-dichIorophen- oxyacetic acid are also capable of potentiating the lipide response). A small promotion of gibberellic acid action is frequently found, but has never reached the level of statistical significance. The enhance- ment of auxin-induced growth is unmistakable, but it is in the presence of both auxin and gibberellic acid that the greatest margin of significance is attained. Hence it appears that the lipides act hormonally, as synergists of the action of auxins and, in the presence of an auxin, of gibberellic acid. Any explanation of the lipide effect thus must be linked to the actions of these hormones. Although the evidence is still far from conclusive, we have ad- Table 4. Comparison of effectiveness of different lipides in enhancing auxin- induced growth of sections of 'Laxton's Progress' pea epicotyls with their ability to restore cytochrome activity of isooctane extracted animal particulate preparations (17). Lipide Relative Effectiv^eness on Pea Sections* Per Cent Restoration of Particulate Cytochrome Activity f Natural lipide extract . . Vitamin E + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 0 0 0 0 0 0 0 100 100 Vitamin Ki 100 Phytol Methyl linoleate Methyl linolenate Triolein Trilinolein Ethyl palmitate Ethyl stearate Monopalmitin 100 100 100 76 50 50 55 0 Tristcarin 0 Tripalmitin 0 Menadione 50 Vitamin A 0 Laurie acid Myristic acid Palmitic acid Stearic acid Oleic acid 0 0 0 0 0 ♦Data of (16). t Compiled from data in (5, 7, 23). Stimulation of Auxin Action by Lipides 425 vanced the hypothesis that the effect of the lipides is due to their activation of the cytochrome system leading to a greater availability of energy for growth (17). The results which have been outlined above bear an amazing similarity to the data obtained by workers with animal systems of isooctane-extracted cell particles (5, 7, 23). In these systems the cytochrome activity of the particles can be re- stored by a number of lipide compounds which apparently facilitate the action of cytochrome-c reductase. Table 4 gives the results of a comparison of a number of compounds which have been tested in both the pea system and with the isooctane-extracted particulates. Of the 19 compounds only 4 show activity in one system and not in the other, and in each of these cases the activity that was observed was submaximal. The analogy probably does not hold in another case, since in a few tests with coenzyme Qio," a lipide-soluble quinone which is found in plants (4) and which acts on mitochondrial electron transport, no convincing effect could be noted in our system. Further- more, the inhibition of section growth caused by antimycin A was not reversed by vitamin Kj, but an irreversible effect of this inhibitor is also shown by some particulate systems (5). Further support to the idea that the respiratory system is involved is given by the data of Figure 2 which show that both methyl myristate and vitamin E increase the respiration of the sections above the values obtained in auxin plus GA3 alone. Although this promotion is not large, it is comparable to that produced by auxin over the con- trols in basal medium, which would be expected from the fact that the growth increments in each case are roughly the same. Obviously, evidence of this kind cannot conclusively implicate the cytochromes. However, the work of Hackett and Schneiderman (11) makes it clear that the auxin-induced growth of pea sections is entirely mediated by cytochrome oxidase. Since this is the case, a limitation of growth imposed by a deficiency in cytochrome-c reduc- tion is all the more plausible. Direct observations of cytochrome-c activation will have to be made to prove this hypothesis. This may be difficult without disruption of the tissue and a consequent loss of the linkage to cell elongation. Additional speculation along these lines might also be made for the case of the growth-promoting effect of cobalt which requires sucrose (14,22). The lipide effect is considerably accentuated by sucrose (18); cobalt has a smaller promotive role (19). In yeast, co- balt can induce respiratory deficiencies, probably due to an effect on Kindly supplied by Dr. F. L. Crane. 426 B. B. Stowe X in >- en Q o ^ u Q. O 12 9 6 3 0 Increase lAA + GA^ + VITAMIN E IAA + GA,o' ASAL „/ B MEDIUM' basal/ MEDIUM J_J \ I I Ly|^_l \ \ L I I I 3 4 5 "^ 4 5 6 TIME IN HOURS AFTER CUTTING Fig. 2. Oxygen uptake of 10 mm. 'Laxton's Progress' pea epicotyl sections during log phase of growth in the presence of lAA 1.8 ^f plus GA3 0.3 fiM and methyl myristate 40 f^AI plus vitamin E 10 fiAI. Basal medium 1.5 per cent sucrose -\- 50 nM CoClo + 5 mAf KH.PO, (pH 5.5) + 0.002 per cent Pluronic F-68. Q, = fi\. Oj/mg dry wt/hr estimated from slope of best straight line through points. Compare Figure 1 in Christiansen and Thimann (3). cytoplasmic particles (12), and in Avena coleoptiles it decreases respir- ation (2). Can it be then, that in peas cobalt suppresses an alternative pathway of respiration, channeling more of the energy derived from sucrose towards growth? As cytochrome-c is known to be the terminus of several respiratory pathways, this suggestion is particularly attrac- tive in the light of the experimental results reported above. Assessment of the merit of these speculations will have to await further experimentation. What has been firmly established is that trace quantities of lipide substances can play an important role in auxin action. But these lipides may not be limiting in all plant tis- sues, since in Avcna coleoptiles they have not as yet been observed to enhance cell elongation (16). This linkage of lipides to hormone action is, of course, not a new suggestion. Crosby and Vlitos (6) have presented evidence that a long-chain alcohol isolated from tobacco can be active in the Avena first internode bioassay. Struckmeyer and Roberts (21) have for some years also been working with a higher alcohol obtained from plant Stimulation of Auxin Action by Lipides 427 tissues which they believe interacts with auxin. In pea sections them- selves, Christiansen and Thimann (3) noted that lipide utilization paralleled auxin-induced growth, and that the same inhibitors which reduced the cell elongation also slowed the lipide decrease. Antedating these reports is the work of English et al. (8) on wound hormones which has been further delineated by Haagen-Smit and Viglierchio (10). Although the wound hormones problem must have some relationship to the present work, inasmuch as in both cases trace quantities of lipides potentiate a hormonal response, there are some striking unexplained differences in the specificity of the two systems. To begin with, traumatic acid has no influence on pea section growth. Next, the most effective substances for wound tissue proliferation were found to include lauric, myristic, and linoleic acids, none of which stimulates epicotyl section elongation. Lastly, cytochrome-c, coenzyme A, and ascorbic acid all increase the wound hormone response whereas all had no influence on the peas (17). There is thus some fundamental divergence in the operation of the two hormonal systems. Unfortunately, none of the compounds now shown to be active in accelerating pea section growth was tested by the above workers on their material. Lipoidal compounds have been linked to auxin action by several workers using several plant systems. Although the mechanisms behind these systems may not be identical, further analysis of these auxin- lipide interactions should prove fruitful for our understanding of hormonal regulation in plants. ACKNOWLEDGMENT This investigation was assisted by Grant G2828 from the National Science Foundation, to Professors K. V. Thimann and R. H. Wetmore. Professor Thimann's interest in this work is deeply appreciated, as is the expert technical aid of Mrs. Irmgard W. Kurland. LITERATURE CITED 1. Brian, P. ^V., and Hemming, H. G. Complementary action of gibberellic acid and auxins in pea internode extension. Ann. Bot. II. 22: 1-17. 1958. 2. Busse, M. Uber die Wirkungen von Kobalt auf Stieckung, Atmung, und Substanzeinbau in die Zellwand bei Avenakoleoptilen. Planta. 53: 25-44. 1959. 3. Christiansen, G. S., and Thimann, K. V. The metabolism of stem tissue dur- ing growth and its inhibition. II. Respiration and ether-soluble material. Arch. Biochem. 26: 248-259. 1950. 4 Crane, F. L. Internal distribution of coenzyme Q in higher plants. Plant Physiol. 34: 128-131. 1959. 5. Crawford, R. B., Morrison, M., and Stotz, E. Studies on the role of lipides in mammalian cytochrome c reductase. Biochim. Biophys. Acta. 33: 543-550. 1959. 428 B. D. Stowe 6. Crosby, D. G., and Vlitos, A. J. New auxins from 'Maryland Mammoth' to- bacco. This volume, pp. 57-69. 7. Donaldson, K. O., \ason. A., and Garrett, R. H. The role of lipides in elec- tron transport. IV. Tocopherol as a specific cofactor of mammalian cyto- chrome c reductase. Jour. Biol. Chem. 233: 572-579. 1958. 8. English, J. E., Jr., Bonner, J., and Haagen-Sniit, A. J. The wound hormones of plants. IV. Structure and synthesis of a traumatin. Jour. Amer. Chem. Soc. 61: 3434-3436. 1939. 9. Galston, A. \V., and Warburg, H. An analysis of auxin-gibberellin inter- action in pea stem tissue. Plant Physiol. 34: 16-22. 1959. 10. Haagen-Smit, A. J., and Viglierchio, D. R. Investigation of plant wound hormones. Rec. Trav. Chim. 74: 1 197-1206. 1955. 11. Hackett, D. P., and Schneiderman, H. A. Terminal oxidases and growth in plant tissues. I. The terminal oxidase mediating growth of Avena coleoptiles and Pisum stem sections. Arch. Biochem. Biophys. 47: 190-204. 1953. 12. Lindegren, C. C, Nagai, S., and Nagai, H. Induction of respiratory deficiency in yeast by manganese, copper, cobalt and nickel. Nature. 182: 446^48. 1958. 13. Lockhart, J. A. Studies on the organ of production of the natural gibberellin factor in higher plants. Plant Physiol. 32: 204-207. 1957. 1-1. Miller, C. O. The influence of cobalt and sugars upon the elongation of etiolated pea stem segments. Plant Physiol. 29: 79-82. 1954. 15. Phinney, B. O. Dwarfing Genes in Zen mays and their relation to the gibber- ellins. This volume, pp. 489-501. 16. Stowe, B. B. Growth promotion in pea epicotyl sections by fatty acid esters. Science. 128: 421-423. 1958. 17. Similar activating effects of lipids on cytochromes and on plant hormones. Biochem. Biophys. Res. Comm. 1: 86-90. 1959. 15. Phinney, B. O. Dwarfing Genes in Zea mays and their relation to the gibberel- lins. This volume, pp. 489-501. 19. Growth promotion in pea stem sections. II. Vitamins E and Kj as synergists of auxin and gibberellin action. (In preparation.) 20. , and Yamaki, T. Gibberellins: stimulants of plant growth. Science. 129: 807-816. 1959. 21. Struckmeyer, B. E., and Roberts, R. H. The inhibition of almormal cell proliferation with antiauxin. Amer. Jour. Bot. 42: 401-405. 1955. 22. Thimann, K. V. Studies on the growth and inhibition of isolated plant parts. V. The effects of cobalt and other metals. Amer. Jour. Bot. 43: 241-250. 1956. 23. Weber, F., Gloor, U., and Wiss, O. Ober den Mechanismus der Reaktivierung der Bernsteinsaure-Cytochrom-c-Reduktase durch die Vitamine E und K. Helv. Chim. Acta. 41: 1038-1046. 1958. 24. Went, F. W. Specific factors other than auxin aficcting growth and root formation. Plant Physiol. 13: 55-80. 1938. DISCUSSION Dr. Jansen: At Bcltsville we have been engaged in a long-term investigation of the effects of surfactants in herbicidal spray prepar- ations. A number of surfactants have been encountered which, in the absence of herbicides, produce some rather striking growth stimu- lations in corn and soybeans. The individual surfactants do not always stimulate both species nor do they always produce stimulation at the same concentrations. Increases in growth up to 25 per cent Stimulation of Auxin Action by Lipides 429 in height have been obtained in 2 weeks. Unpublished data show that 14 of 30 surfactants produced the most striking growth promo- tions in our evaluations. These surfactants are distributed through- out the four major classes, namely those of anionic, cationic, nonionic, and ampholytic natures. They also represent a number of the struc- tural subclasses. In nearly all of the surfactants listed, long-chain aliphatic radicals from either acids or alcohols are integral compon- ents. I think this is a fact which we should probably bear in mind when we use any surfactant in our growth regulation studies. Dr. Crosby: As we pointed out earlier, we found that the acidic inorganic esters of the long-chain alcohols, the 18-, 20-, and 22-carbon sulfates and phosphates, were all extremely active. We, too, feel that these surface-active effects are extremely important. In regard to Dr. Stowe's work, one brief comment for those who might become inter- ested in working in this area. The compounds which may be pur- chased are all natural products. The benzyl alcohol that we used, and the esters which Dr. Stowe used, are initially impure. I think it is necessary to be sure that a rigorous purification is carried out on all these materials. I also notice that Dr. Stowe observed the same interesting concentration-activity relationships that we did, even though the compounds which he found to be active were as insoluble in the bioassay medium as were ours. I wonder if he would have any comment on how it is possible for there to be such a concentra- tion-activity relationship. Dr. Stowe: Well, it's possible, of course, that the concentration here is a bit misleading. These tests were carried out in a standard volume of solution, so we were always adding a known amount of lipide to the bioassay material. I want to emphasize that these are applied as a very good emulsion, and although you cannot, perhaps, speak of a true solution here, specific amounts of the substance were added to each dish, and then you find, as we showed, a nice linear relationship within the right concentration ranges. Dr. Vlitos: Your Figure 1 and Tables 2 and 4 showed activity in the pea internode section test with fatty alcohols at different concen- trations, and then Table 3 indicated that lAA was needed in your solutions to show activity. Now I'm not quite clear whether in the first cases you were dealing with endogenous auxins. Would you clear that up for me? Dr. Stowe: In every case where you saw an effect, there was also added the optimum concentration of lAA and GA. What we were trying to do was to get maximal growth, and we added everything to the pea sections that we knew would promote growth, and then looked for still other growth-promoting factors. -^^ CORWIN HANSCH Pomona College ROBERT M. MUIR University of Iowa Electronic Effect of SubstLtuents on the Activity of Phenoxyacetic Acids In the two-point attachment mechanism of action which has been developed (14, 26, 27), it has been postulated that the plant growth regulators react with a protein substrate in plants by means of a car- boxyl group and a ring position ortho to the attachment of the car- boxyl group. Since the work of Osborne et al. (28, 29) on 2,6-di- substituted phenoxyacetic acids and that of Wain's group (30) on phenylacetic acids, it appears that, stereoelectronic conditions permit- ting, other points on the ring may function in place of an ortho posi- tion. Despite further evidence for the validity of this mechanism (6, 9, 10, 22, 23), considerable doubt still remains (37), and more evidence is necessary for complete understanding. In attempting to correlate the effects of various substituents on the aromatic rings of the different growth regulators, there are three important factors connected with each substituent which must be considered: electronic, steric, and H/L factor (hydrophilic/lipophilic). It is relatively easy to compare molecules with respect to the first and third factors; however, the steric effect of substituents on reac- tivity is extremely difficult to assess since nothing is known of the ge- ometry of the reaction site. Also, the nature of the side chains is such that assuming attachment by the side chain first, the ring with its attached groups could be presented in many ways to a second site. The purpose of this paper is to consider some monosubstituted phen- oxyacetic acids which are simple enough that comparison of the elec- tronic effects of the various groups is not too complicated. First, a quick summary of the more important evidence favoring reaction vs. simple adsorption. Most important is the large amount of evidence indicating that the ring associated with growth regulators [ 431 ] 432 C. Hansch and R. M. Muir must be aromatic in character. Despite a rather thorough search (8, 13,19,36,39), no aliphatic compounds, with the possible exception of one or two thiocarbamates (12), have been found to be active in growth promotion. The only alicyclic compounds which have con- firmed activity are 1-cyclohexenyIacetic acid and its analogues (11, 19). It is not unlikely that these molecules could be dehydrogenated to the quite active phenylacetic acid or one of its derivatives. Such dehy- drogenations are known to occur in biological systems (25). So far, our attempts to establish the occurrence of this dehydrogenation with Avena coleoptiles have been inconclusive. Thus, although there is slight evidence to the contrary, the overwhelming preponderance of evidence from thousands of carefully tested compounds indicates that an aromatic ring is necessary. Most effective are compounds which contain six-membered rings such as benzene, pyridine, and their homologues. However, compounds having the less aromatic five- membered rings, or compounds with one double bond which might easily be dehydrogenated to such, have been reported active (5, 11, 32). If we proceed from the assumption that an aromatic ring is neces- sary, we must answer the question. What is it about such a ring that is necessary? Veldstra (37) and many others have taken the view that it is the lipophilic character of the ring that is important. Important as this character may be, it is of little help in explaining the enor- mous differences in activity obtained with the different substituted phenoxyacetic and benzoic acids. For example, phenoxyacetic acid is at best slightly active. The introduction of a fluorine atom in the 4 position increases the activity at least 200-fold. The fluorine atom would have very little effect on the lipophilic character and no steric hindrance; hence its effect must be electronic in nature. Again the differences between chlorine and methyl groups in size or in lipophilic character would be small, yet when these groups are compared in the 4 position of phenoxyacetic acid, the chlorine atom confers at least 100 times more activity. Another striking example results from the comparison of 3-methyl- and 3-trifluoromethylphenoxyacetic acids. Again, both groups have about the same size so that steric effects are ruled out. If anything, tiie trifhioromethyl group might make the molecule less lipophilic through inductive polarization of the ring, yet it is 100 times more active. Wain's group (7, 35), in a very care- ful analysis of the effect of substituents on phenoxyacetic acids, showed that substitution in both the 3 and 5 positions with either chlorine or methyl groups gave molecules which were inactive or of very low activity. That this was not due to a steric effect or lack of lipophilic Electronic Effect of Substituents on Phenoxyacetic Acids 433 character was clearly shown by the tact that 2,3,4,5-tetrachlorophe- noxyacetic acid was very active. If simple lipophilic adsorption of the ring is not enough to ex- plain its function, one might argue that adsorption of the ring through its pi electrons to give a weak pi complex would help ra- tionalize the situation. Considering the nature of the phenoxy, Table 1 . Activity of phenoxyacetic acids compared to indole-3-acetic acid. * Compound Relative Activity f Compound Derivative of phenoxyacetic acid Derivative of phenoxyacetic acid Relative Activity f 6.00 5.00 1.50 0.03 0.05 0.03 0.00 0.00 0.01 0.07 3-Chloro- \ 2.00 4-Chlnrn- 3-Bromo- 2.50 4-Bromo- 3-TrifluoromethyI- 2-Methoxy- 2-Chloro- 7.00 4-Methoxy- 4-Mpthvl- 0.20 0.06 4-Hydrogen- 2-Bromo- 0.10 2-Iodo 0.10 4-Acctyl- 2-Methyl 0.20 '^-\TptVin XV- 2-Ethyl- 0.00 3-Methyi- * All of the data reported in this table have been previously reported (26) except for 4-fluoro-, 4-acetyl-, and 3-trifluoromethylphenoxyacetic acids. 4-Hydrogenphe- noxyacetic acid is simply phenoxyacetic acid. t Indole-3-acetic acid = 100. phenyl, naphthalene, and indole rings, adsorption to an electron-defi- cient site would seem more reasonable than the assumption of an electron-rich site. The relative activating effect of substituents in the 4-substituted phenoxyacetic acid as shown in Table 1 is exactly the opposite from what one might expect (F>Cl>Br>CH30>I) for such a mechanism. More difficult to rationalize in terms of a non- specific pi complex is the great difference in effect of given substi- tuents at various points on the ring. Thus, for example, 2,4-dichloro- phenoxyacetic is very active, while the 3,5-dichloro-analogue is inert. As mentioned above, the difference in these two molecules cannot be rationalized as being due to steric factors (35). Many other such examples can easily be shown from data published on the phenoxy- acetic acids. If nonspecific adsorptions involving van der Waals forces or pi complex formation do not offer satisfactory solutions, adsorption or 434 C. HanscJi and R. i\I. Muir reaction at a specific point seems to. Such a reaction could vary all the way from weak pi complex formation through Sigfna complex to "normal" covalent bond formation at the particular ring carbon atom. For a working hypothesis we have visualized the following: 0-CH2COOH HoN O-CHoCONH CI m CI CI enzyme V CI + H-enzyme This scheme is drawn showing nucleophilic attack, and thus inter- mediate IV shows delocalization of a negative charge. Of course the same type of intermediate would result from electrophilic or radical attack and delocalization of a positive charge or single elec- tron would be equally important for the stabilization of the inter- mediate. Intermediate III is shown as an amide, although most cer- tainly simple salt formation could function to hold the auxin to the site for the much slower subsequent reaction, be it complex or covalent bond formation. Substance II is assumed to be protein in nature. A consideration of the available information has led us to believe that reaction must occur on one molecule (14). There are three possible ways in which X in the above series of reactions could attack the aromatic ring: X-|-, X- or X:. In each case stabilization of an intermediate through delocalization of a posi- tive charge or electrons will be very important in promoting the reac- tion. Such stabilization would not be possible in an isolated double bond, and therefore it is not surprising that such compounds have invariably been found to be inactive. In still another attempt to find an active aliphatic acid, we tested 4-pentynoic acid and foimd it to be inactive. In view of the relatively electron-rich character of most of the rings in the known auxins, one might expect that X-(- would be the most likely group for two-point attachment. However, if this were true, then one would expect general activation by groups such as methyl and methoxy. That this is not so is readily seen from the Electronic Effect of Substituents on Phenoxyacetic Acids 435 data in Table 1. Aberg (1,2) in carefully controlled experiments has also observed that the substitution of hydrogen by methyl results in lowered activity in a variety of molecules. The deactivating effect of methyl and methoxy groups on phenylacetic acids is also clear in the data of Melnikov ct al. (24). The fact that the activating ability of the halogens follows their increased inductive effect ( — I effect) also points against attack by X-|-. Aberg (1) has observed the same order of activity for the 4-substituted halophenoxyacetic acids which we report in Table 1. He pointed out that the decrease in activity in going from F to I might be due to the increasing atomic radius of each substituent. This hypothesis would not explain the great dif- ference in going from H to F in the 4 position, nor would this ex- plain the difference between CI or Br and CH3, or CH;. and CF3. 3-Trifluoromethylphenoxyacetic acid is the most active biologically of all the monosubstituted phenoxyacetic acids which we have tested. Ftn- ther evidence against attack by X-|- has been fotnid by Fukui et al. (10) for the benzoic acids. They have shown that there seems to be no cor- relation between pi electron distribution on the benzene ring and attack by an electrophilic reagent. It should be noted that both in the phenyl thioglycolic acids (17) and in the phenylacetic acids (24) the order for activity on substitution into the 4 position is Cl>Br>CH3>I, with the latter two substitu- ents giving compounds of very low activity. The only groups which consistently increase activity in rings other than the benzene ring in the benzoic acids are F, Br, and CI. (It seems very likely that CF3 will be added to this group when more such derivatives are studied.) The nitro group sometimes gives weakly active compounds, but amino and hydroxyl groups invariably give low activity. This has usually been at- tributed to the fact that polar groups would greatly redtice the lipo- philic character of the ring (37). Since attack by electrophilic reagents seems unlikely, we have ad- vocated attack by an electron-rich group. Of the two possibilities X: seems more likely than X-. In a recent critical review (3) of the na- ture of free radical attack on aromatic nuclei, Augood and Williams point out that almost all substitution on benzene results in an in- creased rate of attack by radicals at points ortho and para to the sub- stituent. The only groups which do not have total rate factors greater than 1 are isopropyl, tert-butyl and trifluoromethyl. The latter has a value of 0.99, which means essentially no change in over-all reaction from benzene itself. Methyl and methoxyl groups are quite strongly activating. Again this is at odds with the biological activating effects of these groups in phenoxyacetic and phenylacetic acids. More inter- esting is the fact that the order for activation by halogen on benzene for radical attack is I>Br>Cl>F (3). This of course is just opposite to the order found for the biological activating effect of the substi- 436 C. Hansch and R. M. Muir tuents in the auxins. In radical attack substituents have only slight effect on positions rneta to them. In the unsubstitutecl acids one would expect phenoxyacetic acid to be more susceptible to radical attack than phenylacetic acid and theiefore to be more active as an auxin if radical attack were involved. Again, just the reverse is found. Molecular orbital calculations (10) have also indicated that radical attack seems less likely for the benzoic acids than attack by an electron pair. If radical attack and electrophilic attack on the ring do not seem to offer an explanation for the effect of substituents on auxin rings, what evidence is there to support nucleophilic attack? Our early A\ork (14) with the phenylacetic, indoleacetic, and phenoxyacetic acids in- dicated that whenever both ortlio positions were substituted with halogen, alkyl, or alkoxy groups, inactive molecules always resulted. This, plus the relative activating effect of substituents, led us to focus attention on reaction at an ortho position by an electron-rich reagent. This view was modified (27) when it was discovered that a-2,6-di- chlorophenoxypropionic acids were active (28). It seems most likely that a para position must be involved in this reaction since introduc- tion of a group in the para position always destroys the activity of a- 2,6-dichlorophenoxypropionic acids in the Avena test. Some of the 2,4,6-trisubstituted phenoxyacetic acids show weak activity in the slit pea test. The phenylacetic acids seem to follow the same pattern (30), the 2,6-disubstitutecl acids being active and the 2,4,6-trisubstituted ones being inactive. A very interesting and significant difference is apparent in the requirements for activity with these two structures. 2,6-Dichlorophenylacetic acid is quite active, while 2,6-dichlorophen- oxyacetic acid is at best very weakly active. If one assumes two- point reaction by means of the carboxyl and the 4 position of these molecules with a single substrate molecule, a logical explanation is possible. In the phenylacetic acids the two hydrogens on the methyl- ene group interfere with the ortlio halogens, locking the methylene group so that the carboxyl group may be held rather rigidly over the ring near the 4 position. In the phenoxyacetic acids, the relatively small oxygen atom holds the methylene group out far enough so that interference between the methylene hydrogens and the two ortho halogens is slight, and the whole side chain has freer motion. Intro- duction of the a-methyl group thus increases the biological activity because hydrogens on the methyl group would help lock the side chain so that the carboxyl group would be more rigidly held for two-point contact involving the 4 position. All of the data at hand, therefore, would indicate that a specific atom in the ring is involved in the two-point reaction. If we assume then that normally an ortho position in the phenoxy- Electronic Effect of Siibstitueuts on Plienoxyacetic Acids 437 acetic acids is the jDieferred point for 2-point reaction, a study of the substituents in the 3 and 4 positions should throw some light on the nature of this reaction. Substitution in the 2 position will be more difficult to evaluate because of steric interaction with the side chain. As mentioned earlier, there are three important variables con- nected with each substituent: steric, electronic, and H/L factor. The latter factor has two components which one must consider. The rel- ative hydrophilic-lipophilic character of the molecule must be such that rapid penetration to the reaction site is possible. A second point which may or may not be of importance is the possibility that reac- tion may take place on a solid surface and adsorption might be gov- erned by small H/L differences. To try to hold other variables con- stant so that the electronic factor could be evaluated, we chose to look at substitution in the 3 and 4 positions, and to consider groups relatively inert chemically which would have closely related H/L fac- tors. In addition to the above points it must be borne in mind that if a substitution reaction occurred, it could be promoted by enzymatic stretching of a carbon-hydrogen bond. A consideration of the data in Table 1 indicates that in general those groups which are placed so that their electronic effect is to re- duce the electron density at the ortho positions, give an increase in activity over the unsubstituted plienoxyacetic acid. It is interesting to note that the activity of the 4-substituted acids falls off as the — I effect of the substituent on the ortho position decreases. The activity we have found for the 4-halo and 4-methylphenoxyacetic acids is essen- tially that reported by Aberg (1) except that he finds the 4-methyl sub- stituent to be deactivating. Comparison of the 4-chloro and 4-methyl groups is particularly instructive since both groups have about the same size and effect on the H/L factor. The chlorine derivative is about 100 times more active. The fact that iodine in the 4 position gives an inactive molecule is significant and would lend support to Aberg's observation that the size of the group in the 4 position is particularly important. We have noted that 4-iodophenylacetic acid is inactive, and Kato has observed (17) that an iodine in the 4 position of phenylthioglycolic acid essentially destroys the activity of this mole- cule. Aberg has also pointed out that large alkyl groups in the 4 posi- tion also destroy activity. That 4-methoxy substitution gives an active molecule, although it is a bulky group, is probably due to the non- linear carbon-oxygen-carbon bonding which permits the methyl group to assume a skewed position somewhat above the plane of the ring. From Table 1 it is evident that 2 substitution has little effect. The introduction of halogen or methyl groups results in a slight in- crease in activity. No obvious correlation between activity and the three factors — electronic, steric, or H/L — appears. One might ex- 438 C. Hansch and R. M. Mitir pect 2 substitution to cause some steric hindrance of resonance be- tween the electrons of the ether group and those of the ring, with the result that the —I effect of the oxygen would be more important and activation should result. Although 2 substitution almost always results in an increase in activity, the effect is more apparent in the di- and tri-halo derivatives than with the mono. A very important consequence of the two-point attachment hypothesis, especially with the phenoxyacetic acids, is the nature of the spacial arrangement of the side chain with respect to the ring at the time of consummation of reaction at the second point (ring position). If the side chain is held so that electrons from the oxygen can effectively overlap with those of the aromatic ring, the electron density on the ring would be very much greater than if oxygen were twisted so as to prevent this overlap. It would seem most reasonable to assume that at the time of reaction at the second point, the side chain would usually be twisted so that only weak overlap could occur. The great sensitivity of the 2 position to steric factors is indicated by the fact that activity drops to zero in going from methyl or iodine to ethyl. When ethyl is in the 2 position, even introducing a chlorine atom in the 4 posi- tion does not restore activity. 2-Ethyl-4-chlorophenoxyacetic acid is completely inert. A consideration of structure and activity in the phenoxyacetic acids is not complete without some consideration of the disubstituted derivatives. All of the 2,4-halo derivatives with the exception of io- dine are quite active (35). The dramatic lack of activity of 3,5-di- chlorophenoxyacetic acid was first shown by Leaper and Bishop (20) and then confirmed by others to hold for the 3,5-dimethyl derivative as well (26, 35). That this effect is not due to either steric or H/L factors is clearly shown by the discovery that 2,3,4,5-tetrachloro- phenoxyacetic acid is quite active. The inactivity of the pentachloro derivative again points up the importance of an open ortho position (35). Both of these inactive 3,5-substituted molecules have groups which would relay electrons to the ortJio positions via -j-M effects. Moreover, since there is no group in either ortho position, the oxy- gen atom of the ether linkage is able to exert maximum effect on the ortho position tiirough -^M action. The combined effect of three groups directing electrons to the ortho and para positions makes at- tack at these positions by an electron-rich reagent much less likely. Introduction of a chlorine atom in the 2 position should help lower the electron density at the ortlio positions by providing a — I effect and some steric hindrance so that conjugation of the ether oxygen with the ring is not as effective. This is borne out by the fact that 2,3,5-trichlorophenoxyacetic acid is a moderately strong compound (35). Adding another chlorine atom Aviih its — I effect to the 4 posi- Electronic Effect of Sitbstittieuts on Phenoxyacetic Acids 439 tion gives the biologically quite active tetrachlorophenoxyacetic acid. Fawcett et al. (7) have also shown that 2,3,5-trimethylphenoxyacetic acid has very slight activity. Here again, 2-substitution would inhibit the -f-M effect of the oxygen and enhance its — I effect on the ortho position. We have found that 3,5-dimethylphenylacetic acid, al- though less active than phenylacetic acid, is still very definitely active. Although the methyl groups would affect the ortho positions by hy- perconjugation, the -\-M. effect of the methylene group would be very weak compared to the oxygen atom of the phenoxyacetic acid series. A check on the above hypothesis can be made by testing the activity of phenoxyacetic acid substituted in both meta positions with trifluoromethyl groups. On the basis of information now available such a molecule would be expected to be highly active. If a reaction does indeed occur at an ortho position or under spe- cial conditions at another ring position, one would expect that sta- bilization of the charged intermediates, such as IV shown on page 434, would be very important in determining the relative activities of closely related compounds. Differences in the activities of closely related compounds can, in certain instances, be explained in terms of one molecule forming an intermediate more ably than a closely related isomer to delocalize an electron pair. It has long been known that for chemical attack, the a position on naphthalene is much more reactive than the (3 position (16), be the attack by an electron-rich or electrophilic reagent (40). The well-known fact that 2-naphthoxyacetic acid is much more reactive than 1-naphthoxyacetic acid can be explained by assuming better resonance stabilization of the intermediate with the former than with the latter. With 2-naph- thoxyacetic acid, reaction could occur at the 1 position to give an in- termediate stabilized by seven relatively stable resonance structures, four of which would not disturb the benzenoid resonance of the nonreacting ring. In 1-naphthoxyacetic acid, reaction would occur at the 2 position, the intermediate of which would be stabilized by only six relatively stable structures, in only two of which woiild the benzenoid resonance in the nonreacting ring be preserved. The fact that reaction might occur at an a position by reaction at the 8 position in the case of 1-naphthoxyacetic acid must be considered, however. That the 8 position is much less favorably situated for reaction with an -OCHoCOOH side chain is evident from the work of Toothill et al. (35). These workers have amply demonstrated that the introduc- tion of another atom in the oxyacetic acid side chain destroys activity. Such would be the situation if two-point reaction in 1-naphthoxy- acetic acid were required to occur by means of an 8 position. The unsuitability of the 8 position sterically is nicely illustrated by the inactivity of 2,4-dichloro-l-naphthoxyacetic acid (35, 36). An even 440 C. Hansch and R. M. Muir better illustration of tiie greater reactivity for two-point attachment of the a position in naphthalene over that of the (3 comes from the work of Luckwill and W^oodcock (21). These workers confirmed the earlier report (15) that l,3-dichloro-2-naphthoxyacetic acid is com- })letely inactive, as would be expected. The superiority of the a posi- tion over the (3 position for two-point attachment was shown by com- paring l-chloro-2-naphthoxyacetic and 3-chloro-2-naphthoxyacetic acids. The latter isomer, where a attachment is possible, is highly active, while the former compound, where p attachment is demanded, is almost inert. The interesting observation that introduction of chlorine in the 8 position of 1-naphthaleneacetic acid greatly lowers the activity of this molecule (15), can also be interpreted in terms of resonance stabilization of a reaction intermediate. Thus 8 substitu- tion blocks a reaction and forces reaction at the less active (3 (2) posi- tion. One would expect the 8 position in 1-naphthaleneacetic acid to be more favorably placed for reaction than the 2 position. The reasoning behind this is that if reaction occurs at the 8 position, one can consider the structure to be related to cinnamic acid, while if re- action occurs at the 2 position, one must consider the reaction with respect to the side chain to be more like that of phenylacetic acid. C/Vcinnamic acid has been shown to be at least five times as reactive as phenylacetic acid in the pea test (19). Similar reasoning can be used to rationalize the difference in activity between 1- and 2-naph- thaleneacetic acids. Also the fact that partial hydrogenation of the naphthalene ring leads to lower activity (36) may be interpreted as decreasing the ability of the molecule to stabilize an intermediate through charge delocalization. The observation (31) that indole-2- acetic acid is much less active than the isomeric indole-3-acetic acid again lends support to the above hypothesis. Reaction at the 3 posi- tion would not permit as effective charge delocalization as woidd re- action at tlie 2 position. It is noteworthy that delocalization of a negative chaige with reaction at the 3 position would be even less favorable than delocalization of a positive charge. That the 2 position in the indole series is more favorable for reaction than the 4, is indi- cated by the fact that 2-methylindole-3-acetic acid is less active than lAA, while 4-chloroindole-3-acetic acid is more active than lAA (26). Again such reasoning can be used to rationalize the difference in activity found with 2-thianaphtheneacetic and 3-thianaphtheneacetic acids (18). Although the overwhelming preponderance of evidence supports our i^vo-point attachment hypothesis, two important molecules which do not fit neatly into place are 2,4-dichloro-6-fluorophenoxyacetic add anil 3,5-dichloro-2-pyridoxyacetic acid (37). In the latter mole- Electronic Effect of Substituejits on Phenoxyacetic Acids 441 cule, one would have to postulate either displacement of a chlorine or reaction at a meta position. The ring nitrogen atom would acti- vate the substance for attack by an electron-rich reagent at the meta position. Although it has not been shown in other systems that this is sterically favorable, this seems more reasonable than the disj)lace- ment of chlorine. Although it has been postulated that in the 6-fiuoro compound fluorine might be displaced, evidence is still lacking to support this point. LITERATURE CITED 1. Abeig, B. On the effects of Jbara-substitution in some plant growth regulators with phenyl nuclei. In: R. L. Wain and F. Wightman (eds.). The Chemistry and Mode of Action of Plant Growth Substances, pp. 93-106. Butterworth Sci. Publ., London. 1956. 2. . Studies on plant growth regulators. XV. The naphthylacetic and the a-naphthyl-propionic acids. Lantbrukshcigsk. Ann. Uppsala. 25: 221-239. 1959. 3. Augood, D. R., and Williams, G. H. Homolytic aromatic arylation. Chem. Rev. 57: 123-190. 1957. 4. Brain, E. G., and Finar, I. L. The preparation and properties of some pyrazolylacetic acids. Jour. Chem. Soc. 1957: 2356-2359. 1957. 5. Carreras Linares, R., Carreras Bayes, M. D., and Palleiro Cardona, J. Synthesis of furan derivatives for tests as phytohormones. Publ. Inst. Quim. Alonso Barba. 4: 310-315. 1950. 6. Crosby, D. G., and Vlitos, A. J. ?j-Butyl 5-chloro-2-pyrimidoxyacetate — a plant growth regulator analog. Science. 128: 480. 1958. 7. Fawcett, C. H., Wain, R. L., and Wightman, F. Studies on plant growth-regu- lating substances. VIII. The growth-promoting activity of certain aryloxy- and arylthio-alkanecarboxylic acids. Ann. Appl. Biol. 43: 342-354. 1955. 8. , Wain, R. L., and Wightman, F. Plant growth-regulating activity in certain carboxylic acids not possessing a ring structure. Nature. 178: 972-974. 1956. 9. Foster, R. J., McRae, D. H., and Bonner, J. Auxin-induced growth inhibition a natural consequence of two-point attachment. Proc. Nat. Acad. Sci. U. S. 38: 1014-1022. 1952. 10. Fukui, K., Nagata, C, and Yonezawa, T. Electronic structure and auxin activity of benzoic acid derivatives. Jour. Amer. Chem. Soc. 80: 2267-2270. 1958. 11. Gandini, A. Pflanzliche Wuchshormone. Ihre Struktur und physiologische Aktivitat. Ber. 76: 399-405. 1943. 12. Gordon, S. A., and Moss, R. A. The activity of S-(carboxymethyl)-dimethyl- dithiocarbamate as an auxin. Physiol. Plant. 11: 208-214. 1958. 13. Haagen-Smit, A. J., and Went, F. W. A physiological analysis of the growth substance. Proc. Akad. Wet. Amsterdam. 38: 852-857. 1935. 14. Hansch, C, Muir, R. M., and Metzenberg, R. L., Jr. Further evidence for a chemical reaction between plant growth-regulators and a plant substrate. Plant Physiol. 26: 812-821. 1951. 15. Hansen, B. A. M., Burstrom, H., and Tear, J. Root and shoot elongation activity of some naphthalene compounds. Physiol. Plant. 8: 987-1002. 1955. 16. Hine, J. Physical Organic Chemistry. 497 pp. McGraw-Hill Co., New York. 1956. 442 C. Hansch and R. M. Muir 17. Kato, J. Studies on tlie relation between auxin activity and chemical struc- ture. I. On phenyl- and naphthyl-thioglycolic acid derivatives and related compounds. Mem. Coll. Sci. Kyoto. 21: 77-85. 1954. 18. Kefford, N. P., and Kelso, J. M. Sulphur analogues of indoleacetic acid: syn- thesis and biological activity of some thionaphthenacetic acids. Austral. Jour. Biol. Sci. 10: 80-84. 1957. 19. Koepfli, J. B., Tliimann, K. V., and Went, F. W. Phytohormones: structure and physiological activity. I. Jour. Biol. Chem. 122: 763-780. 1938. 20. Leaper, J. M. F., and Bishop, J. R. Relation of halogen position to physio- logical properties in the mono-, di-, and trichlorophenoxyacetic acids. Bot. Gaz. 112: 250-258. 1951. 21. Luckwill, L. C, and Woodcock, D. Relationship of molecular structure of some naphthyloxy compounds and their biological activity as plant growth regulating substances. //;.• R. L. Wain and F. Wightman (eds.), The Chemistry and Mode of Action of Plant Growth Sul«tances, pp. 195-204. Butterworth Sci. Publ., London. 1956. 22. McRae, D. H., and Bonner, J. Chemical structure and antiauxin activity. Physiol. Plant. 6: 485-510. 1953. 23. McRae, D. H., Foster, R. J., and Bonner, J. Kinetics of auxin interaction. Plant Physiol. 28: 343-355. 1953. 24. Melnikov, N. N., 1 uretskaya, R. K., Baskakov, Y. A., Boyarkin, A. N., and Kuz- netsova, M. S. The structure and the physiological activity of substituted phenylacetic and naphthylacetic acids. Dokl. .4kad. Nauk. SSSR. 89: 953- 956. 1953. 25. Mitoma, C, Posner, H. S., and Leonard, F. Aromatization of hexahydrobenzoic acid by mammalian liver mitochondria. Biochem. Biophys. Acta. 27: 156- 160. 1958. 26. Muir, R. M., and Hansch, C. On the mechanism of action of growth regulators. Plant Physiol. 28: 218-232. 1953. 27. , and Hansch, C. Chemical constitution as related to growth regulator action. Ann. Rev. Plant Physiol. 6: 157-176. 1955. 28. Osborne, D. J., Blackman, G. E., Novo, S., Sudzuki, F., and Powell, R. G. The physiological activity of 2:6-substitutcd phenoxyacctic acids. Jour. Exper. Bot. 6: 392-108. 1955. 29. , Blackman, G. E., Powell, R. G., Sudzuki, F., and Novo, S. Growth- regulating activity of certain 2:6-sui3stituted phenoxyacctic acids. Nature. 174: 742. 1954. 30. Pybus, M. B., Wain, R. L., and Wightman, F. New plant growth-substances with selective herbicidal activity. Nature. 182: 1091,1095. 1958. 31. Schindler, W. lndol-2-essigsaurc. Hclv. Chim. Acta. 41: 1111 1113. 1958. 32. Shimizu, S., and Takci, S. SMilhcsis of cyclopcnlaiu' iini\ati\ts Irom k'\u- linic acid. Jour. Agi-. Chem. Soc. Japan. 23: 286-288. 1950. 33. Tamari, K. Studies on the root-forming substances of cuttings. Jour. .\gr. Chem. Soc. Japan. 17: 321-335. 1941. 34. Thimann, K. V. The role of ortho-substitution in the synthetic auxins. Plant Physiol. 27: 392-104. 1952. 35. Toothill, J., Wain, R. L., and \Vighinian, F. Studies on plant growth-regu- lating substances. X. The activity of some 2:6- and 3:5-substituted phenoxy- alkanecarboxylic acids. Ann. Appl. Biol. 11: 517-560. 1956. 36. Veldstra, H. The relation of chemical structure to biological aclivilv in growth substances. Ann. Rev. Plant Physiol. 4: 151-198. 1953. Electronic Effect of Siibstitiients on Phenoxyacetic Acids 443 37. . On form and function of plant growth substances. In: R. L. Wain and F. Wighlman (eds.), The Chemistry and Mode of Action of Plant Growth Sub- stances, pp. 117-133. Butterworth Sci. Publ., London. 1956. 38. , and Booij, H. L. Researches on plant growth regulators. XVII. Structure and activity. On the mechanism of the action. III. Biochem. Biophys. Acta. 3: 278-312. 1949. 39. Went, F. W. Phytohormones: structure and physiological activity. II. Arch. Biochem. 20: 131-136. 1949. 40. Wheland, G. W. Resonance in Organic Chemistry. 846 pp. John Wiley and Sons, New York. 1955. DISCUSSION Dr. Bonner: I think that everybody agrees that for a molecule to be active as an auxin, it has to have some appropriate geometry, presumably because it has to fit into particular kinds of holes, and it has to have two particular reactive groups. It seems clear to me that the reactivity of the aromatic nucleus has an important function in determining the activity of an auxin. Dr. Hansch referred to the strange case of the 2,6-dichlorophenoxyacetic acid and 2,6-dichloro- propionic acid. At low concentrations these compounds behave as auxin antagonists, and at higher concentrations they are active and behave as auxins, but their activity has some very strange character- istics. Dr. Osborne: If 2,6-dichlorophenoxyacetic acid is tested in the Avena straight growth test, one finds that even with relatively low concentrations there is an initial but small stimulation of growth lasting for a few hours followed by a retardation in growth. I am not sure how one would explain this on a 2-point attachment theory. Dr. Bonner: The 2,6-dichlorophenoxyacetic and propionic acids are active, but their activity has the interesting feature that this ac- tivity is manifested for a very short time in contrast to 2,4-D or other auxins. It is a "short-term" auxin; it goes in and does something for only a short time. Dr. Osborne: This appears true for 2,6-dichlorophenoxyacetic acid, but if one does similar experiments with the propionic derivative, the growth stimulation can be continuous for a period up to 24 hrs. One does not find the subsequent retardation of growth. The stimulation with this 2,6-substituted phenoxy compound is apparently there all the time. Dr. Wain: In relation to the 2-point contact theory which we have been hearing so much about, the carboxyl group probably does react chemically in the manner you have suggested. The question is, what is happening in this second contact which you have postulated. It might be reacting with some essential or unessential thiol group in 444 C. Hansch and R. M. Muir a protein. The 2-point contact theory does not explain the speci- ficity of stereoisomers. We have shown that for activity, the molecule should, in general, have at least one hydrogen attached to the carbon adjacent to the carboxyl group. This hydrogen may well be involved in the growth reaction. If you accept this point, then you are able not only to explain the specificity of stereoisomers but, as Dr. Wight- man has shown, you can put competitive antagonism on a logical basis. Dr. Hansch: I feel perfectly easy with respect to the 2-point at- tachment theory and stereoisomerism in the side chain. There are a lot of things that bother me much more than that. There are so many cases in organic and biochemistry where two asymmetric cen- ters react. You can get an enormous difference in reactivity between D and L forms. This difference that you mentioned is very important. It is one of the things that reinforces our ideas about the fact that the carboxyl group does react to form an amide, covalent bond. If it were only an ionic bond, then both stereoisomers could react at the same point with little interference. When there are two optically-ac- tive centers forming a chemical bond, you get into such differences of activity with stereoisomers. Dr. Thimann: With a colleague and student, Mr. W'illiam Porter, we have spent a great deal of time in reviewing the whole problem of structure and activity again, and I woidd like to make a new sug- gestion. It could not be dignified by the name of a fully developed theory yet. First let me say this owes a great deal to various earlier theories; I need not remind you that in the last 20 years a large num- ber of ideas have been proposed, such as that the side chain is at an angle to the ring, that a free hydrogen is needed in the side chain, the concept of an essential distance between the carboxyl and the ring, the concept of lipophilia, and the more recent argument about reactivity at the ortho position. Of course, the idea of studying struc- ture and activity is to get some clue as to what the receptor is like, and I will say nothing about the receptor except to remind you of the suggestion 1 made some time ago (Amer. Naturalist 90: 145. 1956), that the receptor may be a family of closely related bodies; even within one plant there would be a group of slightly different recep- tors—an idea which makes it easily possible to cx])lain some of the phenomena of synergism. Now, if we simply review the accumulated data, what can we de- duce? Let me start with indoleacetic acid and its family of similar isosteric substances, in which instead of nitrogen there occur methy- lene, oxygen, or sulfin- in the same position. It is appropriate to start with these because the compound with a carbon atom in place of ni- Electronic Effect of Suhstituents on Phenoxyacetic Acids 445 trogen indene-3-acetic acid happens to be the first analogue of indole- acetic acid to have been studied. If we look at these four substances, the molecules are the same in general shape. Originally it was thought that they are all active because they all have the same shape. It is now clear that molecular shape is not nearly as simple a criterion as it sounds, for there are compounds like 2,3,6-trichlorobenzoic acid u'hich are highly active, and of greatly differing shape from that of lAA. So we have to ask, What do these molecules have in common other than their shape? One answer is that there is in all four of them a strong fractional positive charge on the atom at the bottom of the 5-membered ring. In the case of nitrogen there is a lone pair of electrons which is drawn into the ring, leaving the nitrogen posi- tively charged; oxygen and sulfur have similar lone pairs that are drawn in and are left positive. Furthermore, there is good evidence that cyclopentadiene and indene have exactly the same property on this methylene atom as the other cycles have on the hetero atom. The cyclopentadiene or indene ring is sensitive to acids; the hydrogen is replaceable by potassium and it combines with NO. Furthermore, the dipole moments of all these rings indicate a marked fractional positive charge. The first thing that is common to these four mole- cules, then, is the fractional positive charge in a characteristic position. Now, let us look at the other end of the molecule. What is char- acteristic of all auxins is the carboxyl group, which can ionize, so that here we have in a characteristic position a potential negative charge. Now, of course, the idea is current that the carboxyl reacts chemi- cally to form an amide type linkage or an acyl group of some sort, but I feel that the evidence points against that. Perhaps the strong- est reason for thinking so is, as Dr. van Overbeek has pointed out, Veldstra's work with the tetrazoles. It is almost impossible to see how a tetrazole could form any kind of an acyl type linkage, and yet the one thing that the tetrazole does do is to dissociate an H+ and thus produce a charge at this point. I suggest that there may be no true chemical reaction in the sense of covalent bonds, and that we have to deal rather with the approach to a receptor, based upon the electronic configuration of the molecule. The distance between these two charges, of course, varies — there is free rotation — but it centers around 5i/2 angstroms. Dr. Wain has presented evidence that long side chains are reactive only after beta oxidation. An exception is made in the case of propionic side chains which are apparently ac- tive in themselves. Correspondingly, in p-chlorophenoxypropionic acid, for instance, the distance between the positive charge at the ortho position and the carboxyl is not much greater than the desired order of magnitude. In p-chlorophenoxyacetic acid it is just right. 446 C. Hansch and R. M. Muir Suppose we introduce a second chlorine atom at one of the ortho positions. It will intensify this charge, and so we get one of our most active auxins. However, if chlorine is present at both ortho positions, we have 2,4,6-trichlorophenoxyacetic acid in which the positive site is occupied and the compound is, as might be predicted, inactive. Recently we have conducted a study of the phenylacetic acids, which we obtained through the courtesy of Drs. Steward and Schantz at Cornell, who got them in turn from Dr. Brian of Imperial Chem- ical Industries in England. I will not discuss them in detail, because Dr. Wain has already mentioned something of their activity, but will make one point, namely that in phenylacetic acid, the side chain is shortened by 1 atom, so that in order to get the most favorable distance to the positive charge, we want it at the meta (instead of the ortho) position. It is interesting that in the phenylacetic acid series, the activities of differently substituted compounds do not vary as widely as they do in the benzoic acid series or in the phenoxyacetic acid series, but the activities are more nearly constant, which suggests that while the meta is the most favored position, still the compound, through its free rotation, is able to react at the ortho or the para posi- tion. Tests with the tri-substituted phenylacetic acids show this very nicely. Substitution in the 2,4,5 positions should force the compound to react at the 3 position, and this tmns out to be the most active. Substitution at 3,4,5 forces reaction at the 2 or 6 positions, and this compound is less active. The same is true when the compound is forced to react at the para position. The effect of methyl substituents was mentioned by Dr. Miur. Methyl substituents do enhance activity. Every English farmer who uses methoxone to kill his weeds knows very well that this is the case; 2-methyl-4-chlorophenoxyacetic acid is an extremely active substance. Now since methyl activates the ring, while chlorine deactivates it, one wonders how two oppositely effective groups can have essentially the same effect. The concept of the spatial distribution of charges ex- plains that perfectly because both are ortho-para directive. That means that, although they do it to different degrees, they essentially act in the same general direction. In a series of substituted phenyl- glycines, which were reported by Takeda a few years ago, it was very characteristic that only the chloro and the methyl derivatives were appreciably active. There are two or three outstanding special compounds; one is 2,6-dichlorobenzoic acid. This compound is blocked in the ortho position; therefore according to Muir and Hansch it should not be active, but of course in benzoic acids the side chain is shortened, and as Dr. Leopold and I pointed out in 1955 (The Hormones, Vol. Ill, Electronic Effect of Substituents on Phenoxyacetic Acids 447 ed. G. Pincus and K. V. Thimann. Academic Press, New York. p. 13. 1955) one would not expect the approach to take place in the ortho position, but further down the molecule, so if a chlorine is present at the 2 position, positivity accumulates at the 4, and if a second one is present at the 6 position, the effect is reinforced. So 2,6-dichloro- benzoic acid would be expected to be very markedly active; 2,3,6- is also active and, as was pointed out a little earlier, 2,5- is active. 2,6- Dichlorophenoxyacetic acid is, of course, the comparable opposite case, for the positive charge is at the 4 position, which is rather far for the group to operate, and correspondingly it is only very weakly active (though it has real activity). Another rather striking fact, brought out by the French biochemist Julia, is that when there are two carboxyl groups in the molecule, activity disappears. Thus, 4- chloro-2-carboxymethylphenoxyacetic acid is like methoxone, 2- methyl-4-chlorophenoxyacetic acid, except that it has a second car- boxyl. One might expect it to partake to some extent of the activity of its parent compound, but it does not at all; its activity is reported to be zero. Presumably an additional negative charge at the wrong point would prevent activity. And lastly, the vexing question of the 3,5-disubstituted compounds. You remember that in the phenoxyacetic acids the optimum site for the positive charge at 5i/4 angstrom units from the negative is close to position 6. A chlorine atom in the 3 or 5 position has the opposite effect, conferring positivity only on the 1 position where it is impos- sible for it to react. So one could deduce that 3,5-dichlorination is doing nothing to increase the reactivity of the molecule. Correspond- ingly we find that it is totally inactive, like the parent compound. This is also true in the case of benzoic acid where again the 3,5-di- substituted molecule is just as inactive as is the parent benzoic acid. In the case of phenylacetic acid, however, the 3,5-disubstituted de- rivative has some activity, and again it is about the activity of the parent molecule, which in the case of phenylacetic acid is quite ap- preciable. Thus a number of the observed phenomena can be ex- plained. Now if this view is correct, the impression it gives us of the site of reactivity of auxin is something like that outlined by Dr. Freed, that the auxin approaches a surface in which charges are placed in characteristic positions and the relatively flat benzene ring is borne down with van der Waals forces to rest upon the surface. There is a similarity here to the discussions about the mode of action of chymotrypsin. Dr. Crosby: For some time, we've been arguing with Prof. Hansch and others about the importance of the ortJio position in simple cases, such as p-chlorophenoxyacetic acid. We decided that perhaps 448 C. Hansch and R. M. Muir we might be able to help finish this argument by making a p-chloro- phenoxyacetic acid which didn't have the usual type of ortlto posi- tions. ^Ve did this by putting nitrogens adjacent to the side chain. Considering that our bioassays w^ere carried on in acid solution, we could conclude that these nitrogens should be positively charged to some degree. Consequently, they would have the tetrahedral con- figuration as do the carbon atoms, and so we would have, in effect, a p-chlorophenoxyacetic ester which had no ortho positions open to electrophyllic attack. We bioassayed this compound in four different tests and found that it was not only completely inactive as a stimu- lant of grow^th, but that it was a competitive inhibitor of />chloro- phenoxyacetic acid and ester. Dr. Bonner: Are these systems pH sensitive? Dr. Crosby: We carried these out actually only at two pH \alues, 4.5 and 6.0. Dr. Aberg: I should like to raise a question which turned out to be a useful touchstone for various hypotheses on the relation between structure and activity of the auxins, at the conferences in Ltuid and at Wye. How does the present hypothesis explain the fairly strong activity of d (-j-)-a-phenoxypropionic acid as contrasted to the very weak activity of phenoxyacetic acid? Dr. Thimann: Dr. Aberg raised a very interesting question. I am not prepared to explain it all, but this applies not only to phenoxy- isopropionic acids. Frequently, alkyl substitution increases or modi- fies activity. 1 think a complete explanation would depend on the availablity of data on the effect of this on the charges of the atom. As you know, in the case of indole, two alkyl groups do not destroy activity, although in the case of phenoxy they do. I think all these points depend on a much more careful evaluation of the properties of the compound than I am now prepared to make. J. VAN OVERBEEK Shell Development Company Modesto, California New Theory on the Primary Mode of Auxin Action The search for the primary action of auxin has so far only given negative results. It appears unlikely that in the primary auxin re- action the molecule undergoes covalent bonding, and it appears also unlikely that the primary auxin reaction involves any one specific enzyme system. Previously it had been assumed that auxin combines with some entity, perhaps a protein, both through an ortlio position on the ring, and through the carboxyl group. This idea was developed with the phenoxyacetic acids in mind. The highly active ones, such as 2,4-D (Figure 1, I) all have at least one unsubstituted ortJio position. When both ortho positions are substituted (II) activity is lost. However, at present we realize that this is not generally true. The di-ortho sub- stituted phenylacetic acid (VI) is highly active, and so is 2,6-dichloro- benzoic acid (IV). On the other hand, 2,4-dichlorobenzoic acid (III) is inactive, while 2,4-dichlorophenylacetic acid (V) is rather weakly active (3, 4, 5). Even if one would suppose that the halogenated ortho position were reactive, this is made entirely unlikely by the fact that 2,6 methyl substituted acids (VII, VIII) show considerable activity (7). The chlorine atom is electro-negative, and the methyl group is electro-positive. For this reason, covalent bonding at the ortho posi- tion of these halogenated or methylated benzoic acids (IV, VII, VllI) would appear impossible. As far as the carboxyl group is concerned, Veldstra et al. (7, 9, 10) have shown that this could be replaced by a number of other acidic groupings including tetrazole (X, XII). Therefore, it also seems im- possible that the carboxyl group could be involved in covalent bonding in the auxin reaction. We are, therefore, forced to conclude that the [ 449 ] CHzCOOH 0 CI + V CHgCOOH 0 CI 0 CI COOH M CI 0 CI COOH CI IZ V ci + 2,4-Dichloro- 2,6-Dichloro- phenoxyacetic acid phenoxyacefic acid CH2COOH CI + CI V CHgCGOH CI m + V CI 2,4-Dichloro- 2,6-Dichloro- phenylacefic acid phenylacefic acid CH2COOH 2,4-Dichloro- benzoic acid COOH HjC CH3 + 2,6-Dimethyl- benzoic acid CH2P(0)(H)0H 2,6-Dichloro- benzoic acid COOH H,C "pm CH3 + CI 3-Chloro- 2,6-Dimethyl- benzoic acid XL CH2COOH + \An/ l-Naphtholeneocetic acid l-Naphthylmethyl- phosphonous acid lndole-3-acefic ocid CH2 — C NH xn H N N COOH xm 5-{3 IndolemethyDfetrozole l-Naphthoic acid COOH H xnr H COOH xz Hg 1, 4-Dihydro-l -naphthoic acid 1,2,3,4-Tetrohydro-l -naphthoic ocid Fig. I. Structural formulae and ;uii\itv of auxins and related compounds. Neiv Theory on Primary Mode of Auxin Action 451 mode of action of auxin must be of a physico-chemical rather than of a chemical nature. All attempts to activate an enzyme system in vitro with auxin have failed, as far as is known. However, the activity of many en- zyme systems is affected after a plant has been treated with auxin. This would lead one to conclude that the primary auxin reaction does not involve a single key enzyme, but that the auxin acts on a number of enzyme systems simultaneously — perhaps via the cyto- skeleton, a membrane system upon which or in which enzymes are located (6). If one accepts this view, one will see that the auxin mole- cule is not a direct participant in the enzyme reactions affected by auxins. One might visualize this by imagining that sorption of an auxin molecule in the lipoprotein membrane of the cytoskeleton may lead, for instance, to a local change in the hydration of the membrane. Such a hydration, in turn, would change the relative distance between the enzyme components on or in the membrane. This would change the relative reaction rates between these enzymes, which ultimately would lead to a changed ratio of metabolites. Such a changed ratio of metabolites is the basis for a changed physiological pattern, as the researches of Skoog et al. have shown. Let us assume that the auxin molecule moves with its ring into a cavity of the membrane of the cytoskeleton, and that the polar side- chain sticks out. This cavity, of course, is a temporary opening cre- ated by the thermal agitation of the molecules of the membrane. The penetration of the ring into it is simply an aspect of the phenomenon of solubilization in surface chemistry. Our next problem is to imagine what the polar side-chain, sticking out of the surface, could accomplish. It has become known that on the surface of polymers, hydrogen bond systems can occur. Under some conditions these hydrogen bonds become coordinated in forming an oscillating system (2). This strengthens the H-bonding capacity of the system. Let us now postulate that the polar group of the auxin molecule becomes part of an H-bond system at the surface of the cytoskeleton, and that this polar group provides the missing link in the system and sets it to oscillating. Providing this missing link and making the H-bond network oscillate would then be the primary auxin function. It is not hard to see that such a strengthening of the H-bond sys- tem would affect the structure of the membrane. It might contract it or, via hydration, expand it. According to this picture then, the polar group of the auxin is the crucial part. In order for it to activate the H-bond network, this polar group must be placed just right to function as the missing link. 452 J. van Overbeek CYTO SKEL£ TON O i Fig. 2. Upper: Comparison Ijclween models of molecules of 2,3,6-trithloio sub- stituted phenoxyacetic (POA), phenylacetic (PA), and benzoic (B) acids as they are envisioned to fit into the cytoskeleton and its accompanying layer of H-bonds. Because of the two ijulkv chlorine atoms on both nrtho positions, the rings have penetrated only relatively shallowly. The chlorine in the 3 position (below the plastic) helps in anchoring the ring. This shallow penetration locates the carboxyl groups of PA and B just right for becoming part of an oscillating H-ijond system at the surface of the cytoskeleton, indicated by the square pieces of foam rubber. PA and B are therefore active. The side-chain of POA is too long for such shallow penetration in the cytoskeleton; it protrudes too far and its carboxyl group is out of reach of the H-bond network. POA is therefore inactive. Lower left: Rear view of the cytoskeleton model showing 2,4-dichlorophenoxyacetic acid and 2,6- dichlorophenoxyacetic acid. 2, ID has a slender ring configuration and passes deeper into the cytoskeleton than the bulky 2,6-D. The carboxyl group of 2,4-D makes contact with the H-bond network, while that of 2,6-D extends ineffectively above it. Lower right: Side view of model showing 2,6-dichlorophenylacetic acid. While the ring is anchored in the cytoskeleton, the side-chain sticks out laterally and its carboxyl group becomes part of the H-bond network. 1l is ihc limclion ol the auxin ring lo anchor tlic molecule in the cytoskeleton and thereby hold the polar grotip in plate. By means of a model (Figure 2) we will see how this explains many questions of auxin physiology that heretofore have remained imanswered. (1) Before the auxin molecule can la II in plate (solubilizc) in the cytoskeleton, it must first arrive there. Since the membrane of the cytoskeleton is probably a lipoprotein, the auxins nuist have a par- tition coefficient favorable for partitioning into fats. It is \\cll known Neiu Theory on Prinmry Mode of Auxin Aetion 453 that all the highly active auxins do have such a partition coefficient (8). Ester and nitrile forms of auxins are more fat-soluble than the acid forms and subsecjuently they often have a higher auxin activity. Chlorination also improves fat solubility of molecules and this is un- doubtedly one of the reasons (although a minor one) for the high ac- tivity of chlorinated auxins. (2) Once the auxin molecule has arrived at the site of action, its ring must sink into the membrane of the cytoskeleton to such a depth that the polar group of the auxin molecule fits into the system of H- bonds at the membrane surface. If it sinks in too deeply or not far enough, the proper contact between the polar group of the auxin molecule and the H-bond network is not made (Figure 2). These con- siderations make it obvious that bulky groups on both ortho positions broaden the molecule to such an extent that the ring will not sink deeply into the cytoskeleton; thus with the phenoxyacetic acids the side-chain is relatively long, and in molecules with bulky groups on both ortho positions, the polar group will stick out above the H- bond network (Figure 2). This, then, is the reason for the inactivity of the 2,6-dichlorophenoxyacetic acid (II). On the other hand, the compounds with relatively short side-chains such as benzoic and phenylacetic acids benefit by the presence of two bulky ortJw groups (1), as it locates the polar group exactly in the right position relative to the H-bond network. It is obvious that methyl groups in the ortho positions would be just as effective as chlorine atoms in preventing the molecule from sinking into the membrane too deeply. (3) In addition to a vertical positioning of the polar groups, there is also a lateral positioning. It seems obvious that the H-bond net- work cannot block the hole into which the ring must slide, so it must be located to the side of it. This explains the well-known re- quirement for auxin activity that the side-chain must be perpendicu- lar to the plane of the ring. It explains why 2,6-dichlorophenylacetic acid is a stronger auxin than 2,6-dichlorobenzoic acid. The phenyl- acetic side-chain sticks out farther laterally than the short carboxyl group of the benzoic acid. (4) Since the polar group has to be held in place, it requires se- cure anchoring of the molecule into the cytoskeleton. This is a func- tion of the ring which is held by van der Waals bonding to the cyto- skeleton. Heavy atoms, such as chlorine, especially on the 3, 4, and 5 positions of the benzene ring, help this anchoring process ma- terially as the strength of the van der Waals forces is a function of the atomic weight. This explains the high activity of 2,4-D and 2,4,5-T; further, why 4-chlorophenoxyacetic acid is highly active, while 4- methylphenoxyacetic acid is poorly active (3). 454 J. van Ovcrbeek (5) Since the polar group of the auxin molecule has to become part of an oscillating H-bond system, this requires it to be undissociated and isolated electronically from the rest of the molecule. If a polar group such as carboxyl is in resonance with the benzene ring, elec- trons are withdrawn from the group and it becomes more highly dissociated. The insulation of the polar group is achieved in the phenyl and phenoxyacetic acids by the carbon atom between the carboxyl group and the rest of the molecule. The insulation of the po- lar group in the benzoic acids is achieved by forcing the carboxyl group out of the plane of the ring by the two bulky ortJio substitu- ents and thereby minimizing resonance interaction between the car- boxyl group and the benzene ring. This simple reasoning also ex- plains the interesting behavior of the naphthoic acids (1). 1-Naphthoic acid is like an unsubstituted benzoic acid. Its carboxyl group is in resonance with the ring; therefore, the acid is relatively strong and poorly fit for H-bonding. 1-Naphthoic acid, therefore, is poorly ac- tive as an auxin. Auxin activity is vastly increased by the simple expediency of saturating the bond next to the carboxyl group (XIV, XV). A tetrahedral structure is thereby achieved, whereby the car- boxyl group is forced out of the plane of the ring, and thus removed from resonance interaction with the double bonds in the ring. It becomes a weaker acid and thus becomes more suitable for partici- pation in H-bonding. In addition, of course, this lateral movement of the carboxyl group places it in a more favorable position to par- ticipate in the oscillating H-bond network (see number 3 above). Let us examine an auxin molecule and try to explain, with the aid of Figure 2, why the molecule is active. Take 2,3,6-tri- chlorobenzoic acid. It is active because: (1) Its chlorine atoms make it partition more into the fat of the membrane of the cytoskeleton than the unsubstituted benzoic acid; (2) The chlorine atoms in the 2 and 6 positions force the carboxyl group out of the plane of the ring. This insulates the carboxyl elec- tronically from the ring system and, in addition, places the carboxyl group in the lateral position needed for becoming a partner in the H-bond network; (3) The clilorine atoms in the 2 and 6 positions prevent the ring from sinking into the cytoskeleton too far; (4) The chlorine atom in the 3rd position helps in anchoring the ring more firmly by van der Waals forces. This makes the 2,3,6- substituted benzoic acid a more active auxin than the 2,6-substituted benzoic acid. LITERATURE CITED I. Hcacock, R. A., Wain, R. L., and Wiglitman, V. Sliulics on plant growth- regulating substances. XII. Polycyclic acids. Ann. Appl. Riol. 46: 352-365. 1958. New Theory oii Primary Mode of Auxin Action 455 2. Hu<^"ins, M. L. Hydrogen bonding in high polymers and indusion com- pounds. Jour. Chem.Ed. 34: 480-488. 1957. 3. Jonsson, Ake. Chemical structure and growth activity of auxins and anti- auxins. Handb. Pfl.-physiol. 14. (In preparation.) 4. Pybus, M. B., Smith, M. S., Wain, R. L., and Wightman, F. Studies on plant growth-regulating substances. XIII. Chloro- and methyl-substituted phenoxy- acetic and benzoic acids. Ann. Appl. Biol. 47: 173-181. 1959. 5. , Wain, R. L., and Wightman, F. New plant growth-substances with selective herbicidal activity. Nature. 182: 1094, 1095. 1958. 6. van Overbeek, J. Auxins. Bot. Rev. 25: 269-350. 1959. 7. Veldstra, H. On form and function of plant growth substances. In: R. L. Wain and F. Wightman (eds.). The Chemistry and Mode of Action of Plant Growth Substances, pp. 117-133. Butterworth Sci. Publ., London. 1956. 8. , and Booij, H. L. Researches on plant growth regulators. XVII. Struc- ture and activity. On the mechanism of action. III. Biochim. Biophys. Acta. 3: 278-312. 1949. 9. Westeringh, C. van de, and Veldstra, H. Researches on plant growth regulators, XXIII. Structure/activity, VIII. Phosphonic and phosphonous acids. Rec. Trav. Chim. Pays-Bas. 77: 1096-1106. 1958. 10. , and Veldstra, H. Researches on plant growth regulators, XXIV. Struc- ture/activity, IX. Tetrazole derivatives. Rec. Trav. Chim. Pays-Bas. 77: II07- 1113. 1958. DISCUSSION Dr. Henderson: We have been studying the mechanism of 2,4-D action at concentrations of 10-^ to IQ-^M. We found that oxygen uptake of oat and pea sections during the time of lAA disappearance was correlated over a six hour period. When one added 2,4-D at 10"^ and 10-5 M, the oat showed a marked decrease in lAA disappearance and the pea showed a more rapid disappearance of auxin. This we called a sparing action of 2,4-D. Recently, a new phenomenon was found which turned out to be the effect of 2,4-D on phototropism in weak blue light. When 2,4-D was sprayed on coleoptiles, phototropic action ceased. When we dipped the coleoptiles in Tween (0.01 per cent) -f 2,4-D the same effect was obtained. In 1957 and 1958 we screened some 30 compounds of six different groups. No single family was necessarily similar in structure and, therefore, there was no func- tional group that satisfied an explanation for this phenomenon. We found that out of some 30 compounds, 15 of these at lO-^M negated phototropism. Only six of these, however, were effective at concentra- tions of 10-5 M or below: lAA, IAN, IBA, NAA, 1-naphthaleneaceto- nitrile, and 2,4-D. Since chemical and physical conditions also must influence this phenomenon, we have experimented with 2,4-D from 10-3 to 10-10 M, ^ith the pH from 3 to 9 in buffer solutions. My question, based on some of the statements that you have made is. Would this evidence fit into the pattern you have presented? Dr. van Overbeek: Offhand, I would certainly think so. You raised two interesting points. One is the antagonism between 2,4-D and > en > V -a I p _S y O u 6 o CO T3 C re u V u re ">- c V Xi Q. O re h ro 1 1 o so in rO CM M M CN en (N ^ M * *J Tj" s ^ "tl- sc o in \o o o o so en vO +-» Si 3 ta re ^— 1 3 O to re h> o O CM O ■* CM en en ■* O vO w T-H CL( t* CA c 1 o c o o c: ^ o o c o o ^ _o re u n coo ijtystjOOijOri i/y^i/m^jOfif^/irjOO'^OO U>i 1 -4— » a o 1 C Tf- in iri r- -^ o — en o o O CI en CN 5«y:iO0iO3 «» | ■t-> 1 ^M ^ Tj- c; o in •^ ni CO vo ^- u OJ o ^— m rn (^ en CM eO en (N — m re '"' '— T— T— ^- "" "o S lO C,O0«>5**^ ZOQiJ00tZij0^ M>= to 3 s be 1 c ON 00 \0 O CN ■^ c^l en ^ ^ o c o ^ c en — CM C^l f^l C: en ^ T— ' ^— < T— < ^- ^— < r— • T— * ^- ^- ^- ■ re > re CO ^— aO^'*~*'ijy:icjOCfJOO '-Cn 1 oc ^ in <— 00 sO en rj- O O en o •4-* V o o o o c T— O ^-^ '— ' ^" C en ■^ ■^ ^ "" '"' XA C I>- 4— ^jOr> o 1 CN CN (M — in c^ en — o r-~ en ex o o o o c o o c o o c> (N T— < r— ^r- •■-^ •^ •r- ■^ rt aOO uysijeKt/^ c/>ow:Xe/Xe/>s*^ •-fK 1 (N O SC \C o ^ o — oc ^ sO o ■^ in •* c~ Tt Tf- ro ■* C C-- '^ ■w "^ '^ r- ^ .^ ■^ ■^ coo tjOfifJ&ifjOn fjo^^jonvjonejorifjyi 'jCK W5 4-' 1 c vD r- c t^ in \o -^ o sO r-~ o CN] in ■* m in Tj- Tt in en c in T— t ^- T— ' ^— « ^- '— ' T3 C ^a cO0t,C»=«>= cjCKUKtO^UKUK sOO 1 c 00 ^ OC c o r-~ t^ o c o ">, O c ■* in >- \0 CM ee^ ■* ■* C xO u T— < ^^ ^— ' ^H t— '— ' ^J re u «o tOtiOa «»= --OS «>= J3 1 oc in o OC t~~ en CM en ■* c- 00 ^ o o CM (N O in o O >— O c^ in ■^ T-H ,-. '"' r- «» «« 1 c (N -* C in o ^ en 00 r-~ CM o c o oc en o O O cv o ^— < r-4 '^ ^ T- T- r-, ^ .^ V- '"' ."H <« c ^ - 'u 5 +-> t: 3 i4 C 1 2 re ■^ O '■ c c . 1 O O 0 o o c C :3 £ E u u u u -_ u _o_o _o _o _o _c .-§ c 1 I I c O 0 c IS 3 !H 15 2 x u -t-l 1-. U l- _o _o _c O O U O O L. h c 4. 2 IS X QQQQCC A u re ouu t 1 1 1 1 1 ee^ Tt in \0 -^ ir en 1 1 1 a. (N r^ ■ •■J c u o a > c u u w G- u > ij • •«-) <1) c •> (U u (! u re a; >, in bo u > o re u .-N re SS CO Neio Theory on Primary Mode of Auxin Action 457 lAA. That, of course, is very readily explained and doesn't differ in principle from the antagonism system of Dr. Bonner and his school. It is a cjuestion of materials competing for the same site. In the case of lAA versus 2,4-D, you have 2,4-D sitting here and lAA can't get in, and vice versa. So that is simply explained by this scheme of solu- bilization adsorption in the cytoskeleton. The other point you men- tioned regarding nitriles and methyl esters — they, of course, are more fat soluble than the acids; therefore, they solubilize better into the lipophilic cytoskeleton so they concentrate there and, therefore, the chances of attaching to a specific auxin site are also increased. The materials are probably hydrolized before they act as auxins (Figure 3 A and B). Dr. Wain: There are quite a number of points I would like to raise on this paper, but wish to deal with four. I want to point out that with mono- and dichlorophenoxyacetic acids you get activity in a number of cases. The 2,6-derivative is active, as has been demonstrated by numerous workers; much less activity is shown by the 3,5-com- pound; indeed, this substance is inactive in all our tests. With the mono- and dimethylphenoxyacetic acids you have inactivity both with the 3,5- and the 2,6-derivatives. Methyl derivatives, in general, are less active than the corresponding chloro-compounds. A full paper on the phenylacetic acids will appear shortly, but in the meantime here are some results obtained in three tests (Table 1). You will observe that phenylacetic acid itself is active, and so are all the chloro- derivatives we examined. Now, the point I want you to notice here is that there is particularly good activity in the 3-, the 2,3-, the 2,6-, and the 2,3,6- derivatives. Bearing in mind what I've just said, I want to go back to Dr. van Overbeek's diagram of phenoxyacetic acid with 2,6 blocking; the molecule is sitting up there with the carboxyl group in the wrong position for activity. If you have 3,5 blocking, the molecule will be still further out of the so-called cytoskeleton. Dr. van Overbeek would say, "That's exactly what I would expect because the compound is in- active." But, in fact, if you make the 2,3,5- or the 3,4,5- compounds, you restore activity; yet the compound is still presumably in the same position as it was before. Secondly, we have the tetrachloro-deriva- tives; the 2,3,4,5-compound, again 3,5 blocked, is quite active, as Dr. Smith has shown. Dr. van Overbeek: Let me answer the easy question as to why the methyl derivative is less active than the chloro compound: The methyl group, being lighter in weight, even though it has the same bulk, naturally sticks less rigidly to the cytoskeleton by van der Waals force. Dr. R. Brian, at the Wye College conference, showed that the behavior of the penetration of these auxin molecules into a mono- 458 /. van Overbeek film is not entirely predictable from their two dimensional structural formulae. After we have studied penetration into membranes, per- haps then we could answer that cjuestion. I am not proposing here a ready-made theory, but I am simply asking the question, "In which direction are we looking?" Are we going to continue to look for an answer to the primary mode of auxin action in the direction of co- valent bonding, activation of a single enzyme system, water soluble systems, etc., or are we now going to try to look at it from the point of view of surface chemistry, membranes, etc., and in the direction of things such as Dr. Freed and I discussed? Dr. Wain: Well, Dr. van Overbeek, I accept that. May I go on to my second point? This is in relation to the phenylacetic acids. Now, according to your concepts, the phenylacetic acids are active when you have 2,6 positions blocked because the 2,6-dichloro atoms sit on top of the cytoskeleton and leave the carboxyl group in precisely the right position. Reference to our results, however, shows that quite good activity is found in 2,3-dichlorophenylacetic acid and there, ac- cording to your idea, the compound should be able to penetrate well down into the cytoskeleton. Exactly the same principles apply to the benzoic acids, where 2,3-dichlorobenzoic acid is active. This com- pound, according to your concept, would penetrate deeply into the membrane, leaving the carboxyl group far too low to join up Avith the oscillating hydrogen bonding network. Dr. van Overbeek: When one studies these problems Avith molecule models and especially when one tries to fit these into 3-dimensionaI holes, one finds that the 2,3-dichlorobenzoic and phenylacetic acids do, indeed, fit the theory very nicely, contrary to what one might expect from 2-dimensional reasoning with structural formulae. This type of 3-dimensional hole is shown in figure 3 and Avas suggested several years ago by Dr. Mullins (cf. van Overbeek, Bot. Rev. 25:300. 1959). The phenylacetic acids have the added feature that the side chain sticks out of the plane of the ring in a very prominent fashion. This gives this type of molecule a tendency to hang itself up, thus favoring activity. Incidentally, the prominent perpendicular side chain may also be a reason why indole-3-propionic acid is highly active, as you have heard dining the discussions on the first day. It, too, has a natural tendency, on account of the angular side chain, to hang itself up and, therefore, stabilize itself in the cytoskeleton system. Not only does my view of looking at the primary mode of action of auxin as a solubilization phenomenon explain puzzles such as why the 2,3-dichlorobenzoic and phenylacetic acids possess activity, but it also gives a plausible explanation for the activity of the structurally totally unrelated 2-heptadecanol, which Dr. Crosby told us the first A^cio Theory on Primary Mode of Auxin Action 459 Fi£. :!. A. Model ot li,:! dichluiubcnzoic acid about to be lowered into a 3-dimen- sional liole. The 3 cylinders are imagined to be 3 molecules of the cytoskeleton. The white pieces of foam rubber indicate part of the hydrogen network on the surface of the cytoskeleton. B. Model of 2,3-dichlorobenzoic acid fits tightly into the hole of the cytoskeleton so that it anchors the polar group in position in the hydrogen bond network. C. Model of 2-heptadecanol fitting into the 3-dimensional hole of the cytoskeleton. Note how the -OH group is held into place for partici- pation in the H-bond network. D. Model of indole-3-acetic acid fitting into a hole in the cytoskeleton. An arrangement of this sort, because of the uniformity of the cylinders (molecules that make up the cytoskeleton) may give the false impression that all holes are of uniform size. This is not intended as the holes into which the auxins fit must be specific (see F. H. Dickey, Jour. Phys. Chem. 59: 695. 1955). day he has isolated from tobacco tissue. If, indeed, as I am proposing, the primary function of an auxin is the participation of the undissoci- ated acid group in a hydrogen bond system, then there is no physico- chemical reason why another polar group, such as the -OH of an alcohol, could not serve equally well. The discovery by Crosby and Vlitos of the alcohol auxin, therefore, supports my views. It will be noted that the -OH group of this alcohol is on the second carbon. Therefore, it is in a lateral position quite comparable to that of a hindered carboxyl group of a benzoic acid. The long hydrocarbon chain fits the auxin binding site in the cytoskeleton (Figure 3C) and is held in position by van der Waals forces. 460 J. van Overheek Dr. Wain: My third point is in relation to the naphthoic acids. The 1 -naphthoic acid has only weak activity; if you reduce the sec- ond ring then the 3,4-dihydro-derivative still has only weak activity. On the other hand, the 1,4-dihydro- is highly active; the 1,2-dihydro- is highly active, and so is 1,2,3,4-tetrahyclro-l-naphthoic acid. The ex- perimental evidence is not in question, but there are other possibili- ties in addition to those put forward by Dr. van Overbeek which ex- plain the activity of these molecules. It will be noted that in all the active compounds there is a hydrogen atom attached to the carbon atom adjacent to the carboxyl group. As you all kno^v, we. at AVye, have repeatedly stressed the importance of the a/p/ia-hydrogen atoms in relation to activity, for I think this is the key to all considera- tions of this kind. I refer to the fact that in those growth substances in which the carbon atom adjacent to the carboxyl group is asym- metric, there is usually activity with one enantiomorph and not the other. To my mind this is one of the most important things of all, and I'm rather surprised that steric considerations of this kind have not been mentioned throughout the whole of this conference, for here, surely, is an important clue to mode of action. AVe have very definite ideas about this, but they are only ideas, and other views, of course, are possible and will be put forward. But, I do think that any proposed theory — and I do congratulate you, Dr. van Overbeek, on the thought you have put into your theory — must take steric con- siderations into account. I come back to what I said this morning, and that is that so complex is the growth response — your molecule must get in, must have the right physical properties to penetrate, to move in the tissues, it must have adequate stability, then it must have the structural requirements for activity at the site of action — that no simple theory on mode of action is likely to be satisfactory. But. let's not overlook this rather interesting clue to the whole situation — this specificity of stereoisomers which, incidentally, was first discov- ered by Dr. Smith and has been developed very considerably by the Swedish school since that time. Dr. Bitancourt: I believe that the site of action of lAA is at the interface between the cytoplasm and the cell wall, and Dr. van Over- beek's theory seems to fit very well into this scheme. But if the hole in the cytoskeleton is so small that only the dichlorophenoxyacetic acid fits in there, how does the indole nucleus fit into this hole? Dr. van Overbeek: Indeed, the indole nucleus does fit. One has to make the stereo models (Figure 3 D) in order to appreciate the 3- dimensional aspects. Dr. Bitancourt: AVell, this can be understood, but then once all the holes are occupied nothing ought to happen, and so you can't New Theory on Primary Mode of Auxin Action 461 explain the part of the action curve of lAA where there is inhibition. This can be explained by the two attachment point theory. Dr. Ray: The crucial point of this theory is one of a specific site involving points at which the auxin combines and brings about ac- tivity. I do not feel that it is fundamentally different from what we have all been thinking about for some years concerning the nature of auxin activity: combination of the auxin molecule with a site of specific shape. I do not think it is at all a question, therefore, of sur- face chemistry or nonspecific adsorption, but as far as the data that this theory is attempting to explain, one primarily of specificity. Dr. Fawcett: If one considers the 2,4-dichlorophenoxy structure, then the acetic acid homologue is a highly active compound, although (3-(2,4-dichlorophenoxy)propionic acid, with its somewhat longer side chain, is inactive in, for example, the split pea curvature test. But, if one considers p-(2,3-dichlorophenoxy)propionic acid, this is more ac- tive than 2,3-dichlorophenoxyacetic acid (Fawcett et ah, Proc. Roy. Soc. 150 B: 95. 1959). Are these examples in line with the present hypothesis? Dr. Osborne: One small point — if one of the hydrogens on the side chain is replaced with a methyl group in certain of the 2,6-sub- stituted phenoxyacetic acids the biological activity is enhanced al- though, supposedly, the ring would still have difficulty fitting into the hole and the side chain would still be too high for activity. Dr. van Overbeek: Yes, but introduction of methyl will improve its partitioning properties so you have more molecules near the site of action and thus a better chance of getting them into position. In addition, the methyl group causes steric hindrance so it brings the side chain over in the effective lateral position. Many things have to be taken into consideration. I disagree with Dr. Ray's comments. This theory is fundamentally different because I am no longer look- ing at a water-soluble system but at an oil-soluble membrane sys- tem; we are not looking at covalent bonding, we are looking at physico-chemical systems. The principle is that of hooking a small polar group into position. What I have tried to do is to show the direction in which we must look for an answer. The Gibberellins B. B. STOWE^ Harvard University F. H. STODOLA USDA Regional Laboratory Peoria, Illinois T. HAYASHI National Institute of Agricultural Sciences Japan P. W. BRIAN Imperial Chemical Industries England The Early History of GibberettLn Research Under the direction of Dr. Stowe, the initial workers with the gibber- ellins on three continents present the original difficulties and prob- lems encountered when they first got into gibberellin work. They represent the Tokyo (Japanese) group, the I. C. I. (British) group, and the United States Department of Agriculture (American) group. DISCUSSION Dr. Stodola: The first suggestion that I could find in the literature that the bakanae effect might be due to the fungus, appeared in 1912 in a paper by the Japanese plant pathologist, Sawada (7). In this paper he said, "On microscopic examination the plant system is found to contain mycelium. It is thought that the plants grow taller due to some stimulation from the mycelium." In the early 1920's, a young graduate of Chiba Horticultural College - Eiichi Kurosawa - came to Formosa to work with Sawada at the Central Research Institute of the Formosa Department of Agriculture. His problem was to work on methods of controlling the bakanae disease which, at that time, was causing severe rice losses on the island. In the course of his in- vestigations, Kurosawa became interested in the unusual symptom of hyperelongation which characterizes the disease, and he undertook to determine the nature of the responsible agent and, if possible, to isolate it. In the summer of 1925 Kurosawa started his experimental ^Subsequently: J. W. Gibbs Laboratory, Department of Botany, Yale Univer- sity, New Haven, Conn. [ 465 ] 466 Slowe, Stodola, Hayashi, and Brian work on this phase of the problem, and by the following year he had published in Japanese his now classical paper, "Experimental studies on the secretion of Fusarium heterosporum on rice-plants" (5). Kuro- sawa showed that sterile filtrates from the bakanae fungus gave marked growth stimulation in lice and grass. In this paper appeared the first photograph found in the literature which illustrated the stimulatory effect of the fungus secretion. It is to Kurosawa, then, that we are indebted for opening up this fertile gibberellin field. I felt that a pioneer such as this deserves to be better known than he is. Kurosawa was born in 1894 in the town of Itabashi in Ibaraki Prefecture, and he died in 1953 at the age of 59. After considerable effort Dr. Hayashi was able to find the in- formal picture shown on the facing page. There you see the man responsible for the work that we are so much interested in. The publication of Kurosawa's paper at once stimulated other workers to take up the problem, of course, and in 1928 papers by Hemmi and Seto appeared from the Phytopathology Laboratory of Kyoto University (4, 8). In 1932 Shimada at Hokkaido University in northern Japan published the first paper on the chemical nature of the growth promoting principle (9). It was at this time that the University of Tokyo workers became interested in the problem. Dr. Hayashi: In 1930 Dr. Yabuta, formerly professor of Agricul- tuial Chemistry of Tokyo University, Dr. Kannbe and I studied the isolation of the growth stimulating substance. In 1934, contrary to what we expectetl at first, we obtained a growth deterring substance, fusaric acid, that is, 5-n-butylpicolinic acid, by extracting the cultmed solution either with benzene or petroleum ether. It is reported that in some cases rice plants infected with bakanae organism show re- duced growth instead of the usual elongation. Judging from this fact, fusaric acid might be responsible for the symptoms of retarded growth. In 1934 Kurosawa changed his position to Tokyo and, with his help, Dr. Yabuta and I again began isolation of the growth stimu- lating substance. Kurosawa furnished us with a fungus -whidi pro- duced the active substance abundanlly and indicated suitable ( uliural conditions. The isolation of the active substance from the culture filtrate was carried out as follows: The fungus was grown in a medium contain- ing ammonium chloride, monopotassium phosphate, and glycerol by the culture method in (lask. After about one month, the culture solution was filtered and the filtrate was treated \\iih activated car- bon. The carbon was then eluted with methanolic ammonia. The eluate was concentrated in vacuo. The resulting residue was dissolved in aqueous sodium bicarbonate solution and extracted Aviili ether to Dr. Eiichi Kurosawa, 1894-1953 468 Stowe, Stodola, Hayashi, and Brian remove neutral and phenolic substances. The bicarbonate solution was then acidified and again extracted with ether. After treating with lead acetate, the ether extract was evaporated, and then the active substance was obtained as a white powder. In 1935 Dr. Yabuta gave it the name of gibberellin on the basis of the scientific name of the fungus, Gibberella fujikuroi (Saw.) A\'ollenw. This was the first use of this term in the literature. Later, in 1938, Dr. Yabuta and Dr. Sumiki reported the isolation of crystalline gibberellin A and B (15). The studies on the chemical structure of gibberellin started at this time. By 1950 we had published 15 papers on the production of gib- berellin and on its chemistry (cf. 10, 13, 14). It was at this time that work outside of Japan began. Dr, Stodola: The first work on gibberellin outside of the Orient was done at the Chemical Corps Biological Laboratories at Camp Detrick, Maryland; in March, 1950, Dr. J. E. Mitchell reviewed this work in a talk before the American Phytopathological Society (6). That summer the Korean War started, and then gibberellin took on a military aspect as far as this country was concerned. A large scale production was needed to provide sufficient material for proper test- ing, and because of our experiences at Peoria with industrial fermen- tations, the problem was brought to us in August, 1951. Dr. Raper, the head of our culture collection section, was assigned to carry out the fermentation studies, and I was to work on the isolation and characterization of the gibberellin. I was very fortunate at the start to be able to talk over the whole problem with Prof. Sumiki, who is the authority on the production and chemistry of gibberellin, while we were at the International Congress of Pure and Applied Chemistry in New York in early Sep- tember, 1951, where Prof. Sumiki gave a talk on gibberellin. In get- ting started on work of this sort, one needs first cultures of the organ- ism, a suitable medium for growing it, assay procedures for esti- mating the amount that is produced, and pure compounds for use as standards. We had trouble with all of these, as you will see from the following excerpts from letters that I picked out. For example, a letter from Dr. Mitchell to Dr. Raper, written in October, 1951, states, "I'm very sorry to hear that the cultures I brought to Peoria did not survive. I can't understand why that should have hap- pened, inasmuch as I have kept them for much longer periods of time in the past on that medium without trouble." Another letter from Mitchell two months later, "I am sorry to hear that you are having difficulty with your assay procedure. I wish that I could give you details of an effective assay that would give the desired results. We likewise have not gotten the results ih;it wc had hoped for." A Early History of Gibberellin Research 469 letter from me to Professor Sumiki in February, 1952, goes in part like this, "In recent experiments we have failed to obtain even mod- erate growth using your medium containing glycerol, dihydrogen phosphate, and ammonium chloride. We have found, however, that when magnesium sulfate is added, in even small amounts, growth is greatly enhanced. We are wondering if you have found it necessary or desirable to add magnesium sulfate to your fermentations." Final- ly, a letter to me from Sumiki in March, 1952, "I am very surprised that the sample of gibberellin A sent to you by my assistant while I was in the United States showed little activity. The activity of that sample was not tested but this is the first time we have heard of crys- talline gibberellin losing its activity so fast. We are now preparing a new sample to send to you." By May, 1952, these difficulties had been straightened out, and in June we had a successful pilot plant run which yielded 12 g. of crys- talline gibberellin. From this I was able to isolate by repeated crys- tallization a sample of pure gibberellic acid with a rotation of -|-90° (12). Later, we developed a chromatographic method that would effi- ciently separate gibberellin Aj and gibberellic acid (11). Our experi- ence was passed on to a number of fermentation companies in this country, and before long there was enough gibberellin available here for everyone. All this time, of course, the British workers were carry- ing out their work on gibberellic acid. Dr. Brian: We started work either in 1951 or late 1950. Undoubt- edly, the stimulus to us was the sudden spate of abstracts of Japa- nese work which hadn't reached us during the war years. Looking back on things, I am very much more struck by stupendous pieces of luck that we had rather than by difficulties. I can just mention a few of these. First of all, our strains of the fimgus, Gibberella fiijikiiroi, were obtained originally simply by getting them from culture collections. By far the best that we found in the early days and by far the best that we still have was a strain which to my certain knowledge had been kept in culture collections for over 30 years, and I believe, in fact, was the type isolated by Sawada. We were very lucky indeed, I think, to come across so stable an organism to work with. The second piece of luck we had was that in carrying out our preliminary fermentations, we completely ignored any previous work and used the kind of media that we had been used to using in our other work on fungal metabolic products. We immediately got yields of a gibberellin in far greater quantities than any previously-recorded yields — again I think purely by accident (1, 2). Very shortly after we got this material, one of my colleagues, Philip Curtis, said that this stuff 470 Stowe, Stodola, Hayashi, and Brian wasn't the same as the Japanese gibberellin A. We, I'm afraid, told him he was talking nonsense, that this was virtually impossible, that it should be something different; but in point of fact, he turned out to be right. We had, as a result of using the strain we did and the culture media we did, arrived straightaway at a pure gibberellin, a material now known as gibberellic acid or gibberellin A3. A further piece of luck that we had was that almost immediately in our bio- logical work we stumbled on this dwarf-tall relationship which had the effect of first making us think of the possibility of isolating na- tural gibberellins and also provided us in the very early days with a very convenient assay, the dwarf pea (3). Looking back on things, I feel that our early history was characterized by quite extraordinary and undeserved pieces of luck rather than the kind of difficulty that Dr. Stodola mentioned. LITERATURE CITED 1. Borrow, A., Brian, P. W., Chester, V. E., Curtis, P. J., Hemming, H. G., Hene- han, C, JefTreys, E. G., Lloyd, P. B., Nixon, I. S., Norris, G. L. F., and Radley, M. Gibberellic acid, a metabolic product of the fungus Gibberella fiijikuroi: some observations on its production and isolation. Jour. Sci. Food Agr. 6: 340-348. 1955. 2. Brian, P. W., Elson, G. \\., Hemming, H. G., and Radley, M. The plant- grou'th-promoting properties of gibberellic acid, a metabolic product of the fungus Gibberella fujikuroi. Jour. Sci. Food Agr. 5: 602-612. 1954. 3. , and Hemming, H. G. The effect of gibberellic acid on shoot growth of pea seedlings. Physiol. Plant. 8: 669-681. 1955. 4. Hemmi, T., and Seto, F. Experiments relating to stimulative action by the causal fungus of the "Bakanae" disease of rice. Proc. Imp. Acad. Tokyo. 4: 181-184. 1928. 5. Kurosawa, E. Experimental studies on the secretion of Fusarium heterospomm on rice-plants. Jour. Nat. Hist. Soc. Formosa. 16: 213-227. 1926. (English translation included in reference 10, p. 111.) 6. Mitchell, }. E., and Angel, C. R. Plant-growth-regulating substances ob- tained from cultures of Fusarium moniliforme. Phytopath. 40: 872,873. 1950. 7. Sawada, K. Diseases of agricultural products in Japan. Formosan Agr. Rev. (Taiwan Nojiho). 63: 16. 1912. 8. Seto, F. The reactions of rice seedlings to infection of the causal fungus of tiie "Bakanae" disease and lo filtrates of its cultures. Mem. Coll. .^gr. Kyoto Imp. Univ. 7: 23-38. 1928. 9. Shimada, S. Further studies on the nature of the growth promoting substance excreted by the "bakanae" finigus. Ann. Phytopath. Soc. Japan. 2: 442-452. 1932. 10. Stodola, F. H. Source book on gibberellin (1828-1957). 560 pp. Agr. Res. Serv. 71-11, USDA, Peoria, Illinois. May, 1958. 11. , Nelson, G. E. N., and Spence, D. J. The separation of gil)bcrcllin A and gibberellic acid on buffered i>artition columns. Arch. Biochem. Biophys. 66: 438-143. 1957. IZ Early History of Gibberellin Research 471 -, Raper, K. B., Fennell, D. I., Conway, H. F., Sohns, V. E., Langford, C. T., and Jackson, R. W. The microbiological production of gibberellins A and X. Arch. Biochem. Biophys. 54: 240-245. 1955. 13. Stowe, B. B., and Yamaki, T. The history and physiological action of the gibberellins. Ann. Rev. Plant Physiol. 8: 181-216. 1957. 14. , and Yamaki, T. Gibberellins: stimulants of plant growth. Science. 129: 807-816. 1959. 15. Yabuta, T., and Sumiki, Y. Communication to the Editor. Jour. Agr. Chem. Soc. Japan. 14: 1526. 1938. CHARLES A. WEST University of California The Chemistry of GibberelUns From. Flowering Plants' The chemistry of the gibberellins derived from culture filtrates of the fungus Gibberella fujikuroi has been extensively investigated primarily by two groups of chemists — one at the College of Agricul- ture of the University of Tokyo and the other at the Akers Research Laboratories of the Imperial Chemical Industries, Limited in Eng- land. The results of the investigations of Grove et al. of the Akers research group have led to the following proposed structures for gib- berellic acid (A3) (2) and gibberellin Ai (Aj) (3). 2 -^ COOH OH tCH. COOH ^This research was supported in part by grants from the National Science Foundation (G-3526) and Merck & Co., Inc. ^ After the preparation of this manuscript, the author was informed of a re- port to be published by B. E. Cross, J. F. Grove, J. MacMillan, J. S. Moffatt, T. P. C. Mulholland, and J. C. Seaton in which the proposed structure of gibberellic acid is revised to the following: COOH [473] 474 C. A. West Sumiki and his collaborators at the University of Tokyo interpret their results to indicate the same structure for Ai and A3 as above, except for the {position of the lactone ring in ring A (4, 8, 10). They report that lithium aluminum hydride reduction of the methyl ester of Aj leads to a product which could not be oxidized by periodate. They also reported the isolation of a degradation product from the lithium aluminum hydride reduction product which was identified as a l,3-dimethyl(luorene derivative. In line with their findings they propose the A rings of A^ and A3 to have the following structures: Sumiki and his co-workers have also proposed structures for gib- berellin Ao (A2) and gibberellin A4 (A4) which they have isolated from the fungal filtrates. Both are thought to have the same A-B ring struc- tures as Aj but are modified in the C-D rings as follows: A4 There is now abundant evidence for the presence of substances with biological properties similar to those of the fungal gibberellins in flowering plant tissues. The obvious implication is that such sub- stances act as natural regulators of growth and development in the plant. It is important, therefore, to isolate these substances and de- termine their structure if we are to approach intelligently questions regarding their role in controlling growth and development, the mode of their biosynthesis, and the mechanism of genetic control of these processes. Chemistry of Gibherellins From Flowering Plants Alb MacMillan and Suter (6) have reported the isolation of Aj from immature bean seed {Phaseolus multiflorns). The identification was based on the identity of the infrared spectra of the free acid and methyl ester of the isolated specimen and those of authentic Aj free acid and ester and the identity of the melting points and mixed melt- ing points of the methyl esters. West and Phinney (11) have reported the isolation of two crystalline compounds with gibberellin-like bio- logical properties, called bean factor I and bean factor II, from imma- ture bean seed (Phaseolus vulgaris). These substances were incom- pletely characterized. Sumiki during this conference reported the iso- lation of Ai from water sprouts of mandarin orange (Citrus unshiu) (9). These are the only reports to date of the isolation of gibberellins from flowering plants in a sufficient state of purity to allow a useful determination of physical and chemical properties. The purpose of this paper shall be to review the properties of bean factor I and bean factor II and their implications for the struc- tures of these materials. EXPERIMENTAL PROCEDURE Isolation The procedure employed for the isolation of bean factors I and II has been described (11). The initial steps included extraction of approximately 25 kg. of immature bean seed with acetone-water (1:1), adsorption of the active substances onto charcoal from aqueous solu- tion and re-elution with acetone (omitted in the second run) and ex- traction of the active substances from aqueous buffer at pH 2 with ethyl acetate. Further purification of this concentrate was achieved by column chromatography on charcoal, column chromatography on silicic acid, and countercurrent distribution. Crystallization was ef- fected from ethyl acetate-petroleum ether solvent mixtures. The pro- gress of purification was followed by bioassay on dwarf mutants of maize. In one run approximately 2 mg. of bean factor I and 2 mg. of bean factor II were recovered. In a second run approximately 1 mg. of bean factor I and 5 mg. of bean factor II were obtained. Silicic acid chromatography has proved the most useful technique for fractionating bean factor II from bean factor I. In a typical col- umn 40 g. of prewashed and oven-dried silicic acid is dry-packed in a column. The material to be chromatographed is adsorbed on a small amount (2 g.) of silicic acid by evaporation from an organic solvent and this mixture is packed on the top of the adsorbent col- umn. The column is developed in succession with 400 ml. chloro- form, 400 ml. of 20 per cent ethyl acetate in chloroform (by volume), 400 ml. of 40 per cent ethyl acetate in chloroform, 400 ml. of 60 per 476 C. A. West cent ethyl acetate in chloroform, 400 ml. of 80 per cent ethyl acetate in chloroform, and 400 ml. of ethyl acetate. Forty ml. fractions are collected and aliquots are tested for activity. Bean factor II is eluted primarily in those fractions obtained with 40 per cent ethyl acetate in chloroform as the developing solvent and bean factor I is eluted by 60 per cent ethyl acetate in chloroform as the developing solvent. Under identical conditions A^ and A3 are eluted with the latter de- veloping solvent. Such columns have been used to demonstrate the presence of gibberellin-like substances in crude extracts and also as a terminal step in purification. Biological Properties The biological properties of bean factor II are discussed more completely by Phinney elsewhere in this volume (7). In quantitative assays on dwarf mutants of maize, bean factor I and A^ show the same activity on a weight basis. The growth response of maize mu- tants dwarf-2, dwarj-3, dwarf-5, and anther-1 to bean factor II is equal to or greater than the response to an equivalent amount of A3, the most active of the fungal gibberellins. However, bean factor II is less than 5 per cent as active as A3 for the dwarf-1 mutant. Thus, bean factor II is quite distinct from A^, Ao, A3, and A4 in its biological properties. Neutral Equivalent A spectrophotometric micro-method for the determination of the neutral equivalent weight of carboxylic acids was developed. A known weight (about 0.05 microequivalent) of acid to be tested was dissolved in 3.50 ml. of a freshly prepared solution of the sodium salt of jjhenolsulfonphthalein (phenol red) (3 mg. per 100 ml. of boiled distilled water). Care was taken to exclude atmospheric carbon di- oxide. The absorbancy at 550 lUfj. was measured in a Beckman B spectrophotometer for (1) a reagent blank (no acid added), (2) the solution of an unknown acid, and (3) the solution of a standard acid. The absorbancy of sodium phenolsulfonphthalein decreases at 550 ni/i, in the presence of an acid due to the conversion of the indicator from the base to the acid form. The magnitude of the decrease is a function of the equivalents of acid added and can be standardized by reference to a standard acid. Determinations of the neutral equivalent of bean factor I by this technique with A3 as standard^ gave values of 370 and 340 (average = 355) and values of 380 and 340 (average =r 360) were obtained for ' Reference samples used in these studies were kindly supplied as follows: A„ A2, and A, — Prof. Y. Sumiki, University of Tokyo, Tokyo, Japan, and A, and A:, -Dr. Frank Stodola, Northern Regional Research Lab., USDA, Peoria, 111. Chemistry of Gibberellins From Floweririg Plants Table 1 . Paper chromatography of the gibberellins. 477 R.a* in Solvent System f Gibberellin A B C A, (1.0) 1.0 1.1 2.3 1.0 2.0 (1.0) 1.0 1.1 1.3 1.0 1.2 (1.0) Ai 2 A^ 4 A4 t Bean factor I Bean factor II 2 9 * Kga = Migration relative to A3. t Solvent systems: A = Upper phase of a mixture of n- butyl alcohol: 1. 5 JV ammonium hydroxide (3:1). B = Upper phase of a mixture of n-amylalcohol:pyridine:water (35:35:30). C = Upper phase of a mixture of benzene: acetic acid (gla- cial) :water (4:1:2) (1). X Migrated off the end of the chromatogram under the con- ditions employed. bean factor II. Theoretical neutral equivalents for Ai and A3 are 348 and 346. Thus, bean factors I and II are clearly shown to have an acidic functional group with a neutral equivalent close to those of the fungal gibberellins. Paper Chromatography It can be seen by reference to Table 1 that Aj, A2, and A3 migrate to approximately the same extent on paper chromatograms in the three solvent systems shown, whereas A4 moves considerably further from the origin. Bean factor I behaves as Ai in these systems. Bean factor II is closer to A4 in its behavior, though not identical with it. Since Aj, Ao, and A3 each have two alcoholic hydroxyl groups and A4 has only one, these results might be interpreted as presumptive evi- dence for the presence of one alcoholic hydroxyl group in bean fac- tor II. Determination of Ethylenic Double Bonds A preliminary microhydrogenation experiment suggested that bean factor II has two ethylenic double bonds per acid equivalent as does A3 and bean factor I has one as does Aj. Confirmation of these conclusions came from the application of a microtechnique for the determination of such double bonds. A sample of 20 to 40 micrograms of gibberellin of known weight was dissolved in 3.5 ml. of freshly prepared potassium permanganate solu- tion (3 mg. per 100 ml. solution in distilled water). After 15 min. the absorbancy of the solution was determined at 415 m/x in a Beckman 478 C. A. West Table 2. Ethylenic double bonds from permanganate reduction studies. Double Bonds per Acid Equivalent Gibberellin Found Theory A3 Ai 2.0 1.3 0 1.3 1.0 1.9 2 1 A2 A4 Bean factor I Bean factor II 0 1 B spectrophotometer. The absorbancy of an unknown relative to that of a standard (absorbancy of blank subtracted from each) was used to calculate the number of double bonds per unit weight. Table 2 shows the results for A^, Ao, and A4, bean factor I, and bean factor II with A3 taken as a standard. Determination of Exocyclic Methylene Groups A procedure adapted from Lemieux and von Rudloff (5) was em- ployed for the micro-estimation of terminal methylene groups of the type reported to be present in A^, A3, and A4. The procedure is based on conversion of the methylene group to formaldehyde by means of a periodate-permanganate reagent followed by the colori- metric estimation of the formaldehyde produced with a chromotropic acid reagent. Table 3 summarizes the results obtained with this method for the fimgal gibberellins and the bean factors. The yields are in the range of those found by Lemieux and von Rudloff. These results suggest the presence of exocyclic methylene groups in bean factor I and bean factor II. However, this interpretation is tentative since A2 seems to yield formaldehyde even though it is thought not to have such a group. Table 3. Determination of exocyclic methylene groups. Moles of Formaldehyde Per Acid Equivalent Gibberellin Found Theory Ai Aj A3 A4 Bean factor I Bean factor II 0.76 0.61 0.51 0.53 0.83 0.59 1 0 1 1 Chemistry of Gibberellins From Flowering Plants 479 Infrared Absorption Spectra The infrared absorption spectrum of bean factor I in KBr pellet is identical with that of a sample of Aj supplied by Stodola. The infrared absorption spectrum of bean factor II in KBr pellet resembles in a general way those of the fungal gibberellins, but there are some distinct differences. Some absorption maxima for bean fac- tor II and tentative structural assignments based on similar features of fungal gibberellins are as follows: 3400 cm-i alcoholic hydroxyl 1750 cm-i y-lactone carbonyl 1720 cm-i carboxylic acid carbonyl 1650, 890 cm-i )C=CHo 1620 cm^ ethylenic double bond DISCUSSION The identity of the infrared spectra of bean factor I and an au- thentic sample of Aj leads to the conclusion that bean factor I and Ai are the same compound. All the other properties determined are consistent with this conclusion. Thus, there are three species of flow- ering plants which have been shown to contain Ai as a natural con- stituent in small amounts — immature seed of Phaseolus midtifloriis, immature seed of Phaseolus vulgaris, and water sprouts of Citrus unshiu. The properties of bean factor II indicate that it is structurally similar to the fungal gibberellins but not identical with any of those reported to date. The presence of a carboxylic acid group is clearly indicated, and the neutral equivalent is in the range of those of the fungal gibberellins. A y-lactone group is also indicated by the infra- red spectrum. The evidence also suggests the presence of one alco- holic hydroxyl group. Two ethylenic double bonds are present per acid equivalent. One of these is most likely bonded to a terminal methylene group. These two double bonds are not in conjugation with other sites of unsaturation since bean factor II does not show an ultraviolet absorption maximum above 220 m/x. If the seemingly reasonable assumption is made (for which there is no direct evidence) that bean factor II has the same carbon skeleton as the fungal gibber- ellins and the exocyclic methylene group is as in A^, A3, and A4, then the following positions seem the most likely possibilities for the sites of unsaturation. 480 C. A. West ^"^ (I) (2) (3) Structure 3 would seem less likely since bean factor II does not fluo- resce when dissolved in sulfuric acid, whereas A3, with a double bond in the A-ring, does. There is no evidence to assist in the placement of a lactone, a carboxylic acid, or an alcoholic hydroxyl, although it would seem most likely they would be substituted in one of the positions occupied by such groups in Aj, Ao, A3, or A4.^ SUMMARY Two crystalline substances, with gibberellin-like biological pro- perties, called bean factor I and bean factor II, have been isolated from acetone-water extracts of immature seed of PJiaseolus vulgaris. The infrared spectrum and other properties of bean factor I dem- onstrate that it is identical with gibberellin A^ isolated from the fungus Gibberella fiijikiiroi. The biological and chemical properties of bean factor II show that it is not identical with the fungal gibber- ellins, Aj, A2, A3, or A4. It has a carboxylic acid group and a neutral equivalent of approximately 360. Evidence is also presented for the * After the preparation of this manuscript the author was supplied with a copy of a report to he published by J. Mac\lillan, J. C. Scaton, and P. J. Suter in which they describe the isohition of a substance (gibberellin A^,) from seed of Pliaseolus miiltiflnrus. They assign to this compoiuid the structure: COOH A comparison of the infrared spectrum of this substance with that of bean factor II shows them to be identical. Chemistry of Gibberellins From Flowering Plants 481 presence of a lactone, an alcoholic hydroxyl, and two ethylenic double bonds not in conjugation with other sites of unsaturation. These properties are discussed in terms of possible structures for bean fac- tor II. LITERATURE CITED 1. Bird, H. L., Jr., and Piigh, C. T. A paper chromatographic separation of gihber- eUic acid and gibbereliin A. Plant Physiol. 33: 45, 46. 1958. 2. Cross, B. E., Grove, J. F., MacMillan, J., and Mulholland, T. P. C. The struc- ture of gibberellic acid. Proc. Chem. Soc. 1958: 221, 222. 1958. 3. Grove, J. F., Jeffs, P. W., and Mulholland, T. P. C. Gibberellic acid. Part V. The relation between gibbereliin A^ and gibberellic acid. Jour. Chem. Soc. 1958: 1236-1240. 1958. 4. Kitamura, H., Takahashi, N., Seta, Y., and Sumiki, Y. Biochemical studies on "Bakanae" fungus. Part 47. Chemical structure of gibberellins. Part XIV. Bui. Agr. Chem. Soc. Japan. 22: 434, 435. 1958. 5. Lemieux, R. U., and von Rudloff, E. Periodate-permanganate oxidations. II. De- termination of terminal methylene groups. Canad. Jour. Chem. 33: 17I0-I7I3. 1955. 6. MacMillan, J., and Suter, P. J. The occurrence of gibbereliin Ai in higher plants: isolation from the seed of runner bean {Phaseolus multiflorus). Natur- wis. 46: 45. 1958. 7. Phinney, B. O. Dwarfing genes in Zea mays and their relation to the gibber- ellins. This volume. 489-501. 8. Seta, Y., Takahashi, N., Kitamura, H., and Sumiki, Y. Biochemical studies on "Bakanae" fungus. Part 45. Chemical structure of the gibberellins. Part XII. Bui. Agr. Chem. Soc. Japan. 22: 429-431. 1958. 9. Sumiki, Y., and Kawarada, A. Relation between chemical structure and physi- ological activity. This volume. 503-504. 10. Takahashi, N., Seta, Y., Kitamura, H., and Sumiki, Y. Biochemical studies on "Bakanae" fungus. Part 46. Chemical structure of gibberellins. Part XIII. Bui. Agr. Chem. Soc. Japan. 22: 432, 433. 1958. 11. West, C. A., and Phinney, B. O. Gibberellins from flowering plants. I. Isola- tion and properties of a gibbereliin from Phaseolus vulgaris L. Jour. Amer. Chem. Soc. 81: 2424-2427. 1959. DISCUSSION Dr. Galston: I don't need to remind you that I am not a chemist, but I had understood from previous reports that when the lactone ring was broken, the evidence indicated that the position of two hy- droxyl groups was ortho to each other on the A-ring. This new struc- ture proposed by the I.C.I, group would not permit this. Is that correct? Dr. West: It is correct that when you hydrolyze gibberellic acid with base to open the lactone ring, the resulting product reduces periodate. Both the British and Japanese groups found this and it was one of the reasons why the British group suggested the structure given. However, the new position is this: when you hydrolyze the 482 C. A. West lactone ring in the newly proposed structure, you obtain an alco- holic group allylic to a double bond. Such alcoholic groups migrate extremely easily and by such a rearrangement in this case you obtain a product with adjacent alcoholic hydroxyl groups which can reduce periodate as observed. (The Japanese had suggested that some rear- rangement does occur during basic hydrolysis.) So there is a good chemical explanation consistent with the periodate data. Dr. Crosby: Actually, I have only one comment to make. I think that those who are not familiar with complicated chemical structures ought to be reminded that this picture that we draw of gibberellic acid actually bears no resemblance to the way that gibberellic acid looks in space. Work is going on in a number of laboratories now to try to determine more closely how this material actually does look. I'm afraid that many of us may be misled if we continue to think of the chemistry of gibberellic acid and the other gibberellins in terms of symmetrical drawings that we can make on a blackboard or on a piece of paper, when actually the configurations are not like that at all. My point is, of course, that when we wish to relate chemistry to growth promoting activity, any previous ideas of these correlations which we have obtained with simple planar structures that we could write on a blackboard may not be adequate for spatially-complex sub- stances such as gibberellic acid. YUSUKE SUMIKI and AKIRA KAWARADA University of Tokyo Occurrence of G 'LbbereUin. Ai in the Water Sprouts of Citrus In 1951, the occurrence of a new phytohormone which greatly stimu- lated stem elongation in immature bean seeds was reported by Mitchell, Skaggs, and Anderson (3). It was the first description of the occurrence of a gibberellin-like substance in higher plants. West and Phinney (8) reported the occurrence of gibberellin-like substances from species of several different families of flowering plants. Since then a number of reports (4, 5, 6, 7) related to gibberellins or gib- berellin-like substances have been published. Last year, MacMillan and Suter (2) obtained gibberellin A^ from the immature seeds of runner bean, Phaseolus multiflorus, which was the first successful isolation of one of the known gibberellins. In our laboratory, the constituents of water sprouts of mandarin orange were examined, and gibberellin A^ was obtained in pure crys- talline form. The material used in this experiment was a bud variation of Citrus imshiu, first found by K. Furusato (1) in Shizuoka Prefecture in 1949. In spring, many long twigs sprout from its apex like a witches'-broom of the cherry tree. The leaves are lighter green and smaller than those of the normal branches and flowering is not initiated. In this paper, the procedure of isolation and identification of gibberellin A^ from the elongated water sprouts is described. EXPERIMENTAL The elongated water sprouts (ca. 1.8 kg.), harvested in November, 1957, were divided into leaves (0.6 kg.) and shoots (1.14 kg.), and the latter portion was cut, ground by the blender, immersed in 1.5 1. of 50 per cent aqueous acetone, and extracted overnight at room tempera- ture. After the extraction was repeated, the eluates were combined [ 483 ] 484 Y. Sumiki and A. Kaiuarada and evaporated to a small volume under reduced pressure, and a dense greenish liquid (pH 5.4) was obtained. It was adjusted to pH 8.0 with sodium bicarbonate, extracted five times with ethyl acetate (total volume 1.5 1.) to remove the nonacidic substances. The aqueous layer was then acidified to pH 3.0 with dilute sulfuric acid, and again extracted five times with ethyl acetate (total volume 1.5 1.). Upon evaporation of the solvent, a dark greenish syrup was obtained. This did not show any physiological activity in the gibberellin bioassay. For the purification of gibberellin-like factor from this crude acidic substance, the countercurrent distribution method was applied [14 plates, ethyl acetate: \M phosphate buffer (pH 5.2) ]. After develop- ment, the buffer layer of each plate was acidified to pH 2.0 with sul- furic acid, extracted three times with an equal volume of ethyl acetate added to the upper layer, and dried with anhydrous sodium sulfate. The results of this method and subsequent bioassay indicated that the peak was near plate 8. After the fractions of plates 6 to 10 were combined and counter- current distribution was repeated, the small portion of plate 8 was spotted on Whatman No. 1 filter paper and developed by ascending method with the solvent system of isopropanol-28 per cent ammonia- water (10:1:1). At the same time, samples of gibberellins A^ and A3 were subjected to the same procedure. When the solvent front as- cended 24 cm., the paper was dried, divided into twelve sections, and assayed. The physiologically active zone from the water sprouts co- incided closely with that of gibberellin A^ or A3; the latter were detected also by spraying bromocresol green indicator. Another collection (7.2 kg.) of water sprouts, harvested in Oc- tober, 1958, was treated with the same procedure, i.e., aqueous ace- tone extraction, ethyl acetate extracion, and two countercurrent distributions. The active fraction was poured onto a column of cellu- lose powder (3 X 32 cm., Whatman No. 1), and eluted with the sol- vent system of isopropanol-28 per cent ammonia-water (10:1:1) at the flow rate of 1 drop per 2 sec. Ten ml. fractions of eluates Avere col- lected and the fractions 4 to 7 were combined and yielded 98 mg. of a colorless oily substance. The material was then spotted on sheets of Whatman No. 1 filter paper (four sheets, 8 X 40 cm.) and developed with the solvent system described above. The appropriate areas, de- tected by guide spots of authentic specimen on both sides of paper sheets, were cut out and eluted with methanol (5 ml. per area). Then, in order to change the above ammonium salt solution to free acid, the eluate was passed through a short column (0.8 X 5 cm.) of Amberlite IR 120 (H^ form). On evaporating the solvent under reduced pressure, 12 mg. of the colorless amorphous powder was ob- 4 6 8 10 WAVE LENGTH, MICRONS Fig. 1. Infrared spectra of the crystals from Citrus unshiu and of the known gibberellins. 100 50 Y n If 1 n 1 Ai - me lOOr 50 ' A4-me 6 8 10 12 WAVE LENGTH, MICRONS 14 Fig. 2. Infrared spectra of the methyl esters of the crystals from Citrus unsliiu and of tiie known gibberellins. Gibberellin Aj in Water Sprouts of Citrus 487 tained which, on crystallization from ethanol-ethyl acetate-ligroin, gave about 2 mg. of colorless prisms. These crystals melted at 230° to 236° C. (decomposition) on a Koffler block, and then infrared spectrum (Nujol) coincided with that of gibberellin A^ (Figure 1). The infrared spectra of the methyl esters of these compounds again dem- onstrated the identity of the Citrus gibberellin with gibberellin Aj (Figure 2). SUMMARY Gibberellin A^ was isolated from the elongated water sprouts of a bud variation of Citrus unshiu. Its identity was established from in- frared spectra and physiological properties. LITERATURE CITED 1. Furusato, K. A new type of bud variation in Citrus. Ann. Rep. Nat. Inst. Genet. Japan. 8: 48,49. 1957. 2. MacMillan, J., and Suter, P. J. The occurrence of gibberellin Ai in higher plants: isolation from the seed of runner bean (Phaseolus multiflorus). Natur- wis. 45: 46. 1958. 3. Mitchell, J. W., Skaggs, D. P., and Anderson, W. P. Plant growth-stimulating hormones in immature bean seeds. Science. 114: 159-161. 1951. 4. Murakami, Y. A paper chromatographic survey of gibberellins and auxins in immature seeds of leguminous plants. Bot. Mag. Tokyo. 72. 36-43. 1959. 5. Radley, M. Occurrence of substances similar to gibberellic acid in higher plants. Nature. 178: 1070, 1071. 1956. 6. , and Dear, E. Occurrence of gibberellin-like substances in the coconut. Nature. 182: 1098. 1958. 7. Simpson, G. M. A colorimetric test for gibberellic acid and evidence from a dwarf pea assay for the occurrence of a gibberellin-like substance in wheat seedlings. Nature. 182: 528, 529. 1958. 8. West, C. A., and Phinney, B. O. Purification and properties of gibberellin-like substances from flowering plants. Plant Physiol. 32 (suppl.): xxxii. 1957. BERNARD O. PHINNEY University of California, Los Angeles Dwarfing Genes in Zea mays and Their Relation to the GibberelUns'' Evidence has accumulated in the field of biochemical genetics to sup- port the hypothesis that the gene acts as a physiological unit through the control of a single chemical reaction (4, 6). Thus, a mutant gene may determine a particular phenotype by interfering with a specific step in a sequence of chemical reactions leading to a particular product. This product may be any one of a number of substances necessary for normal growth. The accumulated evidence also suggests that nonallelic mutant genes concerned with the same growth sub- stance control different steps in the reaction sequence leading to this substance. In Zea mays L. there are more than twenty mutant genes^, each of which results in the dwarf habit of growth (1,2,3). The non- allelism is well established for all but one of the ten dwarfing genes used in the studies reported here; nine of them are simple recessives, one is a simple dominant. The knowledge of the precise genetical basis for the dwarf habit of growth allows for the interesting speculation that the normal allele of each dwarfing gene in some way controls the presence, or at least the availability, of a substance necessary for normal growth. Studies on the physiology and biochemistry of these single gene mutants could lead to specific information on the bio- chemical mechanisms controlling growth. It is the purpose of this ^ Certain aspects of the studies reported here were supported in part by grants from The University of California Reseach Committee, National Science Founda- tion (G-3526), and Merck & Co., Inc. ^The names and abbreviations for the nonallelic dwarf mutants of Zea mays used in this paper are: dwarf 1 (d,); dwarf 2 (d.); dwarf 3 (d,); dwarf 5 (d^); an- ther ear 1 (an,); nana 1 (na,); nana 2 (na,); petite 1 (tiny 4963) (pe,); and midget 2 (midget 8043) (mL). The linkage relationship of the mutant, dwarf 8 (domi- nant dwarf) (ds) , is as yet unknown. [489] 490 B. O. Phinney paper to evaluate certain evidence which leads to an interpretation of the physiological action of the dwarfing genes d^, d^, d.^, dr^, and an I of Zea mays. Both auxins and gibberellins-' have been studied in their relation to the growth of several of the dwarf mutants of Zea mays. AVhile the auxin level from certain of the dwarfs can be shown to be less than that from normals, these mutants, as well as other dwarfs of Zea mays, exhibit no growth response to added indole-auxins such as lAA, lAEE, IAN, IBA, or to the auxin NAA, or to kinetin and numerous individual amino acids (5, 7, 9, 14, 15). In contrast, the five mutants f/j, ^2' ^3» ^5» ^nd rt/ij respond by normal growth to microgram amounts of the gibberellin GA3 (8, 9, 13) (Figures 1 and 2). It has been suggested that these GAg-responding mutants might be controlling different steps in a biochemical pathway leading to the production of a naturally occurring gibberellin which is similar to GA3 and necessary for the normal growth of Zea mays (10, 11). This native gib- berellin would then be limiting in the mutants and its absence or presence in limiting amounts responsible for the d^varf habit of growth. Five other mutants, Jia^, na^, dg, pe^, and m,i2, give no growth response, or only a slight growth response in the early seedling stages, to added GA.^. It has been suggested that these five nonre- sponders could have blocks in a pathway stibsequent to a CrAg-like compound; or they could be due to blocks in a biochemical pathway or pathways unrelated to the gibberellins (10, 11). If the interpretation of the GAg-responding mutants is correct, it is possible to make certain predictions and subject them to experi- mental tests. Some of the predictions that will be considered are as follows: (1) It should be j)ossible to find gibberellins producing differen- tial growth responses for the five GAg-responding mutants. Compounds from different steps in the presumed gibberellin pathway should either be active or inactive for a particular mutant, depending on the position of the mutant block in this pathway. Thus, there should be some gibberellins that produce a normal growth response to four of the five GAg-responding mutants; others that produce a growth re- ^ Auxin will refer lo any native growth regulator found to be active in the Ax)ena curvature test or the Avena straight growth test. The term gibberellin is used for substances active in the d^, d.,, d^, d^„ or aii^ bioassay of Zea mays. As used here, the term is further restrictecl to substances found to contain a fluorene ring system. The term gibbryrlUn-lilie is used for substances ha\ing i)iological proper- ties similar to the gibberellins but for which the chemical properties are unknown. The abbreviations used for certain growth factors considered in this paper are: lAA, indole-3-acetic acid; IAN, indolc-3-acetonitrile; lAEE, ethvl indole-3-ace- tate; NAA, 2-naphthoxyacetic acid: IRA, 4-(indole-.'?-)-)/-butyric acid: GA„ GAo, GA,, and GA^, gibberellin A,, A.„ A.„ and A,: and BF-II, the gibberellin, bean fac- tor II. ANTHER DWARF 1 DWARF- 2 DWaRF-3 DWARF-5 DWaRF 8 .AR~l Fig. 1. Growth response of the mutants, anther ear 1, dwarf 1, dwarf 2, dwarf 3, dwarf 5, and dwarf 8, to gibberelHc acid; treated mutants above, controls below. Dominant dwarf is a nonresponder; the other five nonallelic mutants are GA3 responders. GA3 was added in aqueous solution to the uppermost unfolding leaves at two- to three-day intervals from the earliest seedling stage to maturity; dosages varied from 1 to 10 ^g. per treatment; total amount applied was 300^g. 492 B. (). Phinney gibberelllc acld(0.lwg.) dwarf-2 Fig. 2. Response of the first leaf sheath of tlie mutant, dwarf 2, to a single ap- plication of gibberellic acid. Plants photographed 5 days following treatment. sponse to three of the five; still others that produce a growth re- sponse to two of the five, etc. In this way the order of the dwarfing genes in blocking intermediate steps in the gibberellin j)athway would be established. (2) Any gibberellin found to be active in promoting the growth of a dwarf mutant should produce less growth when applied to normal plants, if native gibbcrellins are the limiting factor distin- guishing the two types of growth. Also, gibbcrellins producing a dif- ferential response for a mutant should not produce a differential re- sponse when applied to normal plants. This would be expected since the normal form of each dwarfing gene would presumably be able to carry out its function of converting a particular intermediate to the next step leading to a final gibberellin product necessary for growtli. (3) Gibberellins should be present in normal plants and absent, or present in reduced amounts, in the dwarf seedlings. However, the Dioarfing Genes in Zea mays and Relation to Gibberellins 493 total amount of gibberellins in normal seedlings may be difficult to evaluate if the active substances represent a mixture of different kinds of gibberellins, some of which are intermediates in the pathway con- trolled by the normal forms of the dwarfing genes. In this event, the choice of the mutant used for bioassay becomes critical. It is also pos- sible that the dwarf mutants may be accumulating gibberellin inter- mediates which would be inactive when assayed on the accumulating mutant, yet active on one or more of the other GA^-responding mu- tants. The dwarf controlled by the mutant gene farthest back in the presumptive pathway would be the best choice for determining total amount of gibberellins. In the final analysis, quantitative crossfeed- ing studies with all five GAg-responding mutants are necessary for the proper evaluation of amounts and kinds of native gibberellins from normal and mutant seedlings. Experimental tests of the above predictions require the use of a specific and quantitative assay for the detection of gibberellins and gibberellin-like substances, and for the estimation of their relative activities. The dwarf mutants of Zea mays have been used for such a purpose because of their specificity, sensitivity, and rapidity of re- sponse to the gibberellins (10, 11). For quantitative studies, standard procedures are used for growing, treating, and measuring the growth of the assay plants. Ten mutant seedlings are used for each dosage level of a particular compound or preparation to be tested for ac- tivity. Mutant seedlings are treated by placing 0.1 ml. of the material to be assayed into the first unfolding leaf as it emerges from the cole- optile. The test plants are grown at temperatures ranging from 25° C. to 35° C. for a period ranging from seven to ten days. The length of the first leaf sheath or the sum of the lengths of the first and second leaf sheaths is used as a measure of response. Dosage-response curves for GA3 have been found to be linear over the range of 0.001 ^g/plant to 1.0 ^g/ plant when the logarithm of the response is plotted against log dosage (Figure 3). The bioassay is used for quantitative studies only when the curves for the standard and unknown(s) are straight and parallel to each other. Estimations of relative activities are made from graphic analyses of the response curves. The statistical signifi- cance of differences in relative activities is determined from analyses of variance of the original response data. Differences in activity of 20 per cent can be shown to be statistically significant at the 5 per cent level. For the qualitative use of the bioassay, single plants are treated repeatedly with the unknown; growth responses 25 per cent over the largest dwarf control are considered as evidence for activity. All unknowns are run in triplicate. The variables of light, photo- period, and temperature are minimized by running the standard and 494 B. O. Phinney < UJ X < UJ X o z UJ o O 0.001 0.01 0.1 I GIBBERELLIC ACID, ^G./PLANT Fig. 3. Dosage response curve for gibberellic acid, using the mutant, dwarf 1, for bioassay. Measurements are for length of the first leaf sheath only. Each point represents the mean of 10 measurements. When tlie log of lengths of both the first and second sheaths is used, the dosage-response curve is linear over the range of 0.001 to 10.0 Mg- per plant. Data from Neely (7). unknown(s) simultaneously as a single bioassay. Surfactant effects have been found to be an important variable. Introduction of a wet- ting agent or organic solvent into the solvent solution (water) will increase the sensitivity of the bioassay fifty-fold (Figures 4 and 5). Be- cause of this variable, all solutions to be assayed contain the wetting agent, Tween-20, at the concentration level of 0.05 per cent by volume. Organic solvents, as well as the wetting agent, Tween-20, have been found to give no growth response by themselves. EXPERIMENTAL RESULTS Relative Activities of the Gibbercllins for Dwarf Mutants of Zea mays The three gibbercllins GAj, GAo, and GA^ have been shown to be active in promoting shoot growth for the mutants d^, d.-,, r/^, d-^, and aui (9, 13). The relative activities of these three gibberellins and the gibberellin, BF-II, have been determined for the GAg-responding mu- tants (Table 1). Each gibberellin was applied to sets of ten mutant seedlings at three dosage levels for the mutants d-^, do, d^, and dr, and 60 < UJ 40 I < UJ X I- e> z UJ 20 - - - o o CVJ o OJ c c Q) (U o O c c c c in c c o o o o o o o o GA^ > GA2 for all five mutants. Preliminary experiments suggest that GA4 is intermediate in activity between GA3 and GA^. In contrast to the similar order of activities of these gibberellins for the GAg-responding mutants, bean factor II is 5 per cent as active as GA3 for the mutant di (Figure 6); it is as active or more active than GA3 for the mutants ^2, d^, d^, and an-^. At dosage levels of 0.1 /xg/plant, BF-II produces no growth response for the mutant d-^, and a normal type growth re- sponse for the other four GA3-responding mutants. Relative Activities of the Gibberellins for Normal Zea mays The relative activities of the gibberellins GA^, GAo, GA3, and BF-II have been determined using normal Zea mays seedlings for bio- assay. The seedlings were treated and measured in the same manner as described for the quantitative use of the dwarf bioassay. GA3 and BF-II were applied at three dosage levels, GAj and GAo at only one dosage level. The activities of GA3 and BF-II were found to be very similar to each other (Figure 6). Comparison of dosage-response curves suggests that the order of one hundred times as much GA3 is necessary for an initial growth response for normal seedlings as com- pared to the GAg-responding dwarf seedlings. Gibbcrellin A^ and GAo were also found to produce a smaller growth response for normal seedlings than for the dwarf mutants. The order of activity for normal seedlings of Zea mays appears to be GA3 = BF-II > GA^ > GA2. Gibberellin-Like Su])stances From Zea mays Gibberellin-like substances have previously been reported from immature normal kernels of Zea mays using the mutant d^ for bio- Dwarfing Genes in Zea mays and Relation to Gibberellins 497 2.4 S S tn I I- < 2.2 UJ X (/) < UJ T3 c CM 2.0 o z UJ o o o Normal ^ y^ ^ 1.6 I ^ L 0 0.01 0.1 I 10 DOSAGE, /xG Fig. 6. Quantitative bioassays for gibberellic acid and bean factor II using nor- mal and dwarf I seedlings. Each point represents the average of ten measurements of the sums of the first and second leaf sheaths. assay (13). Experiments reported here with the mutant dg for bio- assay show the presence of gibberellin-like substances from seedling shoots of normal Zea mays, their absence, or presence in reduced amounts, from the mutants d^, do, dg, d^, and an-,^. Both normal and mutant seedlings were grown in the greenhouse for periods ranging from 2 to 6 weeks. Five hundred gram samples of the shoots were harvested and stored at — 20° C. Acetone-water extracts were obtained from duplicate samples of each type of ma- terial and purified according to methods developed by West (16, 17). Silicic acid chromatography (16) was used as the final step of purifica- tion for one sample, paper chromatography was used as the final step of purification for the second sample. Each fraction obtained from elution of the columns and from elution of the chromatograms was assayed for the presence of gibberellin-like substances. Evidence for activity was based on the qualitative use of the dr^ bioassay. As a measure of the total response from any one extract, the response data for all active fractions from any one extraction were added together. 498 B. O. Phinney The sums obtained from different extractions were then compared with each other as a crude estimate of relative activities. Both methods of chromatography revealed two zones of gibberellin- like activity from extracts of normal seedlings. Activity from silicic acid chromatography appeared following elution with 20 per cent ethyl acetate in chloroform, and again following elution with 60 per cent ethyl acetate in chloroform; likewise two zones of activity were found by paper chromatography, these being at Rf values of 0.15 and 0.43. Gibberellin-like activity was obtained from as little as 100 grams of shoot tissue, fresh weight. Gibberellin A3 controls showed activity from silicic acid chromatography in the fraction following elution with 20 per cent ethyl acetate in chloroform, and at an Rf value of 0.41 from paper chromatography. No gibberellin-like activity was obtained from extracts of the mu- tants d^, dr^, and an-^ either by paper chromatography or by silicic acid chromatography. Additional extractions from 1 kg. samples failed to reveal any evidence of activity. Extracts from the mutants rfi and ^2' however, showed evidence of the presence of gibberellin-like sub- stances from silicic acid chromatography and from paper chromato- graphy. Two regions of activity were present which had positions in- distinguisiiable from those obtained from normal seedlings. Compar- isons of the total responses from the active fractions of c^^, d.^, and normal material suggest that these mutants contain less than half as much total gibberellin as do the normals. The responses were lower whether compared on a fresh weight, dry weight, or per plant basis. Cross-Feeding Studies Only preliminary studies have been made to test for the accumu- lation of unicjue gibberellin-like substances by the dwarf mutants of Zea mays. Qualitative bioassays show the gibberellin-like substances from the mutants <;/, and ^2 ^^d from normal seedlings to be rfi in- active and d^, dr^, and arzi active. All fractions obtained from extracts of the mutants d^, dr,, and an^ have been inactive when tested on the five GAg-responding mutants. While the over-all activity obtained from f/j and c?2 mutants is appreciably less than from normal plants, regardless of the mutant used for bioassay, it should be emphasized that more careful purification followed by the quantitative evalua- tion of each active fraction is necessary for a proper evaluation of the cross-feeding studies. The fractions obtained from extracts of normal seedlings and from the dwarf mutants have not been tested for activity on normal seedlings. DISCUSSION AND CONCLUSIONS Evidence is presented in this paper to support the interpretation that the five mutant genes rf,, dc,, d^, d^, and a/?, arc responsible for Dwarfing Genes in Zea mays and Relation to Gibberellins 499 the dwarf habit of growth through the control of the amount of native gibberellins in Zea mays. It is suggested that these genes in- terfere with different steps in a gibberellin pathway leading to a product necessary for the normal growth of Zea mays. The normal type growth response of the five mutants to the gib- berellins, combined with their lack of response to other known plant growth regulators, implicates gibberellin as the limiting factor re- sponsible for the dwarf habit of growth. The correlation of presence of gibberellin-like substances in normal plants, and the lowered amounts or absence in the mutants are further evidence for a causal relation between gibberellin and the dwarf habit of growth. The rela- tively small response to added gibberellin found for normal plants would be expected if native gibberellins were less limiting in normal plants than in the dwarf mutants. The response of all five mutants to gibberellic acid suggests that GA3 or a compound very similar to it occupies a position in a meta- bolic pathway in Zea mays subsequent to the steps controlled by the five dwarfing genes. While the gibberellins GAi, GA2, and GA3 ex- hibit the order of activity of GA3 > GA^ > GAo, this order is the same for all five mutants and for normal plants. Such similarities in relative activities would exclude any interrelationship between these gibberellins controlled by the five dwarfing genes. The similarity in the relatively high activities of GA3 and BF-II on the four mutants do, ^3, d^, and an^, contrasted with the high activity of GA3 and the low activity of BF-II for the mutant d^, suggests a relationship between these two gibberellins controlled by the d-^ gene; bean factor II would be an intermediate the conversion of which to gibberellic acid is blocked by the d-^ gene. The other four genes could then control steps in the gibberellin pathway prior to compounds having properties similar to GA3 and BF-II. Further support of this interpretation is given by the lack of a differential response of normal seedlings to GA3 and BF-II. The difference in auxin level between normal and dwarf seedlings of Zea mays can be attributed to an indirect effect of the dwarfing genes. A lower auxin level would then be the result of the reduced amounts of native gibberellins in the mutants. The lack of response of the dwarf mutants of Zea mays to a number of auxins is in agree- ment with this interpretation. The accumulation of inhibitors of gibberellin-induced growth could be an obvious alternate explanation for the dwarf habit of growth in Zea mays. Added gibberellin would then be expected to overcome this inhibition effect, resulting in a normal growth response for the GAg-responding mutants. Such an explanation would require a series of inhibitors, one specific for gibberellic acid, another specific 500 B. O. Phinncy for bean factor II, etc. As yet there is no evidence to suggest such an alternate explanation. The evidence presented in this paper suggests that the primary action of the mutant genes c/^, dc,, d-^, dr^, and an^ is to control the amount of native gibberellins in Zca mays by interfering with different steps in a biochemical pathway leading to a gibberellin product nec- essary for normal growth. The present evidence would suggest that the gene d^ is controlling a terminal step in this series. The lower auxin level found in certain dwarf mutants of Zea mays is attributed to an indirect effect of the dwarfing genes. LITERATURE CITED 1. Anderson, E., and Pettem, F. D. Dwarfs of Zea mays. Corn Co-op News Let- ter. 30: 9, 10. 1956. 2. , and Pettem, F. D. Dwarfs of Zea viays. Corn Co-op News Letter. 31: 29, 1957. 3. Emerson, R. A., Beadle, G. \V., and Eraser, A. C. A summary of linkage stud- ies in maize. Cornell E\per. Sta. Mem. 180: 83 pp. 1935. 4. Finsham, J. R. S. The role of chromosomal loci in enzyme formation. Proc. 10th Int. Cong. Genet. 1: 355-363. 1958. 5. Harris, R. Auxin relations in a dicarf-l allele of 7.ea mays L. Ph.D. Thesis, Univ. Calif. Los Angeles. 6 Horowitz, N. H. Biochemical genetics of Neurospnra. Ad\. Genet. 3: 33-71. 1950. 7. Neely, P. M. The development and use of a bioassay for gibberellins. I'liD. Thesis, Univ. Calif. Los Angeles. 1959. 8. , and Phinney, B. O. The use of the mutant dicorj-l of maize as a quantitative bioassay for gibberellin activity. Plant Physiol. 32 (sujjpl.): xxxi. 1957. 9 Piiinney, B. O. Growth response of single-gene dwarf mutants in maize to gibberellic acid. Proc. Natl. Acad. Sci. U. S. 42: 185-189. 1956. 10. , and West, C. A. The growth response of single gene dwarf nuitants of Z,ca mays to gibberellins and to gibbcrellin-likc substances. Proc. Int. Genet. Symp. pp. 384, 385. 1957. 11. , West, C. A., and Neely, P. M. Single gene dwarf mutants of Zea mays and their dilfcrcntial growth response to gibberellins and to gibberellin-likc substances. C^orn Co-op News Letter. 32: 6, 7. 1958. 12. , West, C. A., and Neely, P. M. Biological properties ot the gibberellin, bean factor II. (In preparation.) 13. , West, C. A., Ritzel, M., and Neely, P. M. Evidence for "gibberellin- like" substances from flowering plants. Proc. Natl. Acad. Sci. U. S. 43: 398- 404. 1957. 14. van Overbeek, J. The growth hormone and the dwarf ivpe ot growth in lorn. Proc. Natl. Acad. Sci. U. S. 21: 292-299. 1935. 15. . Auxin production in seedlings of dwarf mai/c. Plant Plivsiol. 13: ,">87- 598. 1938. Dxoarfing Genes in Ze2L mays and Relation to Gibberellins 501 16. West, C. A. The chemistry of gibberelHns from flowerhig plants. This volume. 473-482. 17. ^ and Phinney, B. O. Gibberellins from flowering plants. I. Isolation and properties of a gibberellin from Phaseolus vulgaris L. Jour. Amer. Chem. Soc. 81: 2424-2427. 1959. DISCUSSION Dr. Barton: Dr. Phinney has mentioned that genetic dwarfs are controlled by genes, as of course they are, but we have found that physiologic dwarfs can also be stimulated to elongate by the use of gibberellic acid. These physiologic dwarfs are those of peach, apple, crabapple, etc., dwarfed because the seeds were not given low tem- perature pre-treatment. Is this also controlled by genes? Dr. Phinney: It cannot be said that a particular physiological property is controlled by a single gene until the inheritance pattern is known for this property. Dr. Galston: I would like to ask a question based on the point that Dr. Barton raised, i.e., the whole problem of whether any of the overgrowths noted in pathology or in commensalisms can be explained in terms of abnormal production of GA or its analogs. Several years ago we found that when we applied GA to some dwarf bean plants, the number of nodules on the roots was depressed. This has been confirmed in various ways by Dr. Brian and by Dr. Kefford, the latter using sterile culture. Now, I wonder whether there is any possibility that nodule formation may be in some way related to the fact that the plant has a sub-optimal level of gibberellin, and that the bac- terium causes a localized production of something like gibberellin, which gives to the cells in that region a selective growth advantage. The application of gibberellin to such a plant could remove that se- lective advantage, thus repressing nodule growth. My question should really be addressed either to Dr. Stodola or to Dr. Brian, who have worked with microbial fermentations. Is there any evidence that any organisms related to the nodule-forming forms produce anything like gibberellin, either in culture or in contact with plant cells? Dr. Brian: There is not to my knowledge. So far as I know, there's just one Gibberella fiijikuroi. There is a recent Russian claim of a yeast which produces GA. So far as I know, these are the only two microorganisms ever suspected of producing gibberellins. YUSUKE SUMIKI and AKIRA KAWARADA University of Tokyo Relation Between Chemical Structure and Physiological Activity For the purpose of elucidating the growth regulating activity of gib- berellins, some 20 derivatives or degradation products were prepared and their physiological activity observed using rice seedlings. Gibberellins have recently come to be recognized as important in the growth regulating system of higher plants, and the chemical con- stitutions of gibberellins are almost established (1,2,3,4,5) though much remains to be elucidated about their stereochemistry. The au- thors believe it is of considerable interest to study the relation be- tween the chemical constitution and physiological activity of gibber- ellins. Moreover, the authors expect that the determination of the par- tial structures essential for showing growth response will be a guide for the synthesis of new gibberellin-like substances having more simplified structures than the native gibberellins. The compounds, i.e., the four gibberellins and their derivatives or degradation products, were examined as to their purity by paper chromatography. The physiological activity was observed by measur- ing the length of the second leaf-sheath of rice seedlings on incuba- tion at a concentration of 10 /xg/ml as described in a previous paper. The results are illustrated in Figure 1. The values indicate the relative activity of each compound when that of gibberellin A3 is assumed to be 100. Data in Figure 1 may be summarized as follows: (1) On A-ring — S-lactone ring and secondary hydroxyl group are es- sential, and by the inversion of stereochemical configuration of hy- droxyl activity is lost. On B-ring — physiological activity is much re- duced where the carboxyl group is masked by methylation. On C- and D-rings — (a) exocyclic methylene, or double bond, is not essential [ 503 ] 504 Y. Suiniki tnid A. Kaxvarnda ReUtion Between Chemical Structure ana Physiol ogicdl Activity HO HO H CH, COOM )H (i sp activityi 0 rep, y^ CH, \ OH (^, S6 4) ° HO MO fCQ, COOH \H} A {Mttittr ± ) Hoor 3H (-f-. /OO 0) HO COOH CKj COOH =CH, CH COOH H5 ( — ) OH (-) J-CHj (fie titer ± ) no /\ HOt^C COOH (+, 7J / ) HO-l. "0^ ^^^i CHs />« ejff r ± ) CHiOH ■OH ( — ) =CH, HO x^ CHj COOH J-b'bb A, OH (-) tCH, CIVIH HO CHj ^CO HO CH3 ( + , 2S g) Ch5 COOM HO HO '^5, CH, •OH (+, ,ff,) COOH rc?, HO HO CHj ^^9, COOH to (-) -COOH CHj COOH a-.tb c iO tP, CHj •CHjBr (+, / / /; COOM iC ■CH, ( — ) COOH /JO- S.'»t Ai CH,Br( + , / C f) ' COOH COOH but attiviiy is decreased on catalytic hydrogenation or ozonolysis; (b) also, tertiary hydroxyl (bridge head) is not important; (c) by opening D-ring, the activity disappears. LITERATURE CITED 1. Kitamura, H.. Seta. V., Takaliashi, N., Kawarada. A., and Siimiki, Y. Biochemical studies on "Hakanae" fungus. Part 19. Chemical structure of gibhercUins. Part XIV. Bui. Agr. Chem. Soc. Japan. 23: 408-411. 1959. 2. Seta, Y., Takahashi, N., Kawarada, A., Kitamura, H., and Sumiki, Y. Biochemical studies on "Bakanac" fungus. Part 50. Chemical structure of gibbcrellins. Part XV. Bui. Agr. Chem. Soc. Japan. 23: 412-417. 1959. 3. , Takahashi, N., Kitamura, H., Takai. .\I., Tamura, S., and Sumiki, Y. Biochemical studies on "Bakanae" fungus. Part 52. Chemical structure of gib- bcrellins. Part XVII 1. Bui. Agr. Chem. Soc. Japan. 23: 499-509. 1959. 4. Takahashi, N., Kitamura, H., Kawarada. A., Seta. V., Takai, M., Tamura, S., and Sumiki, Y. Biochemical studies on "Bakanae" fungus. Part 34. (Isolation of gibbcrellins and their properties.) Bui. Agr. Chem. Soc. Japan. 19: 267-277. 1955. 5. , Seta, Y., Kitanuiia, H., and Sumiki, \. Bioduinical stuilics on '"Bakanae" fungus. Part 42. (A new gibberellin, gibberellin A,.) Bui. Agr. Chem. Soc. Japan. '21: 396-398. 1957. M. J. BUKOVAC and S. H. WITTWER Michigan State University' Biological Evaluation of GibbereUins A, A, A, and A, and Some of Their Derivatives Early difficulties experienced by American and British scientists in the production and isolation of gibberellin A,- as announced by Yabuta and Sumiki (20), eventually led to the characterization of several fungal gibberellins. Stodola et al. (14) announced the isolation of gibberellin A and a new gibberellin designated as gibberellin X. Concurrently, Curtis and Cross (7) isolated gibberellic acid, which was found (6) identical to Stodola's gibberellin X, but differed from the Japanese gibberellin A. The Japanese workers then re-examined their product and found a mixture of three gibberellins, which they termed gibberellins Aj, Ao, and A3 (17). Gibberellin A^ was identical to Stodola's A and gibberellin A3 to gibberellin X and gibberellic acid. Takahashi et al. (18) next reported the isolation of gibberellin A4. Henceforth, the four fungal gibberellins will be re- ferred to as gibberellins Aj, A2, A3, and A4. Extensive field and greenhouse experiments have now been con- ducted and the results summarized (1, 15, 16, 19) for a wide variety of plant and crop responses produced with commercial gibberellin preparations consisting largely of A3 or undetermined mixtures of Al and A3. Meanwhile, little attention has been devoted to the bio- logical effects of gibberellins Ao and A4. With the exception of some preliminary reports (3, 5, 9, 12), no critical studies of the effects of the four fungal gibberellins and their derivatives on diverse responses of higher plants have appeared. 'Journal article no. 2445 from the Michigan Agricultural Experiment Station, initially named gibberellin B and changed to gibberellin A (20, 21). [ 505 ] 506 M. J. Bukovac and S. H. IVittwer COMPARATIVE BIOLOGICAL ACTIVITIES OF GIBBERELLINS Ai, A2, A3, AND A4 Vegetative Extension Epicotyl elongation in beans (Phaseolus vulgaris, 'Blue Lake'). Bean seedlings were germinated in quartz sand, and transferred to aerated solution cultures containing a complete nutrient solution when the primary leaves were approximately 50 per cent expanded (4). After 24 hrs. 10 lA. of a 3 X lO-^, 3 X 10% or 3 X 10-^ M solution of gib- berellins Aj, A2, A3, or Aj (Table 1) were applied to the terminal bud (Figure 1) or to the upper surface near the base of one of the primary leaf blades. Epicotyl elongation was determined 48 or 96 hours following treatment. Application of gibberellin Aj or A3 to the leaf blades resulted in plants with significantly longer epicotyls than plants similarly treated with A2 or A4 (Figure 2 and Table 2). Length of epicotyls of plants treated with gibberellin Ao on the leaf blades did not differ signifi- cantly from the controls. All gibberellins when applied to the terminal bud resulted in plants with epicotyls that were significantly longer than those on the controls. Gibberellins A^, A3, and A4, however, were slightly more effective than Ao (Table 2). This same relationship held for all three concentrations of the various gibberellin solutions. Petiole elongation in celery (Apiurn graveolens, 'Utah 10-B'). Seedlings were started in sand, selected for uniformity of fresh weight and petiole length, and transferred to solution cultures at the three to four true leaf stage. Ten jxl. of a 3 X 10^ or 3 X 10^^ M solution of gibberellins A^, Ao, A3, or A4 were applied to the youngest unfolding leaf 96 hrs. after transfer of the plants to solution cultures. The length of the second outer petiole was recorded 14 days later. Treatment Tabic 1 . Characteristics of the gibberellins assayed. Gibberellin Empirical Formula Melting Point * (degrees C.) Optical Rotation (degrees) A, C19H24O6 232-35 [a] 28 + 42 . 3 D A2 CigH'jeOe 235-37 [a] 15+11.7 D A3 C19H22O6 232-35 [a] 20 + 92 . 0 D A,. . CigHojOs 222 [a] 20 - 20.8 D Decomposition. Fig. 1. Placement of solution on the vegetative apex for assay of gibberellin in the bean epicotyl elongation test. Table 2. Comparative biological activities of gibberellins Ai, Ai, A3, and A4 (ex- pressed as per cent of control). Assay Microliters Applied (3 X \Q-K\I) Ai A2 A3 A4 Least Significant Differences (P = .01) Epicotyl elongation in beans Leaf blade application Terminal bud application Petiole elongation in P* celery Stem elongation in cucumbers Acceleration of flower- ing in lettuce Heading type ('Great Lakes') . . . Leaf lettuce ('Grand Rapids'). Growth of tomato ovaries . . 10 10 10 10 30 30 10 40 * 340.0 480.0 219.6 153.8 77.6 84.8 179.0 14.0 455.8 120.0 370.0 160.9 117.5 93.2 96.2 183.0 0.0 402.1 390.0 500.0 217.4 132.9 72.7 80.3 166.0 38.0 481.1 170.0 450.0 187.0 279.1 95.6 96.2 179.0 1.0 465.3 70.0 70.8 17.4 11.5 4.3 3. a 37.0 Overcoming photo- induced dormancy in W'eigela Stimulation of lettuce seed germination . . . 3.4- 7.2 * Two ml. of a 100 p. p.m. solution per 6-cm. petri dish. 508 A/, y. Bukovac and S. H. Wittwer Fig. 2. Growtli ol l)eaii epicotyls following application of 10 ^1. of 3 x 10 ■ M solution of gibbcrellins Aj, A., A:i, or A4 to one of the primary leaf blades (top), or 10 the terminal biul (bottom). Left to right: control, Aj, A,, A3, and A4 (photo- graphed 96 hrs. after treatment). with 3 X 10--^ M of Aj, A3, or A4 produced plants with significantly longer petioles than nontreated controls. Plants treated with A, did not differ from the controls. Petiole elongation was equally stimu- lated by Aj and A.^ at 3 X 10 "' ^^> ^"<1 signifuantly more so than with Ao and A4 (Table 2). Stem elongation in cucumber (Cucutnis sativiis, 'Burpee Hybrid'). Cucumber seeds were germinated in vermiculite and transplanted into soil after the cotyledons had emerged. Twenty-four hours later, a ten ,A. aliquot of a 3 X 10 •"'. 3 X 10 *, or 3 X 10"^ M solution of gib- berellin A^, A2, A3, or A4 was aj)plied to the terminal bud. At all con- centrations, A4 was strikingly more effective than A,, Ao, or A.5 in stimulating stem elongation (Table 2). The comparative stem elonga- tion of the 'Burpee Hybrid' cucumber and 'Blue Lake" bean following application of various amoimts of gibbcrellins A., and .\j to the terminal bud is illustrated in Figure 3. V Fig. 3. Stem elongation of the 'Burpee Hybrid' cucumber and the 'Blue Lake' bean, 120 hrs. subsequent to terminal bud applications of gibberellins A3 (left) and A^ (right). Top to bottom: 10 ^1. per plant of 3 X 10"'. 3 X 10"'. 3 X 10= M, and water (control). 510 M. /. Bukovac and S. H. Wittwer Flowering in lettuce (Lactuca sativa, 'Great Lakes' and 'Grand Rapids'). Plants were grown in pot cultures in a greenhouse under the prevailing winter photoperiod (9 to 1 1 hrs.) and at a night tempera- ture of 18° C. After six to eight true leaves had developed, 3 X 10"^ M solutions of the various gibberellins were applied to the stern apices, and the treatment repeated after two and four weeks. Gibberellins Aj and Ag were considerably more active than Ag and A4, both in accelerating flowering (Table 2) and stimulating seed- stalk elongation (Figure 4). While no significant differences were noted 2 3 4 WEEKS AFTER 5 6 7 TREATMENT 8 Fig. 4. Rate of seedstalk elongation in 'Grand Rapids' leaf lettuce following appli- cation of gibberellins Aj, Ao, A,, and kt to the youngest unfolding leaf. (Three applications of 10 ^^1. of 3 x 10"^ ^ solutions at 2-week intervals.) Biological Evaluation of Gibberellins 511 between Ao and A^, gibberellin A3 was slightly more active than A^. All plants of both varieties treated with any of the gibberellins flow- ered in fewer days (Table 2) than the controls. 'Great Lakes' lettuce plants treated with A^ or A3 did not head and seedstalks commenced to elongate within ten days (Figure 5). Plants treated with Ao and A4 headed, as did the controls, and the seed- stalks, as with controls, later emerged through the heads. At the time of first visible flower primordia, seedstalk heights for plants treated with Ai, Ao, A3, and A4 were 41, 28, 84, and 29 cm., respectively, and 30 cm. for controls. The effects of different levels (two applications of 1, 10, 50, 100, and 200 /xg. per plant) of each of the gibberellins on flowering of let- tuce were next determined. Increasingly greater amounts of A^ or A3 resulted in progressively taller seedstalks, and hastened flowering in Fig. 5. Response of 'Great Lakes' head lettuce to treatment with gibberellins. Left to right: control, A^, A,, A3, and A,. Note the absence of heading in Ai and A3 treated plants. (Three applications of 10 fi\. of 3 X 10"' M solutions at 2-week intervals.) 512 M. ]. Bukovac and S. H. Wittwer both varieties. Increasingly greater amounts, however, of A^ or A4 did not accelerate flowering, but the repeated dosages of 100 and 200 fji^. per plant resulted in seedstalks that were slightly taller than the controls. 'Great Lakes' head lettuce plants treated with gibberellins Ao or A4, up to total dosages of 400 fx%. per plant subsequently pro- duced heads, as did the controls, and seedstalks later emerged through the heads. By contrast, as little as 20 /xg. of A^ or A3 resulted in im- mediate bolting, and head formation was bypassed. Parthenocarpic fruit groivtli of the tomato (Lycopersicon esculen- tum, 'Michigan-Ohio Hybrid'). Tomato ovaries, were emasculated ap- proximately 24 hrs. before anthesis, and treated with ten fx\. of a 3 X 10-5, 3 X 10'^> or 3 X 10"^ ^ solution of each gibberellin. As controls for comparison, ovaries were also treated with indole-3-acetic acid (lAA) and p-chlorophenoxyacetic acid (CIPA). Additional controls consisted of emasculated and non-pollinated, as well as pollinated ovaries. Three single plant replicates, each with 3 ovaries on the first flower cluster comprised a treatment. The diameter of the ovaries was measured after six clays. Ovaries treated with the 3 X 10-^ M concentration showed greater growth with gibberellins A^, A3, or A4 than with lAA or CIPA, but significantly less than the pollinated control. While gibberellin Ao was not active at 3 X 10"^ ^l' it was as effective as any of the gibberel- lins at 3 X 10"^ ^i- At the latter concentration all gibberellins pro- duced ovaries of approximately the same size as the pollinated con- trols and lAA, but smaller than those treated with CIPA (Table 2). Dormancy Dark-induced dormancy in seed of lettuce (Lactuca sativa, 'Grand Rapids'). 2 ml. of a 100 p.p.m. solution of gibberellins A|, Ao, A3, A4, or distilled water were added to 6-cm. petri dishes containing 50 seeds on Whatman No. 1 filter paper. Seeds were germinated in the dark at 26° C. for 96 hrs. All four gibberellins effectively replaced the light requirement for germination (Table 2). Photo-induced dormancy of Weigela ('Vanicek'). Dormancy ^vas induced in Weigela plants by exposure to a short (9 hr.) photoperiod. Ten ^1. (3 X 10"^ ^I) of gibberellins A^, Ao, A3, or A4 were then ap- plied to the dormant terminal buds at weekly intervals for four weeks. Dormancy was partially broken by gibberellins A, and A;, but not with A2 or A4 (Table 2). EVALUATION OF SEVERAL ESTERS OF GIBBERELLIN A3 A homologous series of 7i-alkyl esters of gibberellin A3 (13) was prepared with the appropriate alkyl iodide (10). Physiological activity Biological Evaluation of Gihhcrellins 513 Table 3. Characterization of several ?i-alkyl esters of gibberellin A3 and their effect on lettuce seed germination in the dark (13). Ester Control H (gibberellin A3 Methyl Ethyl n-Propyl w-Butyl . H-Amyl. . . f!-Hexyl «-Heptyl «-Octyl ?i-Nonyl w-Decyl Empirical Formula CigHosOe C20H24O6 C21H26O6 CaoHogOe C23H30O6 C24H32O6 C25H34O6 C2CH36O6 C27H38O6 C28H40O6 C29H42O6 Melting Point, Degrees Centigrade 232-35 202 155 138 145 165-66 188-89 181-82 157-58 131-32 102.5-108. Germination of Lettuce Seed, Per Cent 41.2* 80.6 63.5 78.4 68.9 83.7 79.1 56.8 57.2 48.0 46.7 40.0 L.S.D. at P .05: 11.7; L.S.D. at P .01: 16.4. was then assayed in terms of the promotion of lettuce seed germina- tion in the dark, stimulation of bean epicotyl elongation, and growth of tomato ovaries. Germination was significantly enhanced by the methyl, ethyl, n- propyl, ??-butyl, w-amyl, n-hexyl, and n-heptyl gibberellates at 3 X 10-5 TVf (Table 3). The ?z-octyl, n-nonyl, or n-decyl gibberellates were inactive. Germination from the methyl, ethyl, n-propyl, n-butyl, and n- amyl gibberellates equalled or approached the response from the free acid. By contrast, none of the esters promoted the elongation of bean epicotyls or, over a wide range of concentrations in lanolin (3 X 10"^ to 3 X 10^ M), the growth of tomato ovaries. In separate experiments, however, the butyl cellosolve ester of A3 (Merck & Co., Inc.) was found equally as effective as the free acid in promoting lettuce seed germination, tomato ovary growth, and elonga- tion of the bean epicotyl. COMPARATIVE ACTIVITY OF SEVERAL DERIVATIVES OF GIBBERELLINS Ai, A3, AND A4 Several derivatives of gibberellins Aj, A3, and A4, procured from Dr. Y. Sumiki, were next assayed for their promotion of epicotyl elon- gation in bean seedlings and growth of tomato ovaries. The relative activities of the respective derivatives as related to the parent compounds are given in Figures 6, 7, and 8. Dihydro-gib- berellin Aj (Figure 6, II) and the keto product of dihydro-gibberellin Ai (Figure 6, III) were slightly active. Compound IV, in which the COMPOUND BIOLOGICAL ASSAY HO BEAN EPICOTYL TOMATO OVARY 5\^ ELONGATION GROWTH 6 Active Active CH, ^ COOH " Sligtitly Active Slightly Active Not Active H CH, Sligtitly Active Slightly Active Not Active 0=1 Not Active Fig. 6. Relative aclivily of gibberellin A^ derivatives in elongation of tlic bean epicotyl and growth of tomato ovaries. Biolog-ical Evaluation of Gibberellins 515 HO COMPOUND BIOLOGICAL ASSAY BEAN EPICOTYL TOMATO OVARY ELONGATION GROWTH Active Active n ^^3 " COOH Slightly Sligtitiy Active Active CH^BR = 0 HOOC m 12 CHa Not Active Not Active Fig. 7. Relative activity of gibberellin A3 derivatives in elongation of the bean epicotyl and growth of tomato ovaries. lactone group on ring A was removed, and compound V, in which the lactone group was removed and the product decarboxylated, were not active. The Wagner-Meerwein derivative of A3 (Figure 7, II) was slightly active (less than 20 per cent of Ao), while products III and IV (fl//ogibberic acid) were not active. Dihydro-gibberellin A4 (Figure 8, II) was about 50 per cent as active as A4. When the D rmg 516 M. /. Bukovac and S. H. Wittwer COMPOUND BIOLOGICAL ASSAY ^•^3 ^ COOH BEAN EPICOTYL TOMATO OVARY 5\. ELONGATION GROWTH 6 Active Active n ^^3 " COOH Slightly Active Slightly Active IE ^"'H c^oH " COOH Not Active Not Active Fig. 8. Relative activity of gibberellin A, derivatives in elongation of llie bean cpicotyl and growth of tomato ovaries. was ruptured and a keto group substituted for the hydrogen (carbon 7) the compound was inactive (Figure 8, III). DISCUSSION The diversity of alterable responses following ajiplicaiion of the gibberellins and their derivatives to the intact plant presents unlim- ited possibilities for assessing comparative biological activity. Only a few representative areas of plant behavior — vegetative extension, (lowering, dormancy, and fruit growth — were selected for assay in these studies. As might be anticipated, the biological activities of the four fungal gibberellins and their derivatives varied with the assay, the site of treatment, and the quantity applied. AVitli stem elongation in the bean, terminal bud applications (10 Biological Evaluation of Gibherellins 517 /xl. of 3 X 10 '"^ ^l) oi' all gibherellins resulted in comparable activities with epicotyls significantly longer than nontreated controls. The first measurable response on bean epicotyl elongation was observed with ten /xl. of gibberellin A3 at 3 X ^^'^' M, for Aj and A4 at 3 X 10-"' M, and 3 X 10"* M for Ao. Gibherellins A^ and A3, however, applied to the leaf blade induced significantly greater growth than Ag or A.,. If the length of the epicotyl following application of each of the gibherellins to the terminal bud was divided by the length of the epicotyl follow- ing application to the leaf blade, the ratios for Aj and A3 approached unity (Table 2). Gibherellins Ao and A4, in contrast, were much more active when applied to the terminal bud than to the leaf blade, and growth from the former was 2 to 3 times greater. These data suggest possible limitations in the absorption and transport of A2 and A4 from the primary leaves, and that the rate of penetration and transport of exogenously applied gibherellins from the treatment site to the site of action may be an important consideration in assessing activity by a selected assay. The strikingly greater effect of gibberellin A4, in contrast to A^, Ao, or A3, on stem elongation of the cucumber is an intriguing de- parture from the usual response pattern. Such specificity among the gibherellins for any vegetative elongation response has not heretofore been recorded, and has not been duplicated in other bioassays em- ployed. The response of the cucumber to gibberellin A4 may be uti- lized as a bioassay for differentiation between naturally occurring A4 and Ai, Ao, and A3 in higher plants; and further serves as a guide for the study of the controlling mechanisms in flower sex expression of cucurbits (19). Quantitative as well as qualitative differences were recorded among the four gibherellins. Ao did not stimulate the growth of tomato ovaries at 3 X 10"^ M, but was highly effective at 3 X 10"^ ^^- In con- trast, relatively large quantities (400 ^g. per plant) of Ao and A4 failed to promote seedstalk elongation in 'Great Lakes' head lettuce, whereas 20 ixg. of either A^ or A3 per plant induced bolting without heading. The presence of gibberellin A^ in some higher plants (11, 12) may alter responses to exogenous applications of the fungal extracted gib- herellins. Marked specificity among the latter has already been noted for vegetative extension, the most commonly observed gibberellin ef- fect. As more species and responses are examined these relationships will undoubtedly be multiplied. Esterification of the carboxyl group of gibberellin A3 resulted in a complete loss of biological activity when elongation of bean epi- cotyls or growth of tomato ovaries constituted the assay. For promo- tion of germination of lettuce seed in the dark, however, the ethyl, 77-butyl, and ??-amyl esters equalled the activity of gibberellin A3, while 518 M. J. Bukovac and S. H. Wittiver the methyl, 77-propyl, «-hexyl, and ?i-heptyl esters showed a significant increase above the controls. The pronounced activity of some of the gibberellates in lettuce seed germination may have resulted from their hydrolysis in the germinating medium and/or on the seed surface. In this regard the butyl cellosolve ester of A3 was equally as active as gibberellin A3 in promoting the elongation of bean epicotyls, growth of tomato ovaries, or lettuce seed germination, whereas ??-butyl gib- berellate was inactive except in promoting the germination of lettuce seed. Solubility and penetrating properties may have been favorably altered by the side chain of the butyl cellosolve ester of A3. Other derivatives of gibberellins Aj, A3, and A4 Avere biologically active. Compared with the parent compounds, hoAvever, the activity was less and seldom exceeded 60 per cent. Activity disappeared when the lactone group of ring A was removed. Likewise, activity was lost following rupture of the D ring. Intact A and D rings and a reactive carboxyl group appear to be essential for biological activity. The re- port of Brian et al. (2) that aZ/ogibberic acid is not biologically active has been confirmed. The necessity of ring A for activity has also been confirmed in that gibberellenic acid is inactive (8). SUMMARY Gibberellins Aj, Ao, A3, and A4, the n-alkyl esters of A3, and some derivatives of A^, A3, and A4 were bioassayed utilizing several test systems with intact plants. In promotion of vegetative extension of bean epicotyl and in celery petiole elongation, A3 was most active, followed by Aj, A4, and A2. Reduced epicotyl elongation following leaf blade applications of Ao and A4, as compared with A^ or A3, suggested limitations in the transport of Ag and A4 in the bean plant. A marked deviation from the usual order of growth extension activity among the four gibberellins occurred in stem elongation of the cucumber. Gibberellin A4 was strikingly the most active followed by Aj, A3, and A2. Gibberellins A3 and Ai were more effective than Ao or A4 for ac- celerating flowering of both 'Great Lakes' head and 'Grand Rapids' leaf lettuce. Whereas gibberellin A3 was more active than Aj, there were no differences between Ao and A4. Increasing the dosages of Ao or A4 twenty-fold above Ai or A3 did not compensate for the markedly greater acceleration of flowering resulting from the latter. Gibberel- lins Al, A3, and A4 were more active than A2 in promoting the growth of tomato ovaries at low dosages (3 X 10 "* or 3 X 10'^ ^^)' while growth was comparable with all four gibberellins at 3 X ^^'^ ^^- ^^^ gibberellins stimulated germination of lettuce seed in the dark. Photo- induced dormancy of Weigcla was partially overcome by treatment with gil)berellins A, and A3, whereas Ao and A4 were inactive. Biological Evaluation of (Mbberellins 519 The ;7-alkyl esters (methyl to /?-decyl) of gibberellin A3 were not active in promoting growth of bean epicotyls or tomato ovaries. Let- tuce seed germination, however, was enhanced by the methyl, ethyl, n-propyl, ?2-butyl, n-amyl, 72-hexyl, and n-heptyl gibberellates. The presence or absence of biological activity of several other de- rivatives of gibberellins Aj, A3, and A4 showed that intact A and D rings and a reactive carboxyl group were essential. As of publication date, five additional gibberellins (A5, A„, A-, A,, and A,) have been isolated and characterized — A-„ Ae, and A, from immature seeds of the 'Scarlet Runner' bean and A.- and A9 froin the fungus GibbereUa fujikuroi. No data are available on their comparative biological activity. Also, a revised structure for A;, has been proposed in -which the lactone of ring A is attached to carbons 1 and 1 1 . (See Cross et al. Proc. Chem. Soc. p. 302. 1959.) LITERATURE CITED 1. Brian, P. W. Effects of gibberellins on plant growth and development. Biol. Rev. 34: 37-84. 1959. 2. , Grove, J. F., Hemming, H. G., MulhoUand, T. P. C., and Radley M. Gibberellic acid, part VI. The biological activity of a/Zogibberic acid and its identity with gibberellin B. Plant Physiol. 33: 329-333. 1958. 3. Bukovac, M. J., and Wittwer, S. H. Comparative biological effectiveness of the gibberellins. Nature. 181: 1484. 1958. 4. , Wittwer, S. H., and Gaur, B. K. Some factors influencing the response of the bean {Phaseolus vulgaris L.) to gibberellin. Mich. Agr. Exp. Sta. Quart. Bui. 41: 296-302. 1958. 5. Cathey, H. M. Growth evaluations of four gibberellins and several derivatives. Plant Physiol. 33 (suppl.): xliii. 1958. 6. Cross, B. E. Gibberellic acid. Part I. Jour. Chem. Soc. pp. 4670-1676. 1954. 7. Curtis, P. J., and Cross, B. E. Gibberellic acid. A new metabolite from the culture filtrates of GibbereUa fujikuroi. Chem. and Ind. (London) p. 1066. 1954. 8. Gerzon, K., Bird, H. L., Jr., and Woolf, D. O., Jr. Gibberellenic acid, a by- product of gibberellic acid fermentation. Experientia. 13: 487-489. 1957. 9. Imamura, S., Ogawa, Y., Okuda, M., and Hirono, Y. Bioassay of gibberellin with a dwarf mutant of Japanese morning glory, Pharbitis Nil Chois. Abst. 2nd Meeting Japan Gibberellin Res. Assoc. Tokyo, Japan, p. 69. 1958. 10. Imperial Chemicals Industries Limited. New organic compounds. Accepted patent application No. 10190-1955. Commonwealth of Australia. 1955. 11. MacMillan, J., and Suter, P. J. The occurrence of gibberellin Ai in higher plants: isolation from the seed of runner bean (Phaseolus multiflorus). Natur- wis. 45: 46. 1958. 12. Phinney, B. O., and Neely, P. M. Differential biological properties of gib- berellin-like factors isolated from beans and peas. Plant Physiol. 33 (suppl.): xxxviii. 1958. 13. Sell, H. M., Rafos, S., Bukovac, M. J., and Wittwer, S. H. Characterization of several n-alkyl esters of gibberellin A3 and their comparative biological activity. Jour. Org. Chem. 24: 1822, 1823. 1959. 14. Stodola, F. H., Raper, K. B., Fennell, D. I., Conway, H. F., Sohns, V. E., Lang- ford, C. T., and Jackson, R. W. The microbiological production of gibberel- lins A and X. Arch. Biochem. Biophys. 54: 240-245. 1955. 520 M. J. Bukovac and S. H. Wittwer 15. Stouc, B. B., and \amaki, T. The history and physiological action of gib- berellins. Ann. Rev. Plant Physiol. 8: 181-216. 1957. 16. , and Yamaki, T. Gibberellins: stimulants of plant growth. Science. 129: 807-816. 1959. 17. Takahashi, N., Kitamura, H., Kawarada, A., Seta, Y., Takai, M., Tamura, S., and Siuniki, Y. Biochemical studies on "Bakanae" fungus. Part XXXIV. Isolation of gibberellins and their properties. Bui. Agr. Chem. Soc. Japan. 19: 267-277. 1955. 18. , Seta, Y., Kitanuua, H., and Sumiki, Y. Biochemical studies on "Bakanae" fungus. Part \l\iii. \ new gihberellin, gibberellin \. Bui. Agr. Chem. Soc. Japan. 23: 405-407. 1959. 19. Wittwer, S. H., and Bukovac, M. J. The effects of gibberellin on economic crops. Econ. Bot. 12: 213-255. 1958. 20. Yabuta, T., and Sumiki, Y. (Communication to the editor.) Jour. .\gr. Chem. Soc. Japan. 14: 1526. 1938. 21. , Sumiki, Y., Aso, K., Tamura, T., Igarasi, H., and Tamari, K. Bio- chemical studies of "Bakanae" fungus. Part X. The chemical constitution of gibberellin (1). Jour. Agr. Chem. Soc. Japan. 17: 721-730. 1941. C. SIRONVALi Embourg, Belgium GibbereliLns, Cell DLVLSion, and Plant Flowering Recent studies on the gibberellins indicate that there is an activa- tion of cell division as well as an activation of cell elongation (4, 14, 23, 24, 34). The discovery of the primary effects of gibberellin on stem elongation led to the idea that the action of the gibberellins was similar to that of auxin. Since that time many other activation effects have been observed (6,21,22,25,26,33). Some are concerned with the functioning of the growing point of the stem and particu- larly with a modification of the rate or the direction of cell division. It is now clear that in some cases the effect of gibberellins (GA) on stem elongation is partly due to enhanced cell division activity. Figure 1 shows longitudinal sections of Perilla stems in which the dimensions of the control cells are approximately the same as those of the treated cells, while the internode length of the treated plants was 2.3 times that of the control. Sometimes the stem elongation is pro- moted more easily in the inflorescence than in the vegetative stem. With Iberis amara, for instance, we obtained very little length in- crease in the vegetative stem, but the length of the terminal inflor- escence was markedly increased (Figure 2). Such specific effects have been observed in Begonia (14) and in strawberry (R. Lemaitre, per- sonal communication). Enhanced cell division plays an important role in these effects. The activated cells are those in the zone immediately under the apical meristem, as noted by Sachs and Lang (34) in vege- tative plants of Hyoscyamus niger. Modifications of leaf form and size induced by GA have often been observed. Two very characteristic cases are those of Statice sinuata and Lepidmm ruderale (Figure 2). The continual application 1 Subsequently: Laboratory of Plant Physiology, Centre de Recherches de Gorsem, Gorsem-Saint-Trond, Belgium. [521] 522 C. Sirowal C T j- 0.5 MM. Fig. 1. Longitudinal sections of the external (left) and central (right) parenchyma of stems of Perilla naiikinensis C, control; T, treated with 100 p.p.m. GA. of GA to the growing point does not promote the growth of the stem, but the shape of the leaves is very strongly modified. In these two cases the leaves are larger in the treated plants but their form is more simple. This can only be explained by postulating a modifica- tion of mitotic activity within the leaf initials. The action of GA on flowering of long-day (LD) plants grown in short days is also an effect on cell divisions in the stem apex (24). In short days the functioning of the apex is normally restricted to the formation of leaf initials. The application of G\ enhances cell divi- sion in such a way that the whole meristematic region is activated, giving rise to the "manteau dc Gregoire" (12) from 'which the flower primordium is formed. The occurrence of these different effects fits relatively well with the anatomical and cytological description of distinct zones inside the meristem as proposed by Buvat (7). We can visualize that, depending on the species and upon the circumstances, GA acts selectively on one or another meristematic zone of the stem and on the young tissues initiated by the activity of the meristem. In stem elongation, the "meristeme medullaire" and the zone situated immediately under it would be activated. In modifications of leaf form, the activation would affect the "anneau initial" and the leaf initials. The formation of tlic flower would correspond lo a more complete activation of the Fig. 2. tttecL ol gibberellic acid on morphogenesis. Elongation of the inflorescence of Iberis amara (top), modification of leaf shape in Statice sinuata (middle), and Lepidium ruderale (lower). Plants at right are controls; plants at left were treated with 100 p.p.m. GA. 524 C. Sironval mitotic capacity of the meristem as a whole including the "meristeme d'attente." These three effects could coincide in time. To distinguish between the different meristematic activities of the growing point of the stem, one can compare the differential action of GA on LD and SD plants, \\lien GA is applied to SD plants grown under long days, it cannot induce flowering but acts only on stem elongation. When applied to LD plants grown in short days, how- ever, GA promotes both stem elongation and flowering. Using Bu- vat's concept, this means that the "meristeme d'attente" cannot be ac- tivated by GA in SD plants, while it can be activated in LD plants. Tschailachjan (42) has proposed a theory that accounts for this. He postulates that florigen is composed of two hormones, GA and an- thesin. When the two hormones are both present, flowering is pro- moted, as evidenced by the flowering behavior of SD plants under short days and LD plants under long days. Following Tschadachjan, LD plants synthesize anthesin in short days, and the addition of GA results in the formation of florigen (GA -|- anthesin). On the other hand, SD plants synthesize GA in long days, and a further addition of GA has no effect on flowering because anthesin is lacking. It is clear that these differences in reaction when GA is applied indicate that an SD plant grown under long days is not identical to an LD plant in short days. There is a sort of dissymmetry which Tschailach- jan's proposal attempts to interpret. But this does not change the principal open question: How does GA activate cell division in the young tissues of the stem? BIOCHEMICAL APPROACH TO THE ACTIVATION OF CELL DIVISION BY GA Biochemically speaking, cell division is a very complicated phe- nomenon involving the synthesis of protein for which several bio- chemical conditions must be met. The role of ribonucleic acids in protein synthesis has been shown in animals as well as in plants. Protein synthesis also depends on the availability of a suffic ient source of energy w^ith the resulting adenosine triphosphate (ATP) playing a prominent role (1,3, 8). This ATP may be synthesized in both respira- tion and in photosynthesis (16, 43). It is likely that some of these con- ditions are absent in meristems, particularly in the "meristeme d'at- tente" of an LD plant grown in short days or in that of an SD plant grown in long days. Protein synthesis would, therefore, be at a level insufficient for accelerated cell division. The type of block may well be different for the two groups of plants. It is highly probable that GA can modify the rate of protein syn- thesis in the cells of the growing point of the stem. Further promotion of resj^iration (2, 19), action on several cn/yme systems (40, 41), modifi- Gibberellins, Cell Division, and Plant Flowering 525 cation of sugar content (5, 30, 40), reduced nicotine content in tobacco (45), increased ascorbic acid levels in clover (30), action on chloroplast pigments (5, 14, 30), etc., have been reported to occur after treatment by GA. In many species the effect on the pigments is grossly evident, but it is rather complex. Without a supplementary supply of mineral nutrients, as in a normal garden soil, there is generally a lowering of the pigment content. Table 1 lists nine species we have studied. In some cases the anthocyanin content is also modified. When mineral fertilizers are added in the presence of GA, the chlorophyll content does not drop much or does not drop at all. However, the drop re- mains evident when the treated plant flowers (30). As shown by Moso- lov and Mosolova (30), redox processes are strongly enhanced in the leaves of GA-treated clover plants, and the sugar content of the leaves increases. The assimilation of mineral nutrients also increases. All these facts show that GA profoundly affiects the metabolism of plants. In spite of the fragmentary data, some of these facts clearly indicate that under adequate cultural conditions in which mineral nutrition is not limiting, GA enhances certain essential metabolic processes and increases the availability of some important metabolites. This is likely to be very favorable for protein synthesis inside the meristem and in the young tissues of the treated plants. Another argument supports this conclusion. Photoperiodic induc- tion of flowering, which can be replaced by the application of GA to LD plants grown in short days, seems to induce an immediate change in the capacity of meristematic cells to synthesize proteins. This ap- pears from the following facts: (a) Metzner (27, 28) reported that the proportion of amino acids in the protein fraction of the meristems of Kalanchoe blossfeldiana Table L Effect of 100 p. p.m. of gibberellic acid on pigment content of plants. Direction of Change in Chlorophyll Content of Leaves Anthocyanosides Species Tested Direction of change in anthocyanoside content Locus of effect Statice siniiata Draba aizoides Capsella bursa-pastoris. Iberis amara Lepidium ruderale Beta vulgaris Bellis perennis Perilla nankinensis . . . . Cheirantus cheiri Salvia splendens Ageratum mexicanum . . Arabidopsis arenosa . . . 0 0 0 0 0 0 0 + 0 0 0 0 Stem Petioles Stem Leaves 526 C. Sironval grown in long days undergoes a rapid mollification following several short days. Moreover, modifications in the nucleic acid fraction of the meristems occur during this SD induction. (b) It is well known that photoperiodic induction rapidly changes the type of the gas exchange between the plant (in particular its leaves) and the environment (13, 35). Respiration measurements of very young isolated leaves (including the meristem) of an LD strain of Salvia splendens showed that during the induction phase the respiration is significantly higher under long-day conditions than under short-day conditions (10). (c) Studying the total hematin content of leaves of LD and SD plants of Perilla nankinensis (SD), Caymahis saliva (SD), Sinapis alba (LD), and Salvia splendens (LD), we found (unpublished) that induc- tion always causes a decrease of the molar ratio chlorophyll.hematin of the leaves. This decrease is most evident in young leaves. The modi- fication is very rapid and is measurable a few days after the beginning of induction. We always observed that in the very young leaves the chlorophyll accumulation becomes slower upon induction, ^\•hile hematin accumulates more rapidly. (d) In flowering Fragaria vcsca the vitamin E content of the young leaves is approximately proportional to the day length. In field ex- periments a maximum is found in June to July, coinciding \vith the increase of flower initiation (38). As shown by Nason and Lehman (32), vitamin E acts in vitro as an activator of cytochrome c reductase. Points c and d directly relate to chlorophyll metabolism which is controlled by day length, although the exact site of the photoperiodic control is not yet known (9, 11, 29, 36, 37). Points b, c, and d suggest some inductive change in enzyme systems of the young tissues, a pos- sibility which is very consistent with point a. Taken together, the four classes of facts support the following hypothesis: In affecting chlorophyll metabolism, photoperiodic induction acts on several important metabolic processes; it enhances the respiration of the young tissues of the stem and it provides them with an im- proved system of hydrogen carriers passing through tlie series of cyto- chromes [the cytochrome carriers are known to be regularly associated in higher plants with meristematic activity (15)]. It therefore in- creases the ATP supply which is necessary for the changes in the pro- tein fraction (27, 28) as well as for increased cell division and flower- ing. It would be very interesting to sec il the activation of cell divi- sions by GA follows a scheme of this type. ON A POSSIBLE DIFFERENCE BETWEEN SD AND LD PLANTS Finally, we may ask why GA induces flowering of LD plants grown in short days but is ineffective in SD plants grown in long days. In Gibberellins, Cell Divisioyi, cuid Plant Flowering b21 other words, why does GA activate cell divisions of the whole meristem in the first case and not in the second? Many hypotheses are possible. We can suppose, as Tschailachian does, that in SD plants an activator other than GA is necessary and that this activator is lacking in SD plants mider long days. We can also suppose that the action of GA on metabolism is not exactly the same for LD and for SD plants, or that the necessary level of activa- tion must be higher in SD plants than in LD plants and cannot be achieved through GA application. But there is another possibility which cannot be neglected. It is known that chloroplast structure is very delicate and that it is very rich in many enzyme systems. Within the plastid, chlorophyll is not distributed at random but is in close association with protein and lipide, the spatial organization of which is now under study in some laboratories (44). To some extent the or- ganization protects the chlorophyll from photodestruction. The de- gree of protection varies from one species to another, or in the same species in accordance with the conditions of its culture. This appears evident when one studies photooxidative effects. We have found that Chlorella pyrenoidosa (Kandler's strain K) was relatively resistant to photooxidation, while Chlorella vulgaris (Pirson's strain P) was much more sensitive (39). In some mutants, photooxidation is very easy (17), but chlorophyll destruction appears to be only the final conse- quence of photooxidation. Long before it occurs, photosynthesis has completely ceased in high-intensity light (18,20), phosphorylations are inhibited (18), and oxygen consumption rises probably with at- tendant peroxide formation (18, 20, 31). A general poisoning of metab- olism occurs. Crawford (inipublished) has studied the sensitivity of the LD plant Salvia splendens to photooxidation by intense light. He found that photooxidation (as measured by the inhibition of photo- synthesis in white light) is much more marked in the leaves of Salvia splendens grown in short days; the plants grown in long days are evi- dently more resistant to photooxidation. The high photosensitivity of Salvia grown in short days may be due to an insufficient protection of chlorophyll inside the chloroplasts, possibly resulting from an ab- normal structure of the jDlastids themselves. Indeed, under short days the chloroplasts of Salvia do not accumulate their pigments in a normal fashion. In practice this means that, during a given short day with light of sufficient intensity, the metabolism of an LD plant grown in short days can be partially inhibited through photooxidative processes. It can therefore be concluded that short days do not permit flowering of LD plants for two interrelated reasons: (1) suitable metabolic con- ditions (of the kind described above) for increased cell divisions in the meristem are lacking, and (2) photooxidation products poison metabolism during the light period. 528 C. Sironval It would be very useful to know if such a poisoning also occurs in SD plants grown in long days. In Kalanclioe. blossfeJdinna, for in- stance, the chlorophyll metabolism is undoubtedly different in short or long days (37). If this corresponds to a decreased level of pro- tection, a long day with relatively intense light is likeh to produce a drastic photooxidation proportional to the length of the photo- period. Perhaps the explanation of the dissymmetry revealed by GA between the behavior of LD and SD plants is to be found here. Dur- ing long days SD plants could withstand more severe metabolic in- hibition of a photooxidative nature than coidd LD plants during short days. GA would be able to overcome this inhibition in the last case but not in the first. ACKNOWLEDGMENT The author is greatly indebted to the "Institut pour I'encourage- ment de la Recherche Scientifique appliquee a I'lndustrie et a I'Agri- culture, I RSI A," Beige, for financial aid. LITERATURE CITED 1. Borsook, H. Protein turnover and incorporation of labelled aminoacids into tissue proteins in vivo and in vitro. Physiol. Rev. 30: 206-219. 1950. 2. Bourdeau, P. F. Interaction of gibberellic acid and photoperiod on the vegetative growth of Pinus elliottii. Nature. 182: 118. 1958. 3. Brachet, J. L'action de la ribonuclease sur les cellules vivantes. Publ. Staz. Zool. Napoli. 27: 146-159. 1955. 4. Bradley, M. V., and Crane, J. C. Gibberellin-stimulatcd canibial activity in stems of apricot spur shoots. Science. 126: 972, 973. 1957. 5. Brian, P. W., Elson, G. W., Hemming, H. G., and Radley, M. The plant- growth-promoting properties of gibberellic acid, a metabolic product of the fungus Gibberella fujikuroi. Jour. Sci. Food Agr. 5: 602-612. 1954. 6. Biinsow, R., and Harder, R. Bliitenbildung von Adonis und RndUrrhia (lurch Gil)l)crcllin. Natiirwis. 44: 153, 454. 1957. 7. Buvat, R. Lc meristtme apical de la tigc. Annexe Biol. 31: 596-656. 1955. 8. Chantrenne, H. Problems of protein synthesis. In: P. Fildes and W. F. van Heyningen (cds.) , The Nature of Virus Multiplication. ])p. 1-20. rni\-. Press, Cambridge, England. 1953. 9. Clauss, H., and Rau, W. iJbcr die Biiacnl)ililung von Hyoscyamus niger und Arabidopsis thaliana in 72-Stunden-Zyklen. Zeitschr. Bot. 44: 437-454. 1956. 10. Crawford, R. Respiration ct photop^riodisme. Bui. Sen. Roy. Sci. Liege. 1-2: 54, 55. 1959. 11. Enloe, J. A. Pigment transformations in soybean cotyledons. (Thesis, Univ. Calif., Davis.) 1959. 12. Gr(5goire, V. La niorphogi^'ntse ct I'autonomic morphologique do I'appareil floral. Cellule. 47: 285-452. 1938. 13. Gregory, F. G., Spear, L, and Thiniann, k. V. 1 lie interrelation l)ciwccn CO. meiabolLsm and photoperiodism in Kalanchoe. Plant Physiol. 29: 220-229. 1954. 1 1. Gundcrscn, K. Some cxperimeius with gibberellic acid. Acta Horii Gotol). 22: 87-110. 1958. GibberclUns, Cell Division, and Plant Flowering 529 15. James, W. O. Plant Res])iialion. 282 pp. Clarendon Press, O.xford, England 1953. 16. Kandler, O. t)ber die Beziehungen zwischen Phosphathaushalt und Pholo- synthcse. I. Phosphatspiegelschwankungen bei Clilorella pyreno'idosa als Folge des Licht-Dunkel-Wechsels. Zeitschr. Naturforsch. 5b: 423-437. 1950. 17. , and Schotz, F. Untersuchungen iiber die photooxydative Farbstoffzer- storung und StofFwechselhemmung bei C/z/ore//a-mutanten und panachierten 0(mothcren. Zeitschr. Naturforsch. lib: 708-718. 1956. 18. , and Sironval, C. Photooxidation processes in normal green Chlorclla cells. II. Effects on metabolism. Biochim. Biophys. Acta. 33: 207-215. 1959. 19. Kato, J. Effect of gibberellin on elongation, water uptake, and respiration of pea-stem sections. Science. 123: 1132. 1956. 20. Kok, B. On the inhibition of photosynthesis by intense light. Biochim. Rio- phys. Acta. 21: 234-244. 1956. 21. Lang, A. Induction of flower formation in biennial Hyoscyamus by treatment with gibberellin. Naturwis. 43: 284, 285. 1956. 22. . The effect of gibberellin upon flower formation. Proc. Nat. .\cad. Sci. U. S. 43: 709-717. 1957. 23. . The gibberellins and their role in plant growth and development. Iji: Colloque International sur le Photo-thermoperiodism. pp. 55-73. Parma. 1957. 24. Lebedenko, L. A. The effect of gibberellic acid on the apical meristem activity of certain plants. Bot. Zhur. 44: 215-219. 1959. (In Russian.) 25. Lona, F. Brief accounts of the physiological activities of gibberellic acid and other substances in relation to photothermal conditions. In: Colloque Inter- national sur le Photo-thermoperiodism. pp. 141-167. Parma. 1957. 2!i. , and Borghi, R. Germogliazione di gemme di Fagxis silvatica L. in periodo di quiecenza invernale, a fotoperiodo breve, per azione dell'acido Gibberellico. Aten. Parm. 28: 116-118. 1957. 27. Metzner, H. Veranderungen der Blattproteine bei photoperiodischer Induktion. Planta. 45: 493-534. 1955. 28. . Biochemische Untersuchungen zum Problem des Photoperiodismus. Kulturpfl. I: 185-192. 1956. 29. Mitrakos, K., Biinning, E., and Eberhardt, F. Endogen-tagesperiodische Schwankungen der Chloroplasten-Farbstoffe. Zeitschr. Naturforsch. 12b: 813, 814. 1957. 30. Mosolov, I. v., and Mosolova, L. V. The effect of gibberellin on growth and development of agricultural crops. Isv. Acad. Nauk SSSR. 4: 577-589. 1959. (In Russian.) 31. Myers, J., and Burr, G. O. Studies on photosynthesis. Some effects of light of high intensity on Chlorella. Jour. Gen. Physiol. 24: 45-67. 1940. 32. Nason, A., and Lehman, I. R. Tocopherol as an activator of cytochrome C reductase. Science. 122: 19-22. 1955. 33. Rappaport, L. Effect of gibberellin on growth, flowering and fruiting of the earlypak tomato, I.y coper sicum esculentum. Plant Physiol. 32: 440—144. 1957. 34. Sachs, R. M., and Lang, A. Effect of gibberellin on cell division in Hyoscyamus. Science. 125: 1144, 1145. 1957. 35. Schmitz, J. Uber Beziehungen zwischen Blutenbildung in verschiedenen Licht- Dunkelkombinationen und Atmungsrhythmik bei wechselnden photoperiod- ischen Bedingungen. Planta. 39: 271-308. 1951. 36. Sironval, C. La photoperiode et la sexualisation du fraisier des quatre-saisons a fruits rouges (metabolisme chlorophyllien et hormone florig^ne). Compt. Rend. Rech. IRSIA. 18: 1-229. 1957. 530 C. Sironval 37. Sironval, C. The chloropliyll metabolism, a photoperiodic phenomenon. .\tti 2nd Cong. Internat. Fotoljiol. pp. .^87-395. 1957. 38. , and El Tannir-Lomba, J. Vitamin E and flowering of Fragaria vesca L. \ar. semperjlorens Duch. Nature. 185: 855-856. 1960. 39. , and Kandler, O. Photooxidation processes in normal green Chlorella cells. I. The bleaching process. Biochini. Biophys. Acta. 29: 359-368. 1958. 40. Stowe, B. B., and Yamaki, T. The history and physiological action of the gibberellins. Ann. Rev. Plant Physiol. 8: 181-216. 1957. 41. , and Yamaki, T. Gibberellins: stimulants of plant growth. Science. 129: 807-816. 1959. 42. Tschailachjan, M. C. Hormonale Faktoren des Pflanzenbliihens. Biol. Zentralbl. 77: 641-662. 1958. 43. Whatley, F. R., Allen, M. B., and Arnon, D. I. Photosynthetic phosphorylation as an anaerobic process. Biocliim. Biophys. Acta. 16: 605, 606. 1955. 41. Wolken, J. J. A comparative study of photoreceptors. Atti 2nd Cong. Inter- nat. Fotobiol. pp. 81-85. 1957. 45. Yabuta, T., Sumiki, Y., and Takahashi, T. Biochemical studies of "bakanae" fungus XVI. The effects of gibberellin on special components and special tissues of plants. 4. Action of gibberellin on tobacco seedlings. Jour. Agr. Chem. Soc. Japan. 19: 396. 1943. M. KH. CHAILAKHIAN U.S.S.R. Academy of Sciences Effect of Gibberellins and Derivatives of Nucleic Acid Metabolism on Plant Grov\/th and Flowering' The discovery of gibberellins (GA) is an outstanding achievement in research on plant ontogeny, as mankind has now received a new powerful tool for controlling the growth and development of plants (2, 28). On the other hand, it may serve as a crucial test of the cor- rectness of those hypotheses and of theoretical generalizations which at different times have been made regarding the inner causes of plant flowering since, as is usually the case, the appearance of new facts of outstanding importance revolutionize our theoretical conceptions and excite new ideas. It is quite evident that the discovery of gibberellins is directly related to the concept of flowering hormones in plants or, as it was called, florigen, which we proposed over 20 years ago (8). This is es- pecially true if it be noted that a comparison of data on the effect of GA on plants and the results of previous grafting experiments show that GA is not a flowering hormone which is essentially the same for long-day and short-day species. As may be recalled, the concept of flowering hormones, among others, was based on grafting experiments in which it was shown that flowering of short-day plants under long-day conditions can be induced by substances produced in the leaves of long-day species, and conversely that long-day plants flower under short-day conditions in the presence of substances from the short-day components of the grafts (7, 23, 24, 25, and others). This was the basis for suggesting that in long-day, short-day, and neutral species flowering hormones (flor- igen) are of the same type. ^ Read at the Conference by Dr. Clark A. Porter, Boyce Thompson Institute. [ 531 ] 532 M. Kh. Chailakhian On the other hand, consistent results have been obtained by many authors and have indicated that GA accelerates flowering in many long-day plants, including some winter forms and seedlings of bi- ennials, but does not affect the flowering of short-day species (3, 4, 9, 10,11,14,16,17,18,20,22,30). Thus, an important problem was to clarify the relation between GA and florigen. To tliis purpose we carried out some experiments in 1957, together with L. I. Khlopenkova and T. N. Konstantino^■a at the Institute of Plant Physiology of the USSR Academy of Sciences. The influence of GA on the long-day species of rudbeckia (Rudbeckia bicolor) and tobacco (Nicotiana silvestris), and on the short-day spe- cies of red perilla (Perilla nayikinensis), 'Mammoth' tobacco (Nico- tiana tobacum), and winter rape (Brassica napiis var. oleifera) was studied. The results of these experiments led us to the conclusion (11) that two sets of substances compose the flowering hormones, or florigen, which are common to all plants. These are, on the one hand, GA, which is necessary for the formation and growth of stems and, on the other hand, some substances that are necessary for the formation of flowers and which have tentatively been called anthesins. From this standpoint the absence of flowering of long-day species under short- day conditions is explained by a lack of GA, whereas the absence of flowering of short-day species under long-day conditions is ascribed to anthesin insufficiency. Absence of flowering in winter forms and in seedlings of biennials is due to gibberellin deficiency under long- day conditions and to gibberellin and anthesin deficiency under short- day conditions (Figure 1). This assumption, which is based on new facts concerning the in- fluence of GA on the growth and development of plants and also on data from grafting experiments, requires, of course, fmthcr experi- mental and theoretical study. One proof of this hypothesis would be to extract GA-like sub- stances from the leaves of short-day plants located under long-day conditions and to induce, with the aid of these substances, flowering in long-day plants located imder short-day conditions. A circum- stance which facilitated the solution of this problem was that these substances have been isolated from the seeds and unripe fruits of a number of plants (5, 19, 21, 26). Together with V. N. Lozhnikova we carried out an experiment (13) for extraction of GA-like substances from leaves of short-day plants ('Mammoth' tobacco and Perilla), as well as of long-day plants (Rudbeckia). The plants were cultivated under long (L) and short (S: 9-hr.) days, and after buds began to form under a day length suit- Effect of Ccitain Siibstonces on Growth and Floioering m Short-day plont Neutral plont Long-day summer form O ^ Sh ^ L V Sh jCi> Long-day winter form L Sh G+A 6+A GtA G + A Gibberetlins - hormones necessary for the stem formation. Anthesines - hormones necessary for the flower formation. Florigen= gibberellins + onthesines, necessary for flowering. Fif^. 1. Scheme of tormation of flowering hormones in various plant species. able for development, leaf samples were prepared with the aim of extracting GA-like substances. Acetone was employed, and after evaporation the dry residue was dissolved \\\ distilled water. Determination of GA-like substances by the growth reaction of maize seedlings was performed by the method developed by Boyarkin and Dmitrieva (1) in our laboratory. It was found that GA-like substances were present in all tested extracts, there being more of them in extracts from plants kept under long-day conditions than in extracts from the leaves of plants kept under short-day conditions. The action of GA-like substances on the growth and flowering of Rudbeckia was verified by the drop method for rosette plants sown in the spring and kept under conditions of a short (9-hr.) day. A drop of the tested extract, GA, or water was daily applied to the center of the rosette of each plant. The experiment was carried out accord- ing to the following scheme: (1) 0.02 per cent GA3; (2) an extract from the leaves of long-day (L) 'Mammoth' tobacco; (3) the same for a short-day plant (S); (4) an extract from the leaves of long-day red perilla (L); (5) the same for a short-day plant (S); (6) an extract from the leaves of long-day Rudbeckia (L); (7) the same for a short- day plant (S); (8) the control, water. Four plants were used in each experiment. The plants were treated during three months from De- cember 9, 1958 to March 8, 1959. The experiment was completed on April 4. As usual in plants treated with gibberellin, a fast reaction was observed. They rapidly began to bud and flower, whereas up to the 534 M. Kh. Chailakhian end of the experiments the control phints remained in tlie rosette stage. Two o£ the experimental plants that were treated with an ex- tract from the leaves of long-day 'Mammoth' tobacco developed at an especially high rate, and one of them budded and flowered almost at the same time as the plants treated with gibberellin. The plant treated with an extract from the short-day 'Mammoth' tobacco de- veloped more slowly (Figure 2). Later, growth of the stems of plants treated with an extract from long-day 'Mammoth' tobacco was cjuite intense, and at the end of April many flowers had formed on the thick stem of one of the plants. The stem of the plant treated with a short-day 'Mammoth' tobacco extract was also thick, but much shorter, and had only one terminal flower (Figure 3). Two plants treated with an extract from leaves of long-day Perilla only at the end of April formed small buds on short shoots and did not flower. Plants treated with short-day Perilla extracts did not bud at all. A plant treated with an extract from long-day Rudbeckia bud- ded and began to flower earlier than that treated with an extract from the leaves of short-day Rudbeckia (Figure 4). Fig. 2. Effect of extracts from 'Mammoth' tobacco leaves on ilic growth of Rudbeckia. A, 0.02 per cent GA; B, extracts from lca\cs of Imig-dav phints: C, c\- trnrt from leaves of sliort-day plants; D, control, water. Fig. 3. Effect of extracts from 'Mammoth' tobacco leaves on the growth anci flowering of Rudbeckia. A, extract from leaves of long-day plants; B, extract from leaves of short-day plants; C control, water. Fig. 4. Effect of extracts from Rudbeckia leaves on growth and flowering of Rudbeckia. A, extract from leaves of long-day plants; B, extract from leaves of short-day plants; C, control, water. c o T3 C o o O X! CO Ih U T3 C 3 a 0:; o bo C 'u V o T3 C o bo V ■*-» C o CO > (U C o u c RJ ■*-» en XI 3 V 'be J3 s o O O u V s in P 1^ o r-- -o o ^ ^ CM so in r- OC oc XI be C V o 00 o -i- ^ fsj cs Cs) Date of Flowering CO ^ o ID CS lO o' T- ■* < S if /Feb. 12, '59 \Apr. 30, '59 Mar. 7, '59 cs Cs lO LD ' " ^ CO u u a ex << cs CN LO lO o" csf •4^ is V o Q 6 Q in "- o" v£> fM d d to C3 ^ —5 cs 00 to lf> \D~ CO d ^ 00 00 LO U-l co" r-" CN CM o u O Hi Q Q Photo- period i-J C/) H— * * * J c/) -4-1 C V s h < O Extract from tobacco leaves > t« V ">< a 1 e o nJ V Sh •♦-> 4; Bj U a a 4j Ol J3 u <« u C • >-l tf) •«■> 4-) n G ca a u ■(-> O u 4-* 4-» (1) Si (« (fl u a Ih RJ Xi u R) T3 c $ • I-t o Ih c bo ffj Tl ac u (S hfi be s C u tfl o o « U ^ bn u G ll Ri ? in 3 -ax u u xix: hh Effect of Certain Substances on Growth and Flowering 537 Results of observation of development of plants and also data on stem growth are presented in Table 1. The data of the table show that all tested extracts contained GA- like substances, the amount in extracts from long-day plants being larger than that in short-day plant extracts, which is in agreement with the results of determination of GA-like substances in maize seedlings. The experiment shows that Rudbeckia can be made to flower under short-day conditions by treating it with extracts from leaves of various plants containing GA-like substances. It is especially sig- nificant that flowering of a vegetating plant of the long-day type (Rud- beckia) under short-day conditions could be attained by treating it with an extract from the leaves of the short-day species of 'Mammoth' tobacco which vegetated under long-day conditions. Another way of confirming our hypothesis would be to isolate anthesin, which is of a nitrogenous nature, from the leaves of long- day plants growing under short-day conditions, and to use it to induce flowering in short-day plants growing under long-day conditions. Substances of this type have not yet been isolated, but some data ob- tained by us indicate to a certain extent the nature of the substances which induce flowering in short-day plants located under long-day conditions. The point here is that recently some data have been presented which indicate a relation between the nature of the photoperiodic reaction in plants and the peculiarities of nucleic acid metabolism (6, 29), and also an inverse relationship of flowering of the short-day plant cocklebur on substances of the 5-fluorouracil type, which is an antinucleoside (27). Correspondingly, we undertook some experi- ments to study the effect of adenine, kinetin, and some physiologi- cally active substances on differentiation of flower buds of plants under conditions of cultivation of isolated tips by the technique proposed by Butenko (12). For this purpose, terminal buds 3 to 4 mm. in size were taken from the main or upper lateral shoots of vegetating red Perilla plants grown under long-day conditions. After sterilization the buds were planted, under sterile conditions, in test tubes containing a White's agar medium (31), to which were added microelements according to Heller (15) and 2 per cent sucrose. The test tubes were then placed in a greenhouse under the follow- ing illumination conditions: (1) short, 9-hr. day; (2) long natural day with addition of fluorescent light to daylight; and (3) continuous darkness. The experiments were carried out according to the follow- ing scheme: (1) control, stock White's medium; (2) adenine (0.0001 538 M. Kli. ChaUakhian g/1); (3) kinetin (0.001 g/1); (4) heteroauxin (0.00001 g/1); and (5) GA3 (0.0005 g/1). Aliogether, three sets of experiments were carried out. The results ot the third set, which was started January 14, 1959, are mainly given in the present paper. Two or three days after the isolated tips were transferred to the artificial nutritional medium, growth of leaves began, and during the next 7 to 10 days growth of shoots and formation of calluses were observed. Root formation commenced from the 15th to the 20th days. Those plants in which roots were formed had a rapidly growing main shoot, whereas in the absence of roots, growth of shoots was much slower. Perilla leaves which developed before the appearance of roots were usually red, whereas those on the rapidly growing shoot formed after the appearance of the roots were green (Figure 5). i i D B I'ig. 5. Growlli of young Perilla Jiankinensis plants tullivalcd in ust lul)c-s in Willie's medium under long-day conditions. A, control; B, adenine (0.001 g/1); C, kinetin (0.0001 g/1). D, 1 he terminal buds of Perilla during the first day of plant- ing are shown in the up])cr right part of the figure. Ejjcct of Cotain Siibsta)ices on Groxuth and Flowering 539 A B C D Fig. 6. A, Lower part of Perilla plant under short-day conditions. Flower buds in the axils of the lower leaves. B, Lower parts of control Perilla plant under long-day conditions. Small leaves of the growth shoots are visible. No flower buds are present. C, Lower part of Perilla plant under long-day conditions in White's medium with adenine. Flower buds in the axils of the lower leaves. D, Lower part of Perilla plant under long-day conditions in White's medium containing kinetin. Flower buds in the axils of the lower leaves. Magnification x 10. Under short-day conditions, which were favorable for generative development of the Perilla plants, their growth was quite rapid; ad- dition of GA and kinetin to the medium retarded root formation and, as a result, growth of aerial parts of the plants was also slower. In all experimental plants, except those treated with kinetin, (in which growth was strongly retarded) differentiation of flower buds was observed 25 to 30 days later, mainly in the axillary shoots of the lower leaves formed before the appearance of roots (Figure 6, A). When kept continuously in the dark the plants formed elongated, etiolated shoots consisting of one internode and terminating in two 540 M. Kh. Chailakhian reduced etiolated leaves. Addition of GA and kinetin to the medium led to complete inhibition of root formation and, as a result, to slow- ing down of shoot growth. In all experiments, excluding those with kinetin, differentiation of flower buds on the tips of the etiolated shoots was observed 25 to 30 days after planting. Plant growth was more intense under long-day than under short- day conditions. The growth was somewhat weaker in the heteroauxin, adenine, and kinetin experiments and especially in the GA exper- iments. Differentiation of flower buds under long-day conditions occurred only in experiments in which adenine and kinetin were added 25 to 30 days after planting. With growth of the differentiated buds they became distinctly visible. The data obtained show that when adenine and kinetin are in- troduced into the medium, Perilla plants grown from isolated buds under long-day conditions form flower buds. This permits one to suggest that nucleic acid metabolism as a whole, and some derivatives of this metabolism, are related to processes responsible for the initi- ation of flower organs in short-day species. The experimental data presented here do not, of course, solve the problem of elucidating the nature of the flowering hormones. Never- theless, it seems to us that they confirm the main idea that flowering of very different types of plants is the result of interaction of two groups of substances which compose florigen, or the flowering hor- mone complex, which is the same for all plants. SUMMARY The elucidation of the problem on the interrelation between gibbcrellins and florigen led to the idea that flowering hormones common to all plants (florigen complex) consist of two groups of substances: gibbcrellins, necessary for stem formation and growth, and anthcsins, required for flower formation. This idea finds sujjport in the induction of flowering in a long- day species, Rudbeckia, under short-day conditions by means of gib- berellin-like substances. These latter are present in acetone extracts from the leaves of short-day species, 'Mammoth' tobacco, which vege- tates under long-day conditions. Another proof is given by the in- duction of flower bud formation in plants of a short-day species, red Perilla, reared from isolated tops under long-day conditions mider the effect of atlenine and kinetin. These data prove the idea that flowering of various plants is a result of the interaction of two groups of sidistances which comprise the flowering hormone complex, which is the same for all plants. Effect of Certain Substances on Growth and Flowering 541 LITERATURE CITED 1. Boyarkin, A. N., and Dmitrieva, M. I. Biological test for gibberellins. Fiziol. Rast. 6 (6): 741-747. 1959. 2. Brian, P. W. Effects of gibberellins on plant growth and development. Biol. Rev. 34: 37-84. 1959. 3. Biinsow, R., and Harder, R. Bliitenbildung von Bryophyllum durch Gibberel- lin. Naturwis. 43: 479, 480. 1956. 4. , and Harder, R. Bliitenbildung von Adonis und Rudbeckia durch Gib- berellin. Naturwis. 44: 453, 454. 1957. Penner, J., and Harder, R. Bliitenbildung bei Bryophyllum durch Extract aus Bohnensamen. Naturwis. 45: 46, 47. 1958. 6. Butenko, R. G. Intensity of nucleoproteid synthesis in the terminal shoots of soy and lupine grown under various conditions of day length. Abstr. All- Union Conf. Nucleic Acid Metab. PI. 25, 26. Ufa. 1958. 7. Chailakhian, M. Kb. New facts in support of the hormonal theory of plant development. Dokl. Akad. Nauk SSSR. 4: 77-83. 1936. 8. The hormone theory of plant development. Izd. Akad. Nauk SSSR. pp. 1-198. 1937. 9. . Effect of gibberellins on the growth and flowering of plants. Dokl. Akad. Nauk SSSR. 117: 1077-1080. 1957. 10. . The effect of gibberellin on the growth and development of plants. Bot. Zhur. 43: 927-952. 1958. 11. . Hormone factors in flowering of plants. Fiziol. Rast. 5(6): 541-560; Biol. Zentralbl. 77(6): 641-662. 1958. 12. , and Butenko, R. G. Effect of adenine and kinetin on flower bud differentiation in isolated perilla apices. Dokl. Akad. Nauk SSSR. 129: 224- 227. 1959. 13. , and Lozhnikova, V. N. Effects of gibberellin-like substances extracted from the leaves of various plants on the growth and development of rudbeckia. Dokl. Akad. Nauk SSSR. 128: 1309-1312. 1959. 14. Harder, R., and Biinsow, R. Einfluss des Gibberellins auf die Bliitenbildung bei Kalanchoe bloss^eldiana. Naturwis. 43: 544. 1956. 15. Heller, R. Les besoins min^raux des tissus en culture. Annee Biol. 30: 261-281. 1954. 16. Lang, A. Induction of flower formation in biennial Hyoscyamus by treatment with gibberellin. Naturwis. 43: 284, 285. 1956. 17. . Gibberellin and flower formation. Naturwis. 43: 544. 1956. 18. . The effect of gibberellin upon flower formation. Proc. Nat. Acad. Sci. U. S. 43: 709-717. 1957. 19. , Sandoval, J. A., and Bedri, A. Induction of bolting and flowering in Hyoscyamus and Satiwlus by a gibberellin-like material from a seed plant. Proc. Nat. Acad. Sci. U. S. 43: 960-964. 1957. 20. Lona, F. Osservazioni orientative circa I'effetto dell'acido gibberellico suUo sviluppo riproduttivo di alcune longidiurne e brevidiurne. Aten. Parm. 27: 867-875. 1956. 21. MacMillan, J., and Suter, P. I. The occurrence of gibberellin Ai in higher plants: isolation from the seed of runner bean {Phaseolus multiflorus). Natur- wis. 45: 46. 1958. 542 M. Kh. Cliailakhian 22. Marth, P. C, Audia, W. V., and Mitchell, J. W. Effects of gibbeiellic acid on growth and development of plants of various genera and species. Bot. Gaz. 118: lOG-lU. 1956. 23. Melchers, G. Die Bliihhornione. Ber. Deutsch. Bot. Ges. 57: 29-48. 1939. 24. , and Lang, A. Weilere Untersuchiingen zur Frage der Bliihhormone. Biol. Zeniralhl. 61: 16-39. 1941. 25. Moshkov, B. S. Flowering of short day plants under continuous illumination as a result of grafting. Sotsial. Rast. 21: 145-156. 1937. 26. Phinney, B. O., West, C. A., Ritzcl, M., and Neely, P. M. Evidence for "gibberellin-like" substances from flouering plants. Proc. Nat. Acad. Sci. U. S. 43: 398-404. 1957. 27. Salisbury, F. B., and Bonner, J. Effects of uracil derivatives on flowering of Xanthium. Plant Physiol. 33 (suppL): xxv. 1958. 28. Stowe, B. B., and Vamaki, T. The history and physiological action of the gibberelliiis. Ann. Rev. Plant Physiol. 8: 181-216. 1957. 29. Turkova, N. S., and Zhadanova, L. A. Peculiarities of nucleic acid metabolism in plants prior to flowering. Abstr. Plant Physiol. Sect. All-Union Bot. Congr. pp. 61-^3. 1957. 30. \Vittwer, S. H., Bukovac, M. J., Sell, H. M., and \Veller, L. E. Some effects of gibberellin on flowering and fruit setting. Plant Physiol. 32: 39-41. 1957. 31. ^Vhite, P. R. The Cultivation of Animal and Plant Cells. 239 pp. Ronald Press, London, 1954. JAMES A. LOCKHARTi California Institute of Technology The Hormonal Meckanism. of Growth Inhibition by Visible Radiation' A fundamental problem of plant physiology is to gain an under- standing of the mechanisms by which plants respond to their environ- ment. The response may be any one of three general types: metabolic, tropic, or morphogenetic. Each of these responses is the resultant of an interaction between the environment and the genetic and onto- genetic potential of a given organism. Hormones may participate in the regulation of any of these responses, but they are probably most significant in the last two. We are already familiar with the nature of several plant hormones and can infer the existence of others. This paper is concerned specifi- cally with the control of stem growth. Two hormones (or types of hormones) are known to participate in the control of stem growth. The first is auxin, which now appears to function primarily in the tropic responses. Second is gibberellin, which apparently acts as a con- trolling mechanism in many morphogenetic responses. A third hor- mone, caulocaline, produced in roots, is also required for normal stem growth, at least in many species (40). It has not yet been possible to isolate or characterize this hormone. The present paper will consider evidence purporting to show that gibberellin is, in fact, the controlling factor in regulation of stem growth by visible radiation. MORPHOLOGICAL EFFECTS OF VISIBLE RADIATION Visible radiation has two distinct morphological effects on stem growth. Irradiation reduces total length of stem and generally causes it to become thicker and stiffer as well. This is the most noticeable effect of high irradiances, although measurable effects are often ob- ^ Subsequently: Hawaii Agricultural Experiment Station, University of Hawaii, Honolulu 14, Hawaii. ''This research was supported in part by a grant from the Herman Frasch Foun- dation. [ 543 ] 544 J. A. Lockliart served at very low energy levels as well. The most obvious effect of low intensity or brief high intensity irradiation is usually morpho- logical. Internodes which are elongating at the time of irradiation are markedly inhibited but growth of subsequent nodes may be pro- moted proportionally. In many cases, then, low radiation energies may reduce the length of the first internodes but have little or no effect on total growth rate. This appears to be essentially a problem in rate of node formation, since irradiated plants have more nodes, but the same total stem length as dark-grown plants. Essentially nothing is known as yet about the mechanism which controls node formation, but considerable information is available on the mechan- ism of control of total stem elongation. THE NATURE OF THE VISIBLE RADIATION AFFECTING STEM GROWTH Several distinct effects of visible radiation on stem growth have been observed. Radiation energy level and apparently species as well determine the response of plants to radiation. Early workers utilized filters to isolate broad spectral regions of high-intensity solar radia- tion (cf. 8). They found, generally, that blue radiation produced short, thick stems while green and red radiation yielded relatively tall, slender plants. These responses have been confirmed with higli- intensity fluorescent lamps (25, 37, 39). At low energy levels, on the other hand, red radiation is the most inhibitory to stem growth. As radiation energies are increased, a cross-over point is reached at which red is no longer more effective than blue. At irradiances above the cross-over point, blue radiation is most effective. Thus, red radiation is more inhibitory at low intensities, but maximum inhibition is at- tained with high-energy blue radiation. AMiether or not different mechanisms are involved is not known. The red far-red pigment is the photoreceptor for the low-energy red response (2, 5, 29). Its action may also be manifest in a somewhat diffeient manner. If plants are grown in white light for 8 hrs. a day, their groAvth takes place largely during the 16-hr. dark period (33). Wassink and Stolwijk (39) found that the extent of growth during the dark period was controlled by the spectral distribution of radiation received immediately preceding the dark period. Ihis radiation may be of high or low intensity; growth depends only on the ratio of red to far-red radiation (6). Thus the red far-red pigment system acts to modify growth even when plants are grown in high-intensity artificial or solar radiation. The only results available (33) suggest that the growth inhibition by high- intensity blue radiation is not reversed by far-red irradiation at the beginning of the dark period. Hormonal Mcchanisni of Groiuth Inhibition by Radiation 545 A specific high-energy radiation effect on stem growth has been reported by Mohr (26). Stem growth of Sinapis alba was found to be unaffected by the low-energy red far-red pigment system; thus, the ac- tion spectrum for a higli-intensity growth inliibition could be de- termined unambiguously for this species. Two spectral regions of maximum effectiveness were found. The most effective region in- cludes the red far-red region, from about 660 to 740 m^u,. The second region, in the blue, shows a maximum at about 455 m^u. This effect is presumably comparable to the high energy effects observed in other plants. If so, these results would represent a "composite" action spec- trum near the cross-over point. The cross-over point would be at un- usually low^ intensities in this species, because of its relative insensi- tivitv to red. Recently, Mohr (27) has redetermined the blue far-red action spectrum. One peak is found in the blue (ca. 440 m/x) and a much higher peak in the far-red (ca. 730 ni/x), with a shoulder extending through the red. Ultraviolet radiation (254 ni/x) may also inhibit stem growth, but it does so only at relatively high irradiances. The mechanism of growth inhibition is different from that of visible radiation (see below). INTRACELLULAR MECHANISM OF RADIATION INHIBITION Visible radiation inhibits stem growth primarily through a de- crease in cell elongation. This conclusion is supported by cell counts (33, 35) and by findings that growth inhibition occurs in the morpho- logical region of cell elongation (14; Lockhart, unpubl.). The only known exception to this generalization is in Gramineae (i.e., Avena) where radiation-inhibition of the first internode is due to a reduction of cell division as well as of cell elongation (1). Cell elongation and, thus, to a substantial extent, stem elongation are controlled by the rate and extent of plasticization of the primary cell walls. The cell walls become more plastic while the internal os- motic pressure remains constant. Thus, more water moves into the cell, resulting in an increase in cell volume. In an elongating stem this increase is mostly an increase in cell length. The osmotic pres- sure in the cell is subsequently restored to its original volume by up- take of solutes (or hydrolysis of existing substrates). A further in- crease in plasticity results in a second increment of growth, etc. (28, 34). What phase of this growth process is affected by irradiation? The relation of radiation to various individual factors which might con- 546 ]. A. Lockharl trol growth has been investigated from time to time (e.g., 7, 10, 11, 32). However, the results obtained represented only correlations between these various factors antl the radiation regime. In more recent investigations, however, all factors kno^vn to in- fluence growth rate have been examined in relation to both radiation regime and growth rate (19). Of the various factors which might affect growth in pea seedlings, only one, cell wall plasticity, shows changes which parallel changes in growth rate in response to irradiation. Within two hrs. after the onset of irradiation, when growth rate has been reduced 50 to 70 per cent, cell wall plasticity has decreased by about 75 per cent as compared to dark-grown plants. Plasticity is here defined as the attribute of a tissue which is measured by irreversible bending or stretching. It was measured as the amount of residual bending (that left after removal of the load) after the tissues had been subjected to a standard transverse load for a standard time (Figure 1). Plasmolysis of these curved sections did en 20 UJ LU ca iS> UJ Q CD UJ CO 40 60 La "T" "T" 1 1 1 Gibberellic Acid Treated I 1 r -1 r Untreated o Irradiated • Dark grown 16 16 MINUTES Fig. 1. Plasticity of the elongating region of stems of 'Alaska' peas, dark-grown or after 3 hrs. red irradiation. Plants treated with gil)berellic acid (1 ng. per plant) 3 hrs. prior to time of irradiation. Plasticity is measured either as the slope of linear bending or as the amount of residual bending after the load has been re- moved. Illustrated are the standard deviations of the means at representative points. Seven plants per treatment. Hormonal Mechanism of Growth Inhibition by Radiation 547 not change the relative curvatures. Plasticity has also been measured as the irreversible stretching of freeze-killed tissue under a longitudi- nal force. After 2 hrs. irradiation a decrease in plasticity of about 75 per cent was found by this method also. Gibberellic acid applied to plants shortly before irradiation com- pletely prevents the decrease in plasticity just as it prevents inhibi- tion of growth. It has no effect on the plasticity of dark-grown plants (Figure 1). The participation of gibberellin in radiation inhibition of growth is discussed below. To demonstrate that the change in plasticity is a cause rather than an effect of growth inhibition, the various other factors which could affect growth rate were investigated. No change in any of these fac- tors was found, even after growth inhibition was complete. The os- motic pressure of elongating stem cells was measured by a turgor method developed for the purpose (21) and also by determining the osmotic pressure of expressed sap. No difference was found in the osmotic pressure of dark-grown and irradiated plants by either method. Water permeability of the cells was determined by measuring the rate of opening of plasmolyzed, split stem sections when placed in distilled water. No effect of irradiation was found. Rate of water movement through growing tissue was measured as the rate of plas- molysis of stem tissue sections. No effect of irradiation was found. A possible water deficit in irradiated plants was investigated by deter- mining the rate of water loss from stems of dark-grown and previously irradiated plants. No difference was found. The possibility that a water deficiency might cause growth inhibition of irradiated plants was further tested by measuring growth inhibition in a water- saturated atmosphere. No effect of relative humidity was found on radiation-inhibition of growth. MORPHOLOGICAL REGION OF RADIATION SENSITIVITY Idle (14) has compared the radiation sensitivity of the stem tip with that of the elongating region of Vicia faba. Either the tip or grow- ing region was briefly irradiated and the time-course of the growth response (of the elongating region only) was followed. Substantial growth inhibition was observed in both cases but irradiation of the tip was most effective. When the tip was irradiated, inhibition was rapid and substantial, and growth of the elongating region never re- covered. When the elongating region itself was irradiated, inhibition was equally rapid but somewhat less complete. The most striking effect, as a result of irradiation of the elongating region, was the ob- servation that growth of the irradiated region recovered almost com- pletely after 4 to 5 hrs. 548 ]. A. Lockharl The stem apex is the site of gibberellin production in Pisuin, a closely related species (17). Thus irradiation of the elongating region probably inactivates gibberellin in situ. Recovery would occur when a new supply of gibberellin is received from the nonirradiated stem apex. Irradiation of the apex would inactivate gibberellin in a region of relatively high gibberellin accumulation and would result in a greater and longer lasting effect. Still unexplained is the rapidity of transmission of inhibiting stimulus from tip to elongating region com- pared to the slow recovery on direct irradiation of the growing region. THE ROLE OF GROWTH HORMONES IN THE EFFECTS OF RADIATION ON GROWTH Gibberellins An interaction between gibberellin and visible radiation was sug- gested in some of the earliest work on the effects of gibberellin on higher plants. Treatment of plants with large doses of gibberellin results in elongated internodes and long, narrow leaves in monocots and extensive stem elongation in dicotyledonous plants. Decreases in leaf area have also been reported (42), although this is less common (3,41,43). Direct evidence that application of gibberellin reverses growth inhibition caused by visible radiation has been reported for etiolated plants by Lockhart (16) and for light-grown plants as well by Lona and Bocchi (23). Inhibition of light-grown Perilla was accomplished by following the daily light period with exposure to red radiation, while relatively noninhibited plants were produced by following each light period with far-red radiation. Gibberellic acid treatments completely pre- vented the inhibition of growth due to red radiation. It resulted in equal growth in red or far-red irradiated plants. With a tall variety of Pisum sativum, 'Alaska,' normal stem growth in complete darkness is unaffected by gibberellin treatment. However, when gibberellin is given immediately prior to exposing plants to con- tinuous visible radiation, growth inhibition is completely prevented. Dwarf varieties of Pisum respond to gibberellin treatment even when grown in complete darkness. At saturating doses of gibberellin, how- ever, the growth rate of treated plants is identical, whether irradi- ated or dark-grown (Table 1). In Pisum, application of gibberellic acid will prevent inhibition of stem growth at continuous irradiances as high as 2,000 ergs- cm.-^ •sec.-i, both red and blue (18,38). These arc the highest intensities so far tested and are of the same order of magnitude as the high- energy blue far-red inhibition of stem growth reported by Mohr (26). On the other hand, inhibition of stem growth by ultraviolet radia- Hormonal Mechanism of Growth Inhibition by Radiation 549 Table 1 . Response of Pisum sativum, 'Alaska' to red radiation and gibberellic acid treatment. Dark* Irradiated t 18.5 ± 0.30cm. 19.3 ± 0.30cm. 11.9 ± 0.30 cm. f^ihhpi-pllir Arid Treated! 19.8 =fc 0.30 cm. * Both dark and irradiated plants were kept in rooms in which the air was filtered through activated carbon. The dark plants were kept in a chamber in which the air was also bubbled through alkaline potassium permanganate. t Irradiation consisted of filtered red fluorescent radiation (ca. 150 ergs -cm. ^• sec.^i) for the duration of the experiment. t Gibberellic acid was applied as a 4 /ihethanolic drop giving a dose of 1 Mg- pcr plant. Limits are standard deviations of the means. tion (254 mp) is completely unaffected by gibberellic acid (and auxin) treatments (18). It is possible to measure the level of the nongibberellin factors controlling stem growth. This is done by saturating the growth sys- tem with gibberellic acid. Growth, then, will be limited by the sum of the other growth factors. In this manner, the level of nongibberellin growth factors may be compared in irradiated and dark-grown plants. In Pisum, saturation with gibberellin results in equal growth of irradiated and dark-grown plants. Thus the level of nongibberellin growth factors is unchanged by irradiation. Where N is the effective sum of the nongibberellin growth fac- tors and g z= growth, or growth rate. In dark: N^ • natural GA^ = ga- in light: Nj ■ natural GA^ = gj, and gi < ga. Since g, < ga, then Nj < iV^ or G^^ < GA^ (or both). Now, saturate the plants with gibberellic acid. Growth is now equal •' ■ gi = gd- Since gibberellic acid is saturating: GAj r= GA^- Therefore A^^ must be equal to N/, and the change in growth due to irradiation must be due to a change in endogenous gibberellin. The next question is, then, how irradiation affects the endogenous gibberellin system. In general terms, it could affect (I) synthesis, (2) effective endogenous level of the hormone, or (3) response of the plant to a given level of hormone. Two lines of evidence make pos- sible a partial answer to this question. It has already been pointed out that dwarf peas respond to gib- berellic acid treatment even when grown in darkness. It is possible, then, to compare the responsiveness of irradiated and dark-grown plants to various concentrations of added gibberellic acid. When this was done (20) it was found that the responsiveness under the two 550 /. A. Lockhart 30 1 — 00003 0.001 0.003 0.01 0.03 DOSE OF GA/PLANT (yug) Fig. 2. The height of dwarf Pisum ('Morse's Progress #9') 4 days after treatment with gibberellic acid (at the doses indicated) and transfer of certain plants to con- tinuous red radiation (150 ergs-cm'=-sec"^). Plants 6 days old at time of treatment (ca. 4 cm. above soil level). Also indicated are the standard deviation of the means. Twelve plants per treatment. conditions was equal. That is, the amount of gibberellic acid (or gibberellic A^) recjuired to give a threshold response, a half-maximal response, or a saturating response is equal in irradiated and dark- grown plants (Figure 2). This indicates the effective level of en- dogenous gibberellin is reduced by irradiation, while responsiveness of the plant to gibberellin remains unchanged. Thus, radiation either reduces synthesis, or diverts or destroys existing gibberellin. Phillips et al. (30) have recently shown that the level of extractable, endogenous growth substances in the 'Alaska' pea is reduced by irradiation. Gib- berellins and auxins were not completely distinguished, but all acidic growth factors showed a general correlation with growth rate. These analyses provide further support for the conclusion that visible radia- tion acts on stem growth through a decrease in endogenous gibberellin. Cell Wall Plasticity It was pointed out above that inhibition of growth by radiation acts through a reduction in the effective level of endogenous gibberel- lin. Furthermore, it was shown (p. 545) that cell elongation of irradi- Hormonal Mechanism of Growth Inhibition by Radiation 551 ated plants is reduced as a result of a decrease in cell wall plasticity. This must mean that a decrease in endogenous gibberellin causes a reduction in plasticity. Gibberellin, then, is necessary for the main- tenance of normal (maximum) cell wall plasticity. Experimental measurements have demonstrated that treatment of irradiated plants with gibberellic acid will result in high cell wall plasticity, equal to that of dark-grown plants. Relation of Gibberellin to the Photochemical Reaction The same red far-red photochemical process affects various mor- phological responses of plants in addition to stem length. In the same plants, i.e., Pisnm seedlings, irradiation affects stem length, leaf development, epicotyl hook opening, and rate of node formation. Gibberellin treatments will completely reverse the effect of radiation on stem growth, but it has no effect on these other photomorphogenic responses. Therefore, applied gibberellin does not act directly on the initial photochemical act, or even on subsequent thermochemical processes which are common to all these reactions. Rather, gibberellin must act on the terminal reactions controlling stem growth. It clearly does not influence any reaction common to all these photomorpho- genic processes (16). Gibberellin Reversal of Stem-Growth Inhibition in Various Species Interactions between gibberellic acid and radiation-inhibition of stem growth have been examined in various other species in addition to Pisnm (18). Responses of plant species so far examined appear to fall into four groups, as follows: (1) Gibberellin results in complete reversal of radiation inhibi- tion. Examples: Pisnm sativum 'Alaska' (tall) and 'Morse's Progress No. 9' (dwarf), Helianthns annuus, Hordeum vulgare. (2) Gibberellin results in partial reversal of radiation-inhibition. Examples: Cucurbita pepo, Cucumis sativus. (3) Gibberellin elicits no growth response in either irradiated or dark-grown plants. Example: Sinapis alba. (4) Gibberellin gives no growth promotion in darkness, but pro- motes growth markedly when plants are irradiated. Examples: Phase- olus vulgaris, various dwarf and normal clones. It is not yet known, then, just how widespread is the interaction between gibberellic acid and visible radiation or in how many species radiation acts through the endogenous gibberellin system. Studies on the mechanism of interaction have been pursued with Pisum, since the interaction is complete in this species, and since it is a de- sirable experimental plant in other respects. Just how far the results obtained with Pisum may be extrapolated to other species remains to be seen. 552 J. A. Lockhart Participation of the endogenous gibberellin hormone in control of stem growth by radiation cannot be ruled out in any of the cases listed above (see discussion in reference 18). It may be suggested here that the gibberellin hormone in various plants differs chemically (31). Interaction Between Light and Auxin Meijer (24) showed that when young tomato plants are exposed to far-red radiation a strong epinastic response occurs, just as when the plants are treated with auxin. Furthermore, the radiation-induced epinasty can be prevented by anti-auxin treatment. It must be kept in mind that responses such as this may well be due to a balance be- tween factors. Thus, de Zeeuw and Leopold (44) showed that the epi- nastic response of tomato to auxin can be prevented by ultraviolet irradiation. Since auxin (1-naphthaleneacetic acid) was added after irradiation, radiation must reduce the response of the plant to auxin. Perhaps far-red radiation increases the responsiveness of petioles to endogenous auxin. Then epinasty would occur as a result of irradia- tion and could be prevented by added anti-auxin. One of the few reports of an actual promotion of stem growth in intact plants as a result of added auxin is due to Meijer (24). He found a 60 per cent promotion of growth of irradiated gherkin {Cu- cumis) seedlings as a result of tryptophol treatment as well as a somewhat lesser response to lAA (indole-3-acetic acid). However, the growth elicited by tryptophol was only a small fraction (< 40 per cent) of the growth of these seedlings in darkness. In any case, these results are for hypocotyl length only. Thus, it is impossible to distinguish between true effects on stem elongation and hypocotyl inhibition due to an increase in epicotyl growth. Lockhart (18) found no effect of added lAA on another variety of gherkin, either with or without si- multaneous gibberellin treatment. van Overbeek (36) studied the relation of auxin to inhibition of stem elongation by red radiation in Rnphaniis. Added auxin would partially restore growth of irradiated stems, but a large proportion of the inhibition had to be attributed to "the responsiveness of the cells to auxin," presumably gibberellin (?). Galston and co-workers have observed many correlations between irradiation and auxin metabolism, but have found no evidence for a causal relationship between auxin and the effects of radiation on stem growth in intact plants (9, 10, 13). Other Hormonal Factors Brian and Hemming (4) have suggested that gibberellin acts to promote stem growth by counteracting an inhibitor from some other part of ilie plant. They reach this conclusion by demonstrating that Hormonal Mechanism of Growth Inhibition by Radiation 553 excised internode sections of a dwarf pea (treated with lAA) grow much more than the same internode on the intact plant (even if lAA is added to the plant). Gibberellic acid added to excised internodes had only a relatively small effect. (If the intact plant is treated with gibberellic acid, the growth of the internode is 6 times as great as maximum section growth.) The critical question here is: How can the excised internode grow more than it does on the plant, when no gibberellic acid is added? Their conclusion is that an inhibitor, formed in some other part of the plant, prevents elongation in the intact plant, while excision of the internode removes the source of this inhibitor. Clearly this inter- node is capable of greater extension, even without added gibberellic acid, than normally takes place in the plant. Thus, some form of growth-limiting effect from another part of the plant must act on the intact internodes. RADIATION STEM GROWTH RADIATION RECEPTOR leaf growth node formation y hook opening nnevalonic acid ( ? ) dwarf nnutants GIBBERELLIN + auxin + other growth factors HIGH PLASTICITY water high OP in eel CELL EXTENSION (GROWTH) Fcg. 3. Suggested mechanism by which visible radiation limits stem elongation. 554 /. A. Lockhart This inhibitor concept is not, of course, irreconcilable with the function of gibberellin discussed above. Incorporating the inhibitor, radiation would cither decrease the gibberellin, resulting in a lower gibberellin-inhibitor ratio, or increase the inhibitor with the same effect on the ratio and, thus, the same effect on growth. The mechanism by which visible radiation may limit stem elonga- tion is shown in Figure 3. SUMMARY The mechanism by which visible radiation limits stem elongation as we understand it today may be outlined as follows. Radiant energy is intercepted by the receptor pigment(s). The activated pigment transfers (and amplifies) the signal through one or more thermochemical steps common to all photomorphogenetic pro- cesses. From one or more "master reactions" the signal is divided and passed eventually to some step in the developmental sequence of each of the processes under its control. One of the processes affected by visible radiation is stem elonga- tion. Stem elongation normally proceeds by an increase in plasticity of the young cell walls. For maximum increases in plasticity, evidently, many growth factors are required. Among the growth factors recog- nized to be necessary for increased plasticity are auxin, gibberellin, and probably caulocaline. Visible radiation, through an unknown sequence of reactions, acts to reduce the amount of available gibberel- lin. Thus, plasticity of the cell walls is decreased and growth is re- duced. LITERATURE CITED 1. Avery, G. S., Jr., Burklioldcr, P. R., and Creighton, H. B. Polarized growth and cell studies in the first internode and colcoptile of Avcna in relation to light and darkness. Bot. Gaz. 99: 125-113. 1937. 2. Borthwick, H. A., Hendricks, S. B., and Parker, M. W. Action spectrum for inhibition of stem growth in dark-grown seedlings of albino and nonalbino barley {Hordeum vulgare). Bot. Gaz. 113: 95-105. 1951. 3. Brian, P. W., Elson, G. W., Hemming, H. G., and Radlcy, M. Ihc plant- growth-promoting properties of gibbcrellic acid, a metabolic product of the fungus Gibberella fujikui-oi. Jour. Sci. Food Agr. 5: 602-612. 1951. 1. , and Hemming, H. G. Complementary action of gibberellic acid and auxins in pea internode extension. Aim. Bot. II. 22: 1-17. 1958. 5. Downs, R. J. Photoreversibility of leaf and iiypocotyl elongation of dark grown red kidney bean seedlings. Plant Physiol. 30: 468-473. 1955. 6. , Hendricks, S. B., and Borthwick, H. A. Photoreversible control of elongation of pinto beans and other plants under normal conditions of growth. Bot. Gaz. 118: 199-208. 1957. 7. DuBuy, H. G. Ober Wachstum and Phototropismus von Ai'cna sativa. Rec. Trav. Bot. N6erl. 30: 798-925. 1933. 8. Duggar. B. M. (ed.). Biological Effects of Radiation. Vol. H. McGraw-Hill Book Co., New York. 1936. Hormonal Mechanism of Groivth Inhibitioyi by Radiation 555 9. Galston, A. W., and Baker, R. S. Studies on the physiology of light action. V. Photoinductive alteration of auxin metabolism in etiolated peas. Amer. Jour. Bot. 40: 512-516. 1953. 10. , and Hand, M. E. Studies on the physiology of light action. I. Auxin and the light inhibition of growth. Amer. Jour. Bot. 36: 85-94. 1949. 11. Gessner, F. Wachstum und Wanddehnbarkeit am Helianthus-Hypokoiy]. Jahrb. Wiss. Bot. 80: 143-168. 1934. 12. Heyn, A. N. J. The physiology of cell elongation. Bot. Rev. 6: 515-574. 1940. 13. Hillman, W. S., and Galston, A. W. Inductive control of indoleacetic acid oxi- dase activity by red and near infrared light. Plant Physiol. 32: 129-135. 1957. M. Idle, D. B. Studies in extension growth. II. The light-growth responses of Vicia faba L. Jour. Exper. Bot. 8: 127-138. 1957. 15. Klein, W. H., Withrow, R. B., Elstad, V., and Price, L. Photocontrol of growth and pigment synthesis in the bean seedling as related to irradiance and wave- length. Amer. Jour. Bot. 44: 15-19. 1957. 16. Lockhart, J. A. Reversal of the light inhibition of pea stem growth by the gibberellins. Proc. Nat. Acad. Sci. U. S. 42: 841-848. 1956. 17. .. Studies on the organ of production of the natural gibberellin factor in higher plants. Plant Physiol. 32: 204-207. 1957. 18. . The response of various species of higher plants to light and gibberellic acid. Physiol. Plant. 11: 478-486. 1958. 19. Intracellular mechanism of growth inhibition by radiant energy. Plant Physiol. 35: 129-135. 1960. 20. . Studies on the mechanism of stem growth inhibition by visible radia- tion. Plant Physiol. 34: 457-460. 1959. 21. . A new method for the determination of osmotic concentration. Amer. Jour. Bot. 46: 704-708. 1959. 22. , and Gottschall, V. Growth responses of Alaska pea seedlings to visible radiation and gibberellic acid. Plant Physiol. 34: 460-465. 1959. 23. Lona, P., and Bocchi, A. Interferenza dell'acido gibberellico nell'effecto della luce rossa e rosso-estrema suU'allungamento del fusto di Perilla ocymoides L. Aten. Parm. 27: 645-649. 1956. 24. Meijer, G. The influence of light and of growth regulators on the elongation of gherkin seedlings. Acta Bot. Neerl. 7: 621-626. 1958. 25. . The spectral dependence of flowering and elongation. Acta Bot. Neerl. 8: 189-246. 1959. 26. Mohr, H. Der Einfluss monochromatischer Strahlung auf das Langenwachstum des Hypocotyls und auf die Anthocyanbildung bei Keimlingen von Sinapis alba L. (— Brassica alba Boiss.). Planta. 49: 389-405. 1957. 27. . Der Lichteinfluss auf das Wachstum der Keimblatter bei Sinapis alba L. Planta. 53: 219-245. 1959. 28. Ordin, L., and Bonner, J. Permeability of Avena coleoptile sections to water measured by diffusion of deuterium hydroxide. Plant Physiol. 31: 53-57. 1956. 29. Parker, M. W., Hendricks, S. B., Borthwick, H. A., and Went, F. W. Spectral sensitivities for leaf and stem growth of etiolated pea seedlings and their sim- ilarity to action spectra for photoperiodism. Amer. Jour. Bot. 36: 194-204. 1949. 30. Phillips, I. D. J., Vlitos, A. J., and Cutler, H. The influence of gibberellic acid upon the endogenous growth substances of the Alaska pea. Contr. Boyce Thompson Inst. 20: 111-120. 1959. 31. Phinney, B. O., West, C. A., Ritzel, M., and Neely, P. M. Evidence for "gib- berellin-like" substances from flowering plants. Proc. Nat. Acad. Sci. U. S. 43: 398-404. 1957. 32. Priestley, J. H. Light and growth. II. On the anatomy of etiolated plants. New Phvtol. 25: 145-170. 1926. 556 ]. A. Lockhart 33. Stolwijk, J. A. J. Wave length dependence of photomorphogenesis in plants. Meded. Landbouw. Wageningen. 54: 181-244. 1954. 34. Thiraann. K. V., and Samuel, E. W. The permeability of potato tissue to water. Proc. Nat. Acad. Sci. U. S. 41: 1029-1033. 1955. 35. Thomson, B. F. The effect of light on cell division and cell elongation in seedlings of oats and peas. Amer. Jour. Bot. 41: 326-332. 1954. 36. van Overbeek, J. \Vuchsstoff, Lichtwachstumsreaktion und Phototropismus bei Raphanus. Rec. Trav. Bot. Neerl. 30: 537-626. 1933. 37. Vince, D. Studies on the effects of light quality on the growth and develop- ment of plant. II. Formative effects in Lycopersicon esculentiitn and Pisum sativum. Jour. Hort. Sci. 31: 16-24. 1956. 38. Vlitos, A. J., and Mcudt, W. The effect of light and of the shoot apex on the action of gibberellic acid. Contr. Boyce Thompson Inst. 19: 55-62. 1957. 39. Wassink, E. C, and Stolwijk, J. A. J. Effects of light of narrow spectral re- gions on growth and development of plants. Proc. Akad. Wet. Amsterdam C. 55: 471-488. 1952. 40. Went, F. W. Specific factors other than auxin affecting growth and root forma- tion. ' Plant Physiol. 13: 55-80. 1938. 41. Yabuta, T., and Hayashi, T. Biochemical studies on "Bakanae'" fungus of the rice. III. Physiological action of gibberellin on the plants. Jour. Agr. Chem. Soc. Japan. 15: 403-413. 1939. (In Japanese.) 42. , Sumiki, Y., Aso, K.. and Hayashi, T. Biochemical studies of -'Bakanae" fungus. XIII. The effects of gibberellin on special components and special tissues of plants. 4. Action of gibberellin on tobacco seedlings. Jour. Agr. Chem. Soc. Japan. 17: 1001-1004. 1941. (In Japanese.) 43. Sumiki, Y., and Torii, H. Biochemistry of the "Bakanae" fungus of rice. XVII. The effects of giberellin on special components and special tissues of plants. 6. Action of gibberellin on tea leaves. Jour. Agr. Chem. Soc. Japan. 19: 396-398. 1943. (In Japanese.) 44. de Zeeuw, D., and Leopold, A. C. The prevention of auxin responses by ultra- violet light. Amer. Jour. Bot. 44: 225-228. 1957. DISCUSSION Dr. Burstrom: I would like to ask Dr. Lockhart about his results on the influence of gibberellic acid on cell walls. This is a most in- teresting point, of course, and most important, because if you have a change of growth you should have some direct or indirect effect upon the cell wall. 1 would like to ask if you know any more about what is happening here. You measure plasticity by the bending test, and there are two points which surprise me. Firstly, that this plastic bending is going on at a very slow rate and apparently does not come to an end. That would not be expected from a real plastic bending; it ought to be a fairly rapid change. Secondly, you determine, if I am right, the plastic bending after subtracting the reversible elastic bending which obviously is the same at the beginning and at the end of the tests. You have no change in the elastic tension. This is surprising. If you have a plastic change of this kind, you should have a change in the elastic bending because that's also a function of the Hormonal Mechanism of Growth Inhibition by Radiation 557 changes which take place during the plastic deformation of the cell walls. You shouldn't have a constant elastic tension before and after if the gibberellic acid has not changed elasticity. I don't know what this means quantitatively, but this general point of view should not be overlooked. Dr. Lockhart: I can partially answer this by adding that I have also measured plasticity by imposing a longitudinal load on freeze- killed stem sections. The effect of radiation on plasticity was almost identical as measured by the two methods. No significant change in elasticity was observed during the bending procedure. It's not clear to me whether a substantial change in elasticity would be expected, a priori, or not. The deformation of the tissue is, after all, relatively small. Dr. van Overbeek: May I answer that question about elasticity and plasticity? The earliest research I did in the field of auxin research was to work together with Heyn on elasticity and plasticity of coleop- tiles under influence of the auxins. (A. N. J. Heyn and J. van Over- beek. Proc. Kon. Akad. Wetensch. Amsterdam 34: 1. 1931.) One of the things we found invariably, by the stretching technique of plas- molized material and by the bending technique, was that the plas- ticity always increased, whereas the elasticity was not changed at all. H. R. CARNSi F. T. ADDICOTT- K. C. BAKER R. K. WILSON University of California, Los Angeles Acceleration and Retardation of Abscission by Gibber ellic Acid Although there is a voluminous literature on the growth-promot- ing effects of gibberellic acid, there are very few publications on its abscission effects. The first two investigations found no effect of gib- berellic acid on excised leaf abscission zones of Coleus (5) and none with corollas of Papaver and Origanum (8). However, gibberellic acid when applied to flowers or young fruit increased fruit-set in to- matoes (9, 12), and increased fruit-set in cotton. Gibberellic acid sprayed on branches of deciduous trees in late summer had little or no effect on some species, but retarded leaf abscission in other species and accelerated branchlet abscission in Taxodium (6). In view of the now considerable knowledge of the effects of auxins on abscission (1) and the recent discovery of an abscission accelerating hormone (7), and in view of the differences among the results of the abscission experiments with gibberellic acid, further investigation of the role of gibberellic acid in abscission and of its interrelations with the above hormones appears well justified. This paper reports an investigation in which both accelerated and retarded abscission resulted from applications of gibberellic acid to cotton explants (ex- cised abscission zones) and discusses the seeming contradictions in the literature. MATERIALS AND METHODS Seedlings of cotton (Gossyphim hirsutum, 'Acala 4-42' and 'M8948') were grown in the greenhouse or under fluorescent lamps. No differences in response were noted between the two cultivars. The planting medium was Sponge Rok, a white volcanic ash. The seedlings were watered with tap water fortified with ferric salts to prevent chlo- * Subsequently: USDA, Beltsville, Maryland. = Subsequently: Department of Agronomy, University of California, Davis, California. [ 559 ] 560 Cams, Addicott, Baker, and Wilson rosis. Explains were cut when the seedlings were 2 to 3 w-eeks old. In most experiments the cotyledons were debladed 24 hrs. before the explants were cut. The explant technique was a modification of that described by Addicott et al. (3). The explants consisted of 5 mm. slumps of coty- ledonary petioles and stem, with 10 mm. of the hypocotyl. Explants were placed in plastic or stainless steel holders in petri dishes contain- ing a sheet of moistened filter paper and kept in the dark at 25° C. Gibberellic acid was applied in agar blocks of the size used in the standard Avena coleoptile assay (2.7 X 2.7 X 1-0 mm.). Application was either distal (to cotyledon petiole stumps) or proximal (to stem stump). The blocks w^ere removed after 24 hrs. to facilitate testing for abscission. Abscission was determined by means of an abscissor (a spring instrument) calibrated to deliver a force of 5 g. The gibberellic acid used was provided by Merck and Company as samples No. 57-RTS-931 and No. 57-RTS-lOOO. EXPERIMENTS AND RESULTS in the first experiments, gibberellic acid was applied distally to explants from debladed seedlings and in concentrations of 0.01, 0.1, 1.0, and 10 /^g. per block; blank agar blocks were applied proximally. In the controls, blank agar blocks were applied both distally and 100 tn tn o < iij o a: UJ a., GA, ^g./block o 10.00 o 1.00 • 0.10 A 0.01 A 0.00 DAYS Fg. 1. Acceleration of abscission by distal application of gibberellic acid. Each line is the average of three expcrinienis totaling 100 abscission zones. Acceleration and Retardation of Abscission 561 proximally. Results are summarized in Figure 1. With the lowest concentrations ot gibberellic acid, 0.01 /xg. per block, abscission was appreciably faster than in the controls, and the rate increased with increasing gibberellic acid. In similar experiments higher concentra- tions of gibberellic acid were used; 100 /xg. per block (the highest concentration which would remain in solution) produced somewhat more rapid abscission than 10 /xg. The second group of experiments was similar to the first except that lower concentrations were applied: 0.0001, 0.0005, 0.001, and 0.005 /xg. per block. Results are summarized in Figure 2. Slightly increased abscission followed the 0.005 /xg. application. Results from the other applications were very close to the controls. In the third group of experiments gibberellic acid was applied proximally and blank agar blocks distally; other conditions were identical with those of the first two groups of experiments. The re- sults (Figure 3) were closely similar to those of the first group of ex- periments. The rate of abscission increased with increasing concen- trations of gibberellic acid. In a few experiments the proximal applications included 100 /xg. of gibberellic acid. As with distal applications these produced some- what more rapid abscission than 10 /xg. However, another quite un- expected result also occurred: The stem stump abscised in some iOO 80 - 60 O cn m < o UJ a. GA,^g./block O 0.0050 e 0 0005 • 0.0010 A 0 0000 A 0.000 1 DAYS Fig. 2. Abscission following distal application of small amounts of gibberellic acid. Each line is the average of five experiments totaling 200 abscission zones. 562 Cams, Addicott, Baker, and Wilson 100 80 o V) ^ 60 o {/) CD < Z 40 UJ o tr UJ a. -=&: ^ GA, ^g/block O 1.00 e 10 00 • 0.1 0 A 0.01 A 0.00 DAYS Fig. 3. Acceleration of abscission by proximal application of gibberellic acid. Each line is the average of four experiments totaling 160 abscission zones. (but not all) of the explants. The anatomical changes in this stem abscission were found to be identical with the changes which nor- mally occur in the abscission zones of cotyledonary petioles. In the fourth group of experiments the seedlings were not de- bladed prior to cutting the explants; gibberellic acid was applied proximally in low concentrations. In these experiments (Figiue 4) gibberellic acid retarded abscission. The highest concentrations of gibberellic acid, 0.005 /^g. per block, retarded the rate only slightly, but from 0.001 down, retardation was appreciable. The rate of ab- scission decreased with decreasing concentrations of gibberellic acid. DISCUSSION These experiments show that gibberellic acid can affect abscission when applied close to the abscission zone, and can either accelerate or retard abscission depending on concentration and site of applica- tion. Under the conditions of these experiments, gibberellic acid prob- ably reaches the abscission zone and directly influences the process of abscission. Under other conditions, the effects of gibberellic acid on abscission appear to be indirect. For example, the retarded leaf abscission of sprayed branches (6) and increased fruit-set (9, 12) were probably indirect effects resulting from increased vigor or growth Acceleration and Retardation of Abscission 563 100 80 CO i^ 60 o en 03 < UJ o q: UJ Q. 40 20 GA.^g. /block o 0.0000 e 0.0050 • 0.0010 A 0.0005 ▲ 0.000 1 DAYS Fig. 4. Retardation of abscission by proximal application of small amounts of gib- berellic acid. Each line is the average of three experiments totaling 120 abscission zones. o£ the branch or young fruit, rather than direct effects of gibberellic acid on the process of abscission. In other investigations where no effect of gibberellic acid was found, the results appear due, in some cases at least, to the limitations of the experimental material. For example, the Coleiis petiole ex- plants used by Brian et al. (5) abscised so rapidly that both untreated and treated petioles fell before the first observation was made. Under such conditions, of course, accelerated abscission was not detected, although it could well have occurred. For exploratory experiments on abscission, the plant material should be so grown or so selected that the controls show a moderate rate of abscission, thus permitting the detection of both acceleration and retardation. The anomolous stem abscission following proximal application of 100 ^g. of gibberellic acid is of considerable interest in that abscis- sion of stem stumps has not been previously reported in cotton. (How- ever, abscission of weakened or injured branches is characteristic of a few plant species.) The stem abscission observed here was somewhat similar to the anomolous abscission of bean stems following appli- cation of triiodobenzoic acid to the apical bud, described by Whiting and Murray (11). These unusual types of abscission deserve further physiological investigation. 564 Cams, Addicott, Baker, and Wilson Although our knowledge of the role of gibberellic acid in abscis- sion is still meager, it is consistent with the concept emerging from other fields of research: that gibberellic acid functions through inter- action with auxin (4). The evidence on auxin and abscission accum- ulated since 1935 indicates that auxin is the principal endogenous regulator of abscission (2). Now it is apparent that gibberellic acid sometimes may be similarly involved in the regulation of abscission (although the frequency with which it is a critical or deciding factor under natural conditions is still obscure). Considered together, all the evidence now available suggests a new hypothesis: that three hor- mones — auxin, gibberellic acid, and the abscission-accelerating hor- mone — interact in a common mechanism which regulates the process of abscission. Since knowledge of the physiology of endogenous gibberellic acid, as well as of gibberellic acid's interactions with auxin, is still very fragmentary, speculation on the nature of their interaction in abscission is not yet justified. Thus the recent suggestion that gibber- ellic acid and auxin counteract each other in the regulation of young fruit abscission — gibberellic acid preventing and auxin promoting the abscission (10) — appears premature; and further, it is imsatisfac- tory in failing to account for the numerous investigations which show auxin to be involved in the prevention of young fruit abscission (1, 2). Further experiments are being directed to the definition of the role of gibberellic acid in abscission and to the understanding of its physiological and biochemical interactions with auxin and the abscis- sion-accelerating hormone. SUMMARY Gibberellic acid accelerated abscission in excised cotyledonary nodes of cotton when applied in relatively high concentrations either proximal or distal to the abscission zone. Gibberellic acid retarded abscission when applied in relatively low concentrations proximal to the abscission zone. In these experiments gibberellic acid was probably directly in- fluencing the process of abscission. In some experiments, reported by others, gibberellic acid effects were probably indirect; e.g., increased fruit-set following gibberellic acid application appears to be an in- direct effect on abscission resulting from stimidation of fruit develop- ment. Failure to obtain abscission responses from gibberellic acid applications appears due in some cases to limitations of the experi- mental materials. The hypothesis is advanced that the three hor- mones—auxin, gibberellic acid, and the abscission-accelerating hor- mone — interact in a common mechanism which regulates the process of abscission. Acceleration and Retardation of Abscission 565 LITERATURE CITED 1. Addicott, F. T. Auxin in tlie regulation of abscission. Encyc. Plant Physiol. 14: (In preparation.) 2. . Physiology of abscission. Enclc. Plant Physiol. 15: (In preparation.) 3. , Lynch, R. S., Livingston, G. A., and Hunter, J. K. A method for the study of foliar abscission in vitro. Plant Physiol. 24: 537-539. 1949. ^ 4. Brian, P. W. Effect of gibberellins on plant growth and development. Biol. Rev. 34: 37-84. 1959. A 5. , Hemming, H. G., and Radley, M. A physiological comparison of gibberellic acid with some auxins. Physiol. Plant. 8: 899-912. 1955. 6. , Petty, J. H. P., and Richard, P. T. Effects of gibberellic acid on de- velopment of autumn colour and leaf-fall of deciduous woody plants. Nature. 183: 58,59. 1959. 7. Cams, H. R., McMeans, J. L., and Addicott, F. T. An abscission-accelerating hormone in cotton and some of its interactions with auxin and gibberellic acid. Proc. Ninth Internat. Bot. Cong. 2: 60 (abstr.) . 1959. 8. Laibach, F. Der Einfluss von Auxin und Gibberellin auf die Abstossung von Pflanzenorganen. Naturwis. 44: 594, 595. 1957. 9. Persson, A., and Rappaport, L. Gibberellin-induced systemic fruit set in a male-sterile tomato. Science. 127: 816. 1958. 10. van Overbeek, J. Auxins. Bot. Rev. 25: 269-350. 1959. 11. Whiting, A. G., and Murray, M. A. Abscission and other responses induced by 2,3,5-triiodobenzoic acid in bean plants. Bot. Gaz. 109: 447-473. 1948. ~ 12. Wittwer, S. H., Bukovac, M. J., Sell, H. M., and Weller, L. E. Some effects of gibberellin on flowering and fruit setting. Plant Physiol. 32: 39^1. 1957. ROY M. SACHS and ANTON LANG^ University of California, Los Angeles Skoot Histogenesis and the Subapical Meristem: the Action of Gibberellic Acid, Amo-1618, and Maleic Hydrazide Until the late 1950's most discussions of shoot growth were concerned primarily with cell division at the apical meristem and cell elonga- tion in the more distal regions o£ the stem. The few notable excep- tions in which evidence was presented for cell division below the apical meristem and its contribution to shoot development were ig- nored (1,9, 17). With the discovery of the action of the gibberellins, the quaternary ammonium carbamates and maleic hydrazide on stem growth, subapical meristematic activity assumed new import- ance. Thus, the experiments relating to the re-evaluation of sub- apical cell division represent, at the same time, an account of one mode of action of three plant growth regulators which have been the subject of intensive study. GIBBERELLIN-INDUCED STEM ELONGATION IN ROSETTE PLANTS Cytological examination of Samolus parvifiorus, a long-day rosette plant, revealed a great increase in mitotic figures in the regions im- mediately below the apex within 24 hrs. after the application of gib- berellic acid (gibberellin A3, abbrev. GA) (1 1). Similar evidence is available for other rosette plants [ Table 2 in (18) ], but only the data for Samolus, which has been studied in greater detail, will be pre- sented. As the influence of GA continues, the zone of cell division increases in length, exactly equalling the growth in length of the stem (Figure 1). This new zone of division, comprising the cortical, vascular, and pith tissues, can be considered as a virtual subapical meristem. Another important feature of GA action is illustrated in Table 1. There is no increase in cell length for 72 hrs. following GA * Subsequently: California Institute of Technology, Pasadena, California. [567] 568 R. M. Sachs and A. Lang Table 1. Length of pith cells oi Samolus following application of gibberellic acid. Hrs. After Application Distance Below Apical Meristem in Microns 0 to 200 to 400 to 600 to 800 to 1000 to of GA 200 400 600 800 1000 1200 15 23 32 40 53 64 Controls (8-25) * 12 (20-30) 22 (25-40) 31 (30-50) 40 (45-65) 45 (55-75) 64 24 hrs. (5 18; 11 (15-30) 11 (25-35) 19 (35-45) 23 (35-50) 21 (50-80) 31 48 hrs. (5-15) 11 (10-13) 15 (10-25) 14 (15-30) 18 (15-25) 18 (25-35) 28 72 hrs. (5-18) (10-20) (10-25) (13-25) (10-25) (15-40) Numbers in parentheses indicate range in ceil length for each zone. application. It is clear then that in the initial stages of stem elonga- tion GA causes only an increase in cell number. Equally noteworthy is the fact that more than 80 per cent of the induced cell divisions are transverse; i.e., they are oriented to contribute to stem elonga- tion (18,20). What are the quantitative aspects of GA-induced subapical meri- stematic activity? In a few experiments with Samolus in which the dose was varied, a minimum cell division response was obtained with 0.5 ^g. GA per plant, but for several reasons discussed elsewhere (19) it is difficult to establish the precise relationship between GA 200y 400,1 1000^ I400y DISTRIBUTION OF CELL DIVISION I'ig. 1. The effect of GA upon stem elongation and cell division in Samolus. Num- i)cr and position of mitotic figures per -18 ^ median longiluilinal section at 20° C. There were six plants per group collected at the indicated limes, and tiic diagrams are composites of six median sections (each 8 fi tliick) taken from one plant. Each dot represents a mitotic figure. The boundaries of the vascular tissue and apical meristem are inilicated hv dasiied lines. Shoot Histogenesis and the Subapical Meristem 569 dose and niilodc activity. We have, however, studied in some detail the nature of the cell division res[)onse in Samolus at saturating doses of GA (in excess of 10 /i.g. per plant daily). With these rela- tively large doses, periodic (Uictuations in the number of mitotic fig- ures appearing in the pith tissue could be observed for at least 72 hrs. (I'igure 2). I'hese maxima are unrelated to the time of day or manner of a])})lic:ation of GA (Figure 3). They are, however, related to the temperature at which the treated plants were maintained and the initial application of GA, always appearing at definite times after the initial application, this time depending on temperature (Figure 4). Thus, we concluded that the peaks in mitotic counts reflect a partial synchronization of (cll division and ihat the average pith cell gener- U — I CO LU =) O u I— O Si HRS. AFTER APPLICATION OF GA. Fig. 2. Counts ot uansverse mitotic figures in the pith tissue at various times after the initial application of GA at 26° C. Each point is the average of counts made for six plants. The plants received 25 yug. of GA at 0, 21, and 48 hrs. 570 R. M. Sachs and A. Lang ation time in the subapical pith region of GA-treated Samolus plants is approximately 24 hrs. at 26° C, 32 hrs. at 20°, and 46 hrs. at 17° (Figure 4). The Qj,, for mitosis, then, is equal to 2.1, a value similar to that found for the apical cells of pea roots (3). Approximately 6,300 pith cells are produced by GA-induced sub- apical mitotic activity at 26° C. in the initial 72 hrs. (19). Assuming that the pith cell generation time is 24 hrs., then there are 3 cell gen- erations in 72 hrs.; hence, in the pith region in the initial 24-hr. pe- riod, 900 cells, representing the first generation, divided in response to GA [for calculations see (19)]. Further calculations reveal that the average duration of karyokinesis (in our study equal to the time re- quired for cells to progress from metaphase through telophase) is about 32 min., which is very close to the figure calculated by Brown (3) for the apical cells of pea roots, namely, 40 min. The time required for GA to diffuse to the active sites in the pith cells of Samolus is less than 2 hrs. (19), which is within the limits of accuracy of our determination of cell generation time. Hence, the 24-hr. delay in the mitotic effect of GA is an inherent trait of the mechanism of action of this substance on cell division. Since the delay approaches 24 hrs. and since, in addition, the pith cell gener- 60 1 1 1 o 25 fiq. every 24 hrs. • 100 /ig. single application « 10 fig. every 24 hrs. / GA applied • 9 AM O 9 PM 0 24 48 9 AM HRS AFTER APPLICATION OF GA 9 PM 9 AM 9 PM TIME OF DAY 9 AM 9 PM Vi)^. 3. Periodic fliicdiations in mitotic arlivity in tlic pith tissue as related to the mode of application o£ GA (A) and lime of day of llie initial application (B). In the latter case, 25 fig. of GA were applied at 0 and 24 hrs. to both groups. Shoot Histogenesis and the Subnpical Meristem 571 60 24 48 HRS. AFTER APPLICATION OF GA. 72 Fig. 4. The effect of temperature upon pith cell generation time. Each point is the average count made for six plants. The arrows indicate the point from which the generation times have been measured. All plants received 100 /xg. GA at 0 hrs. ation time is also 24 hrs., it appears that the stage at which GA exerts its first effect is early interphase (12, 19). With regard to shoot growth in rosette plants, there is little doubt that the subapical meristem is responsible for the marked increase in stem elongation following GA treatment. In fact, the cell contribu- tion from this region is so large that the direct contribution of the apical meristem to GA-induced stem growth in rosette plants may be disregarded (18, 19). Furthermore, at least two facts show that GA- induced stem elongation is closely related to the natural case in rosette plants: (A) A gibberellin-like substance extracted from the fruit of wild cucumber (15) initiates the same cytohistological development as GA (18) and, eventually, flowering (13). (B) In rosette plants in which stem elongation is induced by the proper environmental conditions, the subapical meristem develops in the same manner as in the GA- treated plants, although more slowly. 572 R. M. Sachs and A. Lans: AMO-1618-INDUCED INHIBITION OF STEM ELONGATION IN CAULESCENT PLANTS Extensive work in the U. S. Department of Agriculture on qua- ternary ammonium carbamates (5, 14, 16, 24) has shown that these substances, the most readily available of which is Amo-1618 [(5-hy- droxycarvacryl) trimethylammonium chloride, 1-piperidinecarboxyl- ate], cause a striking inhibition of stem elongation in many plants. In connection with our studies, it was of particular interest that GA re- versed the inhibition induced by Amo-1618 in Chrysanthemum (6). Microscopic examination of several caulescent plants (i.e., plants possessing elongate stems throughout their life), including Chrysan- themum, Xanthium, and tomato, revealed a subapical zone of cell division comparable in length to that of GA-treated rosette plants. The relatively great number of mitotic figures observed in the sub- apical regions suggested that in caulescent plants, too, the sub- apical meristem plays an important role in shoot development (18). Thus, it seemed a logical step to observe the action of Amo-1618 (and GA) on subapical cell division in Chrysanthemum. U s 10* Fig. 5. Number and position of mitotic figures in the pith tissue of Chiysanthemum per 60 /x median longitudinal section. Rooted cuttings were immersed for 24 hrs. (continuous light) at 26° C. in one of the following solutions: A, water; B, G.\ (100 mg/1); C, Am()-1618 (5-hydroxycarvacr\i)tiinict]iylannnonium chloride, 1- piperidinecarboxylate, 100 mg/1); D, mixture of Amo-1618 and GA (both at 100 mg/1). The plants were transferred to soil and placed in long-day greenhouse con- ditions, and 4 days later they were collected and examined. The plants of groups E and F were immersed in Amo-1618 (same as C) and transferred to long-day greenhouse conditions; 14 days later 200 ml. of water (E) or of GA (100 mg/1) (F) were added to the soil in two doses of 100 ml. each on two successive days. Four days after addition of water or GA to the soil, the plants were collected and exam- ined. Each group contained four plants, and the diagrams are composites of six median longitudinal sections (10 yx per section) taken from one of the treated plants. Each dot represents a transverse mitotic figure. The boundaries of the vascu- lai tissue and the lower limit of the apical meristem are indicated by solid lines. Shoot Histogenesis and the Subapical Meristem 573 Within 4 days after the application of Amo-1618 the number of mitotic figures and the length of the zone of subapical activity were substantially reduced (Figure 5); as the inhibition progressed mitotic activity practically disappeared in the subapical meristem (Figure 5, Amo-1618, 14 days) whereas the apical meristem remained relatively unaffected. If GA was added simultaneously with Amo-1618, there was no inhibition of cell division and, perhaps more significant, GA applied some time after Amo-1618 almost completely reversed the inhibition within a period of 24 to 96 hrs. (Figure 5, Amo-1618, 14 days -^ GA, 4 days). In every case, the activity of the subapical meri- stem could be correlated with stem elongation: The plants that received Amo-1618 alone assumed a rosette habit of growth; i.e., leaf initiation was normal, or almost so, but stem elongation ceased; plants receiving Amo-1618 and GA at the same time grew normally; plants receiving GA after Amo-1618 reverted from a rosette to a caulescent habit of growth. These results thus strongly support the contention that the subapical meristem is responsible for shoot histogenesis in caulescent plants no less than in rosette species. GA or GA-like sub- stances appear to play an important part in the regulation of the subapical meristem. In rosette plants they seem to be the factors limiting its activity; in caulescent plants they are apparently pres- ent at levels insuring optimum mitotic activity, as application of GA to caulescent plants — at least those with which we have worked — does not markedly increase subapical cell division. By use of Amo- 1618 to reduce subapical meristematic activity in these plants, we have been able to demonstrate that GA participates in the regula- tion of this activity and, thus, of shoot histogenesis, in caulescent as well as rosette plants (21). MALEIC HYDRAZIDE INHIBITION OF SHOOT GROWTH There have been several studies on the inhibition of stem growth by maleic hydrazide (MH) (7, 22, 25); one report on tomatoes (8) cited evidence for a reduction in cell number in the treated plants and suggested that this was the main reason for the inhibition of stem elongation by MH. In view of these experiments, we studied the effect of MH upon the subapical meristem in Xanthium. Our re- sults show that MH, in appropriate doses (0.4 mg. in aqueous solu- tion) completely prevents cell division not only in the subapical re- gions (Figure 6) but also in the apical meristem. MH-treated plants, though no longer capable of stem elongation, do not assume a rosette habit of growth because leaf initiation is also prevented; hence, its action as a regulator of cell division is quite different from that of Amo-1618 or GA, lacking the selectivity of the latter two substances. Furthermore, GA (0.4 mg. in aqueous solution). 574 R. M. Sachs and A. Lang [li/i^yi ^!:°' \\l^\l -- 1 --2 -•-3 Fig. 6. EfFect of 0.1 mg. maleic hydrazide on the number and position of mitotic figures in the pith tissue of Xantluiiiii per 60 n thick median longitudinal section. Each group contained four plants and the diagrams are composites of six median longitudinal sections, each 10 fi thick. Each dot represents a transverse mitotic figure. The boundaries of the vascular tissue and the lower limit of the apical nieri- stem are indicated l)y solid lines. The MH was applied in aqueous solution to the shoot apex (three equal applications on consecutive days). whether applied together with or after MH, does not prevent or re- verse the inhibitory effect on cell division. There is some controversy in the literature on the interaction of MH and GA. Bukovac and Wittwer (4) reported that GA overcame the inhibitory effects of MH on the epicotyl growth of beans, and Kato (10) showed with cuctmi- ber seedlings that CiA partially prevented the inhibition of shoot growth by MH. Brian and Hemming (2), working \\ith a variety of peas not responding to GA, concluded that GA ditl not reverse MH- induced inhibition of stem growth and that MH probably interfered with the normal growth response at some stage before GA exerts its effect. They interpreted Bukovac and Wittwer's experiments as well as their own as showing that MH reduced or prevented the response of GA-sensitive plants lo GA. GA does not reverse MH-induccd in- hibition of cell division, yet the story may be quite different where cell expansion is involved. For this reason it is still difficult to assess MH- GA interactions in the over-all growth of plants. V o V 3 h Si o so Si 3 bo o "a M 3 O X e V B u o C o 'a < o Cv O 00 \0 so J5 I o o r-~ •r-. un CM 00 r~- 00 CM in 00 CN C^ 00 I 00 00 CN CN LD \0 OJ ITI CN CM SO so so -* 00 CN r~~ Cs fsj CM Tj- CS O (N CM so o en so 00 o ■* o r~- CM CM O ^ t~- o CM U-) O 00 CM so O in so -rl- o o o 1 o en so en CM o o o O CN (N CM en o CM CM o in CM CM CM o in cs o cs m CM CM esq 00 o Tj- ^ en r- 00 cs 0 O JO .o 2° >" I CM O ss u .^J V s; CL ~ « C/3 S !i ■ ^ ^ ■^ c i^ ^ S ? 1^ ■~ S" „ * K — ! o c^ S "J t-; -M g-s s . •S C S CM i^ '^ — ^ C^ I a nJ X ^ be 3 IS C < w ^ 5 X « 3 2 o 0 J2 be ^ 0 a c o so Cu CM 1 50 be ^ en .2 S Jj « 1> U ^ Si t. CC < bo u -C -t-J -C '^ bOTJ ^ o T3 G > 4J — . >- y^ bo I- 0 p; in Si J2 ^ (M ij en SS 0 C J3 en 22 !^ *" •^ "^ « -^ >.S i c 0 mg/1) panded • 00 22^ i'S.^2 o y "-"TD In D- 3 03 T— < 1> en ^ ■" 3 ^ .en < _>> -S^ ■": r3 0 i5 o a boO Q 03 0 V o o C w 0 ~ 0 T3 0 00 -:; ■*-> CM 1^ >. >, c S >o-a u a. e o a a -d 4J bcE C 03 u ti -J^ y OS 0 be W en U m 3 s- J==3 |0 Si 0 be so enCsl —1 en Oh >- 1 '^ bOn. <- u en 1 <>J ^ 1 Sa o ^ > \ -0 . Si en ■+-• C en O a 5 0 \0 0 c ^^ 0 03 fq^ Oh SJ U"" rt * "H. --g ^-s:-d 0 c^ (« "0 03 ^ -«-' O-cn -0 v en I- z < _j a. o rO o o o UJ X o - cr o ROOT • O A Control 0» i GA-treoted 2 4 6 8 DAYS AFTER TREATMENT Fig. 5. Changes in dry \veight after treatment with gibberellin. 10 leaves were those which were growing vigorously during the experi- ment, and the adult leaves were those which had already attained the maximum leaf area at the start of the experiment. The difference be- tween young and adult leaves was practically two leaf ages. The table shows that there is no significant difference in the photosynthetic ac- tivity between the control and the treated plants. It seems, therefore, that GA does not affect the photosynthetic activity on a unit leaf area basis. Changes in Content of Sugars and Starch by GA Treatment Only rice plants were used for this experiment. Cultural conditions were the same as in the measurement of the photosynthetic activity. The effects of GA treatment on the morphology of rice plants are remarkable. For example, elongation of the leaf sheath of the youngest leaf during 9 days was 1.2 cm. in the control, while it was 16.5 cm. in the treated plants. Changes in dry weight are shown in Figure 5. The dry weight of the top increased, but that of the root decreased, as the result of the GA treatment. In total, dry weight was increased by the treatment. Changes in the content of sugars are shown in Figure 6. In this figure, the scale for the root is magnified five times relative to that for the top. At the start of the treatment, the sugar content was very low owing probably to the after-effect of transferring plants from soil ciUture to water culture. Four days after the treatment, the content Ejjcct of Gihberellin on Photosynthesis 585 of total sugars in the top is the same in the control and the treated plants, but the content of reducing sugars is higher in the treated plants than in the control. In the root, the contents of both reducing and total sugars was decreased as a result of the GA treatment. Figure 7 shows the changes in the starch content. It is noticeable that GA lowers the starch content in the top of the plants. In the root, the starch content was almost nil in both the control and the plants treated with GA. DISCUSSION The 10 to 18 per cent increase in photosynthetic activity of whole plants about one week after treatment (Figures 2 and 3) may be ac- counted for by any of the following possibilities: (1) photosynthetic ac- tivity per unit leaf area is increased; (2) the activity per unit leaf area remains unchanged, but, owing to the increase in leaf area by the treatment, the amount of carbon dioxide fixed by the whole plant increases; (3) the activity per unit leaf area decreases but the increase in leaf area compensates for the decrease in activity; (4) the surface area of organs other than the leaves, for instance, the leaf sheath in rice plants, is increased by the treatment and the increase in photo- synthetic activity of these organs accounts for the increased activity of the whole plants; (5) the treatments affect the shapes of plants, in- TOTAL SUGARS ROOT w A Control » A GA-treated O o Fiff. 6. 4 6 DAYS AFTER TREATMENT 20 16 < _J d o o 12 Q: or < o CO o I- u. o I- 10 Ld o o Changes in the sugar content of rice plants after treatment with gihberellin. 586 T. Hayashi 12 V) < Q. to <3 X o < O 4 _ O o 1 1 1 1 •,A Control - 0,A GA-treoted ^___^ " - TOP - O ^ — n 1 AA ROOT 1 -1 ^ - 4 6 DAYS AFTER TREATMENT 10 Fig. 7. Effect of treatment with gii)bereiiin on the starch content of rice phtnts. crease the light reception, and, therefore, increase carbon dioxide fixation. Among these possibihties, the second seems to be most feasible in view of the data given in Figures 2 and 3 and Table 2. The photosyn- thetic activity per unit leaf area does not change as the result of the GA treatment; but owing to the increase in leaf area, the photosyn- thetic activity of the whole plant increases. In these experiments, sufficient fertilizer was supplied. If the fer- tilizer supply is insufficient, the leaves may become yellowish by GA treatment, the photosynthetic activity per leaf area should then de- crease and the activity per whole plant may also decrease. In rice plants, the increase in the sheath area by GA treatment is often larger than the increase in the leaf area as in the example given in Figure 3. In this instance that may be important in increasing carbon dioxide fixation. Brian et al. (1) and others have previously referred to this possibility. An increase in the efficiency of light reception due to the change in plant shape may also be working in these experiments. In plants which are grown in groups, and sid)je( ted to mutual overlapping in the field, an increase in the efficiency of light reception by GA treat- ment may be appreciable. Effect of Gibberellin on Photosynthesis 587 Next, referring to the carbohydrates produced, the dry weight in the top increases, while in the root it decreases by the GA treatment as given in Figures 4 and 5. Brian also recognized the growth inhibition of roots when GA was added to the culture solution. He considers this to be due to the direct action on roots of GA in a high concentration. Since the data given in Figures 6 and 7 represent only the steady state of the contents of sugars and starch, it is rather difficult to ex- plain inclusively all these facts. Even if the photosynthetic activity does increase, the content of such carbohydrates does not necessarily increase. Any decrease in these carbohydrates may have resulted from the increase in the activity of the system utilizing the photosynthetic products. The increase of reducing sugar in GA-treated plants corresponds with the increase in the invertase activity which has been described in a previous report (3). The decrease of starch, on the other hand, does not correspond with the decrease in the activity of amylase which has also been described in that report. More studies are re- quired to clarify these observations. LITERATURE CITED 1. Brian, P. W., Elson, G. W., Hemming, H. G., and Radley, M. The plant-growth promoting properties of gibberellic acid, a metabolic product of the fungus Gibberella fujikuroi. Jour. Sci. Food Agr. 5: 602-612. 1954. 2. Haber, A. H., and Tolbert, N. E. Photosynthesis in gibberellin-treated leaves. Plant Physiol. 32: 152, 153. 1957. 3. Hayashi, T., Murakami, Y., and Matsunaka, S. Biochemical studies on bakanae fungus. XXXVI. The physiological action of gibberellin. 8. Changes in the activities of various enzymes in leaf-sheaths of rice plants treated with gibberel- lin. Bui. Agr. Chem. Soc. Japan. 20: 159-164. 1956. 4. Noggle, G. R. A study of the effect of gibberellic acid on the alcohol-soluble constituents of oats. Proc. 55th Ann. Convention Assoc. So. Agr. Workers. 55: 228, 229. 1958. 5. Pucher, G. W., Leavenworth, C. S., and Vickery, H. B. Determination of starch in plant tissues. Analyt. Chem. 20: 850-853. 1948. 6. Somogyi, M. A new reagent for the determination of sugars. Jour. Biol. Chem. 160: 61-68. 1945. 7. Yamada, N. et al. A removable apparatus for measuring photosynthesis. Agr. Hort. Tokyo. 30: 73, 74. 1955. DISCUSSION Dr. Tolbert: Dr. Haber and I have confirmed these results work- ing with other plants. We found that radioactive COo fixation per 588 T. Hayashi unit fresh weight of leaves was not increased by gibberellin treat- ment (Plant Physiol. 32: 152. 1957). Dr. Larsen: I should like to mention an additional point, namely the possible effect of gibberellin on the rate of respiration, an item that also enters the balance sheet for the production of dry matter. Does Dr. Hayashi have data on the output of carbon dioxide in the dark? It would be interesting to know whether leaf sheaths of rice can have a positive net rate of photosynthesis or whether their maxi- mum rate of photosynthesis is just capable of counterbalancing the output of carbon dioxide by respiration. If there are stomata on the leaf sheaths of rice (as there are on those of Avena), the sheaths may contribute to the net increase in dry weight. The effect of gibberellin, not only on the leaf area but also on the sheath area, would thereby become important. Dr. Hayashi: I haven't discussed the respiration rate, but I have determined it in these experiments. The respiration rate is from 10 to 20 per cent of the photosynthesis rate at saturated-light conditions, based upon COo exhaustion and fixation, respectively. Dr. Larsen: Would gibberellin influence the rate of respiration? Dr. Hayashi: Yes, gibberellin increases the rate per plant, but the increase is small. Professor Blackman: May I make a comment and a suggestion in relation to the analysis of the effects of gibberellin on growth? The value of growth analysis is admittedly a hobby horse of mine, but it seems to me that these concepts can help in elucidating how these changes in growth are brought about in the field. It can be shown that the relative growth rate (the rate of gain in dry matter per day) is the product of the net assimilation rate (rate of gain in dry matter per unit area of leaf) and the leaf area ratio (total leaf area to total plant weight). It follows that if gibberellin increases the growth rate but does not bring about much change in the rate of assimilation, then it must increase the leaf area ratio. I suggest that it would be worth- while seeing how far gibberellin has altered this ratio. There is an- other point which needs to be taken into account, namely, that there may be self-shading of one leaf by another and in consequence the relationship between light intensity and photosynthesis \\\\\ not be the same for individual leaves and whole plants. We have been work- ing with Salvinia natans where there is no trouble of self-shading since all the leaves are flat on the surface of the water. Gibberellin has little effect on the net assimilation rate, but it depresses the rela- tive growth rate by decreasing the leaf area ratio. WILLIAM S. HILLMAN and WILLIAM K. PURVES Yale University Does Gibber eltin. Act Through an Auxin- mediated Mechanism? Probably most studies of the chemical control of plant growth have dealt with the action of auxins. Recently, however, an increasing number of other growth regulators have claimed the attention of plant physiologists. These include the gibberellins (2), various sub- stituted purines (13), and compounds obtained from endosperm tis- sue (12). Since the mechanisms of action of all such compounds, in- cluding auxins, are unknown, the question as to whether the gib- berellins act through an auxin-mediated mechanism cannot be an- swered conclusively. The chief motive for this attempt is that, for various reasons, the working hypothesis implied by the question ap- pears to have been somewhat uncritically accepted as established in a few papers. In our view, the evidence is largely negative, and such acceptance unjustified. Our first task will be to define the question more precisely. DEFINING THE QUESTION The term auxin will be used to mean indole-3-acetic acid (lAA) or closely related natural or synthetic compounds presumed to have similar physiological action. This is similar to the definition given by Tukey et al. (14) except that their definition could also be used to include the gibberellins, which would merely confuse the issue here. For gibberellin see the review by Brian (2). Gibberellic acid (GA) was used in the experiments reported. An affirmative answer to the question would mean that the physio- logical action of gibberellin is due to a primary effect on a bio- chemical system participating directly in auxin synthesis, transport, action, or inactivation. The term auxin-mediated thus implies an in- timate relation between gibberellin action and auxin, and the ques- [589] 590 W. S. HUlman and W. K. Pwves tion should not be paraphrased "Do the physiological effects of gib- berellin and auxin interact?" Several possible mechanisms of auxin- mediated gibberellin action (cf. 2) can be summarized briefly as fol- lows: (A) Gibberellin may protect native or exogenous auxin from inactivation within the tissue. (B) It may act by increasing the syn- thesis of native auxin, or its translocation or binding to active sites. (C) It may increase the number of sites available with which auxin molecules can react to cause growth. Such proposals all envisage gibberellin as acting by somehow in- creasing net auxin activity. Actions in the reverse sense are not usually considered, in view of the many similarities between gibberellin and auxin activities, but would still constitute auxin-mediated actions. Most of the evidence available is derived from growth experiments, while some comes from studies of the enzymatic activity or growth substance content of extracts. Our own work has been entirely of the former kind; it will be reviewed in the succeeding section and then considered as part of the total data available. EXPERIMENTAL The etiolated pea epicotyl section test was the experimental sys- tem chosen, since it responds to both GA and lAA. Sections were cut from developing third internodes of 7- to 8-day seedlings of Pisum sativum, 'Alaska,' grown in total darkness. They were incubated in darkness for the next 20 to 24 hrs. in a basic medium consisting of phosphate buffer plus 2 per cent sucrose (except as otherwise noted) and further supplemented with GA or lAA as desired. For details of the methods employed and of data described but not presented here see (8) and (9). If sections are cut from various regions of the third internode and their elongation in buffer-sucrose medium compared, the more apical the section the greater the elongation. Thus SI sections (S standing for short, i.e., 5 mm., and 1 standing for 1 mm. below the plumular hook) elongate more than S4 sections, which in turn elongate more than S7 sections. If lAA or GA is added to the medium, further dif- ferences are found. SI sections under these conditions show a very low lAA optimum (about lO-''' M), and the additional elongation over the endogenous (buffer-sucrose only) caused by optimal lAA is quite small; higher levels of lAA are inhibitory. The lower sections show a higher lAA optimum (about lO" M), and the additional elongation caused by optimal lAA is greater than for SI sections. The situation is quite different with respect to GA. There is no pronounced op- timum for GA activity, and a plateau is reached at about 10-« M. The additional elongation caused by GA is greater in SI sections than in Does GA Act Through Auxin-mediated Mechanism? 591 S4, and greater in S4 than in S7 sections. In summary, the greatest response to GA appears in the more apical sections, while the greatest response lo lAA is in the lower, more mature tissues (8). When similar experiments are conducted in the absence of su- crose, the results are different. Under such conditions, the endogenous elongation of SI sections is greatly reduced. The response to GA of all types of sections is so low as to be frequently undetectable; GA and sucrose are in fact synergistic in promoting elongation of SI sec- tions (9). The interaction between sucrose and lAA in the elongation of SI sections is different and more complex; it has been described in detail elsewhere (7). In brief, concentrations of lAA (> 10-« M) which inhibit elongation in the presence of sucrose will promote elongation in its absence. Since the object here was to measure the physiological effects of GA and lAA separately and together with a view to judging the de- gree to which these might be biochemically linked, the first note- worthy point is the apparent spatial separation between tissues most responsive to GA and those most responsive to lAA under the con- ditions employed. A number of workers (cf. 2) have noted the rough correlation between endogenous growth capacity and responsiveness to GA. It has been suggested that the more apical sections show a greater response to GA because of their higher endogenous auxin level, a factor which might also occasion their lower auxin response. A partial test of this hypothesis can be made by seeing whether the response of more basal sections to GA is increased in the presence of lAA. Results of all such experiments have been negative. A typical experiment is summarized in Figure 1, where it is evident that the increment of elongation caused by GA in SI sections is greater than that in S5 sections; levels of lAA which increased S5 elongation to that of the endogenous SI elongation failed to increase their GA response. Numerous systems have been described in which the GA response is markedly dependent on exogenous lAA and in which synergisms between lAA and GA occur. We have not found any such relation- ships in etiolated pea epicotyl sections. Usually the elongation caused by one substance is approximately additive to that caused by the other under all conditions tried. This is true even if the test sec- tions are starved by incubation in sucrose-free buffer for 24 hrs. be- fore the test proper commences (9). It is also particularly significant that, in SI sections, a marked and only slightly reduced response to GA is obtained even in the presence of highly inhibitory levels of lAA. An experiment showing this is presented in Figure 2. Our further attempts to establish some relation between GA and lAA metabolism took the form of treatments which would affect the 592 W. S. Hillman and W. K. Purves < o 2 E o cr CL E E z o I- < z o _J UJ 0 n. 0 GA S5 10"^ M GA y y y / 0 GA 1^ 10 -7 10' 10 -5 NO lAA lAA. M Fig. 1. Effect of distance from apex and of lAA on response of etiolated pea epicotyl sections to GA. Lower histogram: response of SI sections (apical) to 10-" M GA in the absence of lAA. Curves: response of S5 sections (subapical) to GA in the presence of various lAA concentrations. Upper histograms: magnitude of GA re- sponses (GA-treated value minus corresponding control). Divisions of bars and pairs of points represent replicate lots of ten sections. Control medium: phosphate buffer, pH 6.1 plus 1 per cent sucrose. Incubation: 20 hrs. in darkness, about 30° C. lAA responses, and thus inferentially the auxin relations of the tissues. Two such experiments are shown as Figures 3 and 4. Figure 3 shows the effects of adding a-(p-chlorophenoxy)isobutyric acid (PCIB) to the test medium on the control elongation and on the GA and lAA re- sponses of SI sections. PCIB has been described as an anti-auxin (3), but the interpretation of this experiment does not depend on the correctness of this view. It is evident from Figure 3 that a level of lAA which has essentially no effect in the absence of PCIB greatly promotes elongation in its presence, returning the PCIB-inhibited sections to (or occasionally above) the control. PCIB thus inhibits con- trol elongation and brings about a greatly increased auxin response. The additional elongation conferred by GA, however, is essentially the same in the presence or absence of PCIB, whether or not lAA is present as well. Very similar results are obtained by an entirely different treat- ment which also alters the auxin response of SI sections. This treat- ment consists of decapitating the seedlings several hours before the Does GA Act Through Auxin-mediated Mechanism? 593 4 - E £ z o < z 2 o _i UJ - -J G - 1 c 1 Gl lO'^M GA I = lO-'^M lAA Fig. 2. Effect of inhibitory levels of lAA on response of SI sections to GA. test sections are cut from them. As shown in Figure 4, such treatment again markedly changes the lAA response. The control elongation of sections from decapitated plants is reduced, and a level of lAA for- merly inhibitory now promotes elongation. Again, however, the ad- ditional elongation caused by GA is unaffected by such changes. DISCUSSION Because of space limitations this discussion will have to proceed in general terms; for detailed citations of the relevant literature see (2) and (9), and elsewhere in this volume. Evidence Is Against Auxin-mediated Mechanisms Some possible mechanisms of auxin-mediated gibberellin action were summarized earlier, and that outline will be followed here. The possibility (A) that gibberellin acts by protecting native or exogenous auxin from inactivation in some manner has been raised by a number of workers who have interpreted gibberellin effects on peroxidase or lAA oxidase activities as evidence for this view. How- ever, the literature taken as a whole is somewhat contradictory. Re- ports have been made of decreased, unchanged, or increased peroxi- dase activities in extracts from plants previously treated with gib- berellin; of these, only the first kind would be consistent with an auxin-protecting or auxin-sparing role of gibberellin. lAA oxidase 594 IF. S. HiUmnn and \V. K. Piirves 5 NO PCIB lO'^M PCIB E 4 E m J\ _ ELONGATION C - 1 -- 6 G II — 1 h" -- — — • - 1 C 1 G Gl I*I0"'^M lAA G « lO'^M GA Fig. 3. Effect of a-(p-clilorophenoxy)isobutyric acid (PCIB) on responses of SI sections to lAA and GA. activity has been reported as decreased or as unalTected by gibberellin treatment, and gibberellin has also been found to inhibit the activity of lAA oxidase preparations in vitro. Whether or not such results are meaningful for the question at hand depends on whether lAA oxidase or other peroxidase-based systems known to destroy lAA in vitro are believed to do so in vivo. There is no compelling evidence for such a view at present (cf. 4); while there are frequent correla- tions between certain developmental phenomena and lAA oxidase or peroxidase activity, the body of data is not consistent. The fact that GA can still promote pea section elongation in the presence of inhibitory auxin levels (Figure 2) argues strongly against any auxin-protecting mechanism of GA action. In addition, there are in the literature growth systems in which auxin and gibberellin appear to have ojjposite effects, and these are also inconsistent with such a mechanism. The growth phenomena just mentioned are almost equally valid objections to the suggestion (B) that gibberellin acts by increasing the production or translocation of native auxin. Here again, reports on whether or not native auxin levels increase following gibberellin Does GA Act Through Auxin-mediated Mechanism? 595 4 £ E INTACT < 3 LU 2 - n. DECAPITATED | = 3-I0'^M lAA G = lO'^M GA Fig. 4. Effects of decapitation of test plants on responses of SI sections to GA and lAA. Decapitation 6 hrs. before taking of sections. treatment are contradictory. If indeed they do, it still remains to be shown that such increased levels are causally related to the growth in- duced by gibberellin rather than merely correlated with it. GA pro- motion of elongation in the presence of inhibitory lAA levels (Figure 2) is again difficult to reconcile with a mechanism invoking increased auxin production, transport, or binding as the mechanism of GA action, although it can be argued that exogenously applied lAA is not physiologically equivalent to an increase in native pea auxin, whatever its nature. Proponents of such a view would then have to postulate at least partially different modes of action for native and exogenous auxins. The two preceding hypotheses are relatively clear, and appear un- satisfactory on the available evidence. In contrast, possible actions of gibberellin on the availability of molecular sites for auxin action are more difficult to formulate clearly, and thus to evaluate. The data 596 W. S. Hillman and W. K. Purves of Figure 2, which injured the previous hypotheses, are harmless here: it would be reasonable to expect GA-induced elongation in the pres- ence of inhibitory auxin levels if GA increased the other component of the auxin reaction system rather than increasing or protecting auxin itself. A major source of support for this general view can be found in numerous reports of systems in which a response to GA is dependent upon, or greatly increased by, the presence of exogenous auxin. The most striking such system has been described by Kuse (6). GA-IAA synergisms have also been found for the elongation of sections from light-grown pea seedlings and in starved etiolated sections (2) al- though in our work with etiolated pea sections the effects of GA and lAA under all circumstances have been additive or subadditive. The existence of GA-IAA synergisms, however, does not demonstrate an auxin-mediated action of gibberellin; it may simply indicate that such systems are so strongly auxin-depleted that auxin is absolutely limiting to growth. It is notable that in some systems the action of kinetin (6-furfurylaminopurine) is similarly dependent upon the pres- ence of auxin, yet it has not been concluded that kinetin acts di- rectly on auxin metabolism (13). Returning to oiu" own work, we note that there is an almost absolute dependence of GA-induced (but not of auxin-induced) elongation in SI sections on the presence of sucrose. It would be unwise to conclude from this, however, that GA action is specifically mediated by carbohydrate metabolism. Although none of these conclusions can be rejected, they cannot be accepted on this sort of evidence. It has been suggested (2) in support of auxin-mediated GA action that the lower response of subapical older tissue to GA is due to its lower auxin content. It is certainly evident that such tissue is auxin- limited, since added auxin can increase its elongation. However, the auxin occasions no increase in the GA response (Figure 1). Of course, since the apical and subapical sections differ considerably anatomically, it really is not to be expected that a single factor such as auxin would account for the difference in GA response. 11 GA acts directly to increase a nonauxin component of the auxin reaction system, the relation between lAA- and GA-induced elongation in our experiments is sinprisingly loose. Either previous decapitation of the plants or inclusion of PCIB in the test medium re- duces the control growth and at the same time results in an increased response to lAA, thus creating a strong presumption of a change in the auxin relations of the tissues. In effect, either treatment appears to make auxin more limiting. Yet the elongation induced by GA re- Does GA Act Through Auxin-mediated Mechanism? 597 mains virtually unchanged by these treatments (Figures 3 and 4), a result which seems to render any direct relationship between GA and auxin action unlikely, although it cannot disprove it. Alternative Views If present evidence leads to a rejection of the concept that gib- berellin action is auxin-mediated, it may still be useful to consider ways in which auxin and gibberellin might interact in growth phe- nomena. It has been proposed (2) that GA and auxin interact through some third factor. If this factor is conceived of as an auxin-destruction system, or a direct inhibitor of auxin action, such an hypothesis ap- pears inilikely in view of the above evidence. If, however, it is re- garded as some unspecified complex of unknown reactions, or, to ex- pand it further, the plant tissue itself, the hypothesis is of course perfectly reasonable, if unspecific. It was suggested in the experimental section that the increment of elongation induced by GA in a section is relatively independent of the presence of lAA or PCIB, although it is affected by the presence or absence of sucrose. Similarly, the increment (positive or negative) in elongation caused by lAA is about the same in the presence or ab- sence of GA. Such results, in which the absolute effects of various treatments appear to be independent of each other, are frequently encountered in pea section growth tests, at least in this laboratory, and merit some consideration here. A digression into light physiology is necessary to provide further background. In 1941 Schneider (11) reported that the absolute magnitude of the inhibition of elongation caused by red light in dark-grown Avena first internode sections was more or less constant, even when the total elongation of the sections was varied over a wide range by changing the auxin level. Hillman (5) extended these observations in studying the red light inhibition of the elongation of pea sections from dark- grown plants and the far-red promotion of sections taken from plants grown in red light. The absolute magnitudes of the red light inhibi- tion or far-red promotion were unaffected by any but high levels of lAA, and were also independent of GA, although both growth sub- stances affected total elongation. The conclusion was reached that a portion of "endogenous" growth was light-sensitive while GA- and lAA-induced growth was not. These results were obtained with long, subapical pea sections. Since then, Bertsch (1) has shown that in SI sections, in which elongation is greatly promoted by sucrose, only that increment of elongation attributable to sucrose is labile to red light; the red light inhibition is the same in absolute units as the 598 VV. S. Hillman and IF. K. Pwves sucrose promotion, while the increments obtained in buffer alone, or, again, with GA or lAA, are unaffected by light. This somewhat extended account is presented solely to indicate that it may be unwise to regard the elongation of pea sections, or other organs, as homogeneous and limited by a single system through which all effects are to be explained. It behaves under certain condi- tions as if it consisted of separate "components" which add or sub- tract to give the total but frequently do not interact. This of course does not establish the objective reality of such components. In this connection it is worth recalling that a two-phase mechanism for root cell elongation, in which one phase is promoted while another is in- hibited by auxin, has been proposed by Burstrom (3). Further investi- gation along analogous lines might uncover these and other phases in pea sections as well, and help provide data with which to judge the reality of the components mentioned here. It is well to remember that the only observations made in most work of this kind are on length or weight changes, with no close examination of histological or cytological changes. One cannot assume, for example, that two substances have the same action simply because each can cause a 2 mm. increase over the control. Although the additivity of various effects is often imperfect in the presence of optimal lAA, and al- ways in the presence of superoptimal levels, as observed elsewhere (1,5,11) and as is evident in the few data presented here, it may still be useful to consider possible interpretations for independent elongation components on the assumption that the interactions are secondary. Returning specifically to GA and lAA, this independence may arise from their action (at least when promoting growth) in different, spatially-separated systems. They may be limiting to the growth of different groups or types of cells within a single section. Alternatively, there may be, within a single cell, regions whose elongation, expan- sion, or differentiation is limited by these two different factors, and others as well. Some evidence for this sort of view is given by the different re- sponses, mentioned earlier, of apical and subapical tissues to GA and lAA. The two substances may affect different stages in cell develop- ment. Strong support for such a view can be foinid in "Wareing's (15) report on the effects of GA and lAA on cambium in trees. Although the two synergize in promoting development, GA specifically pro- motes cell division, and lAA, differentiation; together they promote the formation of a large zone of normal wood, but their actions are nevertheless qualitatively distinct, and there is no reason to suppose that only a few biochemical steps intervene between them. Yet if growl h had been measured only in some gross quaniitaiive sense sudi Does GA Act Through Auxin-mediated Mechanism? 599 as bulk or fresh weight increases, one might conclude simply that GA and lAA both promote growth and that the two interact to produce a maximal effect on the same growth system. It should be noted in passing that perhaps one reason for attempts to show close linkage between gibberellin and auxin actions was the belief that the former, like the latter, promoted cell extension or expansion rather than cell division, although this generalization is not even completely valid for auxin. In any case, several papers, such as that cited above and that of Sachs et al. (10), have shown in elegant fashion that GA can act as a potent cell-division factor as well. To associate a single growth factor with one component of growth, as if others were not involved at all, is almost certainly an error, and one which we do not wish to commit. Probably all the known growth factors, and more, are necessary for each plant cell; experimental techniques are such as to identify as participating only those which can be made limiting to a given process. It seems unlikely, however, that all or even several act in a single biochemical process limiting many stages of development, and the value of thinking in terms of some such master growth reaction is questionable. It might be just as well to discard the general term "growth" entirely in such dis- cussions and look in more specific morphological, cytological, and biochemical detail at the phenomena in question before proposing simple mechanisms for their interactions. ACKNOWLEDGMENTS We are indebted to Professor A. W. Galston for his encourage- ment and criticism, and to Dr. B. A. Bonner for reading the manu- script. W. S. Hillman was supported by a National Science Founda- tion grant (G-4433) to Professor Galston, and W. K. Purves by an NSF Predoctoral Fellowship. Part of this material was included in a doctoral dissertation (Purves, 1959) submitted to the Graduate School of Yale University. LITERATURE CITED 1. Bertsch, W. F. Effects of composition of the medium on the Hght sensitivity of etiolated pea section growth. Plant Physiol. 33 (suppl.): xxxii, xxxiii. 1958. 2. Brian, P. W. Effects of gibberellins on plant growth and development. Biol. Rev. 34: 37-84. 1959. 3. Burstrom, H. Auxin and the mechanism of root growth. Symp. Soc. Exper. Biol. 11: 44-62. 1957. 4. Galston, A. W., and Hillman, W. S. The degradation of auxin. In: W. Ruhland (ed.). Encyclopedia of Plant Physiology. 14: Vic, b6. Springer-Verlag, Berlin. (In press.) 5. Hillman, W. S. Interaction of growth substances and photoperiodically active radiations on the growth of pea internode sections. In: R. B. Withrow (ed.), Photoperiodism and related phenomena in plants and animals, pp. 181-196. American Association for the Advancement of Science, Washington, D. C. 1959. 600 W. S. Hillman and IT. A'. Fuwes 6. Kuse, G. Necessity of auxin for the growth effect of gibberellin. Bot. Mag. Tokyo. 71: 151-159. 1958. 7. Purves, W. K. Interaction of sugars and hormones in the control of growth of etiolated pea epicotyl sections. (Ph.D. dissertation, Yale Univ.) 1959. 8. , and Hillman, W. S. Response of pea stem sections to indoleacetic acid. gibberelHc acid, and sucrose as affected by length and distance from apex. Physiol. Plant. 11: 29-35. 1958. 9. , and Hillman, W. S. Experimental separation of gibberellin and auxin actions in etiolated pea epicotyl sections. Physiol. Plant. 12: 786-798. 1959. 10. Sachs, R. M., Bretz, C. F., and Lang, A. Shoot histogenesis: the early effects of gibberellin upon stem elongation in two rosette plants. Amer. Jour. Bot. 46: 376-384. 1959. 11. Schneider, C. L. The effect of red light on growth of the Avena seedling with special reference to the first internode. Amer. Jour. Bot. 28: 878-886. 1941. 12. Steward, F. C, and Shantz, E. M. The chemical regulation of growth (some substances and extracts which induce growth and morphogenesis). Ann. Rev. Plant Physiol. 10: 379-404. 1959. 13. Strong, F. M. Topics in Microbial Chemistry: Antimycin, Coenzyme A, Kine- tin and Kinins. 166 pp. John Wiley and Sons, N. Y. 1958. 14. Tukey, H. B., Went, F. W., Muir, R. M., and van Overbeek, J. Nomenclature of chemical plant regulators. Plant Physiol. 29: 307, 308. 1954. 15. Wareing, P. F. Interaction between indoleacetic acid and gibberellic acid in cambial activitv. Nature. 181: 1744,1745. 1958. JIRO KATQi Kyoto University Physiological Action of GibbereUin With Special Reference to Auxin Since Yabuta et al. (31) isolated gibberellins from the culture medium of Gibberella fiijikuroi (Saw) Wollenweber, it has been observed that gibberellin has the effect of causing hyperelongation in many higher plants. I became interested in the mode of action of gibberellin and began a study comparing it with auxin. At the start I had to use a crude preparation of gibberellin. How- ever, it became evident that gibberellin is a growth substance quite different from auxin. As summarized in Table 1, gibberellin was negative in the Avena test and in the pea test at any concentration up to 1,000 mg/1. A 1 per cent lanolin paste of gibberellin did not inhibit, but accelerated, the growth of lateral buds of kidney bean and of etiolated pea seedlings. Gibberellin was not active in induc- ing callus formation on the cut surface of tomato and sunflower seed- lings. Gibberellin A (GA^) did not promote the root formation of etiolated pea stem, but was rather inhibiting (Table 1). The root growth of cucumber seedlings was not affected by from 1 to 100 mg/1 of GAj. This agrees with the result of Brian et al. (5). Whaley and Kephart (28) have reported that the root growth is sig- nificantly stimulated by gibberellic acid (GA) in a certain genotype (strain 854) of maize but not in another (strain 857). These results led to the conclusion that gibberellin is a type of growth-regulating substance quite different from auxins as represented by indole-3-acetic acid (lAA) (11, 12). In order to confirm this conclusion, the interaction between gib- berellin and auxin was studied. Ten mg/1 of GA^ were added to a concentration series of lAA and 1-naphthaleneacetic acid (NAA). ^Subsequently: Division of Biology, California Institute of Technology, Pasa- dena, California. [ 601 ] 602 J. Kato Table 1. Differences in activities of gibberellin and auxin (11, 12). Test lAA GAi Avena standard . Pea split stem Tomato epinasty Callus formation Bud inhibition Root formation + + + + + + — Elongation of etiolated pea stem sections showed that the effect of GAi was additive to the effect of 0.0001 mg/1 through 10 mg/1 of the auxins (12). Purves and Hillman (23) obtained similar results. Even when auxin inhibited the shoot growth of cucumber seedlings by being supraoptimal in concentration, GA^ added to the auxin solu- tions manifested its own growth-promoting effect independent of the auxin effect (Table 2). As to the root growth of cucumber seedlings, GAj showed no effect; and GA^ with auxin inhibited growth. Inhi- bition by NAA of the lateral bud of kidney bean was reversed com- pletely by 5 per cent GA^-lanolin paste. GA^ is inhibitory to root formation and is antagonistic to the root-forming activity of lAA. All these results show that gibberellin works quite differently from auxin. The inhibition of shoot growth by coumarin (CM) and maleic hydrazide (MH) was reversed by GA^ (Table 2) and the inhi- bition of root growth by CM and MH was not reversed, nor increased (Table 3). Hence gibberellin differs also from these substances in its physiological action. In order further to confirm that the reaction sequence caused by gibberellin is different from that caused by auxin, the effect of GA was observed in combination with various types of anti-auxins. As shown in Figures 1, 2, 3, and 4, 4-chlorophenoxyisobutyric acid Table 2. Effect of gibberellin A on lAA-induced growth inhibition of cucumber seedling (11). Concentration of lAA, Mg/L Concn. of GA,, Mg/L 0.0 0.05 0.5 5 .0 Root* Shoot t Root* Shoot t Root* Shoot t Root* Shoot t 0 50 100 100 103 105 100 125 139 93 81 90 98 121 131 54 54 52 64 110 100 28 28 30 62 100 111 level * In each column, differences among the values arc not significant at the 5 per cent t In each column, differences among the values are significant at the 5 per cent level. Physiological Action of Gibberellin 603 Table 3. Eflfect of gibberellin A on coumarin-induced growth inhibition of cu- cumber seedling (H). Concentration of Coumarin Mg/L Concn. of GAi, Mg/L 0.0 1 20 30 Root* Shoot t Root* Shoot t Root* Shoot t Root* Shoot t 0 50 100 100 100 100 100 135 135 100 105 99 105 124 147 60 55 53 62 87 101 48 48 54 73 * In each column, differences among the values are not significant at the 5 per cent level . t In each column, differences among the values are significant at the 5 per cent level. (4-CIBA), 3-chlorophenoxyisobutyric acid (3-CIBA), 2,4,6-trichloro- phenoxyacetic acid (2,4,6-T), and 2-methyl-l,4-dihydronaphthoquinone (Kg) reduce the effect of auxin when the auxin concentration is low, but not when it is high enough. Hence they seem to be competitive with auxin, as already pointed out by McRae and Bonner (16), In- gestad (10), and Fransson (7). In contrast, the anti-auxins used made GA completely ineffective even up to a high concentration, 300 mg/1. The inhibition of GA effect by anti-auxins shall be considered later. Here it is to be noted that anti-auxins are not competitive with GA, while they are comj^etitive with auxin. 140 » • • GA + 4-ClBA 1 1 1 0.001 0.01 0.1 10 1 10 100 CONCN., lAA.MG./L. CONCN., GA, MG./L. Fig. 1. Interaction of 15 mg/1 of 4-chlorophenoxyisobutyric acid (4-CIBA) with lAA and gibberellic acid in the elongation of pea stem sections (13). o o 140 130 - 120 O no z LLl _ 100' 0' — 0 IAA + 2,4,6-T 0 001 0.01 0.1 CONCN., lAA, MG./L. 11 • • • GA + 2.4.6 -T I 10 100 CONCN., GA, MG./L. Fig. 2. Interaction of 30 mg/1 of 3-chlorophenoxyisobutyric acid (3-CIliA) with lAA and gibberellic acid in the elongation of pea stem sections (13). O q: 140 130 O o U. 120 o l- O 110 z llJ - 100' ,i^ 90' — 0 0.001 0.01 0.1 CONCN., lAA, MG./L. I 10 100 CONCN., GA, MG./L. I"ig. 3. Interaction of 2,4,6-trichIorophenoxyacetic acid (2,4,6-T) with lAA and gibberellic acid in the elongation of pea stem sections (13). Physiological Action of Gibberelli?! 605 I40| — 130 O cr Z O o U. 120 o l" H- O 110 z llJ < - 100 90 AA + K3 v • • GA + K3 j_ 0.001 0.01 0.1 CONCN., lAA, MG./L. I 10 100 CONCN.,GA, MG./L. Fig. 4. Interaction of 2-methyl-l,4-dihydronaphthoquinone (K3) with lAA and gibberellic acid in the elongation of pea stem sections (13). The series of experiments so far presented may suggest that the reaction sequence caused in tissues by gibberellin differs from that caused by auxin, or more specifically, that probably the physiological receptor for gibberellin is different from that for auxin. Curtis (6) found that, using Phaseolus vulgaris 'Black Valentine,' the inhibitory effect of the filtrate from the culture medium of Aspergillus niger could be reversed by GA but not by lAA, and inferred that GA operates through a system different from that of lAA. The complete inhibition of the GA-induced elongation by anti- auxins, as described above, was reversed by the concomitant addi- tion of lAA or indole-3-acetamide. This reversing effect of lAA, how- ever, could not be substituted by other growth factors such as amino acids (L-leucine, L-histidine, tyrosine, L-phenylalanine, L-methionine, DL-threonine, L-hydroxyproline, DL-iso-leucine, aspartic acid, dl- ornithine, L-arginine, L-alanine, L-proline, glycine, L-lysine, DL-va- line, L-glutamic acid), vitamins (thiamine, riboflavin, ascorbic acid, vitamin E, vitamin K, pyridoxine, pantothenic acid, folic acid, nicotin- amide, vitamin B^g), diphenylurea, biotin, casein hydrolysate, and yeast extract. Hence the effect of GA seems to be caused by some process involving auxin, as already suggested by Brian and Hemming (4), Kuse (15), and Galston and Warburg (8). On the other hand, Applegate (2), using seedlings of Zinnia ele- 606 ]. Kato gans, reported no difference between the effect of GA and mixtures of GA and TIBA in various concentrations, and concluded that auxin was not definitely responsible for GA-induced cell elongation. As to the mode of action of gibberellin, Pilet (21), Pilet and Wurgler (22), and Stutz and Watanabe (25) think that gibberellin operates through its effect on the lAA oxidase, namely through rais- ing the auxin level in plant tissues. Nitsch (18) reported that GA treatment of some woody plants increased their auxin content. How- ever, my experiments using combinations of gibberellin with auxins and anti-auxins have suggested that the effect of gibberellin involves a physiological sequence different from that of auxin (12, 13). Brian and Hemming (4) have shown that GA had no stimulatory nor inhibitory effect on the lAA oxidase prepared from etiolated pea seedlings. Kato and Katsumi (14) also tested the effects of GA and GAi on the activity of lAA oxidase prepared from etiolated pea shoots. As is shown in Figure 5, neither was stimulating nor inhibit- ing. GA had no effect even at high concentrations and at various pH values. According to my unpublished experiments, 0.1 to 1.0 mg/1 solu- tions of crude gibberellin accelerated the multiplication of fronds of Lemna paucicostatn, 0.5 mg/1 being the optimum. Fronds grown in 100 3 TIME IN HOURS Fig. 5. Lack of effect of gibberellic acid and gibberellin A on the activity of indole- 3-acetic acid oxidase (14). Physiological Action of Gibberellin 607 the presence of gibberellin were smaller and lighter green in color than normal ones. With lAA, however, multiplication of the frond was inhibited by I mg/1 and stimulated by 0.5 mg/1; fronds grown in the latter concentration were larger in area and somewhat deeper green in color than the controls. Therefore, the hypothesis that the gibberellin action is due to a change in the auxin content in plant tissues should be revised. There are many reports (1, 2, 3, 6, 9, 17, 20, 24,26) which do not conform with the hypothesis of Pilet (21), Pilet and Wurgler (22), and Stutz and Watanabe (25). There remains, then, the problem of the mechanism of action of gibberellin. Without entering into this difficult problem, I wish to mention that some hormone-like factors are needed for the growth effect of gibberellin. The necessity of auxin is already discussed. Vlitos and Meudt (27) demonstrated, by using etiolated pea cuttings, that some factor(s), regarded as existing in the shoot apex, is involved in GA action. Brian and Hemming (4) and Galston and Warburg (8) postulated that a third factor is required for gibberellin to be effective. Since Wittwer and Bukovac (29) and Wittwer et al. (30) showed that the photoperiod was an important factor controlling the plant response to gibberellin, it is presumed that some factor(s) produced under proper photoperiods is necessary for the gibberellin action. The necessity of these factors should be kept in mind when the action mechanism of gibberellin is considered and also when the bioassay of gibberellin is attempted. It should also be noted, on the other hand, that such factor(s) may be contained naturally in certain kinds of strains of plants and not in others (19, 28). SUMMARY Gibberellin is a growth-promoting substance quite different in nature from auxin. Its growth effect is not due to a change in auxin level of the affected tissue. The reaction site of gibberellin in plant tissues is different from that of auxin. ACKNOWLEDGMENTS The author wishes to thank Professor Joji Ashida for his cordial guidance through the preparation of this paper. The crude gibber- ellin and gibberellin A were provided through the courtesy of Pro- fessor Y. Sumiki, Tokyo University, and gibberellic acid through the courtesy of Dr. C. Leben, Eli Lilly Co. The author is greatly in- debted to Professor R. L. Wain, University of London, and Professor H. Burstrom, University of Lund, for kindly providing the author with anti-auxins. 608 ./. Kato LITERATURE CITED 1. Ahrams, G. J. von. Auxin relations of a dwarf pea. Plant Phvsiol. 28: 4J3— 156. 1953. 2. Applegate, H. G. Polarity and gibbercllic acid in intact plants. Bot. Gaz. 119: 76-78. 1957. 3. Brian, P. W., and Hemming, H. G. The effect of gibberellic acid on shoot growth of pea seedlings. Physiol. Plant. 8: 669-681. 1955. 4. , and Hemming, H. G. Complementary action of gibberellic acid and auxin in pea internode extension. Ann. Bot. II. 22: 1-17. 1958. 5. , Hemming, H. G., and Radley, M. A physiological comparison of gib berellic acid with some auxins. Physiol. Plant. 8: 899-912. 1955. 6. Curtis, R. W. General studies on the production of curvatures and malforma- tion on plants by culture filtrates of Aspergillus niger. Plant Physiol. 33 (suppl.): xxxi, xxxii. 1958. 7. Fransson, P. Studies on the interaction of antiauxin and native auxin in wheat roots. Physiol. Plant. 11: 641-654. 1958. 8. Galston, A. W., and Warburg, H. An analysis of auxin-gibberellin interaction in pea stem tissue. Plant Physiol. 34: 16-22. 1959. 9. Hayashi, T., and Murakami, Y. Studies on the physiological action of gib- berellins. I. Bui. Nat. Inst. Agr. Sci. D. 7: 159-197. 1958. 10. Ingestad, T. Kinetic aspects on the growth-regulating effect of some phenoxy acids. Physiol. Plant. 6: 796-803. 1953. 11. Kato, J. Studies on the physiological effect of gibberellin. I. On the differential activity between gibberellin and auxin. Mem. Coll. Sci. Univ. Kyoto B. 20: 189-193. 1953. 12. . Studies on the physiological effect of gibberellin. II. On the inter- action of gibberellin with auxins and growth inhibitors. Physiol. Plant. 11: 10-15. 1958. 13. . Studies on tlie pliysiological effect of gibberellin. VI. On the inter- action of gibberellic acid with antiauxins. (In preparation.) 14. , and Katsunii, M. Studies on the physiological effect of gibberellin. V. Effect of gibberellic acid and gibberellin A on the acti\ity of indoleacetic acid oxidase. Mem. Coll. Sci. Univ. Kyoto B. 26: 53-60. 1959. 15. Kusc, G. Necessity of auxin for the growth effect of gibberellin. Bot. Mag Tokyo. 71: 151-159. 1958. 16. McRae, D. H., and Bonner, J. Chemical structiuc and antiauxin activity. Physiol. Plant. 6: 485-510. 1953. 17. Maltzahn, K. E. von, and MacQuarrie, I. G. Effect of gibbercllic acid on the growth of prolonemata in Splachninu cvupullaceum (L.) Hedw. Natiue. 181: 1139,1140. 1958. 18. Nitsch, J. P. Growth responses of woody |)lants to photoperiodic stimuli. Proc. Amer. Soc. Hort. Sci. 70: 512-525. 1957. {Cited from Stodola, Frank H. Source Book on Gibberellin. 1828-1957. USDA Agr. Res. Service. 1958.) 19. Phinncy, B. O. Growth response of single-gene dwarf mutants in maize to gibberellic acid. Proc. Nat. Acad. Sci. U. S. 42: 185-1S9. 1956. 20. , West, C. A., Ritzel, M., and Neely, P. M. Evidence for "gibherellin- like" substances from flowering plants. Proc. Nat. Acad. Sci. U. S. 43: 398^04. 1957. 21. Pilct, P. E. Action dcs gii)bcrellines sur I'activitc auxines-oxydasique de tissus cultivi^s in i///ro. Compi. Rend. .\cad Sci. Paris. 245: 1327.1328. 1957. (Cited from Stodola, Frank H. Source Book on Gibberellin. 1828-1957. USDA Agr. Res. Service. 1958.) Physiological Action of Gibberellin 609 22. , and Wiiiglci, VV. Action des gihberellincs sur la croissance et Tactivitd auxines-oxydasi(juc du Trifolium ochroleucum Hudson. Bui. Soc. Bot. Suisse. 68: 54-63. 1958. 23. Purves, W. K., and Hillman, W. S. Response of pea stem sections to indole- acetic acid, gibberellic acid, and sucrose as affected by length and distance from apex. Physiol. Plant. 11: 29-35. 1958. 24. Ricard, J. R., and Nitsch, J. P. Natural growth factors for young coleoptiles of wheat. Plant Physiol. 33 (suppl.): xxxii. 1958. 25. Stiuz, R. E., and Watanabe, R. The gibberellins. II. The effect of gibberellic acid and photoperiod on indoleacetic acid oxidase in Lupinus albus L. Semi- Ann. Rep. Biol. Med. Res. Div. Agr. Nat. Lab. ANL-5732: 107-109. 1957. {Cited from Stodola, Frank H. Source Book on Gibberellin. 1828-1957. USDA Agr. Res. Service. 1958.) 26. van Overbeek, J., Racusen, D. W., Tagarai, M., and Hughes, W. J. Simultan- eous analysis of auxin and gibberellin. Plant Physiol. 32 (suppl.): xxxii. 1957. 27. Vlitos, A. J., and Meudt, W. The effect of light and of the shoot apex on the action of gibberellic acid. Contr. Boyce Thompson Inst. 19: 55-62. 1957. 28. Whaley, W. G., and Kephart, J. Effect of gibberellic acid on growth of maize roots. Science. 125: 234. 1957. 29. Wittwer, S. H., and Bukovac, M. J. The effects of gibberellin on economic crops. Econ. Bot. 12: 213-255. 1958. 30. , Bukovac, M. J., McVey, G. R., and Ballard, J. C. Gibberellin modifica- tions of photoperiod controlled growth in herbaceous plants. Naturwis. 46: 117,118. 1959. 31. Yabuta, T., and Hayashi, T. Biochemical studies of "Bakanae" fungus of rice. Jour. Imp. Agr. Exper. Sta. Tokyo. 3: 365-400. 1940. A. W. GALSTON and D. C. McCUNEi Yale University An AnatysLS of GibbereltLn- Auxin Interaction and Its Possible Metabolic Basis It has now become clear that in plant tissues which respond both to gibberellin and to auxin, several kinds of interactions are possible be- tween these compounds. In such objects as the sub-apical etiolated pea epicotyl section, the growth increment produced by a joint ap- plication of the two substances is usually considerably less than, and never significantly more than, the expected sum of the growth in- crements produced by the separate administration of these com- pounds (7, 18). On the other hand, in such objects as the sub-apical green pea stem section (1,7), the sweet potato petiole (14), fruit cells (15, 21), and in starved or otherwise pretreated etiolated tissues (7, 9, 24) a true supra-additivity or synergism has been reported to exist between the growth effects produced by these compounds. In view of the fact that several theories of auxin-dependent gibberellin action have been proposed (1,7), it seemed desirable to analyze this situation more systematically in a single plant tissue, to attempt to determine the conditions under which synergistic interaction occurs. This has been a major aim of the work here described. In brief, we, and Purves and Hillman (19) of this laboratory, have found, in contrast with previously published reports (7, 9, 24), that etiolated pea tissue, no matter how pretreated, does not show a synergistic interaction between auxin and gibberellin. On the other hand, green pea stem tissue de- rived from plants grown under 8-hr. daily photoperiods almost in- variably manifests a marked synergism. Prolongation of the dady duration of illumination to 16.5 or 24 hrs. of high light intensity re- sults in a diminution or even a disappearance of this synergism, ex- cept under special conditions. iPredoctoral fellow of the National Science Foundation. The work on peroxi- dases constitutes a portion of a Ph.D. thesis submitted by D. C. McCune to the Graduate School of Yale University in June 1960. Subsequently: Boyce Thompson Institute for Plant Research, Inc., Yonkers, N. Y. [611] 612 A. W. Galston and D. C. McCune In view of the fact that synergistic interactions between auxin and gibberellin do occur in certain tissues, a second aim of this work has been an attempt to elucidate some metabolic basis for the growth interaction. In this search we have concentrated on the peroxidases, which are known to inactivate auxins in vitro (5) and which have been implicated in certain growth and developmental phenomena (3). In corroboration of previous reports (10) we have found a marked effect of gibberellins on the peroxidase activity of sensitive cells, es- pecially in dwarf plants. We have further studied this effect in de- tail by an electrophoretic separation of peroxidase into its various component fractions, and by delineation of the particular fractions which are affected by gibberellin. GROWTH EXPERIMENTS WITH PEA STEM SECTIONS Materials and Methods 'Alaska' peas, purchased from Associated Seed Growers, New Ha- ven, Connecticut, were soaked in tap water for 4 hrs. and sown in polyethylene containers in water-saturated vermiculite. Etiolated plants were grown for 7 days in a dark cabinet in a dark room main- tained at ca. 27° C, and were exposed only briefly at harvest to a dim green safelight, produced by wrapping a 15 watt Sylvania green flu- orescent tube with three layers of green and three layers of amber du Pont cellophane. Subapical 5 mm. sections were cut with a guillotine, as previously described (6). Ten such sections w^ere permitted to grow overnight in the dark in 5 ml. of growth medium in a 7.5 cm. petri dish. This medium consisted of a 1 per cent sucrose solution con- taining 0.02 M KH.PO^-Na.HPO^ buffer, pH 6.1, plus lO-^ M gib- berellic acid (GA) and 10-c or lO-s M indole-3-acetic acid (lAA) where indicated. The GA was obtained from Dr. P. W. Brian of Imperial Chemicals Industries and the lAA from Nutritional Biochemicals Co. Both were made up as 10=^ M stock solutions and stored in the dark in the refrigerator for no longer than one month. Growth of the sections was measured to the nearest 0.1 mm. under a dissecting micro- scope after about 18 hrs. Group averages and standard error of the mean were computed. In all experiments here reported, the standard error was 5 per cent of the mean or below. Growth was also measured by obtaining fresh weights of the groups of ten sections after gentle blotting. Green pea plants were grown in three controlled-condition rooms maintained at ca. 23° C, and at photoperiods of 8, 16.5, and 24 hrs. The light intensity at the growing tables was ca. 1,500 foot candles, coming from a bank of mixed fluorescent and incandescent lights. Plants on the growing tables were automatically subirrigated twice Gibberellin-Auxin Interaction and Metabolic Basis 613 daily with a nutrient solution composed of 120 g. Hyponex (Hydro- ponics Chemicals Co., Copley, Ohio) per 100 1. tap water. Subapical 5 mm. stem sections were obtained from 14- to 18-day-old plants with a double-bladed cutting tool. Green sections were grown under high intensity (ca. 1,200 foot candles) fluorescent light in a medium con- taining sucrose and buffer as above, and GA and lAA, where indi- cated, at 10-4 ][/[ These conditions have recently been determined in our laboratories to be optimal for the growth of such sections, but space limitations preclude detailed description of such experiments here. Measurement of the growth of green sections was made by the same methods as detailed above. Results Typical results with etiolated peas are shown in Table 1, which gives the growth increments produced by lAA and GA alone, and the two together, when supplied to (a) sections from completely etiolated plants, (b) sections from etiolated plants exposed to weak red light 24 hrs. prior to harvest, (c) etiolated sections pretreated with GA or control media for 30 min. and then transferred to growth solution containing or lacking lAA, and (d) sections from 100 mm. long etio- lated epicotyls treated basally for 30 min. with GA, then excised and placed in growth solutions containing lAA. In the latter two se- ries the pretreatment with GA followed previously published methods (7) of immersion in 1 per cent sucrose and buffer -f 10 ^ M GA for the indicated period. The completely etiolated sections show a large response to GA, a larger response to lAA, and less than additive effects of the two compounds together. Red light pretreatment reduces the endogenous growth and response to auxin, as previously reported (4), as well as the response to GA. GA pretreatment, either to sections or Table 1 . Absence of GA-IAA synergism in etiolated pea epicotyl sections derived from plants treated in various ways. All sections initially 5 mm. long. All figures are averages of two closely checking means, each derived from ten sections growing in a single dish. Data in lower section of table represent a separate experiment. Endog- enous Growth, Mm. AL, lAA, Mm. AL, GA, Mm. A L, GA + lAA , Mm. Treatment Calc. Obs. Obs.- Calc. Etiolated Red pretreatment 1.30 1.11 2.63 2.32 1.15 0.72 3.78 3.04 3.24 2.75 -0.54 -0.29 GA pretreatment (sections) 1.84 1.39 1.49 1,85 1.29 1.21 2.78 3.06 1.92 2.17 -0.86 GA pretreatment (epicotyls) -0.89 614 A. W. Galston and D. C. McCune to 100 mm. long epicotyls, yields below additive growth increments in the presence of lAA. Thus, it is clear that in no instance is there any GA-IAA synergism. This disagrees with some of our previous re- sults (7) for reasons which we cannot at present explain. Drs. ^V. S. Hillman and \\\ K. Purves have also obtained data like those in Table 1, and pointed out the non-synergistic interaction to us. Their results will be published elsewhere. Similar experiments were performed with stem sections derived from green peas grown under 8, 16.5, and 24 hr. daily light periods. The results are shown in Table 2, from which the following conclu- sions can be drawn: (a) Endogenous growth and response to exogenous lAA are lower in sections derived from the 8-hr. photoperiod plants than in those from 16 and 24 hr. plants, (b) GA response is independ- ent of the photoperiod of the parent plant and is markedly lower in green tissue than in etiolated tissue. Although GA response in the Table 2. GA-IAA synergism in green pea stem sections as affected by daily dura- tion of light to which the parent plant was exposed. Details as in Table 1 . Endog- enous Growth, Mm. AL, lAA, Mm. AL, GA, Mm. A L, GA + lAA, Mm. Daily Duration of Light, Hrs. Calc. Obs. Obs.- Calc. 8 16.5 24 0.85 1.11 1.15 1.87 2.72 2.93 0.40 0.52 0.45 2.27 3.24 3.38 2.89 3.44 3.26 +0.62 +0.20 -0.12 absence of lAA is small, it is not completely lacking as in the ex- periments of Brian and Hemming (2). (c) Marked GA-IAA synergism occurs in the 8 hr. sections, none in the other sections. An experiment was next performed to study the effect of passage of GA through various lengths of green stem tissue on its subsequent interaction with lAA administered to sections excised from the pre- treated stems. Previous experiments had reported such effects to be large (8). For this purpose, green plants were harvested from the 8, 16.5, and 24 hr. photoperiod rooms, decapitated just below the ter- minal bud, cut to lengths of 20, 50, or 100 mm., and then freed of all leaves. These leafless stems were then immersed basally in GA (10-* M -|- 1 per cent sucrose -f buffer) or control solutions for 1 hr. After this, apical sections were excised, placed in lAA-containing (or control) solutions, and permitted to grow for 18 hrs. The results are shown in Table 3. It can be seen that in all three groups there was a synergism manifested in the 100 mm. lengths, while only the 8 hr. group showed synergism at the 20 mm. length (and in the 5 Gibberellin- Auxin Interaction and Metabolic Basis 615 Table 3. Effect of length of stem through which GA passes on degree of subse- quent synergism with lAA in section growth. Photo- period, Hrs. Length of Stem, Mm. Endog- enous Growth, Mm. AL, lAA, Mm. AL, GA, Mm. A L, GA + lAA, Mm. Calc. Obs. Obs.-Calc. 8 20 0.86 2.63 0.19 2.82 3.23 -fO.^7 50 0.88 2.77 0.33 3.10 3.14 +0.04 100 1.12 2.12 0.29 2.41 3.20 ^-0.79 16.5 20 0.98 2.38 0.51 2.89 3.01 +0.12 50 1.05 2.75 0.43 3.18 3.44 +0.26 100 1.38 2.41 0.18 2.59 3.11 +0.52 24 20 1.07 2.35 0.20 2.55 2.81 +0.26 50 0.80 3.09 0.68 3.77 3.24 -0.53 100 0.91 2.73 0.28 2.91 3.56 +(9.55 mm. sections themselves, as in Table 2). One puzzling fact is that no series showed synergism at the 50 mm. length of pretreated stem. We cannot at the moment explain this, but it seems not to obviate the conclusion that in stem tissue from the longer photoperiod plants, which did not show GA-IAA synergism in 5 mm. sections, synergism was obvious in the longest stems treated. These results thus corroborate and extend our previous findings (7). The possibility remained that it was the 1 hr. gap in time be- tween GA and lAA treatments, rather than the length of stem, which was responsible for the synergism induced in the previous experiment. This was tested by dipping excised 5 mm. sections from each of the different photoperiod groups into GA-sucrose-buffer or control so- lutions for 1 hr., then transferring them to growth solutions contain- ing or lacking lAA for an additional 17 hrs. The results, shown in Table 4, indicate that the time lapse is not the important factor, in that synergism is clearly manifested once again only in the 8 hr. photoperiod peas. In a subsequent experiment (Table 5) it was found that, in the 8 hr. peas, the growth increments due to GA, as well as the GA-IAA synergism, were induced equally by exposure of the 5 mm. sections to 1, 10, or 60 min. of 10-^ M GA. These effects resemble similar ones for lAA already described (6), and indicate that timing of 616 A. W. Galston and D. C. McCune Table 4. GA-IAA synergism in green pea stem sections pretreated with GA for 1 hr. and later exposed to lAA. Photo- period, Hrs. Endog- enous Growth, Mm. AL, lAA, Mm. AL, GA, Mm. AL, GA + lAA Mm. Calc. Obs. Obs.-Calc. 8 0.93 1.96 0.32 2.28 3.05 +0.77 16.5 1.06 2.26 0.48 2.74 3.03 0.29 24 1.05 2.42 0.32 2.74 3.02 0.29 GA and lAA "pulses" are not important in determining either the growth or degree of synergistic interaction. In summary, it appears that GA-IAA synergism is not demon- strable in etiolated tissue, but shows up in appropriately treated green tissue. The factors that enhance the synergism in green tissue seem to be short duration of daily illumination and possibly passage of GA through a considerable length of stem. THE EFFECTS OF GA ON PEROXIDASES IN DWARF PEAS AND CORN Several previous investigators have reported that dwarf plants in such widely divergent genera as Phaseolus, Epilobium, Zea, and Pisum have much greater peroxidase activity than their normal coun- terparts (8, 12,20,23). In view of the fact that GA is known to alter the phenotype of certain dwarfs to normal (1, 17), it seemed im- portant to discover whether the peroxidase activity of such GA-treated plants was also altered to the normal pattern. The plants used in these investigations were dwarf peas, 'Progress No. 9' and tall peas, 'Alaska,' both obtained from Associated Seed Growers, and a segregating popu- lation of corn, yielding 75 per cent tall plants and 25 per cent dwarf- 1 Table 5. Effect of duration of pretreatment with 10 '' M GA on degree of syner- gism with lAA in green pea sections derived from 8 hr. photoperiod plants. Pretreat- ment Time, Min. Endog- enous Growth, Mm. AL, lAA, Mm. AL, GA, Mm. AL, 1 [AA -t- GA, Mm. Calc. Obs. Obs.-Calc. 1 0.87 1.91 0.23 2.14 2.77 +0.63 10 0.91 2.04 0.34 2.38 2.81 +0.43 60 0.99 2.21 0.14 2.35 2.94 +0.59 GihbcrelUn-Auxin Interaction and Metabolic Basis 617 mutants, obtained from Dr. B. O. Phinney, of the University of Cali- fornia at Los Angeles. The genetic differences between the dwarf and tall peas are partially unknown and probably complex, but the dwarf- 1 Zea mutant is known to differ from the wild type by a single gene (17). The peas were grown in the light as described above, except that the temperature was maintained at ca. 17° C, rather than 23° C. Fourteen days after planting, half of the plants were treated with 1 /xg. GA in 0.003 ml. ethanol, applied to the stipules enclosing the fifth internode. At various intervals after treatment, the plants were har- vested and the fifth internodes excised and homogenized with a pre- chilled mortar and pestle in 0.025 M pH 6.1 phosphate buffer (1 g. fresh wt/10 ml homogenate). The homogenate was then stored in a deep freezer in Lusteroid centrifuge tubes until further use. The peroxidase activity was unchanged by such storage. Seven days after planting, the corn was treated with 1 /xg. GA in 0.003 ml. ethanol applied to the tip of the first leaf as it emerged from the coleoptile. Two and four days later, the basal third of the first leaf sheath (the rapidly elongating region) of 20 to 30 plants was ex- cised, combined, and homogenized and stored as above. Prior to assay, the tissue homogenate was centrifuged at 2,000 X gravity for 10 min. and the clear supernatant made up to standard volume and used for peroxidase and protein nitrogen determinations, according to previously-published procedures (16, 22). The usual sub- strate for peroxidase determinations was pyrogallol, but lAA and guaiacol were also used extensively. Most of the data were obtained with a Klett-Summerson photoelectric colorimeter, but a Spectracord recording spectrophotometer was also employed in later studies, es- pecially with lAA. The results with pyrogallol as substrate are pre- sented in Table 6. It is clear that GA greatly promotes the growth of both dwarfs, while markedly lowering the peroxidase activity per unit protein N. In the dwarf corn, the peroxidase activity of both dwarf and normals rises with increasing age, and the depressive effect of GA appears to result from a prevention of this normal increase. The GA produces much smaller effects on tall corn, and is entirely without effect on tall peas. Thus, based on pyrogallol as a substrate, GA can be said to depress the abnormally high peroxidase activity of dwarf plants. When the experiment of Table 6 was repeated with guaiacol as a substrate, then GA was found to increase, rather than decrease, the peroxidase activity of the dwarf tissue. This corroborates a recent re- port (10) on the effect of GA on the peroxidase of rice, in which guaiacol was used as a substrate. These opposite results forced us to 618 A. W. Gahton and D. C. McCune Table 6. The effect of GA application on the growth and peroxidase acti\-ities of dwarf and tall peas and corn. Peroxidase substrate was pyrogallol; data obtained on Klett-Summerson photoelectric colorimeter with a blue filter. GA Treatment Length of Com Leaf Sheath or Pea Internode in Mm. Peroxidase Acti\-ity in hlfiM Purpurogallin/ /ig Protein N/Min Plant 2 days 4 days 2 days 4 days Dwarf corn Dwarf corn Tall corn Tall corn + + 15 ± 0.6* 32 ± 0.9 42 ±0.9 53 ± 1.2 20 ± 0.5 44 ± 1 . 6 53 ± 1.5 64 ± 25 52 45 45 40 73 1 56 1 55 52 Dwarf pea, 'Progress No. 9' Dwarf pea, 'Progress No. 9' Tall pea, 'Alaska' Tall pea, 'Alaska' + 5,3 ± 0.13 9.1 ±0.43 21.3 ± 0.92 21 .6 ± 0.83 38 1 28 1 21 19 * Standard error. t Difference significant at the 1 per cent level by analysis of variance. the conclusion that GA induces some alteration of the peroxidase complex of enzymes in the plant such that the relative activities to- ward different substrates are altered. This is not altogether surprising, since it is well known that the peroxidases of plants such as horse- radish and sweet potato are resolvable by electrophoresis into about five components (11, 13). We therefore decided to subject the peroxi- dases of the dwarf and normal corn to electrophoretic separation, in an attempt to delineate further the nature of the changes in peroxi- dase activity produced by GA. The procedure was as follows: 75 g. dry potato starch was washed repeatedly with distilled water, then dried by filtration on a Biichner funnel. To the dried starch were added 33 ml. of buffer 0.02 M, pH 6.1, KHoP04-NaoHP04. The resulting slurry was poured into a trough 40 X 2.5 X 10 cm. and ca. 9 ml. of exuded solution removed by fdter paper blotters. A segment 1 cm. long was then removed from the middle of the block, and replaced by a slurry of 1.2 ml. of centrifuged plant homogenate (see paragraph below) in 1.8 g. dry washed starch. The block was then placed in an E-C Co. electrophoresis apparatus and exposed to 400 V (ca. 2.8 mA) for 12 hrs. in a cold room main- tained at 2° C. After 12 hrs., the block was cut into 1 cm. segments, each of which was placed in a centrifuge tube containing buffer. The > I- < liJ < X o q: ui a. J 12mm ft J 22 mm 38mm /I 50 mm *y ri 63mm U - s 0 72 mm 10 10 20 10 10 20 DISTANCE CM. Fig. 1. Electrophoietic patterns of peroxidases in the normal leaf sheath of corn as a function of length of the sheath. Guaiacol substrate. 620 A. W. Galston and D. C. McCune starch was stirred thoroughly and centrifuged down, the supernatant being drawn off with a pipette and used for the peroxidase assay. For each electrophoretic analysis, the second leaf sheaths of ca. 30-40 plants were harvested. The final homogcnate represented ca. 250 mg. fresh weight of tissue per ml. This homogenate was centri- fuged at ca. 20,000 X gravity for 15 min. and the precipitate discarded. The supernatant liquid was saturated with (NH4)2S04, and the pre- cipitate removed by centrifugation (20,000 X gravity, 15 min.) 3 hrs. later. The precipitate was dissolved in 2.5 ml. buffer, then dialyzed for a total of 20 hrs. against three successive volumes of 200 ml. buffer in the cold room. The final residue in the dialysis bag was again clarified o < CO < o o (E UJ 0. GUAIACOL PYROGALLOL 0 10 DISTANCE CM. 20 Fig. 2. Klcctrophoiclic pattern of peroxidases from normal leaf sheath of corn. Guaiacol or pyrogallol substrate. Gibberellin-Auxin Interaction and Metabolic Basis 621 by centrifugation, and 1.2 ml. of ihe total of ca. 2.5 ml. then used in the electrophoretic separation. The electrophoretic peroxidase patterns are shown in Figures 1, 2, and 3. Figure 1 shows the developmental pattern of peroxidases in the normal leaf sheath as it grows from a length of 12 mm. to a length of 70 mm. in the course of 6 days. Clearly, there is initially a single major peak, which gradually becomes more complex until there are ultimately at least five major components. Pyrogallol and guaiacol give the same general spectrum, differing only in the relative height of the various peaks (Figure 2). Figure 3 shows that at the latest stage of development studied. > < iij CO < Q X o a: DWARF NORMAL 10 20 DISTANCE CM. Fig. 3. Electrophoretic pattern of peroxidases from leaf sheath of normal and dwarf corn. Guaiacol substrate. 622 A. \\ . Galston and D. C. McCune the dwarf differs from the normal mainly in elevation of one major peak and depression of another with guaiacol as a substrate. When the dwarf is treated with GA and the peroxidase assayed with guai- acol, these two peaks are reciprocally affected (Figure 4). It thus appears that GA treatment results in a qualitative change in the peroxidase components of the dwarf corn leaf sheath, the changes induced resembling those produced by the normal allele. > H < liJ < Q X o cr. UJ a. DISTANCE Fig. 4. Electrophoretic paUcni of peroxidases from leaf sheath of dwarf corn treated with GA. Guaiacol substrate. GibberelUn-Auxin Interaction and Metabolic Basis 623 DISCUSSION The significance of tlie GA-IAA synergism for the control of growth in certain green tissues, and of the absence of such synergism in etio- lated tissues is at present impossible to ascertain. In another article in this volume, Hillman and Purves present considerable evidence that, in etiolated pea tissue at least, GA and lAA operate through largely independent pathways. A similar analysis has not yet been per- formed for green stem tissue. What is the significance of these findings on peroxidase levels as affected by GA? In view of the uncertainty concerning the in vivo function of peroxidase, little of a definite nature can be stated. How- ever, the following points seem suggestive of some significance, at least, (a) The changes induced by GA on peroxidase in dwarfs are de tectable at least as early as the growth effects can be noted. They are therefore probably not merely distant and secondary consequences of the alteration of growth by GA. (b) The simple nature of the dif- ferences between the peroxidase pattern of the normal and dwarf plants seems consistent with the fact that they differ in a single gene. (c) The relatively simple and specific effect of GA on the peroxidase pattern seems consistent with the biochemical amelioration of a simple genetic abnormality. Considerably more work is obviously needed to assess these in- teresting possibilities more adequately. ACKNOWLEDGMENTS W^e wish to express our gratitude to Miss Mary Lyons, who per- formed many of the pea stem section growth experiments described in the paper. We also wish to acknowledge the generous support fur- nished by the National Science Foundation and the U.S. Public Health Service to one of us (A. W. Galston). LITERATURE CITED 1. Brian, P. W., and Hemming, H. G. The effect of gibberellic acid on shoot growth of pea seedlings. Physiol. Plant. 8: 669-681. 1955. 2. , and Hemming, H. G. Complementary action of gibberellic acid and auxins in pea internode extension. Ann. Bot. II. 22: 1-17. 1958. 3. Galston, A. W. Some metabolic consequences of the administration of indole- acetic acid to plant cells. In: R. L. Wain and F. Wightman (eds.). The Chem- istry and Mode of Action of Plant Growth Substances, pp. 219-233. Butterworth Sci. Publ., London. 1956. 4. , and Baker, R. S. Studies on the physiology of light action. V. Photo- inductive alteration of auxin metabolism in etiolated peas. Amer. Jour. Bot. 40: 512-516. 1953. 5. , Bonner, J., and Baker, R. S. Flavoprotein and peroxidase as compo- nents of the indoleacetic acid oxidase system of peas. Arch. Biochem. Biophys. 42: 456-470. 1953. 624 A. W. Gahton and D. C. McCitne 6. Galston, A. W., and Hand, M. E. Studies on the physiology of light action. I. Auxin and the light inhibition of growth. Amer. Jour. Bot. 36: 85-94. 1949. 7. , and Warburg. H. An analysis of auxin-gibberellin interaction in pea stem tissue. Plant Physiol. 34: 16-22. 1959. 8. Haan, I. de, and Gorter, C. J. On the differences in longitudinal growth of some varieties of Pisiim sativum. Rec. Trav. Bot. Neerl. 33: 434-446. 1936. 9. Hayashi, T., and Murakami, Y. Studies on the physiological action of gibber- ellins (Part I). Bui. Nat. Inst. Agr. Sci. Japan. 7D: 159-197. 1958. 10. , Murakami, Y., and Matsunaka, S. Biochemical studies on bakanae fungus. XXXVI. The physiological action of gibberellin. VIII. Changes in the activities of various enzymes in leaf sheaths of rice plants treated with gib- berellin. Bui. Agr. Chem. Soc. Japan. 20: 159-164. 1956. 11. Jermyn, M. A., and Thomas, R. Multiple components in horse-radish peroxi dase. Biochem. Jour. 56: 631-639. 1954. 12. Kamerbeek, G. A. Peroxidase content of dwarf types and giant types of plant. Acta Bot. Need. 5: 257-267. 1956. 13. Kondo. K., and Morita, Y. Phytoperoxidase. II. Isolation and purification of sweet-potato peroxidases and their absorption spectra. Bui. Res. Inst. Food Sci. Kyoto Univ. 10: 33-45. 1952. 14. Kuse, G. Necessity of auxin for the growth effect of gibberellin. Bot. Mag. Tokyo. 71: 151-159. 1958. 15. Luckwill, L. C. Fruit growth in relation to internal and external chemical stimuli. Symp. Soc. Stud. Devel. Growth. 17: 223-251. 1959. 16. McCune, D. C., and Galston, A. W. Inverse effects of gibberellin on peroxidase activity and growth in dwarf strains of peas and corn. Plant Physiol. 34: 116-418. 1959. 17. Phinnev, B. O. Growth response of single-gene dwarf mutants in maize to gibbercilic acid. Proc. Nat. Acad. Sci. U. S. 42: 185-189. 1956. 18. Purves, W. K., and Hillman, W. S. Response of pea stem sections to indole- acetic acid, gibberellic acid, and sucrose as affected by length and distance from the apex. Physiol. Plant. 11: 29-35. 1958. 19. , and Hillman, W. S. Experimental separation of gibberellin and auxin action in etiolated pea epicotyl sections. Physiol. Plant. 12: 786-798. 1959. 20. Ro.ss, H. tibcr die Verschiedenheiten des dissimilatorischen Stoffwechsels in reziprokcM /•,7^//o/ni////-Bastardcn und die physiologischc-gcnetische IJrsache der reziprokcn Unterschiede. I. Die Aktivitat der Peroxydase in reziproken Epilo- 6ium-Bastarden mit der Sippe Jena. Zeitschr. Abst. Vererb. 82: 503-529. 1941. 21. Schroeder, C. A., and Spector, C. Effect of gibberellic acid and indoleacetic acid on growth of exci.scd fruit tissue. Science. 126: 701,702. 1957. 22. Siegel, S. M., and Galston, A. W. Peroxide genesis in plant tissues and its rela- tion to indoleacetic acid destruction. Arch. Biochem. Biophys. 54: 102-113. 1955. 23. van Overbeck, J. The growth hormone and the dwarf type of growth in corn. Proc. Nat. Acad. Sci. U. S. 21: 292-299. VX^'k 24. Vlitos, A. J., and Meudt, W. The effect of light and of the shoot apex on the action of gibberellic acid. Contr. Boyce Thompson Inst. 19: 55-62. 1957. DISCUSSION Dr. Burstrom: Do you think, Dr. Galston, that the change in j)er- oxidase activity, which amounted to something between 25 and 40 per cent, can account for an increase in growth by 100 per cent or more? It is diflicult to compare the amounts or the enzyme activities, Gibberelliyi-Auxin Interaction and Metabolic Basis 625 since, when you have two pieces of plants growing at 100 per cent difference in elongation, you have a different morphological pattern. You have, ot course, referred activity to amount of protein, which is the most natural way, but it does not necessarily follow that this is the right expression of the real activity of the enzyme when you have such a fundamental difference in the organization of the tissues, as you must have. Dr. Galston: Your point is certainly well taken. Remember, how- ever, that although we produced only a 40 per cent change in over-all peroxidase activity, we have produced very much greater changes in one or two of the electrophoretically separable peroxidase peaks. The changes can be of the order of several hundred per cent when cal- culated for the individual peroxidase components. As for the mechan- ism by means of which a peroxidase could inhibit growth, we do not know. It may destroy auxin, it may make lignin, or it may do other things. All we can say is that we have here a specific reversal by gib- berellin of a biochemical abnormality in a single gene dwarf mutant. Whether this has significance for the control of growth is for the fu- ture to tell. S. HOUSLEY and B. J. DEVERALLi Unilever Ltd. Sharnbrook, Bedford, England The Influence of GibberelUc Acid on lndole-3- acetlc Acid Disappearance From Solutions Containing Excised Pea Stem. Tissues and I ndole-3 -acetic Acid Oxidase Since 1957 it has been widely recognized that the gibberellins can markedly modify plant growth and development including, for ex- ample, changing the quantity of solid matter in various tissues (2, 7, 23), replacing the light requirement for germination of light-sensitive seeds (14), and influencing the time of flowering (17, 26). Their most characteristic property, however, is the ability to promote stem elongation in many plants, including tall and dwarf varieties of pea, the latter showing the greater increase in growth rate (3). Using seedlings of 'Alaska' pea grown in complete darkness, Lockhart (20) obtained little or no response to applied gibberellins. When seedlings the growth of which had been inhibited by red light were treated, their growth rate was restored to that in darkness. On the other hand, with the dwarf 'Progress No. 9,' growth rate in darkness was increased under the influence of gibberellins, but this enhanced growth was also equalled by gibberellin-treated dwarfs grown in light. Rather different results have been obtained by Brian and Hem- ming (4) with segments excised from stems of dwarf 'Meteor' pea. No increased elongation with gibberellic acid (GA) in light was obtained with sections from light-grown plants, whereas in the dark, with sec- tions from dark-grown plants, Brian, Hemming, and Radley (6) ob- ^ Subsequently: Department of Plant Pathology, University of Wisconsin, Madi- son 5, Wisconsin. Botanv Department, Imperial College of Science and Technology, London, S.W. 7. " [627] 628 S. Housley and B. J. Deverall tained small elongations. However, Brian and co-workers appear to have used different techniques in these studies, and it has been re- cently shown by Purves and Hillman (25) that, at least for dark-grown plants, the size of sections used and position on the stem from which they are excised are of importance in determining response. The results of Brian and Hemming (4) are of interest in that they show a synergism between GA and indole-3-acetic acid (lAA). Such synergisms have not always been obtained (15, 25). The less recent literature has been summarized by Stowe and Yamaki (26). In Brian and Hemming's experiment, section growth in light was examined in a basal medium of 2 per cent sucrose -f- phosphate buffer, medium -\~ lAA, medium -|- GA, and medium -f GA and lAA. ^Vhile GA alone had little or no effect, when in the presence of lAA it increased sec- tion elongation over that obtained in lAA alone. Brian and Hem- ming offered no hypothesis to account for these results, but it seemed to the present authors that the synergistic enhancement of IA.\-in- duced growth by GA could result from an lAA-sparing action brought about, for example, by the blocking of an lAA-destroying system with GA. This possibility has been examined in the experiments reported in the present paper by observing the rate of disappearance of lAA from solutions containing excised pea stem tissues or pea breis oE lAA-oxidase enzyme in the presence and absence of GA. Growth of stem tissues was not measured in this study. MATERIALS AND METHODS Pisum sativum, Trogress No. 9,' (Sharpe Seed Co.) ^vas used ex- cept when stated to the contrary. GA (Imperial Chemical Industries, Ltd.) and lAA (British Drug Houses, Ltd.) were used at a concentra- tion of 10 mg/1 in all experiments. Estimation of lAA was made colorimetrically using the Salkowski reaction technique of Gordon and Weber (10) to develop the red Fe- lAA complex which was measured with a Hilger photoelectric ab- sorptiometer (1 cm. cell and Ilford 604 filter). GA with Salkowski reagent gave no absorption (against reagent as a blank in the ma- chine), and no interference occurred with lAA-color development when GA and lAA solutions were mixed and allowed to stand for 5 to 120 min. before addition of reagent. The relationship between ab- sorption and concentration of lAA is linear, and absorptions of four lAA concentrations are given below: lAA Concn. (mg/1) 2.5 5 10 20 Absorption Units 0.044 0.122 0.267 0.528 Enzyme breis for lAA-oxidase experiments were prepared from 7- day-old plants with third internodes extending. Growth was in the dark at 27° C. with occasional brief irradiation with weak green Influence of Gibberellic Acid on lAA Disappearance 629 light from a tungsten lamp for observation. Ten g. of frozen epicotyls were ground in a chilled mortar with M/50 KHoP04-Na2HP04 buffer (pH 6.4), filtered through washed cheesecloth, and made up to 110 ml. with buffer. Storage was at — 15° C. in the dark. Enzyme activity of this preparation was low, so a preliminary experiment, based on a design used by Hillman and Galston (11), was carried out to deter- mine the quantities of MnCU and 2,4-dichlorophenol that had to be added to the reaction mixture to give a suitable rate of lAA destruc- tion. A reaction mixture of lO'^M 2,4-dichlorophenol, lO-'^M MnCU, 0.02i\f phosphate buffer, and enzyme at X 1/10 of the prepared con- centration was chosen. Experiments on rate of lAA disappearance from solutions contain- ing stem sections were carried out with plants grown in the dark in the manner described above. Ten-mm. sections were cut, one per stem, from just below the first node. After randomizing, four lots of 42 sections were withdrawn, and each lot was placed in 14 ml. of test solution in a 250 ml. wide-necked conical flask. Two ml. was previously removed for an absorption reading at zero time: two flasks were controls which were prepared for reagent blanks in the absorptiometer. Preliminary experiments using 0.0166i\jr phosphate buffer (pH 6.4) and no other addendum gave little lAA destruction; therefore lO-^M 2,4-dichlorophenol and 10-^M MnCl^ were incor- porated into the test solution. Flasks were rocked at approximately 90 oscillations per minute at 27° C. Whenever 2 ml. aliquots of solu- tion were withdrawn for lAA determinations, six sections were re- moved from each flask to maintain uniform tissue/solution ratios. Manipulations for experiments in the dark were carried out in weak green light (Ilford G907 filter). Experiments with apical tissues were carried out in a similar man- ner to those with stem sections except that only 0.0166M buffer was used and no addendum as a suitable rate of lAA destruction was ob- tained. To prepare the tissues, as many ensheathing leaves as possi- ble were stripped with fine forceps from around the apex, and the remaining bud together with 10 mm. of attached stem was excised. Closer stripping could be carried out with experiments in the light than in the dark, while dark-grown plants were easier to prepare than light-grown plants. Plants grown in light were raised in a greenhouse and were used after 21 days when five or six internodes were present. In experiments carried out in light, "warm white" fluorescent tubes were used which gave an intensity of 135 foot candles at the level of the plant material in the conical flasks. Under these conditions, this light did not de- stroy any lAA when an irradiated solution (with addenda) was re- peatedly sampled over a period of 212 min. 630 5. Housley and B. J. Deverall RESULTS Experiments With Enzyme Breis In pea and other phmts an enzyme system occurs (lAA-oxidase) which destroys lAA (18,27). It was necessary, therefore, to examine whether GA has any sparing action upon rate of lAA destruction when incubated with this enzyme system. The result of one experi- ment is shown in Figure lA. In all experiments, absorption is plotted against time of sampling aliquots from the reaction mixture. It is seen that as the concentration of lAA decreases, GA has no effect upon the rate of lAA destruction. Similar results were obtained when this experiment was repeated, and also when a third experi- ment was carried out with a brei prepared from 'Alaska' peas. As the activity of crude preparations of lAA-oxidase of peas can be influenced by light (9), it was decided to examine the effect of GA on rate of lAA destruction when reaction mixtures with enzyme brei were light-irradiated throughout the experimental period. A brei from dark-grown plants was used, as light-grown plants contain an inhibitor of the lAA-oxidase system (9). The experiment was simi- lar to that shown in Figure lA except that it was carried out under fluorescent light tubes. The result was similar to those shown in Figure lA in which GA had no influence on rate of lAA destruction, while another two experiments, one of which is shown in Figme IB, 0.3 50 100 150 50 TIME IN MINUTES 150 Fig. 1. Rate of cicstiuciion of iiulolc-IJadtic acid l)v iiidolc-S-acctic oxidase enzyme in the presence (solid lines) and absence (broken lines) of gibbeiellic acid in dark- ness (A) and in lio;lit (B). Influence of Gibbcrcllic Acid on lAA Disappearance 631 showed an enhanced rate of lAA destruction with GA over part of the experimental period. In no experiment did GA exert a sparing action. Experiments With Stem Sections During the summer of 1957 extensive preliminary experiments were carried out with stem sections, following their use by Brian and Hemming (4), to investigate whether GA influences rates of lAA destruction when sections are incubated in solutions of hormone. Sections were cut serially from the entire length of stems of light- grown plants and were randomized before use. Rather variable spar- ing action results were obtained which, apart from variation caused by restricted sampling of sections, suggested that tissues of different physiological ages and states should be separated. This was done in all the following exj^eriments carried out during the summer of 1958. Results of experiments in the dark with sections from dark-grown plants are shown in Figure 2. Results in Figure 2C were obtained with 'Alaska' pea. A sparing of lAA destruction with GA was ob- tained in all experiments; however, in Figure 2B it was appreciable while in Figure 2A it was smaller, and in Figure 2C it was present over approximately only a 3 hr. period. 0.3 TIME IN HOURS Fig. 2. Rate of destruction of indole-3-acetic acid by pea stem sections in the piesence (solid lines) and absence (broken lines) of gibberellic acid in darkness (A, B, C) and in light (D). 'Alaska' peas were used for the experiment in C and 'Progress No. 9' for the remainder. 632 5. Housley and B. J. Deverall One experiment was carried out with dark-grown material in light under fluorescent tubes (Figure 2D). GA treatment resulted in a small but consistent sparing of lAA destruction. Experiments With Stem Apices Results of experiments with apical tissues are shown in Figures 3 and 4. Several curves differ from those of Figure 2 in being convex rather than concave upwards; the rate of disappearance of lAA tends to increase with time rather than decrease. Experiments carried out in the dark with tissues from dark-grown (Figure 3A, B, C) and light- grown plants (Figure 3D) show, with the exception of Figure 3C, no sparing of lAA destruction with GA; in Figure 3C there is a tendency to sparing. In Figure 3A and B it is thought that the separation of the curves after approximately 4 and 2.5 hrs., respectively, resulted from accidental removal of apical tissues from the hormone solutions during shaking in the dark (this occurred only with apical tissues, and when the volume of experimental solution became small). In contrast with the above experiments in the dark, experiments in the light using apices from dark-grown (Figure 4A and B) and light-grown plants (Figure 4C to F) show a greater range of lAA spar- ing. Marked sparing is shown in Figure 4C, while in the remainder of the experiments there is a progressive decrease until, in Figure 4F and possibly in Figure 4D, no lAA sparing occurs. 0.3 TIME IN HOURS Fig. 3. Rate of ilcstiuction of ind()lc-3-acciic acid by apical pea stem tissues with (solid lines) and without (broken lines) gibbeiellic acid in darkness. Plants grown in darkness (A, B, C) and in light (D). 0.3 CO \- z 3 I- Cl. (T O CO 00 < 0.2 - 0.2 0.2 - TIME IN HOURS Fig. 4. Rates of destruction of indole-3-acelic acid by apical pea stem tissues in the presence (solid lines) and absence (broken lines) of gibberellic acid in light. Plants were grown in darkness (A, B) or in light (C, D, E, F). 634 5. Housley and B. J. Deverall DISCUSSION The present study has shown that GA at a concentration of 10 mg/1 has no influence on the lAA-oxiclase system of pea in vitro in the dark, while in the light on some occasions it may result in an en- hanced rate of lAA destruction. Results similar to the former have been obtained by Brian and Hemming (5) and Kato and Katsumi (16) using lAA-oxidase preparations from pea roots and shoots. The present study has also shown that GA can influence rate of lAA destruction by excised pea tissues, and on some occasions the rate is reduced thus producing a sparing of I A A. The sparing effect is frequently not marked but varies considerably in magnitude from a mere trend (Figure 3C) to an appreciable effect (Figure 4C). This sparing accounts for the growth synergisms obtained by Brian and Hemming (4); also, our range of variation is consistent with the range of growth synergisms shown in Brian and Hemming's data, i.e., small synergisms ranging to marked ones. Brian and Hemming (5) state that they have considered the possi- bility that GA might reduce rate of auxin destruction, but have been unable to detect any effect of GA on lAA destruction by pea inter- node sections; however, they do not mention the method and tech- niques used; therefore it is not possible to assess the reason for their failure. On the other hand, Nitsch (24) has shown that prior appli- cation oi GA to an intact plant (Rhus lyphina) causes the level of endogenous auxin in the apical tissues to be raised, an observation that may be accounted for by an auxin-sparing action of the type described in the j)resent work. Nitsch's results (obtained by Avena straight-growth bioassay of ether-soluble materials after separation by paper chromatography) are important for they are a direct approach to the examination of effects of GA on the naturally-occurring hor- mone system of plants. The possible mechanisms whereby an auxin-sparing phenomenon may be brought about by GA are next discussed. Before carrying out our early experiments, the results of Brian and Hemming (A) were considered in conjunction with those of Lockhart (20), and for a pre- liminary hy[)othesis it was suggested that GA acts by interfering in a light-mediated system of auxin destruction; the same hypothesis has been speculatively put forward by Vlitos and Meudt (28). As it is necessary to view hyjx)theses against the background of sparing actions shown in Figures 2, 3, A, and 6, it is helpful first to summa- rize the latter more concisely (Table 1). In assessing the scoring in this table, the consistency of the sj)aring action through time was taken into account as well as the magnitude of the action. It may Influence of Gibberellic Acid on lAA Disappearance 635 Table 1 . A summary of the sparing of indole-3-acetic acid destruction (+....) obtained in Figures 2, 3, and 4. ( — ) Indicates no sparing action occurred. Stem Sections Apical Tissues Dark-grown Light-grown Dark-grown Light exp. Dark exp. Light exp. Dark exp. Light exp. Dark exp. 2D + 2A + + 2B + + + + 2C + + 4C + + + + 4D + + + 4E +(?) 4F - 3D - 4A + + + + 4B + 3A - 3B -(?) 3C + be noted that shades of opinion in rating the score {e.g.. Figure 2C to be -f-? or Figure 3C to be + + ''') ^^^ "ot alter the final deductions drawn. Table 1 shows that with apical tissue experiments carried out in light (columns 3 and 5) there are sparing actions of almost all magnitudes, whereas in the dark (colimms 4 and 6) sparing actions are either absent or not marked. There is no sharp division between experiments carried out in light and in darkness, but the largest spar- ing actions are shown in the light. In contrast with this distribution of sparing action, stem section experiments have an emphasis on sparing in experiments carried out in the dark, while in light a single experiment of low sparing permits no opinions to be formed. How- ever, it may be noted that again no sharp division exists between experiments carried out in light and in darkness. If one now views the light-mediated auxin destruction hypothesis against the results of Table 1, it is evident that at least two disturb- ing points prevent a satisfactory union between the two. The most obvious lies in the sparing actions shown in the dark (column 2), for with the above hypothesis sparing should not occur under these conditions. The second concerns the marked variation in magnitude of sparing actions referred to above. If GA were to intercede directly in a light-operated hormone-destroying mechanism, one might rea- sonably expect sparing actions to be present more consistently and to be less variable in magnitude. Further hypotheses have been considered by Brian and Hemming (5) to account for the enhancement of growth by GA. The possibility that GA may combine with auxin(s) in the plant to form a more active compound is considered and dismissed. Our own data do not readily fit into any scheme based on this idea and it does not appear to be a profitable line to pursue. Similarly, the possibility that GA 636 5. Housley and B. J. D ever all increases auxin in the plant by increasing the amount formed does not readily lend itself to the interpretation of our data in terms of any known scheme. The possibility that GA retards auxin destruction is considered by Brian and Hemming (5), but since they were unable to obtain a sparing action they took the matter no further. To sum up our own views on one aspect of this hypothesis, it is felt that there is no close relationship between light treatment and GA sparing ac- tions, and that any relationship between the two involves another f actor (s). An hypothesis favored by Brian and Hemming is that GA affects some metabolic process which normally limits growth even though auxin is present in non-limiting amounts. They discuss this idea at length in relation to their own and other data and are led to suppose that in the intact plant there is an inhibitory system which limits growth rate and that GA treatment can be envisaged as a neutraliza- tion of this inhibitory system. Thus, GA plays no direct or positive part in cell extension, but that, by neutralizing an inhibition of ex- tension, it releases the full potentialities of the auxins present. This hypothesis is attractive as it is potentially versatile enough to account for our own data; it is compatible with our own views and has some supporting evidence cited by Brian and Hemming (5). A naturally-occurring system which answers to the above require- ments is the lAA-oxidase enzymes and their inhibitor(s). One may raise arguments objecting to this choice; however, it is profitable to discuss the system and to defer objections for later discussion. The facts required to construct a theory have been briefly simimarized by Galston (8), but only limited data have been published in detail (12). In light-grown 'Alaska' pea considerable lAA-oxidase activity can be demonstrated in young stem and bud tissues while in the leaf a large amount of inhibitor is present. Some inhibitor is present also in the stem, the highest concentration occurring in apical tissues and a gradient existing down the plant (presumably the stem too). The level of inliibitor in the youngest leaves (i.e., part of the apical tissues) may be raised by treatment of the entire plant for 2 days with 10-^M GA (administered via the roots), while inhibitor level in leaves may also be raised progressively by increasing the length of exposure of the jilant to light. 1 his light effect on inhibitor level has been further investigated by Hillman and Galston (12) who have shown that in vitro lAA-oxidase activity of dark-grown 'Alaska' plants is greatly inhibited by red light given to the intact plants before harvest. The inhibition is reversible by near infrared radiation, i.e., far-red (29, p. 384), given immediately after the red light, but not more than 1 hr. afterwards. Influence of GibbereUic Acid on lAA Disappearance 637 GA I I RED NEAR INFRA-RED f^E >E/ RED ^/ NEAR INFRA-RED I^E >E/ Fig. 5. Hypothetical schemes showing possible relationships between gibberellic acid (GA) above and gibberellins (G) below and the inhibitor (I) of the indoleacetic acid oxidase enzymes (E). In dwarf 'Laurel' peas, less inhibitor is found when corresponding parts with tall types, e.g., 'Alaska,' are examined (8). To construct a scheme as in Figure 5 (top) one may imagine that when lAA-oxidase inhibitor (/) associates with the lAA-oxidase en- zyme (£), an inactive complex {EI) is formed which will not destroy lAA; the kinetics of the reaction, however, need not concern us. The level of / in a given plant part is influenced by a number of factors. Galston (8) states that GA raises the level of / in the youngest leaves, and we shall assume that this generally holds in other plant parts, in particular the stem. Red light is thought to raise the level of / also, and near infrared radiation to oppose the reaction. These points, shown diagramatically in Figure 5 (top), are linked as follows. X is a precursor of / and does not inhibit E. The reaction X-^I does not take place in a single step but passes through at least one inter- mediate compound, Y. Y but not / can be changed back to X by near infrared radiation, while in the absence of this radiation Y is slowly changed to / by an unknown process. It is necessary to postu- late the intermediate compound, Y, to account for the negative effect of near infrared radiation when applied 1 hr. after red light. 638 5. Housley and B. J. Dcvcrall Before elaborating on the above scheme, it is desirable first to examine how well the whole inhibitor hypothesis accounts for the results of Figures 2, 3, and 4. During the course of experimentation an impression was obtained that large sparing actions were associated with rapid rates of lAA inactivation and conversely smaller sparing actions with less rapid rates. Data were analyzed and the results are shown in Figure 6. Average rates of lAA destruction (no GA present) over the first 3 hr. period arc plotted against differences between aver- age rates when GA is present and absent. This period was chosen as rates of lAA destruction in many experiments were fairly uniform over this interval. Rates are expressed in absorption units to permit data from the 1957 season to be included, while points are joined merely to show trends more clearly. Figure 6 shows that the spar- ing action with GA tends to increase as the rate of lAA destruction increases. Expressing this in terms of Figure 5, as the rate of lAA destruction increases (i.e., as E increases relative to / and EI) so the effectiveness of GA increases shoiving the same effect as an increase in 1. Expressed alternatively, if EI is high and E is low, there wnll be a low rate of lAA destruction and only a small effect of GA provided the effect of GA can be equated to /. Thus, our own data are con- sistent with Galston's statement that GA increases the le\'el of / in the plant. It may be noted that Lockhart (21) has observed a similar effect: intact dark-grown plants which do not respond to applied GA give the greatest GA-induced growth promotion when decapitated (very high / in intact plants results in no growth with applied GA; however, if / is markedly reduced by decapitation of these plants, the GA-induced growth relative to the intact plants will possibly be at a maximum). It is now possible to consider the results of Table 1 against the background of the inhibitor scheme. It will be recalled that concen- tration of inhibitor is high at the apex and a gradient exists down the plant. The concentration in the sections of the second column will be lower than that in the apices of the sixth column, and therefore there will be a tendency for greater sparing actions to occur in the former; this expectation is reflected in the table. Comparison of light- and dark-grown materials (i.e., column 3 with 5, and column 4 with 6) cannot be made readily as inhibitor levels of light- and dark- grown stems do not appear to have been compared. Comparison of experiments carried out in light and in darkness (column 3 with 4, and column 5 with 6) suggests that inhibitor level in the former was less than the latter on some occasions. This variation may have been due to several factors, the most important possibly being insufficient control over the quality of light used during experimentation. A sec- Influence of Gibberellic Acid on lAA Disappearance 639 to tr. X lO >«. CO o. tr. o (O CD < O I- (/> UJ o 0.2 -0.01 0.02 0.04 0.06 lAA SPARING, ABSORPTION UNITS /3 HRS. 008 Fig. 6. Average rate of indole-3-acetic acid (lAA) destruction over the first 3 hr. period (vertical axis) plotted against the difference between the average rates of destruction over the same period in the presence or absence of gibberellic acid (horizontal axis). Curves (a) and (c) represent experiments (1957) with stem sections from light-grown material; curve (b) represents section experiments (1958) from dark-grown material; curves (d) and (e) represent experiments (1958) with apical tissues. Experiments shown in (a) were carried out with "Lincoln' peas; experiments shown in curves (b) to (e) with 'Progress No. 9' peas. Experiments of curves (d) and (e) were carried out with light- and dark-grown material, respectively. The letter over each point indicates whether the experiment was conducted in light (L) or in darkness (D). ond factor was the differing amounts of leaf tissue removed when apical tissues were being prepared for excision. Other factors may be associated with distinbances resulting from excision, a point discussed by Brian and Hemming (5). As the present data are consistent with an inhibitor interpretation in the scheme of Figure 5 (top), it is profitable to consider this system further. The formation of / is influenced by a red/near infrared re- action, and it is constructive to consider the literature. Johnson and Liverman (13) state that near infrared-induced dormancy in summer- grown tomatoes can be reversed by GA producing a striking effect on stem elongation; the same dormancy can be broken also by red light, auxins, or cool weather (19). Lockhart (22) notes that the GA-in- duced elongation of dwarf bean seedlings may be markedly enhanced by a 2 to 5 min. exposure of the plants to red light. This effect may be reversed by subsequent exposure of the plants to near infrared 640 5. Housley and B. J. Deverall radiation. Other literature relating GA to these light effects has been discussed by Brian (1). Thus, it would appear the applied GA in some systems cannot only by-pass the effects of red/near infrared radiations but the two may mutually reinforce each other. To ac- commodate this, an attractive modification of the scheme in Figure 5 (top) is to suggest that red/near infrared radiations are influencing the naturally-occurring gibberellins (Figure 5, bottom). X now be- comes Pa, a gibberellin precursor, while Y becomes G, its correspond- ing gibberellin. The mechanism of increase in I under the influence of G remains an unknown process. It may be noted that Brian (1) has been led to postulate a similar relationship between gibberellin(s), its precursor(s), and red/near infrared radiations as a residt of theo- retical considerations on mechanisms of flowering in plants. Although it is possible to discuss further this scheme (Figure 5, bottom) in relation to published literature, discussion becomes dif- fuse due to lack of adequate biochemical data. Discussion also be- comes diffuse when published data, which do not appear directly to support the scheme of Figure 5, are considered. For example, choice of the lAA-oxidase inhibitor for / may be objected to on the groimd that growth interactions between hormone and GA are observed with 2,4-dichlorophenoxyacetic acid and 1-naphthaleneacetic acid and these compounds are degraded less readily than lAA (5) and may not go through the lAA-oxidase system. If, however, one accepts this criticism without considering further the data in the literature on which it rests, it does not alter the concept that GA operates through an inhibitor system, but merely removes the scheme in Figure 5 with- out replacing it with any known inhibitor system. In view of the statements of Galston (8) on the relationship between GA and /, it would seem preferable to use Figure 5 (bottom) to design biochem- ical experiments to test the validity of the scheme in this figine. SUMMARY An examination has been made of the rates of disappearance of indole-3-acetic acid (lAA) in the presence and absence of gibberellic acid (GA) from solutions containing either lAA-oxidase enzymes from pea or excised stem tissues of pea. lAA destruction with the enzyme system in vitro was not consistently influenced by GA in darkness or in light. With apical tissues (apex, young leaves -\- 10 mm. immature stem) from light- and dark-grown plants, rate of lAA destruction in light was decreased by GA, thus causing an lAA-sparing effect, while in darkness GA had little or no effect upon rate of destruction. How- ever, there was no sharp division between the light and dark treat- ments, some experiments in light showing little or no sparing action and thus resembling the dark experiments. Influence of Gibberellic Acid on lAA Disappearance 641 AVilh stem sections taken from just below the first node of dark- grown plants, rate of lAA destruction in darkness and in light was decreased by GA. The sparing actions obtained varied in size as for apical tissues. The lAA sparing action is discussed in terms of current theories on the mechanism of action of GA-induced growth. The data are consistent with the hypothesis that GA leads to the production of an inhibitor which retards an auxin-destroying sys- tem. Evidence from the literature that this inhibitor could be an inhibitor of the lAA-oxidase enzyme system is considered. The pos- sibility that the effect of red and near infrared (far-red) radiations on growth could be brought about through an inhibitor/gibberellin- mediated system is pointed out. ACKNOWLEDGMENTS The authors wish to thank the Directors of Unilever Ltd. for permission to publish this work, and to thank Mr. N. E. Wynn for assistance with the experiments and preparation of the manuscript. LITERATURE CITED 1. Brian, P. \V. Role of gibberellin-like hormones in regulation of plant growth and flowering. Nature. 181: 1122,1123. 1958. 2. , Elson, G. W., Hemming, H. G., and Radley, M. The plant-growth- promoting properties of gibberellic acid, a metabolic product of the fungus Gibberella fujikuroi. Jour. Sci. Food Agr. 5: 602-612. 1954. 3. , and Hemming, H. G. The effect of gibberellic acid on shoot growth of pea seedlings. Physiol. Plant. 8: 669-681. 1955. 4. , and Hemming, H. G. A relation between the effects of gibberellic acid and indolylacetic acid on plant cell extension. Nature. 179: 417. 1957. 5. Brian, P. W., and Hemming, H. G. Complementary action of gibberellic acid and auxins in pea internode extension. Ann. Bot. II. 22: 1-17. 1958. 6. , Hemming, H. G., and Radley, M. A physiological comparison of gib- berellic acid with some auxins. Physiol. Plant. 8: 899-912. 1955. 7. Bukovac, M. J., and Wittwer, S. H. Gibberellic acid and higher plants: I. Gen- eral growth responses. Quart. Bui. Mich. Agr. Exper. Sta. 39: 307-320. 1956. 8. Galston, A. W. Studies on indoleacetic acid oxidase and its inhibitor in light- grown peas. Plant Physiol. 32 (suppl.): xxi. 1957. 9. , and Baker, R. S. Studies on the physiology of light action. III. Light activation of a flavoprotein enzyme by reversal of a naturally occurring in- hibition. Amer. Jour. Bot. 38: 190-195. 1951. 10. Gordon, S. A., and Weber, R. P. Colorimetric estimation of indoleacetic acid. Plant Physiol. 26: 192-195. 1951. 11. Hillman, W. S., and Galston, A. W. Interaction of manganese and 2,4-dichlo- rophenol in the enzymatic destruction of indoleacetic acid. Physiol. Plant. 9: 230-235. 1956. 12. , and Galston, A. W. Inductive control of indoleacetic acid oxidase activ- ity by red and near infrared light. Plant Physiol. 32: 129-135. 1957. 13. Johnson, S. P., and Liverman, J. L. The control of summer dormancy in tomato by gibberellic acid. Plant Physiol. 32 (suppl.): xlviii. 1957. 14. Kahn, A., Goss, J. A., and Smith, D. E. Effect of gibberellin on germination of lettuce seed. Science. 125: 645, 646. 1957. 642 S. Housley and B. J. Deverall 15. Kato, J. Studies on the physiological effect of gibberellin. IT. On the inter- action of gibberellin with auxins and growth inhibitors. Physiol. Plant. 11: 10-15. 1958. IG. . and Katsumi, M. Effect of gibberellins on lAA-oxidase. Naturwis. 45: 344. 1958. 17. Lang, A. The effect of gibberellin upon flower formation. Proc. Nat. Acad. Sci. U. S. 43: 709-717. 1957. 18. Larsen, P. Formation, occurrence, and inactivation of growth substances. Ann. Rev. Plant Physiol. 2: 169-198. 1951. 19. Liverman, J. L., and Johnson, S. P. Control of arrested fruit growth in tomato by gibberellins. Science. 125: 1086, 1087. 1957. 20. Lockhart, J. A. Reversal of the light inhibition of pea stem growth by the gibberellins. Proc. \at. Acad. Sci. U. S. 42: 841-848. 1956. 21. . Studies on the organ of producton of the natural gibberellin factor ni higher plants. Plant Physiol. 32: 204-207. 1957. 22. . The light requirement for a gibberellic acid response in dwarf bean seedlings. Plant Physiol. 32 (suppl.): xlviii. 1957. 23. Morgan, D. G., and Mees, G. C. Gibberellic acid and the growth of crop plants. Jour. Agr. Sci. 50: 49-59. 1958. 24. Nitsch, J. P. Growth responses of woody plants to photoperiodic stimuli; Pho- toperiodism in woody plants. Proc. Amer. Soc. Hort. Sci. 70: 512-525; 526-544. 1957. 25. Purvcs, VV^ K., and Hiliman, W. S. Response of pea stem sections to indole- acetic acid, gibberellic acid, and sucrose as affected by length and distance from apex. Physiol. Plant. 11: 29-35. 1958. 26. Stowe, B. B., and Vamaki, T. The history and physiological action of the gibberellins. Ann. Rev. Plant Physiol. 8: 181-216. 1957. 27. Tang, Y. W., and Bonner, J. The enzymatic inactivation of indoleacetic acid. I. Some characteristics of the enzyme contained in pea seedlings. .Arch. Biochem. 13: 11-25. 1947. 28. Vnitos, A. J., and Meudt, W. The effect of light and of the siioot apex on the action of gibberellic acid. Contr. Boyce Thompson Inst. 19: 55-62. 1957. 29. Wassink, E. C., and Stolwijk, J. A. J. Effects of light cjuality on plant growth. Ann. Rev. Plant Physiol. 7: 373-100. 1956. DISCUSSION Dr. Waygood: I'd like to point out that there is no inhibitory effect of gibberellin on a purified lAA oxidase system. The effect of lAA and gibberellin shown in the slide by Brian and Hennning is not a synergistic effect. Dr. Housley: Our results do not conflict with Dr. Waygood's first statement that there is no inhibitory effect of gibberellic acid on a pmificd lAA oxidase system. With respect to his second statement, perhaps the use of the term synergistic in the present context is best left for each individual worker to accept or substitute otherwise ac- cording to preference. Influence of Gibberellic Acid on lAA Disappearance 643 Dr. Andreae: I should like to ask what justification is there to equate loss of lAA from solution with destruction? We found with similar experiments that as little as one-fifth to as much as all of the lAA lost from solution accumulates in the tissues as indoleacctylaspar- tic acid. Dr. Housley: One may certainly account for the disappearance of lAA from solution other than by its destruction; the example you mention is conjugation with some other entity. In our preliminary experiments we frequently obtained little or no disappearance from solution (i.e., no decrease in absorption) over a period of 6 or more hours; however, if suitable amounts of manganese ion and 2,4-di- chlorophenol were incorporated in the solution, lAA began to dis- appear rapidly. This dependence upon addition of these addenda for lAA disappearance led us to suppose that the acid was being re- moved from solution primarily as a result of its destruction by the lAA oxidase enzymes. Dr. Purves: I'd like to describe one experiment in connection with the theory put forth by Dr. Housley. In etiolated pea epicotyl sections under certain conditions you can get a very low lAA opti- mum, and can get a concentration curve showing an lAA optimum at lO'^M with concentrations above lO'^M being inhibitory to the growth rate. That is, the growth produced in the presence of high auxin concentration is actually lower than that of the controls. Now, if gibberellin is to act by virtue of an auxin-sparing mechanism, you would expect the treatment under these conditions with GA leading to an increased auxin content would lead to a further inhibition in the presence of inhibitory lAA concentrations. However, we find that the increment of growth produced by gibberellin is almost as great in the presence of inhibitory auxin concentrations as in the absence of auxin, suggesting that gibberellin cannot act by an auxin- sparing mechanism. Dr. Housley: Dr. Purves' work might suggest that gibberellin is not acting by an lAA-sparing mechanism alone and perhaps that more than one mechanism in his experimental system is operating. Such a mechanism, for example, could involve chemical combination of gibberellin with endogenous auxin of pea sections forming a com- plex with novel growth properties as has been postulated by Phillips, Vlitos, and Cutler (Contr. Boyce Thompson Institute. 20, 111-120. 1959) to account for their gibberellic acid — endogenous auxin studies on pea. With respect to Dr. Purves' suggestion that gibberellin cannot 644 S. Housley and B. J. Deverall act by an auxin-sparing mechanism, one is rejecting a biochemical mechanism by considering data derived from a complex growth phe- nomenon, namely auxin-induced inhibition of growth. Such con- sideration requires caution. Our own work consists of examination of a biochemical reaction; no observations on growth are made. We point out that our results are consistent with Brian and Hemming's growth synergisms obtained with lAA and gibberellic acids at opti- mal and sub-optimal concentrations. p. W. BRIAN and H. G. HEMMING Imperial Chemical Industries England Interaction of Gibbereltlc Acid and Auxin In Extension Growth of Pea Stems The garden pea (Pisum sativum) is an excellent plant for study of the role of gibberellins in internode extension. Of particular interest are comparisons of tall and dwarf varieties and comparisons of intact internodes with internode sections floating on substrate solutions. The following observations have been made in the course of our work on this plant (3,4): (a) The rate of internode extension in tall varieties of pea is far greater than that in dwarfs, yet sections from comparable internodes of tall and dwarf varieties extend at approximately the same rate, whether incubated in plain buffer or in solutions containing sucrose or indole-3-acetic acid (lAA). The rate of extension of sections on lAA/sucrose in short-term experiments is much greater than that of comparable tissues in intact dwarfs, but nearly the same as that of internode tissues in intact tall varieties. (b) Though intact dwarf pea plants give a great response to ex- ogenous gibberellic acid (GA), the response of sections is small, and only demonstrable in the presence of an auxin. (c) Intact dwarfs give a much greater response to GA than intact tall peas, yet sections from both kinds respond to nearly the same extent. These observations indicate that the difference in growth rate be- tween the tall and dwarf varieties is not determined in the internode tissue but elsewhere; similarly, the differential response of tall and dwarf peas to exogenous GA is not due to an innate difference in competence of their internode tissues. Indeed, we have evidence that the apical bud is of great importance in this connection. However, we have taken the view that detailed knowledge of the factors governing [ 645 ] 646 P. W. Brian and H. G. Hemming the response of sections to GA is necessary before full value can be derived from comparisons of internode sections with similar tissues in their normal anatomical context. We have used sections from peas grown in light (green sections), studying their extension in light, because we feel that such a system is closer to conditions in the intact plant than is the more usual sys- tem of etiolated sections growing in darkness, and that more valid comparisons between internode sections and intact plants can there- fore be made. The results now reported supplement those given in an earlier publication (4). METHODS Unless otherwise stated, sections have been cut from the dwarf pea, 'Meteor.' The procedure used for study of growth of these sec- tions has been described elsewhere (4). One modification of that pro- cedure has been made: 0.75 ml. medium is used for each section instead of 0.5 ml. We have found this to give slightly enhanced extension. Unless otherwise stated, the initial length of sections was 5.1 mm., and growth took place over 24 hrs. at 15° C. in a light intensity of about 800 foot candles. RESULTS Interaction Between Auxins and Gibberellic Acid We have already shown that GA alone has little or no effect on extension of 'Meteor' pea stem sections, but that it ^\•ill induce in- creased extension in the presence of lAA (4). We have since con- firmed this in many experiments. Similar synergism is exhibited be- Table 1. Interaction between auxins and gibberellic acid in promotion of exten sion of 'Meteor' pea stem sections. Mean final lengths (mm.) of sections. Basal me dium: 1 per cent sucrose in phosphate buffer. Ckjncn., MK ml Mean Final Length of Sections, Mm. Gibberellic acid, Mg/ml Effect of gibberellic acid Auxin 0 10 NAA NAA 2,4-D 2,4-D lAA lAA None None None 4 1 1 0 . 25 10 2.5 1 9.3 8.6 10.4 9.9 9.6 9.0 6.9 6.9 7.1 10.6 9.8 11.6 10.9 10.9 9.9 7.1 7.2 7.0 + 1.3 + 1.2 + 1.2 + 1.0 + 1.3 +0.9 +0.2 +0.3 -0.1 Significant dilfcrence between means: 0.36 (P = .05), 0.48 (P = .on, 0.62 (P = .001). GibberelUc Acid and Auxin in Extension Groxvth 647 Table 2. Interaction between light and sucrose in promotion of extension of pea stem sections, using two pea varieties. Mean final lengths (mm.) of sections. Basal medium: 10 /ig/ml indoleacetic acid in phosphate buflfer. Lighting Conditions . 0.5 Sucrose, Per Cent 1.0 2.0 4.0 'Meteor' (Dwarf) Light 8.6 6.5 +2.1 9.1 7.1 +2.0 9.4 9.0 7.4 7.2 +2.0 +1.8 8.3 6.6 + 1.7 Dark Effect of light 'Improved Pilot' (Tall) Light Dark Effect of lieht 8.1 6.5 + 1.6 8.7 8.7 9.0 7.5 7.7 8.2 + 1.2 +1.0 +0.8 7.9 7.3 +0.6 Significant differences (P = .01) between means: 'Meteor,' 0.42; 'Improved Pilot,' 0.44. tween GA and such auxin analogues as 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) (Table 1). This re- sult is relevant to discussions of the mode of action of GA and is mentioned again later. Effect of Light Galston and Baker (7) showed that light enhanced extension of green sections; they showed that it could be largely replaced by su- crose, though their data also showed that sucrose could not com- pletely substitute for light. In our experiments (Table 2) different varieties of pea differed in their response to light. With sections from the tall variety 'Improved Pilot' the effects of light and sucrose were less than additive (light X sucrose interaction significant at P = 0.001), indicating partial replacement of light by sucrose. Sections from the tall variety 'Alaska' behaved similarly; this was the variety used by Galston and Baker. But with sections from the dwarf pea 'Meteor' the effects of light and sucrose were approximately additive (light X sucrose interaction not significant at P =r 0.05). Thus, par- ticularly in 'Meteor,' light has some effect of importance which cannot be replaced by sucrose. In the presence of optimal sucrose light has the effect of enhancing the response to lAA (Table 3), 2,4-D and NAA (Table 4). In experiments not reported here in detail we have found that this is a high intensity light effect and that the response is proportional to the length of exposure to light. The response to GA also is dependent on light (Table 5). Factorial experiments in which light, lAA, and GA are supplied separately and in combination (Table 6) suggest that the dependence of the GA re- sponse on light is a consequence of the known GA X lAA interaction Table 3. Interaction between Hght and indoleacetic acid (lAA) in promotion of extension of 'Meteor' pea stem sections. Basal medium: 1 per cent sucrose in phosphate buffer. lAA Mg ml Mean Final Length, Mm. Effect of Light Light Dark 0 0.1 1.0 10.0 100.0 6.5 7.0 7.8 9.1 9.3 5.7 6.1 6.5 6.9 6.9 +0.8 +0.9 + 1.3 +2.2 +2.4 Significant difference between means: 0.40 (P = .05), 0.53 (P = .01), 0.68 (P = .001). Table 4. Interaction between light and 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) in promo- tion of extension of 'Meteor' pea stem sections. Basal medium: 1 per cent sucrose in phosphate buffer. Mean Final Length, Mm. Effect of Light Auxin Light Dark NAA, 5 Mg/ml NAA, 0.5 Mg/ml NAA, 0.05 Mg/ml 2,4-D, 1 Mg/ml 2,4-D, 0.1 Mg/ml 2,4-D, 0.01 Mg/ml None None 8.8 7.8 7.0 9.3 8.8 7.5 6.9 7.0 6.9 6.6 6.3 6.9 6.7 6.2 6.2 6.1 + 1.9 + 1.2 +0.7 +2.4 +2.1 + 1.3 +0.7 +0.9 Significant difference between means: 0.28 (P = .05), 0.37 0.01), 0.48 (P = .001). P = Table 5. Interaction between light and gibbcrcllic acid in promotion of extension of 'Meteor' pea stem sec- tions. Basal medium: 10 Mg/ml lAA + 1 per cent sucrose in phospiiatc buffer. Mean Final Length, Mm. Hrs. of Light Gibberellic acid Mg/ml Effect of Gibberellic Acid None 10 U 6 12 18 24 7.4 8.4 9.2 9.7 9.9 7.7 9.1 10.2 10.9 11.1 +0.3 +0.7 +1.0 + 1.2 + 1.2 Significant difference between means: 0.38 (P = 0.05), 0.50 (P = .01), 0.65 (P = .001). Gibherellic Acid and Auxin in Extension Growth Table 6. Interaction between light, gibberellic acid, and indole- acetic acid in promotion of extension of 'Meteor' pea stem sections. Basal medium: 1 per cent sucrose in phosphate buffer. 649 Mean Final Length, Mm. lAA GA Effect of Mg/ml Mg/ml Light Dark Light 10 0 7.5 6.5 + 1.0 0.01 8.5 6.7 + 1.8 0.1 8.7 6.8 + 1.9 1.0 8.8 6.8 +2.0 10.0 8.6 6.7 + 1.9 0 0 6.1 5.7 +0.4 0.01 6.0 5.7 +0.3 0.1 6.2 5.8 +0.4 1.0 6.1 5.8 +0.3 10.0 6.1 5.8 +0.3 Significant difference between means: 0.25 (P = 0.05), 0.33 (P = .01), 0.43 (P = .001). and also the necessity for light if lAA is to have its full effect. It is, of course, equally true to say that light has its greatest effect in the presence of an auxin and that its effect is still further enhanced if GA is supplied (Table 6). One possible interpretation of this light effect is that some product of photosynthesis, other than sucrose, is essential for the growth reaction in which auxin intervenes. A num- ber of possible early products of COo-fixation have been tested, in- cluding glucose-6-phosphate, glucose- 1 -phosphate, fructose-l,6-diphos- phoric acid, fructose-6-phosphoric acid, 2-phosphoglyceric acid, and 3- phosphoglyceric acid, but none of these had light-replacing properties greater than sucrose. Nevertheless, some indirect evidence that a prod- uct of photosynthesis may be involved has arisen from work with specific inhibitors. Effect of L-Azaserine and 6-Dlazo-5-oxo-L-norleucine Two antibiotics, azaserine (AZS) and 6-diazo-5-oxo-L-norleucine (DON), which have marked tumor-inhibiting properties, have been shown by Calvin and his colleagues (1, 11) to have specific inhibitory effects on the COo-fixation pattern of photosynthesizing algae. Both substances are powerful inhibitors of lAA-induced extension of green sections (Table 7). Furthermore, doses which have little effect on ex- tension of sections in darkness cause a marked inhibition of extension in light. Thus these substances appear to inhibit those growth pro- cesses initiated by exposure to light. Specificity, if it exists, is not complete because higher doses will inhibit extension in darkness. Their anti-tumor activity has been attributed (5, 9, 10) to interference with biosynthesis of purines or some amino acids, and their effects can to some extent be reversed by some purines, cyclic amino acids, 650 P. W. Brian and H. G. Hemming Table 7. Effect of azaserine (AZS) and 6-diazo-5-oxo-L-norleu- cine (DON) on response of 'Meteor' pea stem sections to light. Basal medium: 10 Mg/ml indoleacetic acid + 1 per cent sucrose in phosphate buffer. Concn., Mg/ml Mean Final Length, Mm. Inhibitor Light 24hrs. Dark 24 hrs. Effect of Light None AZS AZS DON DON 20 100 1 5 9.5 8.7 7.5 8.1 7.4 7.5 7.0 6.8 7.4 7.2 +2.0 + 1.7 +0.7 +0.7 +0.2 Significant difference between means: 0.36 (P = 0.05), 0.48 (P = 0.01), 0.61 (P = 0.001). and L-glutamine. In their effect on photosynthesis they leave the car- bon cycle untouched but block transamination reactions, and the effect of AZS has been partially reversed by L-glutamine (1). We have examined the possibility of reversal of DON and AZS inhibition of section extension and have tested a large number of purines, pyrimi- dines, nucleosides, and amino acids. The only substance which has shown any evidence of reversal is L-glutamine, but the effect is at most very slight. Thus we have little information concerning the mode Table 8. Effect of starvation of 'Meteor' pea stem sections on response (A) to indoleacetic acid (10 Mg/ml) and sucrose (2 per cent) and (B) to gibberellic acid. Data from three experiments in each case. [Mean final lengths of sections (initial length 5.1 mm. untreated, 5.4 mm. after starvation treatment). Basal media: (A) phosphate buffer, (B) 10 Mg/ml lAA + 2 per cent sucrose in buffer.] Untreated Sections, Mm. Starved Sections, Mm. Medium (i) (ii) (iii) Mean (i) (ii) (iii) Mean lAA S lAA + S 6.8 8.6 7.6 9.1 6.6 8.0 6.6 8.1 6.3 8.1 6.2 8.7 6.6 8.2 6.8 8.6 6.2 7.6 6.6 10.2 6.1 7.2 6.5 8.3 6.8 7.1 6.2 9.2 6.0 7.3 6.4 9.2 B GA, Mg/ml Untreated Sections, Mm. Starved Sections, Mm. (i) (ii) (iii) Mean (i) (ii) (iii) Mean 0 10 Effect of GA •1 1 9.9 +0.8 8.1 10.0 + 1.9 8.7 9.2 +0.5 8.6 9.7 + 1.1 10.2 10.2 0 8.3 8.8 +0.5 9.2 9.0 -0.2 9.2 9.3 +0.1 .Siirnilicant difference between means of all experiments: 0.28 (P = .05), 0.37 (P = .01), 0.48 (P = .001). GibberelUc Acid and Auxin in Extension Growth 651 of action of these inhibitors of section growth, but we consider their effect relevant both because they appear to block the response of sec- tions to light, and, in a number of preliminary experiments, to GA. Effect of Starvation of Sections We have shown (4) that rapid washing of sections in distilled water affects their subsequent growth. This led us to investigate in greater detail the effect of pretreatment of sections in aerated distilled water over longer periods. A 3 hr. treatment has two obvious effects on subsequent section extension. In the first place, it much reduces ex- tension in buffer or buffer plus lAA, but normal extension is restored on addition of sucrose (Table 8A); this is our main justification for interpreting it as a starvation treatment. Secondly, it very strikingly reduces the response to GA even when optimal lAA and sucrose are present (Table 8B). We cannot decide, on the basis of the data at present available, whether this is due to removal of some cofactor necessary for growth, or to removal of an inhibitor normally re- versed by GA. CONCLUSIONS Our knowledge of the mode of action of gibberellic acid has not been greatly advanced by these experiments, but they have shown that several factors are involved in the response of green sections to GA, and that the response of green sections to light merits further in- vestigation. The response to GA is conditional on the presence of an auxin. We previously reported that synergism could be demonstrated not only between GA and lAA but also between GA and synthetic auxin analogues, but we did not then (4) give experimental evidence for the statement. We have since repeated the relevant work several times and it is now quite certain that a response to GA from green pea sections can be induced by supplying either lAA, 2,4-D, or NAA (Table 1). A very tempting explanation of lAA X GA synergism is that GA directly or indirectly inhibits metabolic destruction of lAA, and suggestions have been made that the activity of lAA-oxidase sys- tems may be altered (6, 12), with a good deal of supporting evidence. However, the fact that GA has a synergistic relationship with such substances as NAA and 2,4-D, which are much less susceptible to metabolic breakdown, throws considerable doubt on this theory, unless it be supposed that such substances as 2,4-D and NAA them- selves induce section extension by displacing endogenous lAA from some physiologically inactive complex, as originally envisaged by Skoog (13). There is little evidence that this does occur. The response of green sections to auxins, and therefore indirectly 652 P. W. Brian and H. G. Hemming the response to GA, is dependent on high-intensity light. This can- not be explained purely in terms of photosynthetic formation of su- crose. This effect of light appears to be blocked by the inhibitors AZS and DON, which are known to affect the pattern of photosyn- thetic COo-fixation in algae in pathways other than those concerned in sucrose formation. They also block the response of sections to GA. From what is known of their mode of action in other systems, it can be suggested that the effect of light on green section growth is per- haps concerned with biosynthesis of purines or amino acids, path- ways blocked by AZS or DON by their effect on transamination reac- tions. In this connection it is interesting to note (2) that treatment of germinating barley grain with gibberellic acid results in considerable increases in transaminase content. Starvation of sections before use has little subsequent effect on ex- tension if sucrose is supplied. Such sections no longer respond to ex- ogenous GA, and it seems probable that they have been depleted of some other substance necessary for the response to GA. It has already been suggested (4, 8) that other factors besides auxin are involved in stem extension responses to GA. The results reported above offer further support for this view. ACKNOWLEDGMENT We are indebted to Parke, Davis R: Co., Detroit, for the gift of samples of azaserine and 6-diazo-5-oxo-L-norIeucine. LITERATURE CITED 1. Barker, S. A., Bassham, J. A., Calvin, M., and Quarck, U. C. Sites of azaserine inhibition during photosynthesis by Scenedesmus. Jour. Amer. Chem. Soc. 78: 4632^635. 1956. 2. Bergqvist, G., StensgSrd, A.M., and Nielsen, N. The influence of gibberellic acid on the transaminase content of germinating barley seeds. Physiol. Plant. 12: 386-389. 1959. 3. Brian, P. W., and Hemming, H. G. The effect of gibberellic acid on shoot growth of pea seedlings. Physiol. Plant. 8: 669-681. 1955. 4. , and Hemming, H. G. Complementary action of gibberellic acid and auxins in pea internode extension. Ann. Bot. II. 22: 1-17. 1958. 5. Clarke, D. A., Reilly, H. C, and Stock, C. C. A comparative study of 6-diazo- 5-oxo-i.-norleucine and O-diazoacetyl-L-serine on sarcoma 180. Antibiot. Chemother. 7: 653-671. 1957. 6. Galston, A. W. Studies on indoleacctic acid oxidase and its inhibitor in light- grown peas. Plant Physiol. 32 (suppl.): xxi. 1957. 7. , and Baker, R. S. Studies on the physiology of light action. I\'. Light enhancement of auxin induced growth in green pc.is. Plant Phvsiol. 26: 311-317. 1951. 8. . and Warburg, H. .\n analysis of auxin-gibberellin interaction in pea stem tissue. Plant Pliysiol. 31: 16-22. 1959. Gibberellic Acid and Auxin in Extension Growth 653 9. Hartman, S. C, Levenberg, B., and Buchanan, J. M. Involvement of ATP, 5- phosphoribosyl-pyrophosphate and L-azaserine in the enzymatic formation of glvcinamide ribotide intermediates in inosinic acid biosynthesis. Jour. Amer. Cliem. Soc. 77: 501-503. 1955. 10. Maxwell, R. E., and Nickel, V. S. G-Diazo-5-oxo-L-norleucine, a new tumor- inhibitory substance. V. Microbiologic studies of mode of action. Antibiot. Chemother. 7: 81-89. 1957. 11. Meulen, P. Y. F. van der, and Basshara, J. A. Influence of 6-diazo-5-oxonorleu- cine on the metabolism of Scenedesmus and Chlorella during photosynthesis. Univ. Calif. Rad. Lab. Rep. 3595: 33-35. 1956. 12. Pilet, P. E. Action des gibberellines sur I'activite auxines-oxydasique de tissus cultives in vitro. Compt. Rend. Acad. Sci. Paris. 245: 1327, 1328. 1957. 13. Skoog, F. Growth substances in higher plants. Ann. Rev. Biochem. 16: 529-564. 1947. DISCUSSION Dr. Sachs: Dr. Brian, could you comment on what you meant by your evidence and that of Dr. Vlitos, concerning the role of the apical bud in the intact plant? Dr. Brian: If you excise the apical bud from a pea plant and ap- ply gibberellic acid, you don't get anything like the response that you get when it is there. Dr. Sachs: I meant to ask about the size of the apical bud. Dr. Brian: A pretty large size. Dr. Sachs: In this case you perhaps removed the region where growth actually takes place by cell division. Evidence indicates that the whole region of shoot histogenesis exists within 2,000 to 3,000 mi- crons, and perhaps up to 10,000 microns in some plants, below the apical meristem. The apical meristem itself is completely inoperative in shoot histogenesis. Dr. Brian: I would agree, but certainly the parts corresponding to the section which increases in length by cell extension have not been removed. Dr. Nitsch: I would like to make two comments. The first one bears on what has just been said. Using oat seedlings, we have been able to demonstrate that gibberellic acid acts mainly on the very young, meristematic tissues, whereas auxins act on somewhat older tissues, located further down the coleoptile or first internode (Nitsch, J. P., and Nitsch, C. - Bui. Soc. Bot., France, in press). The other comment concerns the changes caused by gibberellic acid in the metabolism of the endogenous growth substances. We have been studying this question ever since we discovered that the application of GA to a sumac or a bean plant produces a great surge in the amount of endogenous growth substances (Nitsch, J. P., and Nitsch, C. — hi: Photoperiodism and Related Phenomena in Plants and Animals. R. B. 654 P. W. Brian and H. G. Hemming Withrow, ed., AAAS, \Vashington, D.C., pp. 225-242. 1957). Actually, not one, but possibly three to four compounds appear, or at least in- crease in the tips of bean seedlings after the application of gibberellic acid. (Nitsch, J. P. - XVth Interntl. Hort. Cong., Nice. 1958; Nitsch, J. P., and Nitsch, C.-Bul. Soc. Fran^aise Physiol. Veg. 5:20-23. (1959.) Dr. Galsion: I would like to say that, like Dr. Brian, we have re- cently been working almost exclusively with the green sections that we started with 10 years ago, and we feel much as Dr. Brian does that they have a great many advantages for this kind of work. VV^e completely agree with him that sugar replaces part but not all of the enhancing effect of light uj^on growth. We agree also that the extra promotive effect of light is a high-intensity reaction. \Xq also feel that the previous photoperiod to which the plant has been exposed has a very marked effect on the response of these excised green sections to gibberellin. There is one respect in which our results differ from those of Dr. Brian. We do not ever find that the sections fail to respond to gib- berellin in the absence of auxin. We always get a small but signifi- cant increase in growth with gibberellin alone, and this effect is greater, the greater the previous photoperiod to which the plants had been exposed prior to excision of the sections. Dr. Brian and I have also used a somewhat similar approach in attempting to inliibit photosynthesis. I would like to mention one other compound that we have used which behaves rather like DON and azaserine. This is a compound called fenuron whicli belongs to the urea herbicide group. It is 3-(phenyl)-3,3-dimethylurea. The p- chlorophenyl analogue (CMV or monuron) has been widely used in the inhibition of the Hill reaction. We find that with this compound we can almost completely inhibit photosynthetic activity of green sec- tions but not affect their ability to grow heterotrophically in the pres- ence of sugar. W^e have found to our surprise that fenuron acts as an auxin or auxin synergist in the dark in the presence of sugar. This is a rather surprising eftect for this type of compound. Of course, Drs. Steward and Shantz have reported the isolation of 1,3-diphenylurea from coconut milk and have shown tliat this has some growth-promot- ing ability in tissue culture. I would guess, then, that we are dealing here with a new class of auxins. Mr. Barlow: In your starved sections, is it a (jucstion of actually leaching in water, or have you for instance kept them on damp filter paper? Do you also get the same effect of starvation by simj^ly keep- ing the whole plant in darkness before cutting the section out? Apropos of that, how does the growth of the section compare with Gibberellic Acid and Auxin in Extension Growth 655 that of the same region on the phnit, since these increases that you obtained, of 10 or perhaps 20 per cent at the most, seem rather small? Dr. Brian: I can't answer the first question with any certainty. Cer- tainly by just putting them on filter paper you do get the same kind of effect, but it isn't so pronounced. I called it starvation. I'm not at all sure that it isn't starvation plus leaching. The second point is that certainly the response to gibberellin is never as great as the growth the same piece of tissue would make in the intact plant. The over-all extension is, if you are using a dwarf section, however, very much greater than the extension of the same tissue in the intact plant. Dr. Tolbert: Dr. Haber and I have, some years ago, extensively studied the distribution of carbon-14 in the products from long and short term photosynthetic C^^Oo fixation by gibberellin-treated plants. We found no difference, that could be detected by paper chromatog- raphy, in the rate or amount of C^^ incorporated into the sugars, amino acids, or organic acids, of plants treated with gibberellin as compared to controls. Dr. Lockhart: I'd like to make a comment on the importance of the tip in the response to gibberellic acid. I also agree with the other workers that in light-grown plants or plants that have been treated with light, the response to gibberellin is very much reduced if you remove the tip. However, if the plants are grown and maintained in continuous darkness, then the tip appears to be completely unneces- sary for a response to gibberellin — the tip then can be completely replaced in the promotion of elongation by gibberellic acid. This is true with the 'Alaska' - a tall variety, but not when a dwarf variety is used. Dr. Evenari: First of all, do you know anything about the action spectrum of this interesting high-intensity light effect? We know now, as far as germination is concerned, there is definitely a red-far-red low-energy, and a red-far-red high-energy process involved. And it would be very interesting to know what the action spectrum of your own high-energy effect is; that part which is not photosynthetic. Also is there any effect of a dark period? I mean, is there any difference between continuous light or interruption by dark periods. Dr. Brian: I'll take your second point first. So far as we have in- vestigated it, the dark period has no effect except that it isn't light. We've only just started work on the spectral effects and I really can't make any useful comment on this at all. Dr. Gordon: Considering the irradiances employed, may I sug- gest an alternative explanation for the light effect you observed? Your own experiments have demonstrated the interaction between 656 P. W. Brian and H. G. Hemming auxin and gibberellin in increasing tissue extension. Now, similar irradiances will increase the ability of legume shoots to convert trypto- phan to indoleacetic acid. Light will even more markedly enhance the ability of Coleus roots to form auxin or indoleacetate from tryp- tophan. Could we not infer, therefore, that light caused an enhanced auxin biosynthesis in your experimental material, and that you were simply measuring the interaction between the two growth substances? Dr. Brian: 1 don't think that explanation is possible because the effect of light on sections not supplied with exogenous auxin is very small, and the effect of light is greatest when you have optimal exoge- nous auxin. But even when you've gone beyond the optimal of ex- ogenous auxin so that growth is not quite as good as it would be with a somewhat lower concentration, light still has an increasing effect. I don't think, therefore, that the production of further auxin in the tissue itself could explain the results. J. VAN OVERBEEK and L. D O W D I N G Shell Development Co. Modesto, California Inhibition of Gibber ellin Action by Auxin In 1956 we started a search for a simple, yet specific, test for gib- berellins; something comparable to the section test for auxins. We found it in the Avena leaf base section test and first reported on it at the Stanford AIBS meetings (3). In this test, a section of the basal portion of the coleoptile which includes the enclosed embryonic shoot is taken. It is this shoot which emerges from the coleoptile as a re- sponse specific to gibberellin. We have repeated this test more than 100 times, always with the same general results: gibberellin promotes growth, auxins inhibit the growth of this shoot, while kinetins pro- mote it very slightly. In the beginning the variability of the growth of the individual sections was considerable, but gradually we have learned to reduce this to a minimum. TECHNIQUE OF THE LEAF BASE SECTION TEST Planting and Preparation Wooden flats such as are used in nurseries are filled with approxi- mately 2 inches of rather coarse Vermiculite, covered with a tin lid, and steam sterilized at 100° C. for at least 3 hrs. One-half pint of 'Ka- nota' oat seeds are placed in a one-quart jar, 5 to 10 mg. of Ceresan is added, and the jar is filled with water. The seeds are soaked for 1 hr. The oats are drained and spread on the sterile Vermiculite. The flat is then soaked with water, covered with the tin lid, drained, and put to germinate at 30° C. for 24 hrs. After germination, the seeds are covered with 0.5 inch sterile Vermiculite, saturated with water, the lid replaced, and the flat drained. With the lid still on, the flat of oats is taken to a physiological darkroom (27° C. and 80 to 90 per cent relative humidity). Here the lid is removed and the flat placed in a [ 657 ] 658 ]. van Overbeek and L. Dowding dark chamber. The oats are grown in the dark, interrupted only by short periods of red light from a neon tube of ruby red glass. The Test Solution Solutions are prepared j^rior to cutting the sections. The stock solution contains 2 per cent sucrose and 0.02 M phosphate buffer (Na2HP04 and KH0PO4). Double-strength regulator solutions are made so that equal parts of it can be added to the buffer to give a final concentration of 1 per cent sugar and 0.01 M buffer at a pH of 6.2. One ml. of buffer and 1 ml. of test solution thus give the final 2 ml. of sohuion in ^vhich the sections are placed. Preparation of Beakers Fifty-ml. beakers are heat sterilized in paper bags, as are the watch- glasses which later will serve as covers. The beakers and cover glasses are stored in the bags and remain sterile for some time. The test so- lutions are placed in the beakers and kept covered with the watch- glasses. No special effort is made to sterilize the solutions themselves. UJ CD 5 Q. Q- O < 3 CM. -~l 0 0.01 0.1 PPM 6IBBERELLIC ACID I'ig. 1. Avena leaf section icsi. Tlic leal is shown as it grows oui of the basal 5 mm. of the coleopiile. Duration of liic test: 48 hrs. hiliibition of Gibberelliii Action by Auxin 659 The Test On the fifth day the oats are ready for use. The length from the coleoptile tip to the node is approximately 20 to 30 mm. Twenty-four hrs. prior to use, the flat is jarred gently in order to pack the Vermicu- lite and prevent the oats from growing crooked. Seedlings with a coleoptile length between 20 and 30 mm. are selected in red light for the test. The shoots are cut at the base and floated in a dish of water. After all the shoots necessary for a test have been selected and placed in the water, a 5-mm. section is cut from the base of each coleoptile by means of two parallel razor blades motmted in a special holder. The section includes the coleoptilar node, and the lower cut is made just below it. The shoots are laid on a piece of glass under which is placed a paper with a line on it (Figure 1). The node is placed on the line and the lower razor blade is lined up with this line as a cutting guide. New razor blades are always used, as dull blades may injure the leaf, causing it to grow out crooked from the coleoptile. Immediately after cutting, the sections are floated in a dish of distilled water and held there until they are ready to be put into the final test solutions. When a sufficient number of sections has been cut, they are drained through cheesecloth and, using the cheesecloth as a sack, they are dipped 10 to 15 times in a 0.2 per cent Clorox solution (100 p. p.m. active chlorine). The Clorox is not rinsed off. Without this method of BUFFER 6 A O.i P.PA Fig. 2. Basal 5 mm. coleoptile sections of Avena with leaves growing out of them. Leaf growth is promoted l^y the addition of low concentrations of gibberellic acid to the basic medium of phosphate buffer and sucrose. The sections floated in 2 ml. of solution on a shaker in the physiological darkroom at 27° C. and were occasion- ally exposed to red light for observation. 660 J. van Overbeek and L. Dowding disinfection, it was found that microorganisms will grow on the leaves, and when this happens, growth is abnormal and inhibited. The addi- tion of a low concentration of isopropyl alcohol to the test solutions has also been used to keep them relatively free of interfering micro- organisms. The Clorox-dipped sections are then jjlaced in 50-ml. beakers containing 2 ml. of test solution. The beakers are covered with watchglasses and placed on a slow shaker for 48 hrs. All of these operations have taken place in the physiological darkroom (Figure 2). The objective of this new test was not so much to assay extracts of plant material for their gibberellin content, but rather to provide a convenient means of studying gibberellin action. We want to report here on a striking inhibition of the effect of gibberellic acid (GA) by other regulators, specifically indole-3-acetic acid (lAA) (Figure 3). Because inhibition of kinetin by auxin has been reported in the liter- ature (5), kinetin was included in some of the tests.. As the results in Figure 4 show, kinetin does inhibit the effect of gibberellic acid at the same low concentrations at which indole-3-acetic acid is effective. While lAA by itself inhibits growth, kinetin alone appears to promote growth. When low concentrations of indole-3-acetic acid are applied to- gether with gibberellic acid, the effect of the latter is much sup- pressed (Figures 3 and 4). The entire GA curve is dropped. This in- o iLl % Fig. 3. Avena leaf growth from basal coleoptile sections as inlliuiucd 1)\ low con- ceniralioiis of gibberellic acid (GA) and indolcacetic acid (lAA). Gibberellic acid promoted leaf growth, while indoleacetic acid inhibited it. Throe tests were run on consecutive days; each line is the average of 21 sections. Inhibition of Gibherellin Action by Auxin 661 15 - 50 HOURS 5 o q: < UJ _i 10 < 2 ^ LlJ lAA.K ____-- BUFFER lAA 0.01 GA. PRM. 0.1 Fis. 4. Interaction of hormones demonstrated in the Avena leaf section test. Aver- ages of 7 tests comprising 150 sections for each value plotted. Vertical lines: magnitude of error. Duration: 50 hrs. Gibberellic acid (GA, 0.01 and 0.1 p.p.m.) promoted leaf growth. The addition of kinetin (K, 0.1 p.p.m.) and of indoleacetic acid (lAA, 0.1 p.p.m.) suppressed the GA-induced growth. Addition of a mixture of lAA and K (both at 0.1 p.p.m.) suppressed the GA effect even more, so that growth in a solution with all three regulators was nearly identical to that of the buffer in sugar solution without regulators. hibition of gibberellin by auxin has been found in a large number of tests, although the magnitude of the inhibition was variable. If this phenomenon of gibberellin action inhibited by auxin is a general one, it has some interesting consequences (2). Bud Inhibition The promoting agent here is gibberellin and the inhibiting agent, auxin. This was proven by the work of Kato (1). Fruit Drop If one assumes that certain stages of fruit growth are gibberellin dependent, it follows that auxins will inhibit this. This interrelation was demonstrated on cotton by Walhood (4). It also explains why auxin enhances the June drop of apples. Root Growth In the past it had been assumed that elongation of roots is auxin dependent (2). Suppose it were gibberellin dependent — and there is 662 ]. van Overbeek and L. Dowding nothing in the literature that would disagree with such a view — then the well-known inhibition of root elongation by auxins could easily be explained. LITERATURE CITED 1. Kato, J. Studies on the physiological effect of gibberellin II. On the interaction of gibberellin with auxins and growth inhibitors. Physiol. Plant. 11: 10-15. 1958. 2. van Overbeek, J. Auxins. Bot. Rev. 25: 269-350. 1959. 3. , Racusen, D. W., Tagami, M., and Hughes, W. J. Simultaneous analysis of auxin and gibberellins. Plant Physiol. 32 (suppl.): xxxii. 1957. 4. Walhood, V. T. The effect of gibberellins on boll retention and tut-out in cot- ton. Proc. 12th Ann. Cotton Conf. Memphis. Dec, 1957. 5. Wickson, M., and Thimann, K. V. The antagonism of auxin and kinetin in apical dominance. Physiol. Plant. 11: 62-74. 1958. DISCUSSION Mr. Barlow: We have used a leaf base test similar to that described by Dr. van Overbeek. It was a slight modification of the test pub- lished by Margaret Radley (Ann. Bot., N.S. 22: 297. 1958) in which the wheat leaf base is used instead of the Avena leaf base. We didn't observe this depression of GA stimulation by lAA. I'm wondering if it is partly due to the age of the coleoptile which is used. Radley recom- mended rather old, long, coleoptiles and used a basal 1-cm. section. In that case, of course, the coleoptile section itself doesn't extend, but the leaf grows considerably. I also noticed in yoin^ photographs that the leaf only seemed to grow out of one end of the section. In our experience, the leaf expands on both sides, leaving the coleoptile sec- tion in the middle. Could you tell me if the growth of that leaf is due primarily to enlargement of the cells, or is it due to cell division? I have had a very brief look at this and, as far as I could see, most of the increase in length was due to cell extension. Right at the base of the leaf, however, it became extremely difficidt to say whether, in fact, the cells were growing (in Prof. Thimann's sense of the word growth) or whether in fact the leaf base was adding cells. Dr. van Overbeek: I can answer part of those questions. We cut the section below the node; when we cut it above the node, the leaf grows out on both sides. We have found variability at times, and we thought that part of the answer was the age of the seedling. We tried different ages and still found this inhibition. In oat seedlings, age apparently is not the principal lac tor in the inhibition of G.A. stimu- lation by lAA. Whether the elongation was cell division or cell en- largement, I do not know. We do have the complete bud here, and since there is a certain amount of lag in growth response to G\, it is possible, although unlikely, that cell division is involved. I iliink it is growth in the sense of Thimann's elongation. Inhibition of Gibberellin Action by Auxin 663 Dr. Brian: 1 would like to add that the test that Miss Radley uses is basically similar to this, although the plants are cut above the node and wheat is used instead of oats. Certainly we have never encoun- tered any inhibition by lAA as you have suggested. We do regard it as an almost specific test for gibberellins, but occasionally we find that there is a response in leaf extension due to auxin. Although we use this technicjue in our work on screening for natural gibberellins, we always confirm the results by using one of the tests involving a genetic dwarf. Other Plant Growth Regulators G. BEAUCHESNE C.N.R.S. Laboratoire du Phytotron Gif-sur-Yvette (S. et O.), France Separation des Substances de Croissance d'Extralt de Mais Immature Les recherches qui font I'objet de cette note ont port^ sur clifEerents extraits de Mai's immature conserves dans I'ethanol a 50 pour-cent a la glaciere. Dans plusieurs publications (1,4,5,6,7), nous avons deja expose comment nous etions arrives a la conclusion que les extraits de Mais immature contenaient d'autres substances que les auxines deja con- nues. Apres avoir essaye differentes methodes de separation par sol- vants, par chromatographie, ou par dialyse, qui se sont montrees in- suffisantes, nous avons eu recours a des separations fondees sur les fonctions chimiques des corps obtenus dans ces extraits. C'est ainsi que nous avons utilise les echangeurs d'ions synthetiques. SOMMAIRE DES EXPERIENCES Separation en Trois Groupes (A) L'extrait de Mais immature, en milieu ethylique a 50 pour-cent est verse sur une colonne de resine echangeuse d'ions, polystyrene sulfone de formule generale R — SO^H, preparee par un traitement a I'acide chlorhydrique a 10 pour-cent, et rincee jusqu'a neutralite par de I'eau desionisee. Get echangeur d'ions, sous sa forme acide, echange ses ions H+ contre les cations metalliques et organiques qu'il fixe. L'extrait de Mais traite est en particulier debarrasse de tons les cations organiques tels que les acides amines et les petits polypeptides. La resine est ensuite rincee. Les eaux de lavage sont ajoutees a l'extrait deja traite. Une solution d'ammoniaque 2N est alors versee sur I'echangeur pour en liberer tous les cations organiques. Certains, tel I'arginine, ne sont elues qu'avec difficulte. Apres evaporation sous vide entre 30 et 35° C. de cet eluat ammoniacal, les corps sont remis en [667] 668 G. Beauchesne solution dans I'eau, et le volume ramene a celui de I'extrait initial. Ceci constitue le Groupe A qui contient tous les cations organiques de I'ex trait de Mais. (B) Apr^s ce traitement par echangeur acide, I'extrait de Mais est verse sur une colonne de resine synthetique, polystyrene ammonium quaternaire, de formule generale (R')4 N-OH, se comportant comme une base forte, preparee par une solution de soude a 4 pour-cent, et rincee jusqu'a neutralite. En passant sur cette colonne, I'extrait de Mai's, echange tous les corps presentant une fonction acide, meme extremement faible, contre les oxhydriles de I'echangeur. (C) Apres passage de I'extrait de Mais, la colonne de resine est rincee et les eaux de rin^age ajoutees au filtrat qui ne contient alors ni acide, ni base, mais seulement des substances ne presentant que peu ou pas d'activite chimique, dont des sucres (glucose, levulose, sacchar- ose), des lipides, et une ou plusieurs petites proteines solubles dans I'alcool ethylique a 50 pour-cent. Ce filtrat, appele par nous "filtrat total" constitue le Groupe C. L'echangeur basique est traite par une solution de soude a 4 pour- cent, contenant un nombre d'equivalents plus grand que ne I'exigerait la capacite totale de la colonne. L'eluat sodique est ensuite traite par un echangeur acide pour oter toute trace de soude, et le filtrat acide constitue le Groupe B. Etude de rActivite de ces Trois Groupes L'activite de ces trois groupes de substances A, B et C, a ete essayee a I'aide de culture de tissu. Les tissus de Topinainbour rcvclerent que les trois groupes ctaient actifs. Aucun, pris seul, ne montrait cependant une activite semblable a celle de I'extrait total, mais en ajoutant au Groupe A de I'acide in- dole-3-acetique on obtenait des resultats equivalents ou depassant ceux atteints avec I'extrait de Mais total. Le Groupe B, seul ou avec adjonc- tion d'auxine synthetique, montrait une activite analogue a celle de I'auxine synthetique ajoutee sur le milieu. Enfin, le Groupe C ne semblait agir qu'en presence d'auxine. Etudies de cette mani^re, voici les coefficients d'accroissement ob- tenus en Table 1. 11 fallait trouver un essai biologiquc pcrmettant de distinguer entre substances d'elongation et substances de division cellulaire. Pour cela, nous avons utilise le "Test" mis au point par Jablonski et Skoog (3). Ce test utilise le parenchyme mcdullaire de Tabac. En presence d'auxine ce tissus presente peu ou pas de divisions cellulaires, mais un allongement considerable des cellules qui provoque souvent un eclate- ment des tissus (Figure 1). Substances de Croissance d'Extrait de Mais 669 Table 1. Coefficients d'accroisscment de I'activite de trois groupes d'extrait de mais immature. Temoin sans auxine 0,2 Temoin avec lAA (2,8 X 10-M 6 et < 9 donnent des tissus ayant des cellules plus grandes et un tissu moins ferme que ce qu'on obtient avec le Groupe A non fractionne, et le Groupe C. LITERATURE CITED 1. Beauchesne, G. Les substances de croissance de I'extrait laiteux de rnais imma- ture. Compt. Rend. Acad. Sci. Paris. 244: 112-115. 1957. 2. Gyorffy, B., Redei, B., and Redei, G. La substance de croissance du mais laiteux. Acta Bot. Acad. Sci. Hung. 11: 57-76. 1955. 3. Jablonski, J. R., and Skoog, F. Cell enlargement and cell division in excised tobacco pith tissue. Physiol. Plant. 7: 16-24. 1954. 4. Netien, G., Beauchesne, G., and Mentzer, C. Influence du "lait de Mais" sur la croissance des tissus de Garotte in vitro. Compt. Rend. Acad. Sci. Paris. 233: 92,93. 1951. 5. , and Beauchesne, G. Action d'un extrait liquide de graines de Mais immatures (lait de Mais), sur la croissance des tissus de tubercules de Topinam- bour cultives in vitro. Compt. Rend. Acad. Sci. Paris. 234: 1306-1308. 1952. G. , and Beauchesne, G. Differentes substances de croissance decelees dans I'extrait laiteux de graines de mais et etudiees sur cultures in vitro de tissus de tubercules de Topinambour. Compt. Rend. Acad. Sci. Paris. 237: 1026-1028. 1953. 674 G. Beauchesne 7. Netien, G., and Beauchesne, G. Essai d'isolement d'un facteur de croissance present dans un extrait laiteux de caryopses de mais immatures. Annee Biol. 30: 437-443. 1954. 8. Nitsch, J. P., and Nitsch, C. Studies on the growth of coleoptile and first inter- node sections. A new, sensitive, straight-growth test for auxins. Plant Physiol. 31: 94-111. 1956. LOUIS G. NICKELL and WALTER R. TULECKE^ Chas. Pfizer and Company, Inc. Growth. Substances and Plant Tissue Cultures Because of the dramatic effect obtained by applying gibberellins to intact plants and the natural occurrence of at least one of these com- pounds in higher plants (5), it seemed desirable to study the effect of gibberellin in plant tissue culture systems where many environ- mental and growth variables may be controlled, and where many of the complicated influences of morphogenetic development in higher plants are not present. Furthermore, the percentage of cells that are meristematic (or at least nondifferentiated) is quite large compared with that in intact plants. The present paper includes data from experiments with 49 strains representing 25 different species. The effects of various levels of the gibberellins, variations in the media used, type of tissue, source of the tissue, as well as physiological and pathological state of the tissues involved, are considered. The main purpose of the present paper is to survey a large num- ber of cultures to determine if any correlations can be made between the response of tissue cultures to gibberellin and characteristics of the tissues or the conditions under which they are grown, as well as to compare the resultant data with those obtained from intact plants. MATERIALS AND METHODS Gibberellin The gibberellin used was Pfizer lot #76088, which is a mixture of gibberellin A and gibberellic acid (GA). Parallel tests were run in many cases with potassium gibberellate, with similar results. Solu- tions were made up at 10 times the highest test level, the pH ad- ' Subsequently: Boyce Thompson Institute for Plant Research, Inc., Yonkers, N.Y. [675] 676 L. G. Nickell and W. R. Tulecke justed, and the solutions sterile-filtered through sintered glass. The amount necessary to give the desired level was added to the auto- claved agar medium just before solidification. Recent results in our laboratory, however, indicate a surprising stability of this mixture to heat. Only about one-third of the activity was lost after autoclaving for 15 min. at 15 lbs. pressure. The activity was measured in the dwarf pea test and with avocado tissue. Media Three synthetic media (White's, LP, and 24) and modifications of each were used to grow the varied types of tissues included in these tests. The medium (20 ml.) was added to 1 X 6 in. Pyrex test tubes, plugged with nonabsorbent cotton, and sterilized by autoclaving for 15 min. at 15 lbs. pressure. The pH level of all media was adjusted to between 5.0 and 6.0 before autoclaving. For many tissues the media were supplemented by 2,4-D (6 p.p.m.) and coconut milk (18 per cent by volume). The coconut milk added to culture media is prepared by collecting the liquid from 100 mature coconuts. This pooled coconut milk is filtered, dispensed into flasks, autoclaved, and stored at 5° C. Before use, it is filtered to remove precipitated protein. In this processed form it is then added to media at 18 ml. per 100 ml. of medium, and re-autoclaved. Tissue Culture Methods and Experimental Conditions The inoculum for each test level was weighed and divided into 5 tubes. The final w^et weight in the 5 tubes was divided by the inoc- ulum wet weight to give an index of growth, termed the "growth value." This is the only term used in the present investigation to ex- press increments of growth. Growth was in an air-conditioned cul- ture room at 21° C. in diffuse light. EXPERIMENTAL RESULTS Dosage-response experiments were set up with gibberellin levels varying from 0.1 p.p.m. to 100 p.p.m., using several different kinds of tissues. The responses of the 4 tissues selected for this study were either stimulatory or depressive (Table 1). The Riimcx (sorrel) virus tumor tissue was stimulated at 10 p.p.m., while the broad bean cotyledon tissue was stimulated from 0.5 to 5 p.p.m. Taxus pollen tissue was slightly depressed at 10 p.p.m., strongly at 100 p.p.m. Avocado cotyle- don tissue was extremely sensitive, being strongly inhibited at 5 p.p.m., and killed at 10 p.p.m. (Figure lA). Because of the large number of tissues to be evaluated in the pres- ent work, it was necessary to decide on one test level of gibberellin Groivth Substances and Plant Tissue Cultures 677 Table 1 . Types of growth response of plant tissue cultures to various concentra- tions of gibberellin. * Gibberellin Level in P.P.M. Tissue 0 0.1 0.5 1.0 5.0 10.0 100.0 RllTTl^X 4.0 4.6 5.4 4.1 . 6,1 5.8 2.4 2.0 Persea 5.2 5.4 5.2 4.9 2.0 dead Taxus 7.2 7.0 5.5 4.0 X'lCia 2.9 3.1 3.7 3.8 3.7 3.1 *Ratio of fresh weight at end of test over initial fresh weight. for comparative purposes. In view of the results shown in Table 1 as well as similar experiments in this and other laboratories (3, 6, 7, 10), the level chosen was 10 p. p.m. A stimulatory or inhibitory effect at 10 p. p.m. indicates the general response of a tissue. However, a lack of response is not conclusive. Broad bean cotyledon is an example of a tissue which is stimulated at a level below 10 p. p.m. At 10 p. p.m. no effect is apparent, yet there is significant stimulation at 0.5 to 5 p.p.m. The 49 tissues selected for inclusion in this paper represent 17 families and 25 species. The cultures came from diverse origins within the plants: pollen, root, stem, leaf, tuber, prop root, cotyle- don, and petiole. The time in culture of these tissues varies from Fig. 1. A. The effect of gibberellin on the growth of a tissue culture from avocado cotyledon. Left, untreated; right, 10.0 p.p.m. gibberellin. B. Increased growth of avocado cotyledon tissue culture by removal of 2,4-D from the medium. Left, White's medium with coconut milk; right, the same medium with 6.0 p.p.m. 2,4-D. C. Ne- cessity of 2,4-D for growth of 'Pontiac' potato tuber tissue in culture. Left, White's medium with coconut milk and 6.0 p.p.m. 2,4-D; right, the same medium without 2,4-D. Table 2. Effect of gibberellin on growth of various strains of plant tissue cultures. *W = While's basal medium (13); WM = White's basal medium with lOX KH.PO^ (11): CM = coconut milk (20% by volume); Arg = 1-arginine HCl, 100 p. p.m. (11); 2,4-D = 2,4- dichlorophenoxyacetic acid. 6 p. p.m.; LP = modification of medium 24 with low phosphate, 1 [678] Response to Gibberellin Growth Value** Per Cent Change No. of Gibberellin from Comments Expts. Control (lOp.p.m.) Control Culture 81056 4 8.6 10.2 +20 Culture 5857 3 3.1 4.2 +35 Culture from C. D. LaRue 1 7.2 5.5 -25 VVinkleman culture 3 3.1 2.7 -13 Superior culture 1 2.4 1.4 -42 2 3.2 3.1 - 3 'Golden Cross Bantam' with corn stunt virus 1 3.6 3.6 0 'Golden Cross Bantam' 1 2.4 1.5 -35 crown gall 1 5.1 4.1 -20 2 22.5 19.5 -14 1 1.2 1.0 -17 Red pigment, crown gall 3 3.3 dead dead No pigment, crown gall 1 1 .8 1 .3 -13 1 8.0 5.9 -26 2 6.2 dead dead Chemically induced, habituated 6 3.4 3.7 + 17 Cio crown gall (white) 1 3.0 3.7 +23 Cio crown gall (green) 2 1.6 1.4 -10 Cio crown gall (differentiated) 1 2.3 3.1 +34 1 16.8 16.4 - 3 2 4.2 4.9 + 12 Cio crown gall 1 14.3 12.2 -15 Cio chemically induced 1 7.7 5.8 + 3 Cio virus tumor 1 1.9 3.5 +84 Cio virus tumor 1 5.0 5.6 + 12 4 22.7 19.1 -16 1 11.1 10.6 - 5 2 15.5 16.7 + 8 2 19.0 15.0 -21 2 20.0 20.0 0 'Golden Pod' 1 16.0 14.2 + 12 'Golden Wax' 1 20.8 10.1 -56 2 3.9 2.8 -28 'Dwarf Progress' 2 2.4 2.2 - 8 4 2.8 3.6 +28 1 2.6 2.6 0 1 9.9 5.0 -50 virus tumor 4 4.7 5.7 + 18 1 6.2 5.0 -19 2 15.4 9.2 -40 1 12.8 5.6 -57 1 7.2 6.0 -17 3 9.1 7.1 -22 crown gall 6 4.2 4.9 + 12 Hybrid 1 15.0 15.0 0 'Pontiac' (red) 5 8.4 8.9 + 6 'Katahdin' (white) 3 3.7 4.2 + 14 crown gall 5 14.6 7.3 -50 crown gall 1 13.8 8.2 -41 millimole (8); 24 = synthetic medium for virus tumors with high phosphate, 8 millimole (1); Ye = Mead-Johnson yeast extract, 5 mg/1; pCl = parachlorophenoxyacetic acid, 6 p. p.m. * * Ratio of fresh weight at end of test over initial fresh weight. tO.6 p.p.m., 2,4-D. [679] 680 L. G. Nickell and W. R. Tulecke rather recent isolates to tissues which have been maintained in vitro for 15 years. The media used include both synthetic and supple- mented types. The methods of induction that led to the establish- ment of these strains include hormonal, crown gall, virus, and genetic. The results of a large number of experiments incorporating 10 p.p.m. of gibberellin in the nutrient media are shown in Table 2. These show that, while a few tissues are increased in their growth and others show no response, the majority are retarded. For example, all three strains of rose tissue w^ere retarded; avocado cotyledon and the pigmented cactus stem crown gall were killed. The five strains of tissue from monocotyledonous species were either reduced or showed no effect. These strains are from three dif- ferent families and represent leaf, seedling, and root cultures. Severe depression of tissue growth was noted for two crown-gall tissues of sunflower, one from the stem and the other from the petiole. Growth values at 10 p.p.m. gibberellin were approximately one-half the control values. The results of five experiments with petiolar crown-gall tissue of sunflower are presented in Table 3. Variations in growth among these experiments are due principally to differences in the physiological state of the inocula. Nevertheless, the response obtained, in this case depression, is consistent. Of the other crown- gall tissues tested, some were depressed (Vinca and Melilotus stem). Only two were stimulated to any appreciable extent (Melilotus root crown gall, white and differentiated), and these responses were not so striking as the depressions. Growth of three strains of tissue from the Leguminosae was in- creased by gibberellin; Vicia faba (broad bean) cotyledon tissue was Table 3. The variation in growth of Helianthus petiole crown-gall tissue between experiments. All experiments run for 4 weeks on LP medium. Initial Weight, Mg. Final Weight, Mg. Growth Value * Control Gibberellin Control Gibberellin Control Gibberellin 275 280 4,250 2,680 15.4 9.5 230 245 2,060 1,070 13.1 6.5 245 260 1,795 880 7.3 3.1 245 210 5,755 2,715 23.4 13.5 215 215 2,975 755 13.8 4.1 Average growth value of 1 ive experiments 14.6 7.3 * Ratio ul Iicsh weight at end of test over initial fresh weight. Growth Substances and Plant Tissue Cultures 681 most responsive. Growth of eight strains from this family was re- duced; bush bean cotyledon tissue by as much as 50 per cent of the control growth. The four fastest-growing cultures were all inhibited. These are holly root, pole bean hypocotyl, pinto bean root, and bush bean coty- ledon. Of the slowest-growing cultures, sweet clover virus stem tumor was stimulated, while sweet clover root crown gall, the nonpigmented cactus stem crown gall, and Stapelia leaf tissue were slightly inhibited. Over the last several years, we have found a few cultures that change somewhat in appearance. These "variants" have been segre- gated and maintained as separate cultures. Since they are morpho- logically distinct from the parent culture, they were tested to deter- mine their growth response to added gibberellin. Melilotus root crown gall (white and green) and Opuntia stem crown gall (red and white) show that the variants respond differently to gibberellin from the parent cultures. Whether these "variants" are true somatic mutations, merely segregants from mixed cell populations, or represent other phenomena is not known. Because of the possibility that the auxin-gibberellin relationship in specific tissues might play a part in their response to gibberellin, two types of experiments were set up. In the first type, two tissues were selected that grow on a synthetic medium (LP) with no added auxin. One tissue (tobacco stem) is stimulated by added gibberellin, whereas the other (sunflower petiole) is inhibited. Both tissues are crown galls. In the second type, two tissues were used that were maintained on a medium supplemented with 2,4-D and coconut milk. The growth of one of these was promoted by added gibberellin (broad bean cotyledon), the other was killed (avocado cotyledon). These two tissues are both normal. Before discussing the results of these experiments, it is well to point out the effect on growth of the removal of 2,4-D from the medium. The growth of avocado is increased by removal of 2,4-D (Figure IB). 'Pontiac' potato tuber tissue, on the other hand, dies when 2,4-D is removed from the medium, even on the first subcul- ture (Figure IC). Similar results are obtained with holly and yam tissues. The results with the two crown-gall tissues are shown in Table 4. Tobacco, which is stimulated by gibberellin at 10 p. p.m., is in- hibited by 2,4-D at 1 p. p.m. The addition of both substances causes an intermediate response. Sunflower, which is inhibited by gibber- ellin at 10 p.p.m., is also inhibited by 2,4-D, but to a greater extent. Both substances together give the same effect as 2,4-D alone. The responses of the two normal tissues to gibberellin and 2,4-D 682 L. G. Nickell and W. R. Tulecke Table 4. The eff"ect of gibbcrellin and 2,4-D on the growth of tobacco and sunflower crown-gall tissues in LP medium. Concn. P.P.M. Growth Value * Additive Tobacco stem Sunflower petiole None GA GA and 2,4-D 2,4-D 10 10 1 1 5.1 6.2 3.7 2.0 47.3 14.5 2.2 2,2 * Ratio of fresh weight at end of test over initial fresh weight. are presented in Table 5. Avocado is killed by gibberellin alone or in the presence of 2,4-D, and its effect is apparently independent of the level of 2,4-D used. The growth of broad bean is increased by gibberellin. This effect is found in the presence or absence of 2,4-D. DISCUSSION The growth response of intact plants to applied gibberellins is characterized by high sensitivity and considerable variability from one type of plant to another. Similar responses of plant tissue cul- tures are also observed in the presence of gibberellins. No strict cor- relation was found between the response to applied gibberellin and the inciting agent of tissue growth, the age of the culture, the type of medium, or the plant part from which cultures were obtained. Netien (6, 7) and Henderson (3) foimd the gibberellins inhibitory to the growth of plant tissue cultures of Scorzonera, Dauciis, Rubus, Helian- thiis tuberosus, and H. annuus. Schroeder and Spector (10), on the other hand, obtained a significant growth increase at 5 to 25 p. p.m. gibberellin with fresh explants of the mesocarp of Citrus medica. The results from tlie pollen tissues of Taxus and Ginkgo are in agreement with the work of Chandler (2), who found that the pollens of some plants were stimulated in germination and tube elongation, while other pollens were retarded. Growth of the Ginkgo pollen tis- sue was increased by the addition of 10 p.p.m. gibberellin to the basal medium; this was true for two strains of the tissue. Taxus pollen tis- sue, on the other hand, was retarded. An interesting observation is the effect of gibberellin on die color of all bean cultures. As is shown in Table 2, there are bean c ultures that fall into each category: some are stimulated, some depressed, and Groivth Substances and Plant Tissue Cultures 683 some not atfected in their growth. However, the color of all the bean cultures is darkened, from white to light yellow, from light yellow to dark yellow, from dark yellow to brown, depending on the color of the untreated tissue. Apparently there is an effect on one or more enzvme systems involved in pigment formation that is unrelated to the growth effects. Radley and Dear (9) have shown that coconut milk contains gib- berell in-like substances as determined by the dwarf pea test. This is mentioned because many of the tissues used in the present work were grown on media containing coconut milk. The levels reported by Radley and Dear are for concentrated coconut milk. When the gib- berellin-like activity of unconcentrated coconut milk is calculated from their data, the level is below the amount necessary to obtain a growth response by a standard dwarf pea test. This level is also be- low that to which the tissues respond. It is possible that the gibberellin response of plants is related to the auxin state of the particular plant, plant organ, or tissue to which it is applied. Vlitos and Meudt (12) have emphasized this by con- trasting the responses obtained with intact plants and isolated plant parts. Intact plants respond more to applied gibberellins, whereas the auxin response is exaggerated in isolated plant parts. In addition to the effect of isolation from the parent plant, the tissue culture responses discussed here are obscured somewhat by the presence of plant hormones in many of the media. Moreover, the tissues on media that lack such hormones are, with one exception {Melilotus root. Table 5. The effect of gibberellin and 2,4-D on the growth of avocado and broad bean tissue cultures in White's medium plus coconut milk. Concn., P.P.M. Growth Value * Additive Avocado Broad bean None 4.8 3.7 2,4-D 0.6 3.6 3.1 2,4-D 6.0 1.6 2.5 GA 10.0 0.9 4.5 GA and 2,4-D 10.0 0.6 0.8 3.5 GA and 2,4-D 10.0 0.6 0.9 3.3 * Ratio of fresh weight at end of test over initial fresh weight. 68 i L. G. Nickell and IV. R. Tulecke chemically induced, habituated), all crown-gall or virus tumor tissues, and the latter have been shown by Kulescha (4) to have endogenous levels of auxin much greater than homologous normal tissue. The results with tissues cultured on media containing gibberellin and/or 2,4-D suggest that these two substances operate separately. This suggestion is best supported by the results with the crown-gall tissues, normally grown on a synthetic medium without added auxin. Added gibberellin causes a 20 per cent increase in the growth of tobacco crown-gall tissue and more than a 60 per cent decrease in the growth of sunflower crown-gall tissue, whereas the addition of low levels of 2,4-D reduces the growth of both tissues. SUMMARY Forty-nine strains of tissue from 25 species of plants have been tested for their growth response to gibberellin at 10 p. p.m. in tissue culture. Depression of growth is the most pronounced effect, but the growth of several tissues is promoted. Probably the most interesting effect is the lethal action of gibberellin on two of the cultures. As a group, monocotyledonous tissues were depressed in growth. There is no apparent correlation between response to gibberellin and charac- teristics of strains of tissue such as agents inducing proliferation, plant parts from which the tissues were obtained, age of the cultures, or media supporting tissue growth. Tests with 2,4-D and gibberellin added singly and together suggest that these two growth substances act separately. LITERATURE CITED 1. Burklioldci, P. R., and Nickell, L. G. Atypical growth of plants. I. Cultivation of virus tumors of Rumex on nutrient agar. Bot. Gaz. 110: 426-437. 1949. 2. Chandler, C. The effect of gibberellic acid on germination and pollen tube growth. Contr. Boyce Thompson Inst. 19: 215-223. 1957. 3. Henderson, f. H. M. Effect of gibberellin on sunflower tissue culture. Nature. 182: 880. 1958. 4. Kulescha, Z. Croissance et teneur en auxine de divers lissus normaux et tumoraux. Annce Biol. 30: 319-327. 1954. 5. MacMillan, J., and Suter, P. J. The occurrence of gibberellin Aj in higher plants: isolation from the seed of runner bean (Phaseolus mnltiflorus). Naturwis. 45: 46. 1958. 6. Netien, G. Action des gib!)crcllines sur la culture des tissus vcgctaux cultiv& in vitro. Compt. Rend. Acad. Sci. Paris. 244: 2732, 2733. 1957. 7. Action de la gibberelline sur diff^rents types de tissus vcgctaux cultiv<^s in vitro. Compt. Rend. Acad. Sci. Paris. 247: 1645-1647. 1958. 8. Nickell, L. G. Embryo culture of weeping crabapple. Proc. Amer. Soc. Hort. Sci. 57: 401-405. 1951. 9. Radley, M., and Dear, E. Occurrence of gibbcrellin-like substances in the coconut. Nature. 182: 1098. 1958. Growth Substances and Plant Tissue Cultures 685 10. Schroeder, C. A., and Spector, C. Effect of gibberellic acid and indoleacetic acid on growth of excised fruit tissue. Science. 126: 701, 702. 1957. 11. Tulecke, W. Arginine-requiring strains of tissue obtained from Ginkgo pollen. Plant Physiol. 35: 19-24. 1960. 12. Vlitos, A. J., and Meudt, W. Relationship between shoot apex and effect of gibberellic acid on elongation of pea stems. Nature. 180: 284. 1957. 13. White, P. R. A Handbook of Plant Tissue Culture. 277 pp. The Ronald Press Co., New York. 1943. J. p. NITSCH and C. NITSCH Laboratoire du Phytotron Gif-sur-Yvette (S. et O.), France Growth Factors in the Tomato Fruit' The time at which one of us (J.P.N.) first became interested in the juice of tomatoes as a source of growth factors goes back to January, 1949, when an effort was made to grow excised ovaries in sterile cul- ture. At the suggestion of Mr. E. F. Vacin, who had been using to- mato juice (TJ) to improve the growth of orchid seedlings, tomato juice was added to the media which were to receive excised tomato ovaries. This resulted in the successful development of tomato fruits in sterile culture (20,21,22). Tomato juice was tried, then, on stan- dard strains of tissue cultures, namely crown-gall strains of Helian- thus annuus (De Ropp's PHI), of Nicotiana tabacum (Morel's), and of Opuntia monacantha (Morel's). In all these cases, after an initial slow start, growth burst into vigorously proliferating cultures (23). The fact that none of the strains used required auxin to grow indicated at once that one was dealing here with a factor quite different from an auxin. A more extensive study of this factor was indicated. It is clear from the literature that other workers also observed biological effects resulting from the addition of tomato juice. PHYSIOLOGICAL EFFECTS OF TOMATO JUICE Stimulation of Growth in Microorganisms Some 20 years ago, tomato juice was a popular source for "bios" factors for bacteria, yeasts, etc. (see, e.g., 4, 18, 19). Interest in the "bios" substances faded, however, as they were progressively identified ^A part of the work reported here was supported by the National Science Foundation (Grant G-4046) and the National Institutes of Health, Bethesda, Md. (Grant RG-4840). [ 687 ] 688 J. P. Nitsch and C. Nitsch as vitamins, amino acids, and other chemically-defined factors. In 1942 tomato juice was still reported to contain an unidentified growth fac- tor for hemolytic streptococci (6), and the possibility remains that there are, indeed, unidentified growth factors for microorganisms in tomato fruits. Inhibition of Seed Germination In any event, one of the effects of tomato juice which became known first was its inhibitory effect upon seed germination. After the early reports of Oppenheimer (30) and Reinhard and Gorelik (36), Kockemann (11) extended this type of investigation to other fruits and gave a name, blastocholine, to the principle present in the juice of many fleshy fruits which inhibits the germination of seeds. Larsen (14) found that the extract of ripe tomatoes contained a complex of substances which were inhibitory in the Avena curvature test; he could divide the ether extract into three fractions, each of which had inhib- itory properties. Ozorio de Almeida et al (31), on the other hand, re- ported that the factor inhibiting the germination of tomato seeds was insoluble in ether or chloroform, but soluble in alcohol, whereas Sar- tory and Meyer (37) found that the ether or chloroform extracts of tomatoes inhibited the germination of Lepidiiim seeds. Konis (12) ob- served that the inhibitory effect of tomato juice disappeared at low concentrations, under which circumstances, on the contrary, a pro- moting effect became visible. He thought that the inhibitor was a volatile substance, which could be partially destroyed by boiling. Stimulation of the Growth of Immature Embryos Tomato juice has been reported to stimulate the growth of very young embryos of Hordeurn (9). Its effect could be more or less du- plicated by casein hydrolysate or sodium nucleate. Meyer (17) and Vacin and Went (42) added tomato juice to media prepared for the asymbiotic development of orchid seed in sterile culture, which re- sulted in a marked stimulation of growth. The stimulative effect of tomato juice upon immature embryos is reminiscent of that of coco- nut milk (43, 44). Growth of Excised Ovaries The autoclaved aqueous extract of both green and ripe tomatoes was found to stimulate the growth of excised tomato ovaries which had been treated with 2-naj)luhoxyacetic acid to stimulate parthenocarpy (24), as shown in Figure lA. Confirmation of these results was reported with the use of a different, nonsterile technique (16). Growth of Roots Roots often form on the pedicels of tomato ovaries cultivated in vitro. The tomato juice which was added to the sterile medium was EXTRACT OF GREEN TOMATOES 0 5 10 25 50 75 0 5 10 25 CONCENTRATION (%,V/V) 50 75 Fig. 1. Effects of juice of green (left) and red (right) tomatoes (Lycopersicon escu- lentum) on the following processes: A: growth of excised, unpollinated tomato ovaries planted on artificial media at bloom, after having received 1 drop of an aqueous solution containing 100 mg/1 of 2-naphthoxyacetic acid; measurements after 9 weeks. B: percentage of peduncles of the above fruits which formed roots after 2 and 3 weeks. C: average number of roots produced per rooted fruit after 3 weeks. Each point is the average of 5 to 10 replicates. (From 24) 690 ]. P. Nitsch and C. Nitsch found to stimulate the development of these roots in an astonishing manner. Sucrose at a concentration of 5 per cent generally inhibits the elongation of adventitious roots produced on the floral peduncles, as contrasted with lower concentrations which allow perfect growth of the same primordia. The addition of tomato juice to the medium, however, allowed roots to develop profusely, even though the sucrose concentration was 5 per cent (24), as shown in Figure 1, B and C. Growth of Tissue Cultures Investigations over a period of years with various strains of tissue cultures showed that tomato juice stimulated the growth of normal tissues, tissues habituated to auxin, crown-gall tissues, and also tissues derived from pollen or endosperm. Crown-gall tissues. Since crown-gall tissues are known to grow well without any added auxin, no synthetic auxin was added to the media. Under these conditions, De Ropp's PIII strain of crown-gall tissues of Helianthus annuus and Morel's crown-gall strains of Nicotiana tabacum and Opuntia monacantha (23) and Parthenocissus tricuspi- data (3) all responded to the addition of tomato juice to the medium by an increase in fresh weight up to five times that of the controls, for the optimum concentration of 10 per cent (v/v) of TJ (Figure 2). Gau- CONCENTRATION [% , V/ V ) Fig. 2. Increase in fresh weight on addition of tomato juice to tissue cultures of Morel's crown-gall strain of Nicotiana tobacum (NT) and Opuntia monacantha (OM), after 5 weeks and 9 weeks, respectively. Each point represents the average of 6 (NT) or 10 (OM) replicates. (From 23) GroxL'th Factors in Tomato Fruit (")9 1 1.000 C A C A Fig. 3. Synergistic effect of a syntfietic auxin and 10 per cent (v/v) of tomato juice (TJ) on the growth of the cultures of: normal Helianthus tuberosus tuber tissues (HTN), and Gautheret's strains of habituated (SHH) and crown-gall root tissues of Scorzonera hispanica (SHG). C: controls without auxin. A: = 2,4-dichlorophenoxy- acetic acid (0.1 mg/1). The numbers on top of the black bars give the actual in- creases in mg. of fresh Aveight. (From 25) theret's crown-gall strain of Scorzonera hispanica, however, was in- hibited by TJ when small explants were used (3), but stimulated by TJ when larger explants were used (13). It was shown later by Nitsch and Nitsch (24) that this particular crown-gall strain also could be markedly stimulated by TJ even when small explants were used, pro- viding an auxin was added to the medium (see Figure 3). Habituated tissues. A preliminary experiment with Morel's ha- bituated strain of Rubus fruticosus indicated a stimulatory effect of canned TJ. Although their fresh weight was much greater, the tissues grown on TJ were not as healthy looking as the controls; they were lightly coloied, granular and friable, instead of being white and com- pact. New experiments, in which habituated strains of Parthenocissus tricuspidata of Morel and Scorzonera hispanica of Gautheret were used, showed only a marked growth inhibition by TJ (3). In these ex- periments no synthetic auxin had been added to the media, because it had been demonstrated that the habituated tissues proliferate very well in culture without added auxin (see 5). However, it was found later that the particular strain of habituated Scorzonera hispanica which had been used had, in fact, reverted to the auxin-requiring type. When a synthetic auxin was added to the media together with TJ, then the growth-stimulating effect of TJ became evident on the 692 J. P. Nitsch and C. Nitsch strains of habituated Scorzonera Jiispanica (Figure 3) and of iiabituat- ed Parthenocissus tricuspidata (25). Normal tissues. Normal tissues, such as those of Morel's normal strain of Parthenocissus tricuspidata (3) and the tuber tissues of Heli- anthus tuberosus (24) responded to TJ by a clear-cut increase in growth, providing a synthetic auxin was added to the medium (Figure 4). Tissues from roots of Scorzonera hispanica, from tubers of Solanum tuberosum or from fruit parenchyma of apples did not grow on media containing TJ plus a synthetic auxin. Special tissues. LaRue (15) mentions tomato juice among other addenda tried in order to stimulate the proliferation of endosperm tissue in sterile culture, and Tulecke (41) used tomato juice in some of his cultures of Ginkgo pollen, which led to the formation of un- differentiated masses of tissue. Induction of Bud Formation Cultures of Gautheret's habituated strain of Scorzonera hispanica occasionally formed small buds when cidtured on 5 per cent TJ. These buds could be made to develop until 1 or 2 small leaf primordia be- came visible. Later on, however, the buds died.- The effects which have been listed here are varied, and they may well be due to different constituents of the tomato juice. We will re- strict ourselves, therefore, to that effect which more specifically stimulates growth by increase in fresh weight in undifferentiated tis- sue cultures. GENERAL PHYSIOLOGICAL PROPERTIES OF TOMATO JUICE Looking for an explanation for the stimulative effect observed in tissue cultures, we have come to the following conclusions: Active Principle in TJ Not an Auxin Added auxins have no stimidatory effects on crown-gall cultures (10), in contrast to TJ. Moreover, to obtain a stimidation of growth with TJ on normal and certain other strains of tissue cidtines, an auxin must be added to the medium (25). "Using Skoog's technique, it lias since been found that a purified fraction of the tomato juice which, alone, had no growth-promoting properties on tobacco pith tissue, produced a voluminous and healthy callus when combined with 1 mg/1 of lAA. This is exactly what kinctin did to the same tissue. After some 6 weeks, manv buds developed on the growing cultures, just as they developed on cultures grown on kinetin plus lAA. It can be concluded, therefore, that TJ contains a natural kinin. [Sec Nitsch, J. P. Presence dime substance du type "cineline" dans Ic jus dc tomates. Bui. Soc. Bot. France. 107. 1960. (In press.)] Growth Factors in Tomato Fruit 693 0 2 4 8 16 CONCENTRATION (% ,V/V ) Fig. 4. Effect of various concentrations of tomato juice (TJ) and coconut milk (CM) on tfie growtli of cultures of Helianthus tuberosus tuber tissues, 'Blanc commun' in the presence of lAA (1 mg/1). (Curve TJ from 24, curve CM, new data.) Necessity of an Auxin As a matter of fact, there seems to be a synergistic effect between auxins and TJ (Figure 3). This effect became very striking when various fractions were bioassayed (Figures 5 and 6). Comparison With Coconut Milk Tomato juice often produced a larger increase in fresh weight of various cultures than did a comparable concentration of coconut milk, as is shown in Table 1 and in Figure 4. With coconut milk, however, the cultures were generally more compact and healthier than with TJ. Presence of an Inhibitory Principle Often, however, the tomato juice became highly inhibitory, whereas coconut milk did not. In cultures of Morel's normal strain of Parthenocissus tricuspidata, for example, the toxic effects of TJ became evident as soon as the TJ concentration was increased above 5 per cent (v/v), whereas coconut milk promoted growth over that of the controls even at a 50 per cent concentration (3). Thus, it soon appeared that, in addition to growth-promoting factors, TJ also con- tained substances inhibitory to growth in tissue cultures and to a much greater degree than coconut milk. 1,000- o X UJ 5 X UJ li. LlI < LlI o 500 - 1 993 - c 1 TJ EA 1 w - - 1 464 - - 68 ■ 1 1 59 e ■ 10 1 0 4 c A C A C A C A Fig. 5. Separation of an inhibitory principle from a growth-stimulating one by partitioning tomato juice between ethyl acetate and \\'ater. The fractions were tested on Gauthcret's strain of habituated Scorzonera hispanica without (C) and with 5 X 10'' M indole-3-acetic acid (A). TJ = whole tomato juice (10 per cent, v/v). EA ^ ethyl acetate fraction. W ^ aqueous fraction. The numl)ers on top of the bars give the exact growth increments. (From 25) Fig. 6. Aspect of the cultures of Srorzoiwrn liispaiiiai (hai)ituaicd stiain) giown for 11 weeks on the media indicated in Fissure 5. Growth Factors in Tomato Fruit 695 Table 1 . A comparison between the effects of tomato juice and coconut miliv upon the increase in fresh weight of various tissue cultures. Concn. of Addendum (v/v), % Increase in Fresh Weight, % of Control Tissue with TJ with CM Reference Nicotiana tabaciim (Morel's crown-gall strain) 10 202 107 Nitsch, unpublished Helianthns tuberosus 8 328 120 Nitsch, 'Blanc commun' (normal tuber tissue) unpublished Parthenocissus tricuspidata (Morel's normal strain) 5 297 138 3 Parthenocissus tricuspidata (Morel's crown-gall strain) 10 238 186 3 CHEMICAL WORK Bioassays To measure the biological activity of the different fractions which have been isolated, the following two assays were used: The Jerusalem artichoke test. The difficulty of securing large quantities of crown-gall tissue which would be homogeneous prompted us to use the xylem parenchyma of tubers of the P-17 strain of Je- rusalem artichoke {Helianthiis tuberosus) with which we had worked previously (27, 28). According to the procedure outlined (27), cylin- ders of tissue, generally 15 mg. in fresh weight, were removed asep- tically from the tubers and planted on sterile media. The increase in fresh weight of the cultures was determined 21 days later. The basal medium contained the following substances: the min- eral salts of the N^ solution (27) or those of an N2 solution which had the same composition as the Nj one, except that the sodium diphos- phate had been replaced by an equivalent amount of commercial sodium hexametaphosphate (Calgon); sucrose (50 g/1); lAA (5 X ^^'^ M); and Difco Bacto agar (10 g/1). The pH was always adjusted to 5.5 with HCl or NaOH before autoclaving. The media were auto- claved for 15 to 20 min. at 15 lbs. pressure. The auxin was added to the medium routinely because (a) Je- rusalem artichoke tuber tissues need an auxin in order to proliferate, (b) the aim was to pick up substances other than auxins, and (c) the factors present in TJ seemed to have a synergistic effect with auxins. 696 J. P. Nitsch and C. Nitsch Auxin tests. To determine the auxin activity of various fractions, the first internode test and the sensitized coleoptile tests were per- formed as described previously (26). Stability of the Tomato Juice Factor The tomato juice factor (TJF) was found to be relatively stable when autoclaved with the medium at pH 5.5 for 20 min. at 15 lbs. pressure. On the other hand, an experiment showed that autoclaved TJ (pH 5.0) left at room temperature under sterile conditions but ex- posed to air through a cotton plug had lost its activity one month later. Extraction Procedures The TJF was extracted from both dried and fresh material. After many preliminary experiments, the two following methods were found to give good results: From dried material. Fresh tomatoes were frozen at — 21° C, then broken into pieces while in the frozen state, and lyophilized. The re- sulting dry powder was extracted stepwise with the following sol- vents (Figure?): 1. Petroleum ether (b.p. 30 to 60° C): This produced a yellow liquid which fluoresced red in UV light and contained an in- hibitory principle which was partially destroyed by autoclaving. 2. Benzene: The extract did not seem to contain any active sub- stances. 3. Ethyl acetate: The dark green extract did not seem to contain active substances. 4. Cold methanol: The light green extract was partitioned between ether and water containing 0.5 per cent concentrated HCl. The ether fraction, which picked up all the chlorophyll, had little biological activity. The golden yellow aqueous fraction, on the contrary, contained both growth-promoting and growth-inhibit- ing substances. 5. Boiling methanol: This fraction had the greatest growth-pro- moting activity, together with some growth-inhibiting activity. 6. Boiling water: This fraction still had appreciable growth- promoting activity. In short, the stepwise extraction procedure showed that: (a) pe- troleum ether extracts an inhibitor which is partially destroyed by boiling, and (b) the growth-promoting factor is preferentially water- soluble, together with a strong inhibitor which is also water-soluble. From fresh material. Fresh tomatoes were stored frozen until needed. They were autoclaved for 10 to 15 min. at 15 lbs. pressure, in order to inactivate all the enzymes. The juice was then pressed out, filtered, and the remaining pulp washed with enough hot water to obtain a Growth Factors in Tomato Fruit 697 total weight of juice equal to the initial weight of the tomatoes. The pH of this juice was generally around 4.0. Separation of the Stimulating and Inhibiting Principles A difficulty arose from the fact that both the growth-promoting principle and the strong inhibitor were generally extracted together. Among the various methods tried to separate them, two techniques gave satisfactory results, namely partitioning between water and ethyl acetate and adsorption on activated charcoal. Extraction with ethyl acetate. When the crude juice or an aqueous solution containing the active principles was shaken with ethyl ace- tate, the growth-promoting principle moved, at least in part, to the ethyl acetate layer, the inhibitor remaining in the water layer (25). Experiments showed that more of the growth-promoting factor moved to the ethyl acetate layer at acid pH values (2.0) than at slightly alka- _i o q: I- z o o 200 X LlI X CO 50 MhS McE Controls ^ 100 r 1 "FT < - B tr o z " EA Fig. 7. Effects on Helianthus tuberosus cultures obtained from stepwise extraction of lyophilized tomato fruits with the following solvents: P: petroleum ether (b.p. 30 to 60° C), B: benzene, EA: ethyl acetate, Mc: cold methanol, Mh: hot methanol, W: hot water. The Mc extract has been partitioned between ether (McE) and water (McW) under acid conditions. The Mh extract, upon concentration and standing in the cold, has separated into a white precipitate (Mhp) and the su- pernatant (MhS). All fractions were tested on Helianthus tuberosus, strain P-17, at concentrations equivalent to 20 per cent (v/v) of the original juice. Each value is the average of 12 replicate cultures. Differences less than 15 per cent are not statis- tically significant. 698 /. P. Nitsch and C. Nitsch line ones (8.0). Thus a first method to separate the water-sohible in- hibitor from the growth-promoting principle is to partition the aque- ous extract with ethyl acetate at an acid pH (Figures 5 and 6). Adsorption on charcoal. A second method, which gave even slightly better results in that it apparently isolated a greater quantity of the growth-promoting material, made use of activated charcoal. The crude juice, or an aqueous extract, was treated with activated char- coal and filtered. The inhibitory principle remained in the filtrate while most of the growth-promoting activity remained adsorbed on the charcoal. The growth-promoting principle was eluted with boil- ing methanol. Since this eluate contained also substances of the auxin type, it was further partitioned between ether and water at pH 3.0 (HCl), the auxins moving to the ether layer and the TJF remaining in the water layer. Further Purification of the TJF Further purification of the growth-promoting principle was achieved through the use of paper and column chromatography. \\'ith the mixture ethyl acetate (9) -\- glacial acetic acid (3) -|- HoO (4) (v/v), three different active components could be separated on either paper strips or cellulose columns, the Rf values being: substance I (0.15), substance II (0.45), and substance III (0.95). GROWTH FACTORS IN TOMATO FRUITS The various techniques which had been briefly mentioned here have not yet led to the identification of any single compound which would be responsible alone for the biological properties of tomato juice. Instead, the TJF seemed to split into numerous constituents, many of which had a small activity on one or the other biological tests. Inhibitors Even the inhibitory effect of TJ was found to be due to at least three groups of substances, namely: Petroleum ether-soluble inJiibitor. This was extracted with pe- troleum ether from either dried or fresh material and was present in green and in ripe tomatoes. As was already reported by Konis (12), this inhibitor is partially eliminated by autoclaving. It is probably an essential oil. Ether-soluble inhibitors. This fraction Avas reported by Larsen (14) and Hemberg (7) to contain substances which inhibit curvature in the Avena test. One of these inhibitors may be salicylic acid which was found to move at the same position as the inhibitory ether frac- tion on paper chromatograms and which was found to inhibit the Groiuth Factors in Tomato Fruit 699 elongation of coleoptile sections. Another might be ferulic acid which was reported by Akkerman and Veldstra (1) as being one of the com- ponents of Kockemann's blastocholine. In our tests, ferulic acid had an inhibitory effect upon the growth of the first internodes of Avena seedlings in the presence of small amounts of lAA. Upon the growth of Jerusalem artichoke tissues, however, ferulic acid had no visible effect, at least at concentrations ranging from lO-e to lO-^ M. Ferulic acid, on the other hand, has been reported by Hemberg (7) and by Reinders-Gouwentak and Smeets (35) to have a synergistic effect with auxins. Water-soluble inhibitors. The aqueous fraction was the one which contained the most powerful inhibitors after autoclaving. Their chemical nature has not yet been determined. Chlorogenic Acid and Its Derivatives In addition to ferulic acid, Akkerman and Veldstra (1) extracted caffeic acid from tomatoes and claimed that it was a component of the blastocholine complex. Later on, however, it was reported (7, 35) that caffeic acid not only was not an inhibitor but was actually an auxin synergist. In our tests with Jerusalem artichoke tissues, caffeic acid (at least between lO^ and 10 ^ M) gave a small synergistic effect with lAA. Since caffeic acid is actually a component of chlorogenic acid, which has been reported in tomatoes (8), we have also tested chlorogenic acid^, quinic acid, a mixture of caffeic and quinic acids, and cynarine^ which contains two molecules of caffeic acid per mole- cule of quinic acid (32). None of these compounds gave evidence for a stimulation of growth at greater than 10 to 15 per cent concentra- tions ranging from lO-s to 10-* M. Toxic effects occurred at higher concentrations. On the contrary, quite unexpected results were obtained when these compounds were tested on the Avena first internode and coleop- tile bioassays for auxins. None of them had any auxin activity when used alone. In conjunction with low concentrations of lAA, however, they showed a marked synergistic effect, a rather pronounced re- sponse being obtained with chlorogenic acid (29). The effect of chloro- genic acid could be ascribed to its caffeic acid moiety, the quinic acid part having no growth-promoting effect (Figure 8). Similar results were obtained with coleoptile sections. Chlorogenic acid had an ef- fect even when it was not given at the same time as lAA to the first internode or coleoptile sections. Thus, these sections could be first soaked in chlorogenic acid for 1 hour, then taken out of this solution ^Kindly supplied by Dr. A. C. Hulme. * Kindly supplied by Dr. L. Panizzi. 700 J. P. Nitsch and C. Nitsch LOG. -6 MOLAR -5 -4 -3 CONCENTRATION Fig. 8. Synergistic effects of chloiogenic acid (Ch), carteic acid (Ca), quinic acid (Q), and caffeic + quinic acids at equimolar concentrations (Ca -|- Q) upon the elongation of Avena first internodes. The concentration of lAA was 10 fig/h initial length 4 mm. and transferred to tubes containing lAA only. An enhancement of the lAA effect could be observed under these conditions. Such a re- sult may perhaps be ascribed to the reported inhibitory effect of chlorogenic acid on lAA oxidase (33). Carboxylic Acids Since the fraction containing the TJF Avas acid, the two most prominent acids of tomato juice, malic and citric acid, were tested on tissue cultures. The effect was slight (Table 2). Amino Acids Tomato juice contains, of course, many amino acids, such as glutamic acid, glutamine, arginine, tryptophane (2), y-aminobutyric acid (38), etc. It has been shown (28) that glutamic acid and gluta- mine have a stimulatory effect upon the growth of Jerusalem artichoke tissue cultures in the presence of lAA. A mixture of amino acids, such Table 2. Activity of various known components of tomato juice and related com- pounds on the increase in fresh weight of tissue cultures. (Differences smaller than 15 per cent are generally not statistically significant.) Autoclaved Molar Concn. Fresh Weight, or Species and X 10^5 or Per Cent of Compound Filtered Strain Ml/LJ Control Malic acid autoclaved Helianthus 10 112 tuberosus. 1 100 'Blanc commun' 0.1 95 Malic acid filtered H. tuberosus, 100 106 P-17 10 97 Malic acid and autoclaved Nicotiana 220 citric acid tabacum * 190 100 Casein hydrolysatef autoclaved N. tabacum * 1 ml. 1 ml. 1 ml. 103 113 114 Casein hydrolysate filtered H. tuberosus 200 (N) 136 autoclaved P-17 200 (N) 141 Glutathione filtered 5.2 208 Glutathione and // JV. tabacum * 5.2 216 ascorbic acid It 14.0 Glutathione autoclaved 5.2 73 Glutatliione and // 5.2 casein hydrolysate // 1 ml. 119 Ascorbic acid filtered JV. tabacum * 14 100 10 188 134 99 1 83 Epicatechin filtered H. tuberosus 10 139 ff P-17 1 100 II 10 104 II 1 92 II 0.1 126 autoclaved , 1 107 Catechin filtered H. tuberosus 10 116 II P-17 1 109 II 10 100 II 1 107 II 0.1 92 autoclaved 1 100 Quercetrin filtered H. tuberosus 2.2 125 P-17 0.2 0.02 112 119 * Morel's crown-gall strain of W. tabacum. t Enzymatic casein hydrolysate (vitamin free) 0.5 per cent solution obtained from the Nutritional Biochemicals Corp., Inc., Cleveland, Ohio. X When the same concentrations appear twice in this column the experiments have been repeated at different times. 702 ]. P. Nitsch and C. Nitsch as those contained in casein hydrolysate, was found to promote the growth of the Jerusalem artichoke tissues also as well as that of crown- gall tissues (Table 2). Thus, it is possible that part of the TJ effect is due to its amino acid content. Reducing Compounds Tomato juice also contains reducing compounds, such as ascorbic acid, glutathione (40), phytoene (34), etc. Ascorbic acid and glutathi- one were found to enhance the growth of the strains of tissue cultures which we stimulated by TJ (Table 2). Epicatechin'', at the concentra- tion of 10"^ M, also gave some growth stimulation, more than cate- chin.5 Flavones The yellow color of substances I, II, and III, and the fact that these colors deepened markedly under alkaline conditions suggested to us that these compounds could be flavones. Although the precise chemical nature of these three substances has not yet been determined, we suspect that they are glycosides, at least substances I and II. Quer- cetrin was found to stimulate slightly the growth of Jerusalem arti- choke tissues (Table 2). The presence of biologically active glycosides of flavones in TJ would be of special interest in view of the report that a leucoanthocyanin is one of the active components of coconut milk (39). Sugars The fraction which produced the most intense growth effects upon the growth of Jerusalem artichoke tissues was, when concentrated, of the consistency of a sugar syrup. From this fraction a whitish com- pound could be precipitated out with ether or acetone. This substance, called substance S, was biologically active. Auxins Methanol extracts of tomatoes at various stages of development yielded several growth substances of the auxin type which were separated by paper chromatography. These substances, with their relative biological importance, Rj values, and color reactions are listed in Table 3. It should be noted that, in addition to biologically active substances, several other spots giving a color with the Ehrlich re- agent for indoles were found to have no growth-promoting effect upon the elongation of Avena first internodes. Kiiully sii]>pliccl by Dr. K. Frciulcnlicrg. Growth Factors in Tomato Fruit 703 Table 3. Substances active on the Avena first internode test obtained from methanol extracts of lyophilized, immature tomatoes chromatographed on Whatman No. 3 MM paper in 80 per cent isopropanol redistilled over Zn and KOH. Intensity of Color With Biological Ehrlich's Substance R. Response Reagent A 0.05 + B 0.20 + + + purple * C 0.35 + + D 0.50 + + purple * E 0.65 + + + F 0.90 + * It is not yet known if the growth-promoting substances are actually EhrUch-positive. One can say only that their position coincides with an area which reacts with Ehrlich's reagent. SUMMARY At the present time, the chemical identification of all the active components of tomato juice is not yet completed. The work pre- sented here, therefore, is more a progress report than the account of a completed task. The wide range of biological effects exerted by to- mato juice may well be due to different, unrelated constituents. In any case, we have been able to separate an inhibitory principle which can be split further into three categories: petroleum ether-soluble, ether-soluble, and water-soluble compounds. In the growth-promoting fraction of tomato juice, some compounds, such as chlorogenic acid and derivatives, were found to act as auxin synergists on the first inter- node and coleoptile sections of Avena. Other compounds promoted the growth of Helianthus tuberosiis tuber tissues, but only when an auxin was added simultaneously. Such results gain interest when one considers that the tomato is one of the fruits which can be set easily with applied auxins. It is conceivable that the applied auxins work in conjunction with the synergists which are naturally present in the ovary of this species. LITERATURE CITED 1. Akkerman, A. M., and Veldstra, H. The chemical nature of Kockemann's blastocholine from Lycopersicon esculeiitum Mill. Rec. Trav. Chim. Pays-Bas. 66: 411,412. 1947. 2. Carangal, A. R., Jr., Alban, E. K., Varner, J. E., and Burrell, R. C. The in- fluence of mineral nutrition on the organic acids of the tomato, Lycopersicum esculentum. Plant Physiol. 29: 355-360. 1954. 3. Demetriad^s, S., and Nitsch, J. P. Jus de tomate et croissance des tissus et organes vegetaux. Action sur trois categories de tissus de vigne-vierge et de scorsonere. Comparaison avec le lait de coco. Compt. Rend. Soc. Biol. 147: 1711-1713. 1953. 704 j. P. Nitsch and C. Niisch 1. Elder, M. L. Preparation of crude bios V and its influence on the reproduction of certain microorganisms. Trans. Roy. Soc. Canada. III. 30: 89-97. 1936. 5. Gautheret, R. J. The nutrition of plant tissue cultures. Ann. Rev. Plant Physiol. 6: 433-184. 19.55. 6. Grossowicz, N. A new growth factor for hemolytic streptococci. Proc. Soc. Exper. Biol. Med. 49: 8-11. 1942. 7. Hemberg, T. Establishment of acid growth-inhibiting substances in plant extracts containing auxins by means of the Avena test. Physiol. Plant. 4: 437- 445. 1951. 8. Herrmann, K. tjber das Vorkommen von Kaffeesaure und Chlorogensaure im Obst und Gemiise. Naturwis. 43: 109. 1956. 9. Kent, N., and Brink, R. A. Growth in vitro of immature Hordeum embryos. Science. 106: 547,548. 1947. 10. Klein, R. M., and Link, G. K. K. The etiology of crown-gall. Quart. Rev. Biol. 30: 207-277. 1955. 11. Kockemann, A. tJber eine keimungshcmmende Substanz in fleischigen Friichten. Ber. Deutsch. Bot. Ges. 52: 523-526. 1934. 12. Konis, E. On germination inhibitors. II. On the action of germination inhibit- ing substances in the tomato fruit. Palest. Jour. Bot. Jerusalem. 2: 6-27. 1940. 13. Kovoor, A. Action de quelques substances stimulantes d'origine naturelle sur le developpement des tissus vegetaux cultives in vitro. Annee Biol. 30: 417-429. 1954. 14. Larsen, P. tJber Hemmung des Streckungswachstums durch natiirlich vorkom- mende, atherlosliche Stoffe. Planta. 30: 160-167. 1939. 15. LaRuc, C. D. Cultures of the endosperm of maize. Amer. Jour. Bot. 36: 798. 1949. 16. Leopold, A. C, and Scott, F. I. Physiological factors in tomato fruit-set. .\mer. Jour. Bot. 39: 310-317. 1952. 17. Meyer, J. R. The use of tomato juice in the preparation of a medium for tiie germination of orchid seeds. Amer. Orchid Soc. Bui. 14: 99-101. 1945. IS. Miller, W. L. Wildiers' bios. Trans. Roy. Soc. Canada. III. 30: 99-103. 1936. 19. . Wildiers' bios. Trans. Roy. Soc. Canada. III. 31: 159-162. 1937. 20. Nitsch, J. P. Obtention de fruits charnus en culture in I'itro. Conipt. Rend. Acad. Sci. Paris. 229: 445, 446. 1949. 21. . Culture of fruits in vitro. Science. 110: 499. 1949. 22. Growth and development in vitro of excised ovaries. Amer. Jour. Bot. 38: 566-577. 1951. 23. . Action du jus de Tomate sur la croissancc des tissus de crown-gall cul- tives in vitro. Compt. Rend. Acad. Sci. Paris. 233. 1676, 1677. 1951. 21. . Action du jus de Tomate sur la croissance de certains tissus et organes v^g^taux. Bui. Soc. Bot. France. 101: 433-440. 1954. 25. , and Nitsch, C. Action synergique des auxines et du jus de Tomate sur la croissance de tissus vegetaux cultives in vitro. Bui. Soc. Bot. France. 102: 519-527. 1955. 26. , and Nitsch, C. Studies on the growth of coleoptilc and first internode sections. A new, sensitive, straight-growth test for auxins. Plant Physiol. 31: 94-111. 1956. 27. , and Nitsch, C. Auxin-dependent growth of excised Heliantlius tuber- osus tissues. I. Amer. Jour. Bot. 43: 839-851. 1956. 28. , and Nitsch, C. Auxin-dependent growth of excised Heliantlius tuber- osus tissues. II. Organic nitrogenous compounds. Amer. Jour. Bot. 44: 555-564. 1957. Growth Factors in Tomato Fruit 705 29. , and Nitsth, C. Synergic d'action entre les d6riv& de I'acide chloro- genique et I'acide 3-indolylac(^tique. Bui. Soc. Bot. France. 106: 414-417. 1959. 30. Oppenheiiner, H. Keimungshemmende Substanzen in der Frucht von Solarium lycopersicum und in anderen Pflanzen. Sitzungsber. Akad. Wiss. Wien Math.- Nat. Abt. I. 131: 59-65. 1922. jl. Ozorio de Almeida, A., Goulart, M. D., lelpo, M., and Vieira Pinto, A. Inhibi- tory action of tomato (Solanum lycopersicum) fruit juice on germination of seeds and growth of plants. Revistra Brasil. Biol. 1: 345-354. 1941. 32. Panizzi, L., and Scarpati, M. L. Isolamento e costituzione del principio attivo del carciofo. Gazz. Chim. Ital. 84: 792-805. 1954. 33. Rabin, R. S., and Klein, R. M. Chlorogenic acid as a competitive inhibitor of indolcacetic acid oxidase. Arch. Biochem. Biophys. 70: 11-15. 1957. 34. Rabourn, W. J., and Quackenbush, F. W. The occurrence of phytoene in various plant materials. Arch. Biochem. Biophys. 44: 159-164. 1953. 35. Reinders-Gouwentak, C. A., and Smeets, L. Growth hormone relations in the tomato. Konink. Ned. Akad. Wetenschap. Proc. 56C: 354-358. 1953. 36. Reinhard, A. W., and Gorelik, M. Zur Frage der Samenkeiraung bei Solanum lycopersicum. Planta. 20: 792-794. 1933. 37. Sartory, A., and Meyer, J. Sur un facteur bacteriostatique des tomates fraiches et dessechees. Compt. Rend. Acad. Sci. Paris. 222: 151-153. 1946. 38. Shinano, S., Kaya, T., and Kaneko, H. Isolation of 7-aminobutyric acid from fruit juice. Jour. Agr. Chem. Soc. Japan. 30: 175-178. 1956. 39. Steward, F. C., and Shantz, E. M. The chemical induction of growth in plant tissue cultures. lu: R. L. Wain and F. Wightman (eds.), The Chemistry and Mode of Action of Plant Growth Substances, pp. 165-186. Butterworth Sci. Publ., London. 1956. 40. Tombesi, L., Baroccio, A., Cervigni, T., Fortini, S., Tarantola, M., and Venezian, M. E. Attivita ossidasica, catalasica, carbonanidrasica, perossidasica e contenulo in glutatione ridotto ed acido ascorbico nel corso della maturazione di frutti e semi. Ann. Sper. Agron. II. 6: 857-874. 1952. 41. Tulecke, W. The pollen of Ginkgo biloba: in vitro culture and tissue forma- tion. Amer. Jour. Bot. 44: 602-608. 1957. 42. Vacin, E. F., and Went, F. W. Use of tomato juice in the asyrabiotic germina- tion of orchid seeds. Bot. Gaz. Ill: 175-183. 1949. 43. van Overbeek, J. Hormonal control of embryo and seedling. Cold Spring Harbor Symp. Quant. Biol. 10: 126-134. 1942. 44. , Conklin, M. E., and Blakeslee, A. F. Cultivation in vitro of small Datura embryos. Amer. Jour. Bot. 29: 472-477. 1942. DISCUSSION Dr. Siegel: The reference made by Dr. Nitsch to the chemical re- ducing activity of growth factors prompts me to call attention to the generality of this relationship ancl to the manner in which it ties to- gether many points discussed here. The main concept in our paper in this volume is that most organisms must, of necessity, live in an environment which can and does attack continuously their metabo- lites, catalyses, and architecture. Oxygen is the only common electron acceptor which can support bioenergetic processes effectively, yet the hazards to life of its aerobic environment have been well documented. Our contribution to this 706 J. P. Nitsch and C. Nitsch volume contains a number of recent references to oxygen toxicity, but a fuller appreciation of the phenomenon can be gained by examina- tion of older studies (Clements, Carnegie Inst., \V'^ashington, 1921). The toxic effects of oxygen on plants were known to Scheele (1777), Huber and Senebier (1801), etc. The benefits of reduced (sub-atmospheric) oxygen levels were appreciated by Sedebeck (1881) and Jaccard (1893). We have, on the whole, relegated such nonchemical effects and ob- servations to the physiology of our earlier day, and pursued the study of chemical growth factors without regard to nonmetabolic oxidation. Now we are confronted with observations which suggest that the functions of growth factors be re-examined, keeping in mind the duality of oxygen as part of the biological milieu. Dr. Stowe was concerned about the discrepancies in growth rate between sections and intact organs. He sought to narrow the gap, finally by application of unsaturated lipids. Drs. Crosby and Vlitos discovered that fatty substances could supplant conventional auxins as growth factors. Dr. Crosby recognized the antioxidant character of some indolic auxins. Dr. Marre discussed growth inhibitions which he associated with oxidized ascorbic acid, and speculated that ascorbic acid might also be a growth promoter (but only, we suggest, in re- duced form). Dr. van Overbeek alluded to the importance of acid and the reducing environment of archeozoic times but did not dis- tinguish between oxygen uptake, respiration, and energetic processes, thus leaving the issue unsettled. Drs. Muir and Hansch, and Dr. Thimann considered the electronic features of organic compounds as directly related to their auxin or auxin-like properties. From their comments we would assume that electron delocalization, polarization, and local electron densities are important in relation to attachment of the hormone to its acceptor. Now, the significance of electrical forces in the localization of hormones is obvious, but it was somewhat sur- prising that those who treated these matters wuth such facility did not carry their consideration of electron mobility beyond the primary events of auxin-acceptor interaction. The true mechanism of action of auxin or other growth factors is hardly to be found in a consideration of attachment, nor in delibera- tions on the biophysics of the cell wall. Thus, a knowledge of the factors which enable auxin to be delivered to the right place in the cell still leaves unanswered the question: "What does the auxin do when it gets there?" No laboratory has yet answered this cjuestion, but we contend that important elements of the answer reside in those electronic properties which are particularly meaningful when con- sidered together with oxygen toxicity. "Antioxidant" and "reducing" are operational terms. They a})ply to molecules, or ions, which are characterized more fundamentally by their high electron availability. Growth Factors in Tomato Fruit 707 The property we seek resides in pi-electron donors and atoms with nonbonding electrons, singly or in combination, hence, in the indoles, aryloxy compounds and other aromatic substances which have occu- pied so much of our interest. It does not follow that all antioxidants (or electron donors) will be growth promoters, or even regulators. The growth-promoting po- tentialities of these compounds can only be realized if they are pre- sented to the cell in suitable form. Antioxidants of different structure which promote growth may operate through different limiting path- ways, some acting as auxins, some as auxin-sparing agents, and others as more uniquely localized protectants or cofactors. ; \ y- ■-. r '.; i . . ULRICH N AF The Rockefeller Institute On the Physiology of Antherldlum Formation in Ferns' CONTROL OF ANTHERIDIUM FORMATION IN DIFFERENT GROUPS OF FERNS Dopp (5) demonstrated that an extract from mature prothalli of Pteridium aqiiilinum hastened the onset of antheridium formation in young prothalli of this fern species by a few days and in the pro- thalli of Athyrium filix-mas by a few weeks. Dopp envisaged the pos- sibility that the promotion of antheridium formation was the result of nonspecific growth inhibition. Subsequent investigations (9), though, led to the conclusion that the activity of the extract must be attributed to a specific factor which controls antheridium forma- tion during normal development. An assay was devised which took advantage of the observation that the prothalli of Onoclea sensibilis failed to form any antheridia spontaneously under the prevailing conditions of culture but re- sponded readily if extract from mature prothalli of Pteridium aquili- niim was added (see Figure lA, B). Conditions were further defined under which the extract from 7-week-old prothalli of Pteridium nquilinum was active to a dilution of 1:30,000. This increased the activity obtained by Dopp by a factor of about 300. Under these same conditions of culture the active substance accumulated to almost as high an activity in the medium (9). The available data show that this substance must be active at a dilution of 1.6 X 10"^ o^' l^^s (9). Studies on the activity spectrum of the factor disclosed that it was active toward the tested representatives of seven out of the nine subgroups of the family Polypodiaceae listed by Eames (7): The As- *This investigation was supported in part by research grants (NSF G-3225 and NSF G-6144) from the National Science Foundation. [ 709 ] 710 U. Ndf plenioids, Pteroids (5), Onocleoids, Blechnoids, Dryopteroids (9), Gymnogrammoids (6), and the Woodsioids (11). Among nonpoly- podiaceous species, only Dennstaedtia pinictilobula (Dicksoniaceae) was found to be responsive. The substance failed to promote anther- idium formation even at the highest available concentration (i^ strength Pteridium medium that was active toward the prothalli of Onodea sensihiUs to a dilution of 1:30,000) in the following fern species: Polypodium aureum (Polypodiaceae), Lygodiinn japonicum, Anemia phylUlidis (Schizaeaceae), Osmunda claytoniana, and O. cin- namomea (Osmundaceae). Studies on fern species that were unresponsive toward the Pterid- ium factor led to the demonstration of a substance that controls ■^\ A B "h' \ t v- 9 D ^■^T^:^:- Fig. 1. A. Gametophytc ol Oiioclea sejisibilis, 19 days old, 33 X, gr()\\n in tlie presence of Pteridium medium 1:250. B. Same as A, except grown in the absence of Pteridium medium. C. Incipient ameristic prothallus of Pteridium aquilinum, 20 days old, 55 X. D. Gamctopliytc of Anemia pliyllitidis, 12 days old, 91 X, grown in the presence of Anemia medium 1:30. E. Same as D, except grown in the ab- sence of Anemia medium. F. Ganictopliyte of Anemia plixllitidis, 25 days old, !! X. G. Ganu'i()|)li\tc of Anemia phylUtidis, 35 days old, 30 X. Physiology of Anthcridium Formation in Ferns 711 antheridium formation in Anemia phyllitidis (11) but is inactive to- ward the species used to assay for the Pteridium factor. In Lygodiiim japojiicii7n antheridium formation seems to be controlled by still another substance (12) even though this species belongs to the same family as A. phyllitidis (Schizaeaceae). The application of the Anemia factor to young cultures of Anemia phyllitidis led to the onset of the antheridial phase while the pro- thalli were still at a juvenile (filamentous) phase (Figure ID, arrows indicate antheridia). Control prothalli (Figure IE) were invariably free of antheridia even at the much more advanced stage of develop- ment pictured in Figure IF. It is apparent from these studies that antheridium formation is controlled by different substances in different groups of ferns. It should also be stressed that within the wide range of species respon- sive to the Pteridium factor, the minimally effective concentrations vary widely. Thus the prothalli of Dennstaedtia punctilobula failed to respond unless they were supplied with the Pteridium factor at a concentration about 125 times higher than was necessary in the prothalli of Onoclea sensibilis and of Pteridium aquilinum itself. In the prothalli of Woodsia obtusa the minimally effective concentra- tion of the Pteridium factor exceeded that required for antheridium formation in Onoclea sensibilis by a factor of about 25 (11). The possibility must therefore be considered that the factors con- trolling antheridium formation in these species are actually different but structurally so closely related that the factor produced by Pter- idium aquilinum is capable of bringing about antheridium forma- tion also in Dennstaedtia punctilobula and in Woodsia obtusa if it is supplied at a high enough concentration. Raper (14) also considers the possibility that hormonal specificities account for the failure to obtain oospores in some of the attempted interspecies and interge- neric crosses of water molds. The above results raise a question of biological specificity. The studies of Kluyver and Van Niel have drawn attention to the simi- larity, even identity, of basic biochemical patterns in taxonomically widely separated organisms. It is tempting to postulate that the metabolism associated with antheridium formation, i.e., an event that we conceive of mainly in morphological terms, is also similar in different fern species. The above results may be reconciled with such a postulate if we consider that the induction of an antheridium is likely to involve many reactions and compounds. Antheridium for- mation in Pteridium aquilinum. Anemia phyllitidis, and Lygodium japonicum might thus be controlled by different factors because dif- ferent reactions became rate-limiting during evolution. Alternatively, we might be witness to evolution on a molecular level. On this as- 712 U. Ndf sumption the inducing molecule has undergone a gradual structural modification probably concomitantly with changes in a receptor molecule. The isolation and characterization of the three factors should yield pertinent information. In the meantime, an attempt is being made to assay for similarity between the various factors based on the postulates that one factor may be a precursor of the other or that one factor may behave as a chemical analogue of the other and thus interfere with its synthesis or with the function it performs in the initiation of antheridia. LOSS OF SENSITIVITY TO THE ANTHERIDIUM-INDUCING FACTOR Many individuals in a gametophyte population of Pteridium aquilinum, and of many other fern species, have an early antheridial phase which is, however, terminated as the prothalli attain the arche- gonial phase. Why do these prothalli discontinue the formation of antheridia? The hypothesis may be proposed that they discontinue forming the antheridium-inducing factor. It was shown, however, that the active substance becomes avail- able at progressively higher concentrations as more and more pro- thalli stop forming antheridia and begin to produce archegonia in- stead (10). This suggests that the gametophytes continue to elaborate the antheridium-inducing factor while they form archegonia. It must be pointed out, however, that even mature cultures contain a small percentage of gametophytes which form antheridia only throughout the life of the culture (the so-called ameristic prothalli; see section beginning on page 716). Accordingly, it may be these ameristic pro- thalli rather than the archegonium-bearing prothalli which account for the continued elaboration of the active substance by maturing cultures of Pteridium aquilinum. Dopp (5) effected a separation, ne- cessarily incomplete, of the two types of prothalli and found the ac- tive factor to be present in the extract of either type if at somewhat greater concentration in the archegonium-bearing prothalli. This evidence does not unequivocally support Dopp's conclusion that archegonium-bearing prothalli produce antheridial factor. Rather, the active substance found in the archegonium-bearing prothalli might have been produced at an earlier, antheridial stage. Moreover, the active substance found in either type of prothallus might have been taken up from the medium into which it was secreted by the other type of prothallus. These objcc tions were met by isolating arch- egonium-bearing prothalli, one per flask, and assaying the media for antheridium-inducing activity at intervals over a period of time. Such studies showed (see Table 1) that archegonium-bearing prothalli actu- ally produced large amounts of the antheridal factor (10). Physiology of Antheridium Formation in Ferns Table 1 . Number of media (out of 20 at each in- terval) that have antheridium-inducing activity to the indicated maximal dilutions. 713 Dilution of Medium Time in Days 3 13 23 1/2 17 3 2 16 2 1/10 1/50 2 1/250 12 1/1,250 6 Discontinuance of antheridium formation could not, therefore, be ascribed to a discontinuance of antheridial factor production. The alternative hypothesis suggested itself, that the maturing prothalli become insensitive to antheridial factor, i.e., that they lose the ability to respond with antheridium formation to the presence of that factor. This hypothesis was tested first on the prothalli of Onoclea sensibilis which were particularly suited to such an investigation because they failed to form antheridia spontaneously under the prevailing condi- tions of culture but formed them readily in response to added anther- idial factor. Prothalli of this species were transferred at intervals of 2 days, starting 8 days after inoculation, to new medium containing the active factor at a concentration series ranging from 1/2 to 1/31,250 full strength Pteridium medium. Table 2 shows that, whereas nearly all 10-day-old prothalli were sensitive to the antheridial factor, nearly all 14-day-old prothalli were insensitive to it. Subsequent investiga- tions on isolated prothalli showed that the individual prothallus be- came insensitive within a period of about 2 days or less. Following this interval, a gametophyte fails to form antheridia even at the highest concentration of the antheridial factor, i.e., a concentration Table 2. Number of prothalli (out of 20) that forrned antheridia when transferred from basic medium to medium containing antheridial factor at different intervals following inoculation of the spores. Dilution of Pteridium Medium Interval in Days 8 10 12 14 16 1/2 1/10 1/50 1/250 1/1,250 1/6,250 1/31,250. . . . 20 20 20 20 20 20 7 20 19 20 20 20 19 6 7 6 5 6 5 5 1 2 0 1 2 2 1 0 O OOO OOO 1 Control 0 0 0 0 0 714 U. Ndf Days a'ter inoculation of the spores Fig. 2. Sequence of developmental stages in prothalli of Onoclea seiisibilis. (a) Num- bers of prothalli (out of 40) that have attained heart shape, (b) Numbers of prothalli (out of 40) that have become insensitive to the antheridial factor, (c) Num- bers of prothalli (out of 10) that have initiated one or more archegonia. 15,000 times higher than that sufficient to induce antheridia in pro- thalli just 2 days younger. Figure 2 relates the onset of this loss of sensitivity to two other major events in the development of Oiioclea gametophytes: attain- ment of heart shape and of the archegonial phase. The results re- corded in this figure show that the prothalli became insensitive to the antheridial factor about 2 days after attaining heart shape and 6 to 7 days prior to the attainment of the archegonial phase. In cultures of Pteridium aquiUniim the first insensitive prothalli were observed in 7-day-old cultures. The studies on this fern species further showed that the organization of the meristem, the activity of which results in the attainment of heart shape, again precedes the loss of sensitivity to the antheridial factor (10). Similar studies were carried out on Anejnia pJiylUtidis. Figures IF atul IG show that the prothalli of this fern species have a lateral meristem, a phenomenon encountered in only few fern species. The first antheridium is invariably initiated by a marginal cell a short distance back of the meristematic initial. Marginal cells cut off subsc(|ucntly by the meristematic initial also give rise to antheridia so that ultimately a whole row of them can be seen (marked h in Fig- ure IG). In contrast, the marginal cells given off by the initial toward the tip of the prothallus fail to form antheridia even in the presence of added Anemia factor. Instead, some of them give rise to hook- shaped hairs (arrow r). Once the first antheridium has been initiated Pliysiology of Antheridium Formation in Ferns 715 marginally, others will arise also on cells inside the prothallus (three of ten such antheridia are indicated by arrows). The area of spontaneous antheridium formation always remains restricted, however. Thus, antheridia never arise in the part of the prothallus anterior to the lateral meristem. Again the zone of an- theridium formation in the posterior region does not stretch across the whole prothallus; instead, it extends about half way or less inside the prothallus from the margin of the cell plate which bears the lateral meristem (zone is marked by broken line on Figure IG). The other half of the posterior region remains free of antheridia except for the occasional occurrence of one to three antheridia in marginal cells. Observations following the application of the active substance to cultures of various ages showed that the very young prothallus re- sponded with antheridium formation throughout most of its body. As the age of the prothalli increased, though, the responding area gradually contracted to that described for spontaneous antheridium formation. The loss of sensitivity in the anterior part of the prothal- lus preceded the contraction in the responsive area of its posterior region (11). Dopp (6) showed that the excision of the meristem from arche- gonium-bearing, i.e., insensitive, prothalli of Pteridium aquilimim led to the formation of antheridia in the regenerating fragments. The same phenomenon was encountered in Onoclea sensihilis (au- thor's results). The studies which were carried out on wings, cut from insensitive, archegonium-bearing gametophytes, further showed that antheridium initials in these meristemless fragments did not appear, even at the highest available concentration of antheridial factor, until 7 to 14 days after the wings were removed. In contrast, 9-day-old, i.e., still sensitive, whole prothalli will give rise to anthe- ridium initials within a period of between 21/2 and 3 days after the antheridial factor has been applied. Thus the removal of the meri- stem leads to the restoration of sensitivity with a considerable time lag. Further observation showed that antheridia were formed only in those regions of the meristemless prothalli which had undergone a considerable amount of cell division. This correlation between antheridium formation and cell division indicates that something is diluted out before the cells become sensitive again. As already mentioned, the application of the antheridium-induc- ing factor to 9-day-old whole prothalli led to the appearance of an- theridium initials within a period of between 2i/9 and 3 days at all tested concentrations. If the antheridial factor was withdrawn from the medium li/o days after it was applied, then no antheridia were formed. 716 U. Ndj THREE TYPES OF GAMETOPHYTES IN CULTURES OF PTERIDIUM AQUILINUM Mature gametophyte cultures of PtericUum aquilinum, and of many other homosporous, leptosporangiate fern species, contain two types of prothalli. Some bear anthcridia only and are commonly designated the male prothalli. They lack a meristem (hence their diffuse growth habit and random shape) and are for this reason occa- sionally also referred to as the ameristic prothalli. This latter desig- nation is used in this report for reasons that will become apparent below. Others bear only archegonia and are commonly called the female prothalli. Such observations on mature cultures led to the con- cept that the female prothalli failed to form antheridia under normal conditions of culture or formed them only rarely, especially under atypical conditions of culture (2,4,5,8,16). In contrast, Czaja (3) came to the conclusion that the female gametophytes formed anther- idia regularly at an early stage of development but discontinued their formation as they attained the archegonial phase. It could be demonstrated that the archegonial phase is, in many prothalli of Pteridium aquilinum, actually preceded by an antheridial phase. At the same time it was shown that some individuals of the gametophyte population, to wit, the most rapidly growing and devel- oping ones, attained the archegonial phase without a prior antheridial phase (10). The occurrence of the two types of archegonium-forming prothalli led to the hypothesis that the prothalli of Pteridium aquilinum be- come insensitive to the antheridial factor before they begin to pro- duce it at effective concentrations. This hypothesis was considered established when it was demonstrated that all individuals of the gametophyte population failed to form antheridia if they were grown one per flask, and that all prothalli formed antheridia if they all were exposed to antheridial factor while they were still sensitive to it (10). The antheridia a gametophyte forms thus arise in response to antheridial factor that is secreted into the medium by more rapidly developing individuals of the gametophyte population (which them- selves have already become insensitive to it). Accordingly, the most rapidly developing gametophytes in a culture of Pteridium aquili- num attain the archegonial phase without a prior antheridial phase because they are without a supply of antheridial factor while they are still sensitive to it. A gametophyte population of Pteridium aquilinum thus contains three types of individuals: Archegonium-forming prothalli with a Physiology of Antlieridium Foriuation in Ferns 717 prior antheridial phase, archegonium-forming prothalli without a prior antheridial phase, and the ameristic prothalli which produce only antheridia even in mature cultures. The mechanism underlying the formation of ameristic prothalli remains to be determined. Why do they fail to form archegonia even in mature cultures? The search for the factors concerned with the formation of these ameristic prothalli must also take into account the striking differences between them and the archegonium-forming prothalli with regard to both size and shape. Thus, the surface area of ameristic prothalli amounts to less than a hundredth that of the archegonium-bearing prothalli in mature cultures; again their shape is highly irregular due to their diffuse growth habit while the arche- gonium-bearing prothalli have the well-known heart shape that re- sults from the activity of a characteristically organized meristem. Prantl (13) observed that the formation of ameristic prothalli is favored by conditions that interfere with growth, e.g., poor mineral supply, crowding, and poor lighting. He accordingly advanced the hypothesis that male prothalli are gametophytes retained at a juvenile stage due to adverse conditions of nutrition. This hypothesis accounts at first sight for most of the distinguishing characteristics of ameristic prothalli. Closer observation, though, reveals a number of discrepan- cies. Thus, young prothalli pass through a fairly regular sequence of cell divisions and quickly organize a growing region long before they attain the size of the average ameristic prothallus. The hypothesis, therefore, does not account for the diffuse growth habit and random shape of ameristic prothalli. Again, Prantl's hypothesis calls for a con- tinuum in sizes among the individuals of the gametophyte population. While such a continuum can be readily observed in younger cultures, it gives way in maturing cultures to a steadily widening hiatus between the size range of ameristic prothalli, on one hand, and that of arch- egonium-bearing prothalli on the other hand. A clue to the mechanism that underlies the formation of ameristic prothalli derived from an attempt to detect such prothalli at an in- cipient stage of development. Observations on young cultures of Pteridium aquilinum convinced the author that prospective ameristic prothalli did not begin to differ until they attained the antheridial phase. At this stage of development they gave rise to antheridia not only in the maturing region of the gametophyte but in the meriste- matic region itself (frontal row of antheridium-bearing cells, Figure IC). In contrast, the meristematic region of antheridium-bearing prothalli that subsequently proceeded to form archegonia remained free of antheridia at all stages of development. As indicated, such incipient ameristic prothalli still had a clearly recognizable meristem 718 U. Ndf and their shape conformed to that of other individuals at a compa- rable stage of development. The diffuse growth habit and random shape of ameristic prothalli is thus acquired subsequently. The questions arose: AVhat are the conditions that favor the formation of antheridia by meristematic cells, and can such anther- idium formation account for the distinctive characteristics of amer- istic prothalli? The described incipient ameristic prothalli were observed to be among the smallest individuals of the gametophyte population. The hypothesis may thus be proposed that antheridium formation by meristematic cells is favored by conditions of slow growth. It was demonstrated that all individuals of the gametophyte pop- ulation will give rise to antheridia in their meristematic cells and subsequently acquire the characteristics of ameristic prothalli if, firstly, they are cultivated under conditions of slow growth and if, secondly, they are all supplied with antheridial factor while they are still sensitive to it. The requirement for added antheridial factor is understood if it is recalled, firstly, that the formation of ameristic prothalli is related to antheridium formation (in the meristematic region) and, secondly, that the most rapidly developing individuals (archegonium-forming prothalli without a prior antheridial phase) are not otherwise exposed to antheridial factor during their sensitive period. The question as to how slow growth favors the formation of an- theridia by meristematic cells has not as yet been answered. It may be pointed out, however, that the meristematic cells of rapidly grow- ing prothalli divide at a rate of at least one per day. On the other hand, the appearance of antheridium initials follows the application of antheridial factor with a delay of between 21/2 and 3 days. It is possible, therefore, that the meristematic cells of rapidly growing prothalli remain free of antheridia because they divide before the induction of an antheridium can take effect. It was emphasized above that incipient ameristic prothalli are of no more irregular shape than archegonium-forming prothalli at a comparable stage of development. The formation of antheridia in the meristematic region is, however, soon followed by a breakdown in meristematic growth. Ihe meristem ceases to operate as a func- tional unit and cell division resumes in the basal region of the game- tophyte. The formation of antheridia in the meristematic region thus releases the potentiality of maturing cells for cell division which is suppressed in the presence of an actively functioning (antheridium- free) meristem. Antheridium formation by meristematic cells thus brings forth the same response as the excision of the meristem itself (1). The assumption of heart shape is preceded by, and dependent on, Physiology of Antheridium Formation in Ferns 719 a change in the organization of the meristem. In turn, the attainment of heart shape invariably precedes the loss of sensitivity to the anther- idial factor and the attainment of the archegonial phase (10). The failure of ameristic prothalli to assume heart shape (and their re- sulting diffuse growth habit), their failure to become insensitive to the antheridial factor and to form archegonia may thus be traced to the breakdown in meristematic growth at an early stage of develop- ment. The wide hiatus in sizes between the ameristic and the arche- gonium-bearing prothalli of mature cultures remains to be explained. Clearly, the observation that ameristic prothalli arise from the most slowly growing individuals cannot account for this hiatus. Antheridium formation entails a diversion of growth potential from the formation of vegetative cells to that of antheridial cells (9). Also, it has long been known that a vegetative cell bears not infre- quently two or even three antheridia, mostly at different stages of development. A vegetative cell might thus give rise to new antheridia repeatedly after the previously formed, ephemeral structures have fallen apart. The resulting diversion of growth potential from the formation of vegetative cells to that of antheridial cells could fully account for the ever widening hiatus between the sizes of ameristic prothalli which form antheridia even in mature cultures and the archegonium-forming prothalli which either lack a prior antheridial phase or discontinue it at an early stage of development. It was stressed above that the antheridia a prothallus forms are initiated in response to antheridial factor produced by more rapidly developing individuals of the gametophyte population which them- selves have already become insensitive to it. The ameristic prothalli, like the archegonium-forming prothalli with a prior antheridial phase, thus are the result of interaction between gametophytes. The above results are further in agreement with the postulate that the several distinctive characteristics of ameristic prothalli are all the con- sequence of antheridium formation (in the meristematic region). These distinctive characteristics should therefore disappear if the pro- thalli are removed from the interaction with other individuals of the gametophyte population. Ameristic prothalli taken from mature cultures actually gave rise to heart-shaped lobes, became insensitive to the antheridial factor, and attained the archegonial phase after they were washed and trans- ferred, one to a flask, to new medium. ON THE PHYSIOLOGY OF REPRODUCTION IN PTERIDIUM AQUILINVM It should be of interest to consider the question as to how these three types of prothalli relate to sexual reproduction. If in all pro- thalli an antheridial phase were followed by an archegonial phase, 720 U. Ndf then there would be little overlap between prothalli at an antheridial stage and prothalli at an archegonial stage of development. Accord- ingly, sexual reproduction would be hindered. Ameristic prothalli which form antheridia indefinitely thus appear to be functional by supplying male gametes when the other two types of prothalli have already attained the archegonial phase. With re- gard to archegonium-forming prothalli without a prior antheridial phase, the following argument seems pertinent. Antheridium forma- tion diverts growth potential from the formation of vegetative cells to that of antheridial cells, thus delaying the onset of the archegonial phase (see section beginning on p. 712). The occurrence of arche- gonium-forming prothalli without a prior antheridial phase thus minimizes the time lag between the first appearance in the culture of antheridium-bearing and of archegonium-bearing prothalli and thereby hastens the onset of sexual reproduction. Several characteristics of prothallial development combine to inter- fere with the simultaneous occurrence of male and female sex organs on the same prothallus, especially the failure of ameristic prothalli to form archegonia and the lack of a prior antheridial phase in one of the two types of archegonium-forming prothalli. The more rapid attainment of the archegonial phase by prothalli without a prior an- theridial phase results, during an initial period of time, in the pres- ence of prothalli that bear either antheridia only or archegonia only. As the prothalli ivith a prior antheridial phase attain the arche- gonial stage, the last-initiated antheridia do not fall apart until one to three archegonia have been initiated (10). It is unlikely, though, that the first-initiated archegonium matures before the last-initiated antheridia have fallen apart. The simultaneous occurrence of male and female gametes on the same prothallus is, however, occasionally observed later because antheridia may arise on the basal outgrowths which some of the archegonium-bearing gametophytes form at late stages of development. These characteristics of prothallial development which interfere Avith the simultaneous occurrence of male and female sex organs on the same prothallus would seem to be functional by interfering with self-fertilization. This interpretation, though, appears invalidated by Wilkie's report that the prothalli of Pteridimn aqiiilinum are self- sterile (15). If this finding can be confirmed, then the mentioned characteristics of development could still be considered functional in terms of preventing wasteful contact between incompatible ga- metes. In self-fertile sjjecies, however, these characteristics of develop- ment Avould clearly serve to minimize the diance of self-fertilization. Physiology of AnUieridixim Formation in Ferns 721 Preliminary investigations actually indicate that Polypodium aiireum (personal communication from M. Ward, 1959) and Onoclea seii- sibilis (author's result) are self-fertile. ACKNOWLEDGMENT The author wishes to express his gratitude to Dr. Armin C. Braun for his encouragement of these investigations and for the stimulating discussions on the subject of this report. He is also indebted to Drs. Armin C. Braun, Francis O. Holmes, and Tom T. Stonier for criti- cally reading the manuscript and to Dr. C. V. Morton for his help with the identification of the plants. LITERATURE CITED 1. Albaum, H. G. Inhibitions due to growth hormones in fern prothallia and sporophytes. Amer. Jour. Bot. 25: 124-133. 1938. 2. Campbell, D. H. The Structure and Development of Mosses and Ferns. 3rd ed. 708 pp. Macmillan Co., New York. 1918. 3. Czaja, A. T. Zur Frage der habituellen Diozie bei Onoclea struthiopteris Hoffm. Ber. Deutsch. Bot. Ges. 42: 300-304. 1924. 4. Dopp, W. Gestaltung und Organbildung innerhalb der Gametophytgeneration der Polypodiaceen unter besonderer Beriicksichtigung genetischer Gesicht- spunkte. Beitr. Biol. Pfl. 24: 201-238. 1936. 5. . Fine die Antheridienbildung bei Farnen fordernde Substanz in den Prothallien von Pteridium aquilinum (L.) Kuhn. Ber. Deutsch. Bot. Ges. 63: 139-147. 1950. 6. . Uber eine hemmende und eine fordernde Substanz bei der Antiieridien- bildung in den Prothallien von Pteridium aquilinum. Ber. Deutsch. Bot. Ges. 72: 11-24. 1959. 7. Fames, A. J. Morphology of Vascular Plants. Lower Groups. (Psilophytales to Filiales). 433 pp. McGraw-Hill Book Co., Inc., New York. 1936. 8. Mottier, D. M. Notes on the sex of the gametophyte of Onoclea struthiopteris. Bot. Gaz. 50: 209-213. 1910. 9. Naf, U. The demonstration of a factor concerned with the initiation of antheridia in polypodiaceous ferns. Growth. 20: 91-105. 1956. 10. On the physiology of antheridium formation in the bracken fern [Pteridium aquilinum (L.) Kuhn]. Physiol. Plant. 11: 728-746. 1958. 11. . Control of antheridium fonnation in the fern species Anemia phy ni- tidis. Nature. 184: 798-800. 1959. 12. . On the control of antheridium formation in the fern species Lygodium japouicum. Proc. Soc. Fxper. Biol. Med. (In preparation.) 13. Prantl, K. Beobachtungen uber die Frniihrung der Farnprothallien und die Vertheilung der Sexualorgane. Bot. Zeit. 39: 753-758, 770-776. 1881. 14. Raper, J. R. Sexual hormones in Achlya. VII. The hormonal mechanism in homothallic species. Bot. Gaz. 112: 1-24. 1950. 15. Wilkie, D. Incompatibility in Pteridium aquilinum (bracken). Abstr. Hered. 7: 1504. 1953. 16. Wuist, F. D. The physiological conditions for the development of monoecious prothallia in Onoclea struthiopteris. Bot. Gaz. 49: 216-219. 1910. 722 U. Ndf DISCUSSION Mr. Barlow: What do you think happens in natural conditions? You say that this factor is required from elsewhere, rather than the prothallus itself. Dr. Naf: It has been demonstrated that a prothallus does not begin to produce the antheridial factor at effective concentrations until after it has become insensitive to it. The prothallus will there- fore fail to form antheridia unless it is supplied with it from "else- where," i. e., with antheridial factor secreted into the substrate by the more rapidly growing and developing individuals of the gameto- phyte population. I would think that this is the case also in nature. Mr. Barlow: This is rather curious from the biological point of view, because many mechanisms are available for distributing plants so they get away from each other. This would seem to be one in which it is essential that two or three individuals developing from the spores of the same fern should be present together before they can complete their life cycle, which is rather unusual. Dr. Naf: It is perhaps not too rare that the completion of a life cycle depends on the proximity of two or more individuals. Raper's investigations on Achlya may be recalled in this connection. He showed that the formation of both male and female sex organs is a function of hormones produced not by the individual that forms the sex organ but by individuals of the opposite sex. Raper's name also recalls the myxomycetes, a quite different type of organism. The work of various investigators has led to the conclusion that the aggre- gation of the amoebae depends on a substance, termed acrasin, which is secreted predominantly by one type of individual and is effective on another. It would not be too difficult to add to these examples in which the completion of a life cycle depends on interaction between individuals by a chemical messenger, and therefore on proximity be- tween them. Returning to the bracken fern, it must also be emphasized that there are a number of developmental mechanisms which tend to pre- vent the simultaneous occurrence of male and female gametes on the same prothallus. In addition, \Vilkie has recently demonstrated that this fern species is self-sterile. Thus, both the formation of the zygote and of the antheridium are consequent upon the occurrence of the prothalli in clusters. Dr. Galston: I'd like to ask two questions. Is the formation of archegonia under the control of any such factors as you have de- scribed or only antheridial formation? Can you tell us anything at this time about the stabih'ly of the anlhcrich'uni-pronioting factor in vitro? Physiology of Antheridium Formation in Ferns 72^ Dr. Niif: Preliminary investigations are in agreement with the hypothesis that archegonium formation is also under the control of a demonstrable factor, although it is probably not secreted into the medium. The experimental results as well as certain theoretical con- siderations further indicate that this factor can be demonstrated only if it is assayed against the prothalli of species which have special characteristics with regard to archegonium formation. I should like to emphasize that many more experiments will be required before this can be considered established. The Pteridium factor is quite stable. Specifically it is stable to autoclaving at the pH of the medium and to boiling for 10 min. at pH 2, but labile to boiling for the same length of time at pH 12. Dr. Morel: When an adult gametophyte is cut into small pieces and put into a medium, it regenerates new gametophytes. What is the situation with this new gametophyte; is it the same as when you start with a spore? Dr. Naf: I cannot give a definitive answer to this question because this problem has not been thoroughly investigated. Some incidental observations suggest, however, that the situation in regenerating gametophytes is the same as in gametophytes grown from spores. S. TONZIG and E. MARRI University of Milan, Italy Ascorbic Add As a Growth Hormone According to the generally accepted definition, a growth regulating hormone is a substance which is produced within an organism, can be translocated far from its site of synthesis, and is active, at low concentration, in influencing the rate or the modalities of the process of growth. There is no doubt that ascorbic acid (AA) satisfies in the higher plants the two conditions of endogenous synthesis and of translocation. As to the third condition, considerable evidence has accumulated in recent years, showing that the concentration of AA and of its derivatives is an important factor in the control of the rate of growth. In this survey of the contributions of our laboratory in this field, the following points will be considered: (1) The effects of treatment with AA on the growth of whole plants and of isolated plant parts; (2) the effects of treatment with AA on some physiological processes different from growth; (3) some metabolic changes accompanying the effects of AA on growth and related processes; and (4) the effects of auxin on the AA system. THE EFFECTS OF TREATMENT WITH ASCORBIC ACID Intact Plants In 1950 Tonzig and Trezzi (20) showed that the growth of the shoot organs of oats, peas, beans, lupine, and castor beans is markedly inhibited by treatment with AA, applied either as lanolin paste to the base of the shoot or added as a solution to the nutrient medium (20). This effect was mainly due to the inhibition of cell elongation, al- though also the rate of cell division was decreased. Determinations of AA contents in the treated tissues showed that the experimentally [725] 726 5. Tonzig and E. Marre induced increase of this compound was, in most cases, within what can be considered a physiological range (20 to 100 per cent increase). It is well known, in fact, that large changes of the AA level may occur under physiological conditions, as a function of light intensity or of temperature (3). The inhibiting effect of AA treatment on cell elonga- tion was confirmed by experiments in which geotropic and phototropic bending of stems and coleoptiles, as well as bending caused by uni- lateral application of auxin (indole-3-acetic acid, lAA), was inhibited by the application of a lanolin-AA paste to the shoot bases (21). In further experiments Tonzig and Bracci have shown that in some cases cell division can also be markedly inhibited by treatment with AA. In fact, no root nodules developed on the roots of pea plants in- fected with Rhizohium leguminosarum when AA at concentrations ranging from 0.1 to 0.05 per cent was added to the medium. Similarly, treatment with AA prevented the effect of the bacteria on the induc- tion of calluses in wounded pea stems (19). On the whole, these experiments on intact plants have shown tliat an increase of the internal level of AA effectively inhibits growth in a number of species and under various experimental conditions. Moreover, experiments in which AA changes in the different plant parts were determined confirm that AA is easily translocated in the plant, translocation upward being much more rapid than transloca- tion downward. Isolated Plant Parts Growth by elongation of etiolated pea internode segments and Avena coleoptile sections appeared strongly inhibited by AA at con- centrations higher than 10^ M (12,20). Very low concentrations of AA occasionally induced a slight stimulation, an effect which has been recently investigated by Chinoy et al. (I). The fact that the stimulating effect of AA at very low cencentra- tion ajjpears relatively weak and scarcely reproducible, when com- pared with the much more consistent inhibitory effect of AA concen- trations high enough to induce a significant increase of the internal level of this compound, seems to indicate that the endogenous con- tents of AA in the materials investigated are very close to or even higher than the concentration rccjuired for maximal gro\\th. This condition obviously makes the study of the growth inhibiting com- ponent more susceptible to experimental investigation. On the other hand, there seems to be no ground to assume that the inhibitor) com- ponent of the AA effect has a less important physiological role, in the intact plant, than the eventual stimulating component. Ascorbic Acid As a Growth Hormone ni THE EFFECTS OF ASCORBIC ACID ON PROCESSES DIFFERENT FROM GROWTH Plasma Viscosity Plasma viscosity, measured as change of time of plasmolysis in hypertonic solutions, was shown by Tonzig and Trezzi (23) to be de- creased by AA treatment under a number of experimental conditions and in quite different plant materials. Also the water-holding capacity markedly decreased in the AA treated tissues (22). This suggested that changes of the physico-chemical state of the cytoplasm could play an important role in mediating the effects of AA on water uptake and on growth (18, 22). Respiration The oxygen uptake of pea internode segments was markedly in- hibited by the presence in the medium of AA at concentrations higher than 3 X 10"^ ^- At concentrations which inhibit growth by 40 to 50 per cent, an apparent 20 per cent decrease in respiration rate was observed; this value rose to above 35 per cent inhibition when the data were corrected for the Go uptake due to the enzymatic oxidation of AA in the medium (Figure 1). A very strong AA oxidase activity ap- peared to develop at the cut surfaces of the segments (16, 25) (Table 1). 300 X en UJ CNJ O 200 - 100 120 MINUTES Fig. 1. The effects of ascorbic acid treatment on O^ uptake of pea internocie seg- ments. A, control; B, ascorbic acid (2 X 10"^ M) corrected for autooxidation only; C, ascorbic acid (2 X 10"^ M) corrected for autooxidation and enzymatic oxidation. 728 S. Tonzig and E. Marre Table 1 . The effects of ascorbic acid (AA) on growth and internal contents of AA and dehydroascorbic acid (DHA) in pea internode segments. (E. Marre and G. Laudi, unpublished.) Molar Concn. of AA in the Growth As Per Cent A A, fji Moles /g DHA, M Moles/g Medium * Increase of Fresh Wt. Initial Fresh Wt. Initial Fresh Wt. 0 25 4.5 0.72 5 X lO-i 21 5.1 0.88 2 X 10-3 18 5.3 0.95 6 X 10-3 17 5.5 1.15 10-2 14 5.3 1.35 5 X 10-2 13 4.7 1.65 * Medium buffered with 0.05 M phosphate, pH 6. For experimental conditions see (5). Glutathione Oxidation/Reduction Equilibrium In the pea internode segments, AA at growth inhibiting concen- trations consistently increased the ratio of oxidized to reduced gluta- thione (7). This effect seems quite interesting, as it has been shown that the oxidation-reduction state of glutathione is an important fac- tor in growth regulation (6, 7). It has to be emphasized that all of these physiological parameters (plasma viscosity, water holding capacity, respiration, reduced to oxi- dized glutathione ratio, as Avell as growth by cell extension and cell division), are also influenced — under identical experimental condi- tions — by auxins. However, the auxin induced effects are always op- posite in direction to those induced by treatment with AA. METABOLIC CHANGES ACCOMPANYING THE EFFECTS OF ASCORBIC ACID ON GROWTH AND RELATED PROCESSES The fact that AA seems to behave, at least in most cases, as an antagonist of auxins, suggested the possibility of some kind of bio- chemical competition between the two types of substances or between their active derivatives. This hypothesis appears consistent with the results of experiments showing that the amount of free (easily ex- tractable) auxin increases, while bound auxin decreases in pea stem segments treated with AA. Moreover, diffusion of auxin from isolated oat coleoptile sections was accelerated by treatment with AA (24). A different approach to the problem of the mechanism of action of AA was suggested by some investigations on its fate when ex- ternally supplied. Moreover, the effect of AA and its derivatives on metabolic systems in vitro was investigated. A first series of experiments showed that AA, when supplied as Ascorbic Acid As a Growth Hormone 729 such, accumulated in the treated tissues largely in the oxidized form, dehydroascorbic acid (DHA) (Table 2). It was also observed that the inhibition of respiration and growth is much better correlated with the internal concentration of DHA than with that of AA (12) (Figure 2). Moreover, pretreatment of the pea internode segments with 0.5 per cent diethyl dithiocarbamate (an inhibitor of AA oxidase), which markedly inhibited the accumulation of DHA within the treated tis- sues, consistently reduced and in some experiments completely sup- pressed the inhibiting effect of AA treatment on growth (17) (Table 3). These results were interpreted as evidence that DHA, rather than AA, is the real inhibitor in the case of the treatment with AA. In sup- port of this view, a series of experiments on the effects of AA, DHA, and 2,3-diketogulonic acid on metabolic systems in vitro, show that DHA at concentrations between 10"^ and IQ-^M markedly inhibits several dehydrogenase systems (14), as well as the oxidative and phos- phorylative activity of mitochondrial preparations (2, 10) (Figure 2). This seemed to provide a satisfactory explanation for the inhibitory effect of AA on respiration in vivo, and also to suggest that at least part of the growth effect could depend on the inhibition of some step of respiratory metabolism. Table 2. The effects of diethyldithiocarbamate (DIECA) pretreatment on growth mhibition by ascorbic acid (A A) (11). AA in the Medium, Mg/Ml Growth As Per Cent Increase of Fresh Wt. in 3 Hrs. Per Cent Inhibition Due to AA No pretreatment Pretreatment with DIECA No pretreatment Pretreatment with DIECA 0.0 14.6 10.7 13.3 13.4 2.0 26 0 0.0 9.5 3.0 8.7 6.2 2.0 68 29 0.0 6.3 4.6 6.7 6.7 2.0 37 0 0.0 12.9 6.4 11.3 7.4 2.0 46 35 0.0 9.3 5.8 8.4 7.8 2.0 38 7 0.0 6.0 4.7 6.5 5.6 2.0 22 14 Average 0.0 2.0 9.6 5,8 9.1 7.8 40 14 730 S. Tojizig and E. Marre UJ 1 T 1 ^^ ^ ^° ^ " H Q- ID P — '^SUCCINATE Q. U- O 40 / - Z o 1- m I ■z. — 20 / ^ ' ^ rs / ^^,^.^-'^*^^'^^ MALATE 1/^ 1 J —A 1 ID MOLAR CONCN. OF DHA xlO Fig. 2. The effects of dehydroascorbic acid (DHA) on phosphorylation conpled to malate and succinate oxidation of mitochondria from pea internode tissues (Forti, 2). In addition, the decrease of the reduced to oxidized glutathione ratio in the AA treated tissues appeared fully consistent with the hy- pothesis that the real inhibiting agent is DHA, as the enzyme gluta- thione-dehydroascorbic acid reductase was found present in the Avena coleoptile and pea internode tissues (11, 27). THE EFFECTS OF AUXIN ON ASCORBIC ACID METABOLISM According to the working hypothesis suggested by the above- mentioned results, AA induced inhibition of growth and respiration might be conveniently interpreted as due to the rise of the DHA:AA ratio, followed by a corresponding decrease of the reduced to oxidized glutathione ratio, and by secondary effects such as changes of the physico-chemical state of cytoplasm and the activity of enzyme sys- Table 3. The effects of increasing auxin concentrations on AA and DHA contents in pea internode segments (23). lAA Concentration in the Medium P. P.M. Effect Observed 0 10 100 1,000 3,000 Growth as per cent elongation . AA, /xg/gm initial fresh wt DHA, /ig/gm initial fresh wt. . 4 403 78 16 429 56 12 415 63 4 391 89 1 381 99 Ascorbic Acid As a Growth Hormone 731 terns. The observation that — besides growth — plasma viscosity, res- piration, and glutathione are also influenced by auxin, although in an opposite direction than by AA, indicated a close connection be- tween the mechanisms of action of these substances. This hypothesis found further support in experiments showing that lAA markedly influences in vivo the oxidation-reduction state of AA (4, 9, 25). When added to the medium at concentrations up to the optimal for growth stimulation (10 p. p.m. for the pea internode segments), lAA increased the AA content and decreased the DHA content in the tissues. At higher, relatively growth inhibiting concentrations, lAA induced an opposite effect, that is, a decrease of the AA:DHA ratio (Table 4). It was also found that in etiolated pea seedlings the removal of the apex, a center of production for native auxin, causes a rapid in- crease of DHA and a decrease of AA. In all of these cases, the de- crease of DHA paralleled growth stimulation, and its increase paral- leled growth inhibition (13). The possibility that auxin could affect growth and metabolism at least in part through a direct effect on enzymes controlling the oxidation-reduction state of the AA system prompted a series of in vitro investigations on this aspect of the problem. A relatively modest but significant inhibition of AA-oxidase activity of cell-free extracts or of partially purified preparation from pea internodes was observed in these experiments (5), which is in agreement with the previous re- stdts of Wagenknecht et al. (26) on different materials. Some inhibit- ing action of auxin on AA oxidase from lettuce seeds has also been recently reported by Mayer (15). However, in other experiments this in vitro effect of auxins on AA oxidation appeared poorly reproduc- ible. It appears that further investigations on the mechanism of oxi- dation of AA in the intact tissues are needed before a fruitful studv of a possible direct effect of atixins on AA oxidase can be demonstrated. On the other hand, some investigations on the enzyme systems in- volved in the reductive metabolism of the AA system led to interesting Table 4. The internal concentration of as- corbic acid (AA) and of dehydroascorbic acid (DHA) in segments of pea internodes treated with ascorbic acid. lj.M/g fresh wt. Time of Treatment AA DHA 0 120 min.. . 1.7 2.0 0.37 0 61 732 S. Tonzig and E. Marre and reproducible results. Monodehydroascorbic acid reductase, an enzyme catalyzing the transfer of electrons from the reduced pyridine nucleotide coenzymes to a partially oxidized form of AA, was mark- edly inhibited by concentrations of indole-3-acetic and 2,4-dichloro- phenoxyacetic acids higher than 10'^ M. This provides a good basis for the interpretation of the effect of superoptimal concentrations of auxin on the level of DHA in the intact cells. It also gives a first indication that a pyridine coenzymes-monodehydroascorbic acid reductase-AA system could play a significant role in electron transfer from respiratory substrates to oxygen (8). On the whole, these attempts to demonstrate in vitro an action of auxin on AA metabolism, though not yet leading to definitive con- clusions, seem to support the hypothesis that a direct control by auxin of the oxidation-reduction state of the AA system could be of importance in mediating the final physiological effects of the hormone. SUMMARY AA is considered to be a hormone involved in growth regulation. Almost all of the available evidence indicates that a rise in the AA supply of a tissue (or of its endogenous synthesis) induces a depres- sion of the growth rate. This does not exclude that low amounts of AA could be promotive, or perhaps necessary, for giowth. The increase of AA in a tissue seems to inhibit growth essentially through the concomitant larger increase of DHA, which appears to be the real inhibiting substance. The inhibiting effect of DHA on respira- tory enzyme systems in vitro provides a basis for the interpretation of the in vivo effects on respiration and glutathione oxidation-reduction state, as well as those on plasma viscosity and growth. On the other hand, the mechanism of action of AA appears strictly connected with that of auxins. In fact, treatment in vivo with AA in- fluences the distribution of auxin in the tissues; and, correspondingly, treatment with auxin affects the oxidation-reduction state of the AA system. LITERATURE CITED 1. Cliinoy, J. J., Giovci, R., and Sirohi, G. S. A study of tlie interaction of as- corbic acid and indole-3-acctic acid in the growth of Avena coleoptile sections. Physiol. Plant. 10: 92-99. 1957. 2. Forti, G. Studi sulla fisiologia dcH'acido ascorbico. XXIII. Azione inibente dcH'acido deidroascorbico sulla fosforilazione ossidati\a di mitocondri vcgctali. Atti. Accad. Naz. Lincci Rend. 21: 70-70. 1958. 3. Lona, F., and Porzio Giovanola, E. Ricerche sulla fisiologia dell'acido ascorbico. VII: Contenuto in acido ascorbico dcllc piante in relazionc al fatiore termoper- iodico. Nuovo Gior. Rot. Ital. 58: I(i2-171. 1951. Ascorbic Acid As a Groiotli Hormone 733 4. Marr^, E. Riccrche sulla fisiologia deH'acido ascorbico. X. Variazioni quantita- tive dell'acido ascorbico in coleoptili di Avena e in segmenti di internodio di «Ptsum» tiattati con acido indolacetico. Atti. Accad. Naz. Lincei Rend. 16: 758-763. 1954. 5 ^ and Ariio^oni, O. Ulteriori ricerche suU'azione inibente dell'auxinanei confronti dell'ossidasi dell'acido ascorbico. Atti. Accad. Naz. Lincei Rend. 18: 539-546. 1955. 6. , and Arrigoni, O. Interazione tra glutatione e auxina nello stimolo della crescita. Atti. Accad. Naz. Lincei Rend. 22: 641-649. 1957. 7. , and Arrigoni, O. Metabolic reactions to auxin. I. The effects of auxin on glutathione and the effects of glutathione on growth of isolated plant parts. Physiol. Plant. 10: 289-301. 1957. 8. , and Arrigoni, O. Reazioni metaboliche all'auxina. IV. Su di una possibile base enzimatica dell'inibizione della crescita da parte di concentrazioni sopraottimali in auxina. Atti. Accad. Naz. Lincei Rend. 23: 454-459. 1957. 9. ^ Arrigoni, O., and Forti, G. Reazioni metaboliche all'auxina. II. Effetto di concentrazioni sopraottimali di auxina sui sistemi del glutatione e dell'acido ascorbico e sul metabolismo energetico in segmenti di internodi di pisello. Atti. Accad. Naz. Lincei Rend. 22: 85-91. 1957. 10. , Forti, G., and Pece, G. Ricerche sulla fisiologia dell'acido ascorbico. XVIII. Inibizione da acido deidroascorbico dell'attivita fosforilativa di preparati mitocondriali. Atti. Accad. Naz. Lincei Rend. 20: 646-651. 1956. 11. , and Laudi, G. Trasporto di idrogeno all'ossigeno per la via ossido- riduttiva: trifosfopiridinnucleotide-glutatione-ascorbico in estratti di i- Q a: d I- o < o u. o LU Q. cn TOTAL J. 4 ROOT IMMERSION 0.5 - 2 3 4 LEAF AGE, POSITION 2 4 7 GROWTH STAGE, NUMBER OF LEAVES Fig. 2. A. Radioactivity in cotton leaves after absorption by roots immersed to different depths. B. Influence of leaf age on ethylene-C" fixation in cotton. C. Ab- sorption of ethylene-C" by leaves and stems of cotton plants at three growth stages from yoimg vegetative (2-leaf) to young fruiting (7-leaf). 756 Hall, MilleVj ajid Herrero A second series of cotton seedlings with two true leaves were treated in the same manner (2,000 p.p.m. of ethylene-C'^ in the root medium). Plants were harvested for radioautographing 1, 2, 4, and 8 hrs. after introducing the ethylene. The intact plants were pressed between blotters and frozen. They were lyophilized while pressed, mounted, and radioautographed against No-screen X-ray film for one month. The radioautograms indicated that ethylene was ab- sorbed rapidly by the roots and translocation of its metabolites oc- curred almost uniformly throughout the plant within the first hr. Maximum activity in the leaves was reached in 2 hrs.; however, after 4 hrs. activity decreased in the leaves indicating that the metabolites were being converted to a gaseous form and emitted. The nature of the released radioactive gas is being studied currently by means of the vapor fractometer and vibrating reed electrometer. Tentatively, the gas does not appear to be carbon dioxide. Single Leaf Treatments Single, detached, fully-expanded cotton leaves were treated in the light (2,000 foot candles) and in the dark (< 1.0 foot candle) in a "lol- lipop" flask. The trials were conducted to determine the feasibility of short-term treatments in a manner similar to those used to study photosynthetic intermediates of carbon dioxide fixation. Considerably less fixation occurred in 3.5 to 5 hrs. in the detached leaf than when the single leaf was attached to the plant during treatment. There was three to five times more ethylene fixed in the dark than in the light, suggesting that photosynthesis is probably not involved directly in ethylene fixation by leaves. When the treated leaves were oven-dried overnight at 60° to 70° C. about 79 per cent of the fixed radioactivity was lost. This sug- gests that a major portion of the fixed ethylene is in loose combina- tion in the leaf. Therefore, lyophilization was used in most of the subsequent studies. Influence of Leaf Age on Ethylene Fixation The tops of six cotton plants in the early-fruiting stage were treated with 6.9 ml. of ethylene-C^^ in an eleven-liter chamber. The atmosphere inside the chamber contained about 600 p.p.m. of ethy- lene. Light intensity was reduced to less than one foot candle during the 16 hr. exposure period. Leaves were harvested separately, num- bered from 1 to 6 from the apex to the base, dried, ground, and pressed into uniform briquettes for coiniting. The residts given in Figure 2B show that, in general, the younger the leaf the more ethy- lene it fixed. The number one leaves which were about 2 cm. in studies With CJ'>-labeled Ethylene Ibl width were the exception. The number two leaves contained ap- proximately three times more activity than the oldest leaves (No. 6). Coleus plants with four leaf pairs were treated with the ethylene recovered from the previous experiment with cotton. The experi- mental conditions were the same as used for cotton except 25 g. of sodium hydroxide pellets were placed in the chamber to absorb res- piratory carbon dioxide and moisture. The youngest (No. 1 leaf pairs) and the oldest (No. 4 leaf pairs) fixed the most activity, while the intermediate aged leaves (No. 2 and 3) fixed the least, indicating a species difference from cotton. Influence of Plant Age on Ethylene Fixation Two cotton plants of three different age groups (early-fruiting the oldest) were treated with 6.20 ml. of ethylene-Ci^ to give about 350 p.p.m. of ethylene in the treatment chamber. The plants were main- tained in the dark for 17 hrs. After treatment the leaves and stems of each plant were harvested separately and assayed for radioactivity. The results (Figure 2C) indicate that the older plants absorbed more ethylene per unit dry weight and that the foliage contained higher levels than the stems. METABOLISM OF EXOGENOUS ETHYLENE-C^^ "Fresh" Versus "Aged" Ethylene In the absorption and translocation experiments as well as in other metabolite experiments it was noted that greater amounts of Ci^ were incorporated when "aged" ethylene was used compared to "fresh," pure ethylene, or the gas used immediately after releasing it from a light-proof, sealed vial. "Aged" ethylene denotes ethylene that had been recovered after experiments by mercuric-perchlorate absorption and regenerated from the complex for use (21), or ethy- lene that had been stored for a considerable period and then ex- posed to light, high humidity and temperature. The regenerated ethylene, which appears to be the most active, has been studied in some detail. The difference in absorption and fixation is illustrated by the following experiments. Cotton plants were exposed to 10 ml. of "fresh" ethylene contain- ing 0.4 mc. of ethylene-Ci4 (1,000 p.p.m.) for 15 hrs. in July, 1958. The leaves of the treated plants contained about 4 cpm/mg of C^^. Im- mediately following the experiment the unused ethylene was evacu- ated into mercuric-perchlorate and the complex stored in the dark at 0° C. for one month. The radio-ethylene was liberated from the com- plex (21) into a chamber containing cotton plants of the same age used in the July experiment. The plants were allowed to absorb the 758 Hall, Miller, and Herrero ethylene for 15 hrs. and upon radioassay were found to have incor- porated approximately 100 cpm/mg of C^^, or a twenty-five fold in- crease. The unused ethylene was again complexed by mercuric-per- chlorate and was stored in the light in a glass vial at ambient tem- peratures for one year in the laboratory. Cotton seedlings were treated in the dark in an enclosed chamber for 15 hrs. with the regenerated ethylene. The treated plants possessed a specific activity of about 4,000 cpm/mg, or about a thousand-fold increase in the amount of C^'' incorporated over that from the original "fresh" pure ethylene. Gas chromatography of nonradioactive ethylene and of the original and regenerated samples complexed for various periods of time indi- cate that ethylene is the only main constituent. Only minor amounts of carbon dioxide, which increases with the age of the regenerated ethylene samples, have been detected. The rate of incorporation into cell wall constituents and fixation by plants atypical of carbon di- oxide indicate tentatively that the C^^ from the "aged" samples is not contributed by Ci^02. There is no doubt that C^^ fixation from the "aged" ethylene is increased, but the significance of this must await the results of further tests. Water-soluble Metabolites Two experiments, one using "fresh" ethylene and the other using "aged" or recovered ethylene, were conducted with Coleiis and cot- ton. After 15 hrs. exposure to ethylene-C^^ the tissues were lyophilized, extracted, fractionated, purified, and the identification of some of the radioactive metabolites attempted by various procedures including column and paper chromatography, radioautography, and conven- tional chemical techniques. In general the radioactive metabolites were the same whether "fresh" or "aged" ethylene was used. Only the "aged" ethylene, however, gave sufficient activity in the metabolites for detailed studies. Therefore, all of the results with metabolites pre- sented in this paper are those obtained using "aged" ethylene. During extraction of cotton tissue it was found that one of the major radioactivity-containing fractions was water soluble. The bulk of the total radioactivity, however, remained in the residue and was not extractable by water, ether, alcohol, or other common solvents. Characterization of the water soluble radioactive fraction was at- tempted first. Ten grams of lyophilized cotton or Coleus material were extracted by refluxing for two and one-half hrs. with 100 ml. of boiling water. This was followed by four 30-min. successive extractions using 25 ml. of water each time. The total extract was combined to give a final volume of 200 ml. and was used for subsequent fractiona- tions. Aliquots of the stock solution were purified by fractional elu- Studies With C^^-Iabeled Ethylene 759 Aqueous extract Effluent -<- (discard) Adsorb on Dowex-2 (CO3-) Elute with O.IAT formic acid + O.OSiV NH4CHO0 -^ Eluate I (chromatograph) Elute with 0.15iV NaCl + 0.05N HCl > Eluate II (chromatograph) Fig. 3. Fractional elution scheme of water-soluble cotton metabolites. tion from an anion exchange column as shown in Figure 3. The radio- active fractions were neutralized, reduced in volume, and chromato- graphed in two solvent systems. Inspection of the histograms presented in Figure 4 shows at least two radioactive fractions were obtained by elution from the anion exchanger. The activity in the area of the second peak is almost 14 times that of the first peak. Strong adsorption on the column denoted a fairly acidic compound. Chromatograms of the fractions, sprayed with Hanes and Isher- wood's reagent (6), gave a positive test for phosphates and the dupli- cate radioautograms (Figure 5A, Sh 39 and 40) showed that the main radioactive spots corresponded with the molybdenum blue areas, but with two additional spots above them. The main metabolite from the anion exchanger was chromatographed in two solvent systems and the respective radioautograms are shown in Figure 5B, Sh 41 and 42. The same fraction after purification was chromatographed in Ban- durski and Axelrod solvents (3) and sprayed for phosphate esters (Figure 5B, Sh 43 and 44). The presence of phosphate-containing compounds was demonstrated in all cases and they corresponded with o X q: cJ > < 50 ML. 100 THROUGH 150 THE 200 COLUMN 250 to 'O a: d >- < 30 20 10 - J 1 Part 2 50 ML. 100 THROUGH 150 THE 200 COLUMN 250 Fig. 1. .^nion exchange chiomatogram of watci soluble fractions from cotton. Parts i and 2 are a continued clution spectrum. 760 Fig. 5. A. Radiochromatograms of water-soluble fractions 1 and 2 from anion ex- change column; Sh 39 and 40 are fraction 1 in two different solvents; No. 47 is frac- tion 2 in one solvent. B. Main metabolite chromatographed in two solvents before purification (Sh 41 and 42) and after purification (Sh 43 and 44). C. Ch 61-a and Ch 61 -b — radiochromatograms of crude water extract from cotton showing seven radioactive bands. Ch 60 — same extract chromatographed in different solvent and b band eluted and re-chromatographed. D. Sh 44b — two dimensional radiochro- matogram of purified cotton metabolite developed in Bandurski and Axelrod solvents. [761] 762 Hall, Miller, and Herrero the radioactive spots on the chromatograms. Another aliquot of the purified unknown was chromatographed in two dimensions with the same solvents (3) and gave a single, molybdenum blue spot; its autoradiogram showed only one spot (Figure 5D, Sh 44b) overlap- ping the molybdenum blue area of the chromatogram. The Rf values of the phosphorylated metabolite in six solvents are given in Table 1. Comparison of the Rf values of the two phosphorylated, radioactive spots with those of known phosphorylated compounds gave incon- clusive residts. Table 1. Rf values of phosphorylated cotton metabolite. Hanes- Isherwood Rf No. Solvent System Reaction Values 39 95 per cent EthanoliNHsiHjO (8:1:1) + 0.35 40 Amyl alcohol:5 M Formic acid:H20 (1:1) + 0.27 41 Pyridine:Formamide:Ethyl acetate (1:2:1) + 0.22 42 Phenol :H.,0 (72:28) + 0.10 43* Methanol:88 percent formic acid: H.O (80:15:5) + 0.59 44* Methanol:NH3:H20 (6:1:3) + 0.63 * Developed at 2° C. Radiochromatograms made with the crude water extract from cotton showed at least seven radioactive compounds, but only two important ones coinciding with two fluorescent bands in the paper (Figure 5C, Ch 61-a and Ch 61-b). The principal metabolite exhibited a brilliant blue fluorescence and the less active one a dark blue flu- orescence. Two water soluble, phosphorylated radioactive metabolites were also demonstrated in Coleus tissue processed in the same manner. The second active fraction, after purification on the anion exchange col- umn, contained more radioactivity than found in cotton. The radio- chromatograms in six solvent systems (Figure 6) showed two radio- active spots on each chromatogram. Rf values of the two metabolites, Tabic 2. Rf values of Coleus metabolites. Chromatopram Compound Compound Number Solvent System X Y 89 Ethanol:NH3:HoO (8:1:1) 0.08 0.19 91 r<'r/butanol:NH3:H20 (60:5:35) 0.29 0.42 92 Amyl alc.:5.\/ formic acid (1:1) (org. phase) 0.13 0.04 93 Isopropanol: Formic acid: H2O (35:5:10) 0.73 0.86 Studies With C'-labeled Ethylene 763 designated X and Y, in four of the solvents giving good separation are simimarized in Table 2. When the aqueous extract was radio- chromatographed in two dimensions following Bassham and Calvin's technique (4, pp. 17-24), at least 9 radioactive spots, with a major metabolite in the general area of the phosphorylated compounds, were demonstrated (Figure 6, 100). Two minor spots in the region where organic acids migrate and three others where amino acids usually appear were found (4). Fig. 6. Radiochromatograms of the radioactive fractions from Coleus in six solvent systems (Nos. 94, 95, 96, 97, 98, 99). In each case No. 3 is the water-soUible extract and No. 4 is the fraction soluble in petroleum ether (pigments). No. 100 — two dimensional radiochromatogram of aqueous extract from Coleus, according to Bassham and Calvin's technique, showing multiple radioactive metabolites. 764 Hall, Miller, and Herrero Table 3. Distribution of radioactivity in Coleus material. Fraction Volume, Ml. Counts/ Min/ Ml Total Activity in Fraction, c.p.m. Per Cent 80 per cent ethanol extract Pet. ether extract Aqueous residue Second aqueous residue Solid residue 200 75 14.5 60 wafer 7,030 10.210 28.820 370 1 . 406 . 000 766,000 418,000 22.200 10,700 189,100 99.2 54.5 29.7 1.5 0.8 Not accounted for ... ... 13.4 Total 1.416.700 100.0 Petroleum Ether-soluble Fraction When lyophilized Coleus material was extracted with 80 per cent boiling ethanol, the alcoholic extract reduced in volume and ex- tracted with petroleum ether, 54.5 per cent of the total activity re- mained in the petroleum ether-soluble fraction (Table 3). Radio- assay of the chloroplast pigment fraction (Table 4) showed that caro- tene retained most of the activity. Single dimensional radiochromato- grams in six solvent systems showed only one radioactive spot through- out the series, tentatively identified as carotenoids (Figure 6). Radio- chromatography of the pigments in 1 per cent petroleum ether in n- propanol exhibited one radioactive spot superimposed on the carotene spot in the paper with an Rf value of 0.87. A number of detailed experiments have been conducted with cotton and the results confirm those obtained with Coleus. Ether ex- traction of lyophilized cotton leaves, reduction in volume and ex- traction with petroleum ether (Figure 7), and radiochromatographing in five solvent systems have demonstrated that most of the radioactiv- ity is associated with the carotene fraction. Strip counting of the chromatogram illustrated in Figure 8 showed that the radioactivity coincides with the carotene spot on the chromatogram. Tabic 4. Distril)ution of radioactivity in Coleus pigments. Fraction Pet. ether extract . Chlorophyll a . . . , Chloropiiyll b. . . Carotene Xantho|)iiylls . . . Not Mccoiintcd f()i- Volume, Ml. 60 30 30 50 50 Counts/ Min/Ml 4.045 446 205 3.138 767 Total Activity 242,700 13,400 6 . 1 50 156,900 38,350 27 , 920 Per Cent 100.0 5.5 2.5 64.6 15.8 11.5 2.71 gms. Lyo])hilized Cotton Leaves Containing 10.8 X 10*' c. p.m. '(100%) Ether Extraction Residue (59%) Filtrate (1 1 %) (I, II, III, IV, V) 80%, Ethanol extract Nao CO., Residue (53.1%) Filtrate (5.6%) \A^ater extraction (6.9%) Amino acids (2.4%) (VI) Organic acids Residue (31.7%) (2.9%) ; Filtrate (9.6%) Anion Takadiastase Soluble proteins exchange hydrolysis (IX and X) Dowex 2 Residue (12.9%) HCI hydrolysis Filtrate (5.1%) HCI Autoclave Soluble sugars (2.5%) Residue Hemicellulose I H2SO4 hydrolysis Evaporate to small volume, dissolve in pet. ether, partition with 90% EtOH Organic acids Sugars, etc. (VII) (2.4%) (VIII) Xanthophylls Other and lipids pigments (3.6%) I Cellulose (0.3%) Lignin (0.1%) Fig. 7. Fractional extraction survey scheme of radioactivity in cotton leaves after 15 hrs. exposure to ethylene-C". * The materials which adsorb to the anion exchange column cannot be eluted with IN NH4OH. 2N HCI causes a loss of the radioactive carbon. 766 Hall, Miller, and Herrero Chlorophyll B Xanthophyll Chlorophyll A Carotene Fig. 8. Chronialogiam of the pigment fraction of cotton (1 per cent petroleum ether in n-propanol) and strip count showing major metabolite coincides with carotene. Survey of Activity in Cotton Leaves Cotton leaves from plants exposed to ethylene-C^^ for 15 hrs. in different experiments were extracted as shown in Figure 7 and the extracts chromatographed in five solvents. At least 18 radioactive me- tabolites were found in the different fractions. It can be noted that con- siderable activity remains in the residue after ether extraction and that approximately two-thirds of the activity is lost tiirough the scheme by heating and hydrolysis. Three radioactive metabolites were foinid in the ether-soluble fraction, the major one being as- sociated with carotene. Eight metabolites were present in the alcohol- soluble fraction, one of which has an amino acid-like structme which will be discussed in detail below. An alcohol-soluble metabolite was found in the sugar fraction but does not appear to be any of the known sugars; the other metabolites soluble in alcohol are probably organic acids. From five to nine radioactive, water-soluble metabolites have been isolated in different experiments. Two of the compoinids containing high a( ti\ ity possess a phosphorus moiety and a tliird com- pound apjjears to be a protein. A( it! or enzyme hydrolysis of the residual tissue shows a number of metabolites associated with tlie cell wall constituents. Comparison of the amoinit of C^"* from ethylene fixed in the vari- ous (onijjoncnts of cotton lo the amount of C^' fixed from C^-^Oa in Studies With C^^-labeled Ethylene 767 Table 5. Comparison between fixation of C'-'02 by barley in the light (2) and ethylene-C" fixation by cotton in the dark. Dried whole plant Ether soluble (I) Chlorins (II) Phytol (III) Carotenoids (IV) Other lipids (V) Amino acids (VI) Acids (VII) Sugars (VIII) Soluble protein (IX) ... Nonprotein aqueous (X). Cellulose (XI) Lignins, etc. (XII) Barley, 2.223 g. with 2 X 10" c.p.m. Cotton, 2.71 g. with 10.8 X 106 c.p.m. (per cent) 100* 11 * Loss of radioactivity was primarily due to oven-drying of residues for counting purposes. the light by barley (2) shows a relatively higher fixation in the cell wall constituents in the case of ethylene (Table 5). Amino Acid Fraction Chromatography of the amino acid fraction extracted by ethanol (Figure 7) revealed that the radioactivity appeared primarily in one amino acid-like compound. Only a trace amoimt of radioactivity was found in two or three other amino acids as indicated by two dimen- sional chromatography. Based on Rf values in several solvents and other tests, the major radioactive compound was originally believed to be tryptophan. Con- siderable attention was given this compound because of its possible significance to indole-3-acetic acid biosynthesis. However, as shown in Table 6, the conclusion that the compound was tryptophan is not fully substantiated as shown by the Rf values in other solvents and the color reactions performed. DISCUSSION The present experiments were designed primarily to develop basic background information for more detailed experiments on the bio- genesis, metabolism, and physiological functions of ethylene in plants. It was also hoped that the results would serve as a guide to means of increasing the amount of ethylene fixed, and by shortening the fixa- tion period, to facilitate determining the sequence of metabolite formation. 768 Hall, Miller, and Herrero Table 6. Chromatographic characteristics of the radioactive tryptophan-like me- tabolite from cotton leaves. .Sohent System Butanol:acetic acid:water (100:22:50). . . Ethanol:butanol:water (4:1:1) Methanol :ammonia:vvater (80:5:15). . . . 77 per cent Ethanol 70 per cent Isopropanol with NHj atmos, Butanol:pyridino:water (1:1:1) Butanol:acetic acid:water (60:15:25). . . . Isopropanol :NH3:H20 (8:1:1) Isopropanol:NH3:H20 (20:1:2) Isopropanol :NH3:H20 (80:5:15) Rf \'alues I Tryptophan 0.51 0.35 0.65 0.37 0.82 0.52 0.55 0.50 0.24 0.44 Color Reactions U.V. Fluorescence Spray Reagent Unknown Tryptophan Unknown Tryptophan None It. blue dk. purple none none dk. purple none It. blue Ninhydrin Salkowski /(-Dimethyl aminobenz- aldehyde Ninhydrin in acetic acid Isatin purple no color no color purple gray purple yellow purple purple blue-gray dk. purple none none dk. purple none The results obtained shed additional light on the hormonal nature of ethylene. Quantitative evidence that ethylene is produced naturally by plants (5, 13, 19), that it is translocated and metabolized, and that it affects the basic reactions of plants (16, 17, 20) supports Crocker's contention (8) that ethylene shoidd be classed as a phytohormone. It is apparent from the data that ethylene is absorbed readily by the vegetative organs of the plant. The accumulation of radioactive carbon from ethylene apparently reached a maximum about two hrs. after administration. The amount absorbed increases with the dosage administered and is greatly influenced by the physiological activity of the plant or tissue and whether "fresh" or "aged" ethylene is used. Comparatively, only a small amount of "fresh" ethylene was fixed by Colciis or cotton plants. The changes occurring dining the "aging" of ethylene are presently unknown. Based on field observations (13) apparently similar changes occur in ethylene that increase its reac- tivity for plants wiien it is exposed to atmospheric conditions. Ethylene was absorbed in higher amounts in the dark than in the light under aerobic conditions and with lowered carbon dioxide con- tent of the surrounding atmosphere. A large amount of the ethylene absorbed is unreacted or in loose combination in the plant since two- studies With C^i-labeled Ethylene 769 thirds or more can be removed by oven-drying or other techniques and when introduced through the roots it is partially lost via the foliage. The fixed ethylene apparently does not enter readily into the normal metabolic pathways that result in respiration to carbon di- oxide. More ethylene is fixed by actively growing older plants but not necessarily by older leaves. Translocation of ethylene-C^^ or its metabolites was demonstrated by its movement from treated roots or leaves to other parts of the plant. When applied to roots, the highest concentration of radio- activity was found in the terminal meristem. The cotyledons of cot- ton plants in the two true leaf stage contained more radioactive metabolites than the true leaves. When applied to a single leaf, move- ment of radioactivity occurred to nontreatd leaves where it accu- mulated in higher amounts than in the treated leaf. Intact leaves ab- sorbed and fixed larger amounts of ethylene-C^^ than detached leaves. Although more fixation occurred in the dark, more metabolites were translocated under conditions permitting photosynthesis and trans- piration. The aqueous extracts from both cotton and Coleus plants con- tained at least two major radioactive metabolites with acidic prop- erties since they were absorbed by and could not be eluted from anion exchangers with electronegative reagents. The major radioactive frac- tion from both sources eluted from the exchange column as the sec- ond peak in the elution sequence. After exposure to radioactive ethyl- ene, as many as seven and nine metabolites were detected by radio- chromatographing the water-soluble fraction of cotton and Coleus, respectively. This suggests either the reactivity of ethylene towards certain native plant materials or the entrance of the ethylene mole- cule into some major metabolic pathway. At present the former pos- sibility seems more logical. Although the amount of radioactivity in- corporated from "aged" ethylene-C^^ compares favorably to the quan- tities of Ci^ from carbon dioxide fixed in the dark by tobacco (18) and by barley in the light (2), the distribution and proportion of the metabolites contained in the various soluble fractions of cotton sug- gest that the C^^ was not incorporated as carbon dioxide. Fixation atypical of carbon dioxide is also indicated by higher radioactivity in the cell wall constituents but lower amounts in the soluble com- pounds than would be expected in 15 hrs. It is highly probable that ethylene is translocated both as the gas and in the form of water- soluble metabolites. The major water-soluble metabolites in addition to being strongly electronegative possessed a phosphate ester moiety as indicated by the 770 Hallj Miller, and Herrero positive Hanes and Isherwood reaction. It has been reported that certain phosphorylated organic compounds, mainly insecticides, are able to elicit ethylene-like symptoms in cotton, tomato, carnation, and other plants (11, 12, 13). All of these compounds have free or potential ethyl phosphate groups as parts of more complex molecules. It is possible that these ethyl phosphate-containing compounds are metabo- lized by plants to ethylene, or, more logically, ethylene enters into direct combination with one or more of the many fundamental phos- phorylated compounds known to exist in plants. This suggests a re- lationship between ethylene and phosphorylation reactions and a feasible explanation of some of the striking effects of ethylene treat- ment on plants. For example, it would be attractive to propose as suggested in the literature (9) that ethylene functions by uncoupling phosphorylation from oxidation. This, however, is not the case since significant effects of ethylene upon phosphate uptake or on the transfer of phosphate from ATP to glucose could not be demon- strated with cytoplasmic homogenates or with mitochondria (17). Neither was ethylene-C^^ incorporated by mitochondria (17). Other theoretical implications of ethylene upon phosphorylation could be postulated, but any hypothesis based on the available evidence would be largely speculation. The observation by Buhler et al. (7) concerning the incorporation of ethylene specifically into succinic and fumaric acids has not been confirmed from the present material under study. Labeled organic acids do exist in the alcohol-soluble fraction. In both Coleus and cotton the major portion of the labeled carbon in the ether-soluble fraction was found in association with the caro- tenes. Radioactivity in the xanthophylls suggests turnover from the carotenes by oxidation. Genevois in 1954 (10) expressed the opinion that carotenoids may arise from ethylene. Since some 65 per cent of the ether-soluble radioactivity was present in the carotenoid fraction, it may be assumed that ethylene has reacted with /^-carotene or served as a precursor in its formation. The function of ethylene in inducing chlorosis, coloring, and ripening changes in green organs attaches physiological significance to these observations. A corollary observa- tion is that catalase is inhibited by ethylene (15). The effects of ethyl- ene upon the pigments and catalase, all found in close structural proximity on the chloroplasts, may have more significance than cir- cumstantial evidence and speculation now permits. Rapid fixation and a relatively high retention of the radioactivity from ethylene-C^^ by cell wall constituents may prove to be important in explaining cell wall modifications induced by ethylene, including cellular growth, ripening, and abscission. The preliminary results ob- tained to date do not permit any positive conclusions in this regard. Studies With C^''-labeled Ethylene 111 In the same manner, the association of ethylene-C^^ with a trypto- phan-Hke compound suggests an explanation for ethylene in auxin- controlled responses, especially apical dominance and abscission. By altering or blocking indole-3-acetic acid synthesis, the cause for loss of apical dominance and the stimulation of abscission in plants ex- posed to ethylene becomes apparent. It has also been found that ethyl- ene inhibits catalase but stimulates peroxidase activity (16). This may be important in explaining the effects of ethylene upon auxin- controlled responses. True assessment of the significance of possible ethylene-auxin interactions, however, awaits the results of experiments now in progress. SUMMARY Experiments with Coleus and cotton plants exposed to various levels of ethylene-Ci^ have provided quantitative evidence supporting the hormonal nature of ethylene. Both radioautography and direct counting of the radioactivity have confirmed that exogenous ethylene is absorbed and transported readily by the vegetative organs of the two species. The amount of ethylene absorbed in general increases with the dosage administered and is greatly influenced by the physio- logical activity of the plant or tissue and according to whether "fresh" or "aged" ethylene is used. The causes for the changes occurring dur- ing the "aging" of ethylene, as described in this paper, and the reasons for the increase in its biological reactivity, are presently unknown. Considerable of the ethylene-C^^ absorbed is unreacted or in loose combination in the plant since two-thirds or more of the absorbed radioactivity can be removed by oven-drying or other techniques and when introduced through the roots it is partially lost via the foliage. The transport form of ethylene is unknown, but the evidence sug- gests that it is translocated both as a gas and as water-soluble metabolites. The "fixed" ethylene apparently does not enter readily into normal metabolic pathways that result through respiration in carbon dioxide. More ethylene is fixed by rapidly growing flowering plants than by younger plants, but not necessarily by the older, mature leaves. In- tact leaves absorbed and fixed higher amounts of ethylene-Ci^ than detached leaves. Although more fixation occurred in the dark, more metabolites were translocated under conditions permitting photosyn- thesis and transpiration. At least 18 radioactive metabolites were formed in leaves of plants after exposure to ethylene-Ci^ for 15 hrs. From 5 to 9 radioactive, water-soluble metabolites have been isolated in different experiments; two of these having acidic properties and giving a positive phosphate reaction contained the majority of the water-soluble radioactivity. 772 Hall, Miller, and Herrero Three metabolites were found in the ether-soluble fraction, the major one being associated with carotene. Eight metabolites were found in the alcohol-soluble fraction; a peptide, because of its similarity to tryp- tophan, was studied in some detail. After solvent extraction of the treated tissue with various reagents, the bulk of the fixed radioactivity remains associated with the cell wall constituents. Evidence shoAving that fixation of the activity atypical of carbon dioxide was presented. The physiological significance of the findings and the possible re- lationship of ethylene to phosphorylation reactions, chlorosis, color- ing, and ripening changes, interaction with auxin and cell wall modi- fications are briefly presented and discussed. ACKNOWLEDGMENT This work was supported in part by Contract No. AT(40-l)-2456 United States Atomic Energy Commission and PHS Research Grant RG-6398. LITERATURE CITED 1. Addicott, F. T., and Lynch, R. S. Physiology of abscission. Ann. Rev. Plant Physiol. 6: 211-238. 1955. 2. AronofT, S., Benson, .'\., Hassid, W. Z., and Calvin, M. Distribution of C" in photosynthesizing barley seedlings. Science. 105: 664, 665. 1947. 3. Bandurski, R. S., and Axelrod, B. The chromatographic identification of some biologically important phosphate esters. Jour. Biol. Chem. 193: 405—410. 1951. 4. Bassham, J. A., and Calvin, M. The Path of Carbon in Photosvnthesis. 104 pp. Prcntitc-Hall, Inc., New Jersey. 1957. 5. Biaie, J. B., Young, R. E., and Olmsted, A. J. Fruit respiration and ethylene production. Plant Physiol. 29: 168-174. 1954. 6. Block, R. J., Durrum, E. L., and Zweig, G. A Manual of Paper Chromatography and Paper Electrophoresis. 2d ed. 710 pp. Academic Press, Inc., New York. 1958. 7. Buhler, D. R., Hansen, E., and Wang, C. H. Incorporation of ethylene into fruits. Nature. 179: 48,49. 1957. 8. Crocker, W. Growth of Plants. 459 pp. Reinhold Publishing Company, New York. 1918. 9. Fidler, J. C. Volatile organic products of metabolism of fruits. Jour. Sci. Food Agr. 6: 293-295. 1955. 10. Gcnevois, L. Formation des carotenoides chez les veg^taux. VIII* Cong. Int. Bot. Rapp. Comm. Sect. 11-12: 403,404. 1954. 11. Hacskaylo, J., and Ergle, D. R. An experimental organic phosphate insecticide wliicli produces 2,4-D-type symptoms in the cotton plant. Proc. 11th Cotton Dcfol. Physiol. Conf. pp. 43-45. 1956. IL". Hall, \V. C. .Morpliological and physiological responses of carnation and tomato to organic pliospliorus insecticides and inorganic soil phospiiorus. Plant Physiol. 26: 502-524. 1951. 13. Physiology and biochemistry of abscission in the cotton plant. Texas Agr. Expcr. Sta. Misc. Publ. 285: 3-23. 1958. 14. , Truchelut, G. B., Lcinweber, C. L., and Herrero, F. A. Ethylene pro- duction by the cotton plant and its effects under experimental and field condi- tions. Physiol. Plant. 10: 306-317. 1957. Studies With C^i-labded Ethylene llS 15. Herrero, F. A., and Hall, W. C. Preliminary results on absorption and metab- olism of ethylene-C" in plants. Proc. 56th Assoc. So. Agr. Workers. 1959: 224,225. 1959. 1(3 ^ and Hall, W. C. I. General effects of ethylene on enzyme systems in the cotton leaf. (In preparation.) 17. , and Hall, W. C. II. Effects of ethylene and ethylene derivatives on plant mitochondria and some other enzyme systems. (In preparation.) 18. Kunitake, G., Stitt, C., and Saltman, P. Dark fixation of CO, by tobacco leaves. Plant Physiol. 34: 123-127. 1959. 19. Pratt, H. K. Direct chemical proof of ethylene production by detached leaves. Plant Physiol. 29: 16-18. 1954. 20. Ulrich, R. Postharvest physiology of fruits. Ann. Rev. Plant Physiol. 9: 385- 416. 1958. 21. Young, R. E., Pratt, H. K., and Biale, J. B. Manometric determination of low concentrations of ethylene with particular reference to plant material. Anal. Chem. 24: 551-555. 1952. DISCUSSION Dr. Thimann: As a footnote to Dr. Hall's studies of what happens to ethylene in the plant, I would like to mention some work we have been doing for the last three years on the conditions for the formation of ethylene. This work was carried out in collaboration with Dr. Stan- ley Burg, with the valuable assistance for instrumentation of Dr. Jan Stolwijk who joined us from Holland. I would like also to ac- knowledge the continued advice and criticism of Dr. Bruce Stowe. The first part of the work was largely devoted to improving the methods for determining ethylene, which are very insensitive. Experiments which require having three or four whole apples in a container for several hours are very unsatisfactory for the study of biogenesis, but fortunately Burg and Stolwijk were able to overcome this and, by skillful adaptation of gas chromatographic methods, were able to determine amounts of ethylene only about 1/ 1,000th of that required with the existing procedure of absorbing first in mercury perchlorate and then releasing and measuring the gas volume. I will mention only three of the points which have become clear from this study. The first is the very close relationship between the production of ethylene in plugs or slices of apple, and their respiration. It is possible with these small pieces of tissue to follow in parallel their oxygen consumption and their ethylene production, as a function of oxygen tension. The production of ethylene and oxygen consumption both have pOo^" at between 1.5 and 2 per cent oxygen. Further, numerous respiration in- hibitors all inhibit production of ethylene. Dinitrophenol, iodoacetate, fluoride, fluoroacetate, or, a little less satisfactorily, arsenite, all show the same thing: namely, that the influence on oxygen consumption is paralleled extremely closely by an influence on ethylene production and the level of the inhibitor which just causes a threshold effect is exactly the same for the two processes. These data taken together con- 774 Hall, Miller, and Herrero vinced us that ethylene is produced by an oxidative reaction which is very closely related to the total consumption of oxygen. The second point is that there evidently is a sequence of reactions in the forma- tion of ethylene of which oxidation is only a part. In pure nitrogen there is a very small production of ethylene, limited to the first 20 minutes or so, and after that it ceases. If tissues which have been anaerobic for 4 or 5 hrs. are exposed to oxygen for 5 min. only and again returned to nitrogen, those few minutes of oxygen allow a very considerable production of ethylene afterwards in nitrogen. There is evidence for the accumulation of an intermediate in nitrogen which afterwards is converted in oxygen to a precursor of ethylene.^ Some experiments with tritiated water strongly suggest that one of the terminal reactions is a reversible dehydration because the tritium is incorporated into ethylene just about as rapidly as into the tissue water. Lastly, there are very peculiar osmotic phenomena concerned with ethylene production. W'e early found that slices of apple tissue are very different from slices of potato tissue or indeed from slices of any other tissue with which I am familiar in that when placed in water they do not become turgid. On the contrary, they leak and lose a considerable quantity of their constituents. This has some advan- tages in that they can take up complex organic substances. But their turgor diminishes and simultaneously they lose to a great extent their ability to produce ethylene. On the other hand, in low concentrations of a solute they become tingid, and this is particularly well shown in glycerol. In higher concentrations of solute they become flaccid again. A plot of the increase or decrease in fresh weight of the tissue, as a function of the glycerol concentration, shows that in water, they ac- tually lose water. They gain water in moderate concentrations of solute, and in high concentrations they are plasmolyzed. The effect of these changes on the ethylene production is unexpected; in water, ethylene production goes down just as does the water-holding capacity, in the intermediate concentrations it goes up, and finally in the high concentrations it remains up. That is to say, the tissue can be per- fectly plasmolyzed and flaccid and yet it produces ethylene at the full rate. On the other hand, if it is flaccid due to soaking in water it produces ethylene at less than half the control rate. Thus although the system is osmotically very sensitive, it is not sensitive to the water content of the cells themselves, but rather to the osmotic state of something inside the cells. KCl reacts exactly like glycerol, and CaClo is a little less effective. This phenomenon and the very close relationship to oxygen consiunption strongly suggest that the osmotic ^See S. P. Burg an.l k. V. Thimann, Proc. Nat. Acad. Sci. 45: 335-344, 1959; Pl;ini Pliysiol. (In preparation.) Studies With C^i-Iabeled Ethylene 775 system which produces ethylene is probably the mitochondria and that when the solute concentration of the cell declines, as during water soaking, these particles become swollen and the swelling dam- ages this particular enzyme system. High solute concentrations, such as would occur after plasmolysis, do not adversely affect the enzyme system. Dr. Hitchcock: First, I would like to comment on the ethylene molecule and then on some of the responses induced by ethylene. Many growth regulator structures have been drawn on the blackboard, but I think this is one of the simplest for a growth regulator (CHo:CHo drawn). Carbon monoxide with the simple structure C:0 also has growth regulating properties. Indirect evidence for the translocation of ethylene, also carbon monoxide, throughout plants was obtained a number of years ago. When one leaf on a tomato plant was sealed in a flask containing ethylene or carbon monoxide, all leaves showed an epinastic response. Hall's work with radioactive ethylene now gives direct proof of the translocation of ethylene throughout the plant. Hall's finding that the stem contains only trace amounts of ethylene as compared to large amounts in the leaves appears to explain why there is no stem bending — only epinasty of leaves on plants exposed to ethylene. Ethylene induces many responses characteristic of growth regulators — epinasty of leaves involving cell elongation in the petiole or in the midrib, differences in flowering and the ripening, develop- ment and coloration of fruit, inhibition of nutational movements, and interference with the correlative movements of leaves and leaflets of plants such as Mimosa piidica. The last two are anaesthetic effects. Thus ethylene is unquestionably a growth regulator, but there is dis- agreement as to whether ethylene is a plant hormone. Dr. Nitsch: I'd like to ask Dr. Hitchcock if the response to CO is observed in the light or only in darkness? Dr. Hitchcock: I don't recall whether we had ever run tests in darkness alone as against light, so I can't answer that question. Dr. Galston: I would like to ask several questions of Dr. Hall. First, do you know whether the entrance of ethylene into the leaf is through the stomata? For instance, if you permit a leaf to wilt completely, will the ethylene still enter? Dr. Hall: We have not conducted experiments as you describe but ethylene is absorbed by the petiole. That is, if you remove the blade ethylene is still absorbed by the debladed petiole. Ethylene also enters through the stem or by the roots. So I would judge that it en- ters in other ways in addition to entering through the stomata. Dr. Galston: I was struck by the fact that the ethylene appeared not to enter the very youngest leaf very effectively. I wonder whether 776 Hall, Miller, and Herrero this might not be a reflection of the different ratio of surface area to mass of the leaf at this particular stage. If the youngest leaf were not spread out and the stomata exposed, the ethylene would not be able to enter. This could possibly explain the difference that you have noted in penetration of ethylene into leaves of different ages. Dr. Hall: The youngest leaf you refer to, our No. 1 leaf, was partially expanded. In other words, it had emerged from the apical bud. Based on our work I believe that entrance of ethylene does not depend entirely on the stomata, but it may also penetrate the epi- dermis directly. Dr. Forti: I would like to ask Dr. Thimann if he has any explana- tion about the inhibition of oxygen uptake and ethylene production by dinitrophenol at the level of 10-'^ molar. Dr. Thimann: The oxidation rate in many tissues is inhibited by dinitrophenol at somewhere near that. Actually apple is a little more sensitive than coleoptile or pea tissue or potato tubers by about a power of 10. Dr. Shantz: Dr. Hall, as I understood your remarks with the "aged" ethylene, this was taken up by the plant at many times the rate of fresh ethylene. Have you also found by any method for the quantita- tive estimation of growth effects of ethylene, that the "aged" ethylene induced a correspondingly higher growth response than fresh ethylene? Dr. Hall: The "aged" ethylene acts biologically similar to fresh ethylene. One response that would indicate that it has essentially the same effect as the fresh ethylene is that it induces rapid abscission of leaves, the same as the fresh ethylene. The "aged" ethylene also pro- duces epinasty, chlorosis, and coloring. I might comment briefly about the possible relation of ethylene to smog. We have an air pol- lution problem in Texas where ethylene is being liberated from an industrial plant near the Gulf Coast causing rather serious damage to cotton and other crops in the area. Apparently "aging" of ethylene from this source also takes place in the atmosphere because the ex- tent of the damage to plants depends upon certain atmospheric con- ditions. I might add. Dr. Thimann, that we have already conducted ex- periments with castor bean mitochondria. It was thought that our finding that ethylene reacts with a phosphorylated compound and its well known stimulating effect upon respiration might be more than coincidentally related. In other words, it would be attractive to think that ethylene might be uncoupling oxidation from phosphorylation. However, we have not been able to demonstrate this with castor bean mitochondria. In fact, when using radioactive ethylene, we have not been able to demonstrate any measurable radioactivity in the mito- studies With C^''-labeled Ethylene 111 chondria, yet they do undergo swelling in the presence of ethylene. Although we were not able to demonstrate any significant effect of ethylene upon oxidative phosphorylation, ethylene does affect mi- tochondria in a physical manner. Dr. Bitancourt: I want to refer briefly to some experiments carried out several years ago with Miss Rossetti. We worked with the fungus, Mucor spinosus, which produces a gas which stimulates the growth of Phytophthora. When the air above cultures of Mucor spinosus was passed through water, sulfuric acid, or sodium hydroxide before pass- ing over cultures of Phytophthora, growth stimulation still occurred. The gas produced by Mucor spinosus did not induce epinasty in tomato plants. Dr. Ray: I, too, am a bit disturbed by the "aging" effect. I wonder ^vhether even the original samples of ethylene contain trace amounts of this material derived by aging. It causes one to doubt whether any of the "metabolites" (so-called) of ethylene which are observed are in fact derived from ethylene and not from this other material since it apparently is fixed so much more readily by the plant. I also won- dered whether you had thought of trying to wash the ethylene through towers of alkali, acid, or adsorbents of various kinds to see whether this couldn't be gotten rid of. I think this is a rather basic problem. Even the initial samples could be contaminated with trace amounts, could they not? Dr. Hall: Yes, even a fresh sample of radioactive ethylene may have trace amounts of ethylene oxide present, but the content of ethylene oxide does not increase with age nor is ethylene oxide respon- sible for the increased reactivity of the "aged" sample. Scrubbing of the "aged" ethylene as you propose does not decrease its enhanced reactivity over that of fresh ethylene. Also in experiments where we have used both fresh and "aged" ethylene, we found essentially the same metabolites being formed after absorption, but much higher activity in the metabolites from the "aged" sample. To date we have not been able to show by gas chromatography and the vibrating reed electrometer that any other radioactive material is present in the "aged" sample except ethylene. Dr. Teubner: First, I'd like to comment on some work at Michi- gan State on ethylene production in stored apples. I realize Dr. Thi- mann has some objections to working with intact pieces of tissue, but at any rate as horticulturists we must do this. Reviews by Biale (Ann. Rev. Plant Phys. 1: 183. 1950) and by Smock and Neubert (Apples and Apple Products. 486 pp. Interscience, New York. 1950) discuss the delay of the climacteric with high levels of carbon dioxide (5 per cent) and a low level of oxygen. Respiration of the fruit under these 778 Hall, Miller, and Herrero conditions is quite drastically repressed. On removal and exposure to atmospheric conditions there is an increase in respiratory activity and concomitant with this is an increase in ethylene production. Us- ing this response of apples to these conditions of storage, C^^ labeled carbon dioxide was incorporated into the storage atmosphere for sev- eral days. Upon exposure to a normal atmosphere and collection of the evolved ethylene in mercuric perchlorate, it was found that a considerable portion of the fraction was active. I would also like to direct a question to Dr. Hall which is. How do you distinguish ^vhether or not ethylene is metabolized before or after transport? Since only radioactivity is measured the ethylene could be metabolized within the leaf and the labeled material transported as some other metabolite. Dr. Hall: At the present time we have not distinguished exactly when the ethylene is metabolized. We do not know whether it's before or after transport. However, it appears that most of the metabolism takes place in the leaf following absorption, and before translocation out of the leaf occurs. Apparently the leaves are the principal organs of metabolism, since much less metabolism occurs after absorption by roots or stems. Dr. Thimann: I assure Dr. Teubner I have no objection whatever to intact tissue or anything else that works. It is only that if one needs to feed organic substances to the tissue, the slices are much better. But I would like to ask him if he can give any further details since his experiment suggests that carbon dioxide is converted to ethylene in some way. And so far our efforts to get evidence for this have sig- nally failed. Not only carbon dioxide but many compounds that could be metabolized to give carbon dioxide seem not to transfer any label to ethylene, at least in short-term experiments. It is true that in long- term experiments, labeled sugar gives rise to labeled ethylene but the significance of that is hard to assess. Dr. Teubner: I'm not sure how much further these studies have been carried. Complications arose in attempting to examine potential precursors before the apples released their ethylene. We feel sure there is an anaerobic fixation of carbon dioxide and incorporation into organic acid fractions which are then metabolized. The acceler- ated carbon dioxide production upon restoration to a normal atmos- phere is reminiscent of an uncoupling effect. AVe attempted to exam- ine the organic acids, but the quantity of malic acid in apples pre- sented diffic ulties in specific activity determinations. N. E. TOLBERT Michigan State University {2-Chioroeihy[)tnmethy[ammoniam. Chloride and Related Compounds As Plant Growth. Substances' (2-Chloroethyl)trimethylammonium chloride and certain other struc- turally related compounds act as plant growth substances (4). These compounds are analogues of choline, and trivial names have been formed from choline. Thus, (2-bromoethyl)trimethylammonium bro- mide has been called bromocholine bromide. The most characteristic growth change after treatment with these compounds is the develop- ment of stockier plants with shorter and thicker stems (5, 6). In most respects the appearance of plants after treatment with derivatives re- lated to (2-chloroethyl)trimethylammonium chloride is the opposite from that obtained with gibberellin, and the effects can be reversed by gibberellin. CHEMICAL SYNTHESIS AND BIOASSAY The synthesis and analysis of the compounds have been reported (4). In the procedure an amine was reacted with a halogenated hy- drocarbon under controlled conditions to produce a specific product, the structure of which was confirmed by C, H, N, and halide analyses, melting point, and the picrate salt melting point. Thus, trimethyla- mine and 1,2-dibromoethane at 40° C. formed (2-bromoethyl)tri- methylammonium bromide, and (2,3-r7-propylene)trimethylammonium bromide was prepared from allyl bromide and trimethylamine. Since these salts were infinitely soluble in water, aqueous solutions were simple to prepare and to use for treatment of the plants. The activity of the compounds was assayed in a procedure with 'Thatcher" wheat seedlings (4). Excess aqueous solution of the chem- icals was poured onto the soil once, 1 1 days after planting the seeds. ^Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No. 2480. [ 779 ] 780 N. E. Tolbert Two weeks later the distance in millimeters between the base of the first leaf blade to the base of the second leaf blade was measured, and 4 weeks later the total stem height and the weight of the plant were recorded. After some chemical treatments, negative values for the measurement of height were recorded to indicate the distance in millimeters that the base of the second leaf blade lay below the base of the first leaf blade. In these cases the base of the second leaf blade had forced its way out through the sheath of the first leaf blade. RESULTS Active Structure A general structure for the active derivatives is (CH3)3N+CH2- CH2X where X is CI, Br, or the ^CHo group. The chemical names for the three most active compounds are (2-chloroethyl)trimethylam- monium chloride, (2-bromoethyl)trimethylammonium bromide, and (2,3-n-propylene)trimethylammonium bromide. For the growth of wheat and tomatoes these compounds were effective from 10"^ to IO-2 M when applied to the soil; from 10-^ to lO-^M when applied as a spray; from 10 " to IQ-^M when applied in nutrient solution to toma- toes; and from less than 10-3 to lO-^M when applied by seed treat- ment. For (2-chloroethyl)trimethylammonium chloride a lO^'Af solu- tion is the same as 0.13 p. p.m. A large number of chemicals with related structure were either synthesized or purchased and tested by the wheat bioassay. All com- pounds which were effective on reducing the height of 'Thatcher' wheat plants were also effective in the same proportion when used to treat a variety of other plants. From the screening program certain correlations between struc- ture and activity were developed: (a) A trimethylamine at one end of the molecule was essential for activity, (b) The carbon chain which contains the constituent X should be 2 carbons in length for maxi- mum activity. When this chain was 1 carbon long, as in (bromo- methyl)trimethylammonium bromide, the compound Avas less than I/IO as active. When this chain was 3 carbons long, as in (3-bromo- propyl)trimethylammonium bromide, the compound was 1/100 as active, and if this chain were 4 carbons long or branched, the deriva- tive was inactive, (c) The derivatives which were found to be active have had a CI, Br, or ^CHo group substituent for X of the general structure. Other derivatives are still being tested. It is interesting to note that the natural-occurring derivatives, choline, betaine, and phosphorylcholine, were completely inactive. An iodo derivative was toxic. When X was an H, as in (ethyl)trimethylammonium bromide, there was only a trace of activity. (2-Cliloyoethyl)trimethylammonium Chloride 781 Effect on Growth of Wheat When wheat seedlings were treated with one application of either of the three most active derivatives, they grew with much shorter and thicker stems than untreated plants (Table 1) (5). An example of this type of growth is shown in Figure 1. The treated plants were darker green in appearance; this greening phenomenon was particu- larly noticeable after treating tomato and tobacco plants (6). Leaves of the treated wheat plants were shorter in length and broader. There was no decrease in fresh, wet weight of wheat plants after soil treat- ments of lO-^M or less, but higher concentrations of the chemical re- duced total plant growth. Tillering of treated plants occurred shortly after treatment rather than later during maturation of untreated plants. The tillers developed and headed at about the same rate as the main stalk and produced the appearance of a more bushy plant. At maturity there was more uniformity in height of the treated plants, and heading in the treated plants occurred several days later than in the controls. Thus, wheat plants treated with (2-chloroethyl)trimethyl- ammonium chloride developed into sturdier, shorter, and bushier plants. Effect on Growth of Tomatoes Growth and fruiting of tomatoes have been extensively tested with the three most active derivatives (6). Best growth results have been obtained with (2,3-n-propylene)trimethylammonium bromide, though the halogenated compounds were also very active. Plants grew short and sturdy with intensely green leaves and stems that were thicker Table 1 . Length of 'Thatcher' wheat plants after soil treatment with (2-chloroethyl)trimethyl- ammonium chloride. * Weeks after Treatment Molarity 2 4 0 {Mm.) 34 23 15 7 1 -3 {Mm.) 260 10-6 200 10-6 153 10-4 139 10-3 91 10-2 78 * Twenty 'Thatcher' wheat seedlings in 8-inch pots of sand and loam soil were treated by pour- ing 500 ml. of solution of the chemical on the soil at 1 1 days after planting. The second leaf vvas visible at time of treatment. See text for descrip- tion of the measurement of the plant height. 782 N. E. Tolhert Fig. 1. A. 'Thatcher' wheat seedlings 2 weeks after one soil application of 10"^ M solutions: (left to right) none, (2-bromoethyl)trimethylammonium bromide, (3-bromo- n-propyl)triniethylammonium bromide, and (2,3-n-propylene)trimethylammonium bromide. B. Wheat plants from seed treated (left to right, 0, 10"\ 10'-, 10"^ M) with (2-chloroethyl)trimethylammoniinn chloride before planting. and with more chlorophyll. Tap-root and leaf-stem ratios were re- duced. Flowering was 3 to 10 days earlier and height of the first flower clusters was reduced. Thus, earlier and more prolific flowering and fruiting of market tomatoes, both under greenhouse conditions and in the field, were promoted. Effect on Other Plants The three active chemicals are being tested on a variety of other plants, and preliminary results only are available at the time of this symposium. A similar response has been obtained wnth other vege- table crops such as pepper, eggplant, cucumber, beets, and lettuce (6). Solutions of 10"'' to lO-'^M, when applied to the soil, induced darker green growth of yoimg sugar beet plants.- \Vhen cucinnbers were treated by IQ-'M solutions, the internodes were very much shorter and tendril formation was abolished. ^ High concentrations, lO^^M ^Snyder, F. \\\, Tolbert, N. E., and Wittwer, S. H., unpublished. 'Mitchell, W. D., and Wittwer, S. H.. unpublished. (2-Chloroethyl)trimethylammonii(m Chloride 783 (2-bromoethyl)trimethylammonium bromide, were necessary to shorten the stem and peduncle of chrysanthemums during the summer*; how- ever, it did not damage the plants or delay flowering at concentra- tions less than IQ-^M. In this respect (2-bromoethyl)trimethylammo- nium bromide was not as active as AMO 1618 (3), Combined Action of (2-Chloroethyl)trimethylammonium Chloride and Gibberellins The action of the (2-chloroethyl)trimethylammonium chloride derivatives in altering plant growth was the opposite from the action of gibberellin (GA). GA promoted stem elongation, spindly growth, and lighter green coloration of the leaves. (2-Chloroethyl)trimethyl- ammonium chloride, on the other hand, induced growth with shorter stems, stockier plants, and darker green leaves than the untreated plants. When excess gibberellin and (2-chloroethyl)trimethylammo- nium chloride were applied together to wheat seedlings, the action of the gibberellin at first predominated, but later the (2-chloroethyl)tri- methylammonium chloride became effective. When limited amounts of gibberellin and large amounts of the choline analogue were used, a complete variation in height from an elongated, to a normal, to a short and bushy plant could be obtained. The type of growth ob- tained was dependent upon the amount of each chemical applied. Plants may also be treated with either chemical separately for a per- iod of time and then the first growth pattern reversed by treatment with the other chemical. These mutually antagonistic effects between (2-chloroethyl)trimethylammonium chloride and GA have been dem- onstrated with wheat and tomatoes (5, 6). The chemical structures of the two types of compounds are so entirely different that it is diffi- cult to see how the two chemicals could be affecting the same growth- controlling processes. The normal appearance of plants after treat- ment with amounts of both chemicals which would produce their in- dividual effects, however, must be explained. Light Intensity Plants grow tall or elongated in low light intensities or in re- stricted portions of the visible spectrum. The growth of plants after treatment with (2-chloroethyl)trimethylammonium chloride deriva- tives is similar in appearance to growth in full sunlight, and the growth after gibberellin treatment is similar to that obtained in low light. These qualitative comparisons provide thought for future ex- periments with (2-chloroethyl)trimethylammonium chloride as a sub- stitute for high light intensity in the control of plant development. Lindstrom, R. S., and Tolbert, N. E., unpublished. 784 N. E. Tolbert This phenomenon, however, becomes a problem in reproducing the results. All of our first experiments were carried out in the green- house in the winter when the available daylight intensity was low. Later, when the experiments were redone in April and May, the percentage decrease in stem length from the chemical treatments was less. The effects of the chemical had not changed markedly, but the length of the stems of the control plants was less since the plants were then growing in high light intensity. However, the general pattern of shorter and bushier growth after treatment with (2-chloroethyl)tri- methylammonium chloride was still obtained during the summer months. Effect of Temperature The (2-chloroethyl)trimethylammonium chloride derivatives were most effective at lower temperatures. This was true for the growth of wheat which was tested at temperatures of 56° F. night and 70° F. day, 65° F. night and 75° F. day, and 80° F. night and 85° F. day. Also, wuth tomatoes in the greenhouse the effects were more pro- nounced under relatively cool (55 to 60° F.) temperatures and during the short days of fall and winter. Favorable responses have, however, resulted for both plants at higher temperatures as well as for the other plants so far tested. Thus, although the compounds are most effective near the optimum growth temperature, they may be val- uable when applied to plants at temperatures where satisfactory growth is not readily obtained. (2,3-n-Propylene) trimethylammonium Bromide Only recently has it been realized that the propylene derivative ^\as active. At this time it appears to be the most promising com- pound, since growth of sturdy plants with broad and green leaves without as great a reduction in over-all height of the plant was ob- tained when it was applied over a range of lO-^M to less than IQ-^M. Further, this (()m[)ound on tomato plants appears to be very effective at the elevated temperatures and light intensities obtained in the summer (6). Similarity of Action to Other Chemicals Several other chemicals have been reported to cause plants to grow with shorter stems when applied to plants in low concentra- tions, and it was of interest to compare their structure and activity with the (2-chloroethyl)trimcthylammoniiun chloride derivatives. AMO 1618, 2-is()propyl-4-dimethylamino-5-methylphenyl-l-piperidine carboxylate methykhloride has been reported to promote shorter stem growlli of chrysanthemums and other plants (1, 3). AMO 1618 (2-Chloroethyl)tri7nethylammoniiim Chloride 785 is not structurally related to (2-chloroethyl)trimethylammonium chlo- ride except that both are quaternary ammonium salts. Both com- pounds promote short stem growth, and in both cases these changes are reversed with gibberellin. AMO 1618 was not active on wheat, but on the other hand (2-chloroethyl)trimethylammonium chloride was not very active on chrysanthemums. Thymohydroquinone and thymoquinone have been reported to induce wheat to grow with a shorter stem (2). The structure of thymohydroquinone 2-isopropyl- 5-methylammonium chloride is also not related to chlorocholine chloride. Thymohydroquinone in our tests on wheat was toxic at \0'"M, caused dwarfing at lO'^M, and induced little effect at \Q'*M. Mode of Action The effect of the (2-chloroethyl)trimethylammonium chloride de- rivatives as chemicals to induce growth of plants with shorter and thicker stems seems characteristic of a new type of plant growth sub- stance. There is so far no evidence that these chemicals are naturally occurring nor any evidence as to their mode of action. They are not particularly toxic to either plant or animal. Since a minute amount of the chemicals, when applied once to young plants, affected the further growth and development of the plant during an entire grow- ing season, it is likely that the compounds were not rapidly metabo- lized or bound into a part of the cell structure. The high degree in specificity of structure for biological activity suggests that they may affect an equally specific enzyme site. This can be postulated as a protein with one binding site for the trimethylamine end of the mole- cule and one site for the chloro, bromo, or r^CHo group. The two active sites must be about the length of the ethyl, carbon chain apart. (2-Chloroethyl)trimethylammonium chloride is structurally related to choline and betaine in that the number 2 carbon of the ethyl chain is halogenated instead of being an alcohol or acid group. From this similarity it may be postulated that these derivatives may be influenc- ing lipid metabolism or transmethylation reactions. Alteration of either of these processes might cause altered cell development which would result in shorter plant growth as has been observed. SUMMARY Compounds of the structure (CH3)3N+CH2-CH2X are active as plant growth substances when X is CI, Br, or r=CHo group. Solutions of (2-chloroethyl)trimethylammonium chloride, (2-bromoethyl)tri- methylammonium bromide, and (2,3-n-propylene)trimethylammonium bromide were active at IQ-^M when poured on the soil. Treated wheat plants grew with shorter and thicker stems and greener leaves 786 A^ E. Tolhert than control plants. These growth changes were similar to those produced by high light intensity and the opposite from those caused by gibberellin. The opposite types of growth induced by gibberellin or (2-chloroethyl)trimethylammonium bromide counteracted each other on the same plant. LITERATURE CITED 1. Cathey, H. M. Changing growth and flowering of mums. Florist's Rev. 122: 25,26,30. 1958. 2. Flaig, Von W., Scharrer, V., and Scholl, G. J. Zur Kenntnis der Huminsauren XV, XVI and XVIII. Zeitschr. Pflanzenernahr. Dung. Bodenk. 76: 193-212. 1957. 8. Krewson, C. P., Wood, J. W., Wolfe. W. C, Mitchell, J. W., and Marth, P. C. Synthesis and biological activity of some quaternary ammonium and related com- pounds that suppress plant growth. Jour. Agr. Food Chem. 7: 264-268. 1959. 4. Tolbert, N. E. (2-Chloroethyl)trimethylammonium chloride and related com- pounds as plant growth substances. I. Chemical structure and bioassay. Jour. Biol. Chem. 235: 475^79. 1960. 5. . (2-Chloroethyl)trimethylammonium chloride and related compounds as plant growth substances. II. Effect on the growth of wheat. Plant Physiol. 35: 380-385. 1960. 6. Wittwer, S. H., and Tolbert, N. E. (2-Chloroethyl)trimethylammonium chloride and related compounds as plant growth substances. III. Effect on the growth of tomatoes. Amer. Jour. Bot. 47: 560-565. 1960. Improvement of Growth Regulator Formulation A. S. CRAFTS University of California, Davis Improvement of Growth Regulator Formulation In the use of growth regulators we are constantly attempting to ob- tain maximum effectiveness from minimum dosage. This high effi- ciency is desirable from two standpoints: first, economy, and second, distribution. Economy may or may not be important. With chemi- cals such as the indole derivatives cost is an appreciable factor; with 2,4-D and other phenoxy compounds used as regulators, the price is so low that cost is only nominal. Distribution within the plant is essential to the function of most growth regulators which, by definition, act at a distance from the point of application. And with regulators used as herbicides distri- bution is imperative, as in the control of perennials from foliar ap- plication. In such use we are faced with the paradox of requiring living cells of the phloem to conduct a toxic chemical through leaves and stems at concentrations that must, possibly after some local ac- cumulation, prove lethal in roots. In the foliar application of regulators there are two mechanisms of uptake into the living mesophyll, cuticular and stomatal. However, the differences between these are not basic but rather relative. That is, cuticular absorption involves movement across a relatively thick fatty layer from an environment that may have a low relative humid- ity whereas stomatal absorption involves uptake across cell walls hav- ing a thin cuticle from an environment approaching water saturation. Both include diffusion of the chemical across cells walls, partition to the cytoplasm, and migration via the symplast to the vascular tissues. Earlier work with dinitro compounds and pentachlorophenol (2) proved that buffering the spray solution on the acid side greatly in- creased penetration of the chemicals. This has been taken to indi- [ 789 ] 790 A. S. Crafts Table 1 . Absorption and translocation of 2,4-D as shown by bean bending. Degree of Curvature After X'arious Hours Following Application of 2,4-D pH 1 2 3 4 5 6 7 8 2 0 0 0 0 0 0 0 0 0 0 13 5 0 0 0 0 0 0 0 0 78 50 18 21 20 9 4 0 0 0 90 71 52 50 54 40 13 2 8 0 97 77 72 71 78 62 21 10 15 0 96 80 89 86 86 82 28 16 18 6 88 74 84 87 86 85 35 29 28 18 86 3 3 3 83 75 4 85 5 84 6 7 92 37 8 38 9 37 10 30 cate that repression of dissociation enhances uptake via a lipoidal route by making the applied chemical more lipoid soluble. More recent work (3) has shown this same relation to hold for 2,4-D, Table I. Endothal is another compound that benefits from acidification of the formulation. Work with maleic hydrazide (MH) by Zukel and associates (13) soon proved that acidification of this compound was of no avail, Table 2. Trials with the parent compound and with ester formulations like- wise failed to improve uptake (7), Table 3. The fact that at present the only way to increase uptake of MH is to place the plant in a saturated atmosphere would seem to indicate that there are two dis- tinct routes by which chemicals applied to leaves may move into the vascular channels, a lipoid route and an aqueous route. MH moves via the latter. Considering first the lipoid route, it seems evident that the spray solution must wet the cuticle. From this solution the regulator mole- Table 2. Weight in grams of growth of 'Bermuda' grass shoots sprayed January II, 1951, with 0.5 per cent MH (DEA) adjusted to a series of pH values. (Values are averages of duplicate cultures.) Weights At Various Intcr\als After Application of MH pH 7/3/51 9/25/51 12/3/51 3/7/52 7/23/52 9/2/52 Average Control 2 3. . 4. . . 5. . 6. . 7. . , 8. . . 9 51.5 16.7 17.1 18.5 19.5 17.1 13.9 15.8 20.2 18.5 81.9 59.0 58.0 40.2 18.7 7.7 7.0 0.6 25.2 37.1 12.5 15.2 15.4 16.6 11.7 7.9 5.9 7.9 19.3 11.6 14.0 18.2 18.5 25.3 21.3 9.3 0.0 14.0 24.7 13.7 24.2 22.2 19.2 22.0 28.3 10.9 0.0 15.5 27.9 31.8 10.0 9.5 8.0 8.6 8.6 3.9 0.0 4.1 11.6 10.6 32.3 23.9 22.7 21.9 18.0 9.5 4.5 9.7 21 5 10 20 6 Improvement of Growth Regulator Formulation 791 cules must adsorb to the cuticle (10), dissolve in the cuticle, diffuse through the cuticle, penetrate the ectopias! (plasmalemma), and pass into the mesoplasm. Here the molecvdes may migrate by symplastic movement (diffusion and protoplasmic streaming) to the vascular tis- sues where, experience indicates, they move rapidly in the phloem. Auxin (12), phenoxy compounds (4), dalapon (8), and some other regulators apparently traverse this route. There is evidence that they may also be actively absorbed from the phloem during the process of rapid transport (5, 6). If 2,4-D is applied in solution as the acid, the pH is around 3.3. As the molecules cross the ectoplast they probably dissociate partially at the prevailing pH of 5 to 6. Entering the sieve tubes of the phloem they are present in the assimilate stream at a pH of around 7.0; thus dissociation must be almost complete. This dissociation process un- doubtedly steepens the diffusion gradient and accelerates uptake. Table 3. MH formulations rated according to the amount of regrowth shown by 'Bermuda' grass cultures. Formulation * MH, diethanoleamine salt, in 95 per cent ethanol MH, diethanoleamine salt, in 5 per cent glycerine MH, diethanoleamine salt, in 5 per cent Triton B-1956 MH, diethanoleamine salt, in 0.16 per cent benzene MH, diethanoleamine salt JV-acetyl MH MH, diethanoleamine salt, in 1 per cent KCNS MH technical MH, K salt MH, dodecylamine salt MH, Nasalt MH, Mn salt A''-caproyl MH MH, Mg salt MH, Zn salt ./V-benzoyl MH MH, diethanoleamine salt, in 50 per cent glycerine MH, Al salt MH, Ca salt MH, Cu salt MH technical, no Vatsol 2-Ethyl hexoyl MH JV-benzoyl MH, no Vatsol Control Necrosis, f Per Cent 85 70 95 75 70 65 60 60 60 60 60 60 40 40 40 30 25 25 20 10 10 0 0 0 Regrowth J None None Trace Very slight Slight Slight Slight Moderate Moderate Moderate Moderate Dense Dense Dense Dense Dense Mod. -dense Dense Mod. -dense Dense Dense Dense Dense Dense Flowering J 0 0 0 0 0 0 0 0 + 0 + + + + + + + + + + + + * Spray applied March 22, 1950. MH was applied at 0.5 per cent active basis in all instances. All solutions except those noted contained Vatsol OT at 0.1 per cent. t Data taken May 24, 1950. {Data taken July 11, 1950. 0 = no flowers, + = some flowers present. 792 A. S. Cxifts Because in the use of regulators as herbicides one is dealing with toxic compounds, loinudation should be aimed at bringing about wetting of the cuticle, ordered uptake into the mesophyll, controlled concentration to a\oid injury to sieve tubes, and accumulation to toxic levels in the roots. Balanced solubility in lipoid and aqueous phases is essential, and proper partitioning is required. If regulators are absorbed via an aqueous route, there must be pores from the interior of the leaf to the outer surface which under saturated conditions are water filled. Electron microscope views of the cuticle and of cellulose indicate that such pores exist (9, 11). The aqueous medium in the leaf is undoubtedly a continuum with the saturation of the surface dependent upon the water balance of the Fig. 1. Apoplastic iiioxciuciii ol .niniiol in l)tan leaf as sliown by the dark wedge- sliapcd patlcrn ol an aiiloiadiograin. Symplastic movement also is shown by label- ing of stem, roots, and iiifoiialc leal. nosa'j;c, ]'2') ^ug, as a drop at base of primary leaf. Treatment period 8 hrs. Fig. 2. Symplastic movement of 2,4-D as shown by labeling of stem, roots, and bud. Dosage 50 ng. Treatment period 8 hrs. 794 A. S. Crafts leaf. Under saturated conditions the micro-capillaries must be filled to the surface; under stress, menisci must recede in the capillaries to depths proportional to the extent of the deficit. Applied under conditions of saturation regulator molecules in the solution are able to diffuse directly into the living symplast via the water continuum. Applied under conditions of stress the micro-capil- laries are blocked by entrapped bubbles and entry of the regulator is prevented. Inclusion of a liquid surfactant in the formulation en- ables the applied solution to creep around the bubbles and make contact with the water continimm. Strugger showed some time ago (14) that dye molecules could move rapidly along the cell walls of mesophyll cells. Some molecules, such as amitrol and MH, may move in this way from the point of application to the periphery of a leaf. Figure 1 shows such movement 4 Days 8 Doys 2 Days 2,4 -D m : W'm At #. m • MH # • # Urea » Mon « t tAA m m * 2,4-0 At ') MH ') Urea i # A ^ * ^ *••# ^### • m m ^##« * # • • Mon 'AA ^ JL V hg h4 he V ^2 h4 he V he h4 hg Fig. 3. Symplaslic and apoplastic movemcius of coinpouiuls in potato tiibci tissue as shown by autoradiograms after 2, 4, and 8 days. Top, autoradiograms; bottom, nionnlrd tissue. Com|)onn(Is tested were 2,1-0, amitrol (At): maleic hydra/ide (MH). urea, moiunon, ami lAA. Columns represent median vertical slices through one set of tuber blocks (v); horizontal slices 2 mm. in thickness cut 2 mm. (h2), 4 mm. (h4), and f) nun. (hO) from the top of another treated block. 2,1-D, IA.\, amitrol, and MH show symplaslic movement of compounds of increasing mobility. Monurou shows apoplastic movement. Urea was evidently hydrolyzed and the C^O, lost by dilfiision. Improvement of Growth Regulator Formulation 795 as contrasted with rapid uptake and movement in the symplast (Fig- ure 2). Calcium also moves in this way when applied to the leaf surface. Monuron and simazin applied to leaves move in this way. Figure 3 shows contrasting symplastic and apoplastic movement in potato tuber tissue. Recent studies by Clor (1) have shown that not only MH, but amitrol, urea, and 2,4-D are all subject to accelerated uptake and Fig. 4. Urea translocation in cotton; autoradiograms, top and mounted plants, bottom. The plant on the left of each pair was unringed, the one on the right was steamringed below the cotyledons. The left pair of plants were covered with a polyethylene bag and developed a saturated atmosphere soon after treating. The right pair of plants were left in the greenhouse in a fairly dry atmosphere. Phloem movement to the roots was prevented by the ring in each environment. In the saturated atmosphere the urea moved via the xylem to the opposite cotyledon and into the top leaves. 796 A. S. Crafts 2 4,0 Amitrol Maleic Hydroride Fig. 5. Comparative mobility of 2,4-D (2,500 p.p.m.), amitrol (5,000 p.p.m.), and maleic hydrazide (5,000 p.p.m.) in Zebrina pendula. Mature plants in depleted soil. 'J reatment 4 days with 0.5 jxc; top: radioautogram; bottom: mounted jjlants. movement when ihe treated j)lant is placed in a saturated atmos- phere. Not only is phloem movement increased; there is apparently a temporary reversal of the transpiration stream so that these com- pounds are taken into the xylem and moved down into the stem and then upward into untreated stem and leaves above the treated leaf. Also the imtreated opposite cotyledon of cotton becomes labeled when C^'*-labeled compounds are used. Figure 4 shows the results of such treatments. One additional phenomenon needs consideration at this point; this is the possible transfer of molecules from phloem to xylem dur- ing their movement in the vascular channels. Figure 5 shows three Zebrina plants treated with 2,4-D, amitrol, and MH, respectively. The 2,4-D has jjenctrated the cuticle, entered the symplast, moved to the jihloem, and on inio adjacent stem tissue. However, because of bind- ing in all living cells along the route it has moved only a few centi- meters in llie slem. Improvement of Groioth Regulalor I-'oi innldtion 797 Amitrol after entering the phloem has moved to root and shoot tips; its movement has been limited to phloem. Under like condi- tions MH has moved throughout the phloem; it also has transferred to xylem and entered every transpiring leaf. This compound, like phosphorus, is evidently able to circulate in the plant. Dalapon seems to resemble MH in this respect. Keeping these various responses in mind it is interesting to exa- mine the constituents of a spray formulation and seek out their var- ious functions. The surfactant plays several roles: it promotes wetting of the cuticle and hence may bring about stomatal uptake when stomata are open. The surfactant may serve as a filming agent and cosolvent holding the regulator molecules in a liquid layer in inti- mate contact with the cuticle when the water in the spray has evapo- rated. And if the surfactant dries to a liquid film of low surface ten- sion and highly lipophilic properties it may creep along the surface of the cuticle, pass the entrapped bubbles in the micro-capillaries, and make contact with the water continuum. These various func- \ I 1 2^4,0 lAA Amitrol Fig. 6. Comparative mobility of 2,4-D, lAA, and amitrol in barley. Pair of plants on left in each group treated on leaf no. 1; right-hand pair treated on leaf no. 4. Dosage, 0.05 fiC. Treatment time, 24 hrs. 798 A. S. Crafts Fig. 7. Comparative uptake of nine labeled compounds by roots of barley seedlings. Autoradiograms above, mounted plants below. Dosage 0.01 fxc. per 4 ml. per plant. Treatment times as indicated. Compounds from left to right: 2,4-D, amitrol, MH, urea, monuron, dalapon, simazin, P"- O^, lAA. tions explain the virtues of a surfactant for use in sprays on plants operating under water stress. Humectants such as glycerin (Table 3), glycols, and CaCL aid in the attainment of liquid-liquid contact between the spray solution and the water continuum in the cells. They may do more harm than good imder saturated conditions as they bring about run-olT of the s}Dray solution. Oils are used as filming agents where 2,4-D is used in controlling woody plants. It holds the toxicant in intimate contact with the cuticle or bark and serves to regulate penetration to a slow ordered movement. Recent work by Leonard (personal communication) has shown that amniDiiiiiin ihiocyanate included in an amitrol formulation (.Amiirol-T) protects the treated foliage from rapid contact action Improvement of Growth Regulator Formulation 799 and hence makes for a slow but continued uptake of the toxicant. The treated foliage remains green for several days after control plants have been completely killed, yet regrowth is almost completely in- hibited. Relative mobility is a property inherent in the regulator mole- cules. Figure 5 shows one series, Figure 6 a similar series in barley. If we could find a molecule that translocates as freely as MH and is as toxic as 2,4-D, many of our problems of perennial weed control would be solved. Finally, turning to soil application, there are inherent differences in molecules with respect to uptake and distribution. Here we have very little basic information. The following observations have been made. When Ci^-labeled 2,4-D, lAA, amitrol, MH, urea, dalapon, monuron, or simazin, or P^^ as phosphate are applied to roots of bar- ley seedlings, 2,4-D, montiron, simazin, and lAA are absorbed suffi- ciently to autograph strongly in 30 min. Monuron and simazin move into the tops in 2 hrs. and continue to accumulate to the 8th day. 4 Hrs. 8 Hrs. Fig. 8. Same as Figure 7, treatment times 4, 8, and 16 hrs. «()() A. S. Crafts '.V; ( h S 2 Days 4 Days 8 Days Fig. 9. Same as Figure 7, treatment times 2, 4, and 8 days. Next to move to the tops, in order of appearance are: amitrol and PO4 in 4 hrs.; by 8 hrs. dalapon, lAA, and MH; after 2 days 2,4-D at very low concentration; after 4 days urea, or some compound carry- ing its Ci^. After 8 days, concentrations in roots from high to low are in the following order: PO4, lAA, simazin, monuron, amitrol, 2,4-D, MH, dalapon, urea. In tops the order is simazin, monuron, amitrol, PO4, dalapon, MH, lAA, 2,4-D, and urea. Figures 7, 8, and 9 show the re- sults of this experiment. Formulation of soil-borne chemicals usually aims at solubility as related to penetration and leaching, at particle size as related to rate of solution, at pelleting as a means of distribution and of selectivity, and at pellet composition as related to partitioning. From these various observations it seems that formulation holds much promise for obtaining maximum usefulness of regulator mole- cules. Synthesis and screening arc fmnishing new compounds and Improvement of Growth Regulator Formulation 801 should continue to do so. The conscious design of more effective plant growth regulators for agricultural use is an ever present chal- lenge to jDCsticide workers and biochemists. ACKNOWLEDGMENT Much of the work reported here was supported by A.E.C. Con- tract AT(ll-l)-34 Project No. 9 and Project No. 38. LITERATURE CITED 1. Clor, M. A. Comparative studies on translocation of C"-labeled 2,4-D, urea, and amino-triazole in cotton and oaks. Ph.D. thesis, Univ. Cahf., Davis. 1959. 2. . A theory of herbicidal action. Science. 108: 85,86. 1948. 3. . Herbicides, their absorption and translocation. Jour. Agr. Food Chem. 1: 51-55. 1953. 4. . I. The mechanism of translocation: methods of study ^vith C^*-lal)eled 2,4-D. Hilgardia. 26: 287-334. 1956. 5. . Studies on translocated herbicides. Span No. 3. pp. 5-11. Oct., 1958. 6. Crafts, A. S. Further studies of comparative mobility of labeled herbicides. Plant Physiol. 34: 613-620. 1959. 7. , Currier, H. B., and Drever, H. R. Some studies on the herbicidal prop- erties of maleic hydrazide. Hilgardia. 27: 723-757. 1958. 8. Foy, C. L. Studies on the absorption, distribution, and metabolism of 2,2- dichloropropionic acid in relation to phytotoxicity. Ph.D. thesis, Univ. Calif., Davis. 1958. 9. Frey-Wyssling, A., Mtihlethaler, K., and WyckofE, R. W. G. Mikrofibrillenbau der pflanzlichen Zellwande. Experientia. 4: 475, 476. 1948. 10. Orgell, W. H. Sorptive properties of plant cuticle. Iowa Acad. Sci. Proc. 64: 189-198. 1957. 11. Scott, F. M., Hamner, K. C, Baker, E., and Bowler, E. Electron microscope studies of the epidermis of Allium cepa. Amer. Jour. Bot. 45: 449-461. 1958. 12. Skoog, F. Absorption and translocation of auxin. Amer. Jour. Bot. 25: 361- 372. 1938. 13. Smith, A. E., Zukel, J. W., Stone, G. M., and Riddell, J. A. Factors affecting the performance of maleic hydrazide. Jour. Agr. Food Chem. 7: 341-344. 1959. 14. Strugger, S. Fluoreszenzmikroskopische Untersuchungen iiber die Speicherimg unci VVanderung des Fluoreszeinkaliums in pflanzlichen Geweben. Flora. 132: 253-304. 1938. DISCUSSION Dr. Henderson: What form of 2,4-D was used and where was the label? Do you believe that 2,4-D does not enter as the whole molecule? Dr. Crafts: We used 2,4-D acid, labeled in the carboxyl position. We're convinced that with this compound the bulk of the labeling in our autograms is 2,4-D. With maleic hydrazide, and some of the substituted benzoic acids we're fairly certain that these molecules are stable enough that our autograms show the distribution of the orig- 802 A. S. Crafts inal compound. Now with lAA I must admit that I have no idea what it is that's moving, but we hope eventually that we can find out. I suspect it may be a conjugate. Dr. Thimann: Could you say why you are convinced that radio- activity in the 2,4-D autograms represents unchanged 2,4-D? Dr. Crafts: My conclusions are based on several observations and on Dr. Weintraub's work showing that the decarboxylation of 2,4-D goes on at a relatively slow pace. We can also extract these com- pounds from the roots and get bud suppression and leaf deformation typical of 2,4-D. We are now engaged in chromatographing the ex- tracts of these and hope to follow this much further. Professor Blackman: We have heard a good deal about the effects of surfactants per se. Perhaps I might point out that if you take a series of different types of wetting agents and increase the concentration past the point where there is any further and appreciable lowering of the surface tension of water then, though the surface tension is not changed, there will still be effects of concentration on the level of retention following a spray application. Moreover there will be sig- nificant interactions between the species and the type of surfactant. One of the many queries that arise is whether measurements of sur- face tension by accepted methods employing static conditions are ap- plicable to droplets impinging on the leaf surface. DONALD P. GOWING Pineapple Research Institute of Hawaii Some Comments on Growth Regulators With a Potential in Agriculture' The topic for discussion is the design of more effective plant growth regulators for agricultural use. This paper will propose that a number of effective growth regulators for agriculture are already designed and that they simply await the recognition that will follow their wider test- ing. Although a full range of variously substituted aryloxyalkyl car- boxylic acids has been studied in the laboratory in biological tests of relative potency, only a few have thus far found horticultural use. If one names the 2-methyl-4-chloro-, the 4-chloro-, the 2,4-dichloro-, and the 2,4,5-trichlorophenoxyacetic, alpha-propionic, and gamma-butyric acids, he has very nearly covered the field. It is interesting that although the phenoxyacetic series has been in use for some time, the phenoxypropionic and butyric analogues are much more recent arrivals on the commercial scene, usually for a higher degree of selective weeding, and the propionic derivatives also for crop control in circumstances where the acetic derivatives would be quite injurious. Wain (6) and co-workers have adequately pointed out that the relative activities of the butyric and acetic analogues derive from merely quantitative considerations in some plants, but qualitative differences in others. It is the qualitative aspect that I wish to stress, for in this may lie the key to greater usefulness of other phenoxyalkyl carboxylic acids in crops too sensitive to those mentioned. For instance, the pineapple plant may be forced into unseasonal flowering by 2,4-dichlorophenoxyacetic acid (2,4-D) at rates from 5 to 10 g/acre (rates such as would be encountered in spray drift from herbicidal applications of 2,4-D at 1 to 2 lb/acre). This can result ^Published with the approval of the Director as Technical Paper No. 268 of the Pineapple Research Institute of Hawaii, Honolulu, Hawaii. [ 803 ] 804 D. P. Gowing Table 1. Some substituted phenyl- and phenoxyalkyl carboxylic acids with hcrbi- cidal properties on young broad-leaf weeds. Acid* Herbicidal Rate, Lbs/Acre f Pea-Stem Test, Per Cent of Activity of NAAJ Induces Flowering in Pineapple 2,4-Dichlorophenoxyacetic 2-Methyl-5-bromophenoxyacetic 2-Methyl-5-chlorophenoxyacetic 2,5-Dichlorophenoxyacetic a-2 4-Dichlorophenoxyvaleric 0.25 2 2 2 2 2 2 8 2 8 2 4 8 2 4 96 137 115 57 68 7 0 110 287 98 111 150 75 29 0 yes no no no no 3-Chloro-5-methylphenoxyacetic 3,5-Dimethylphenoxyacetic no no 3-Ethylphenoxyacetic 2-Methylphenylacetic 2,5-Dimethylphenylacetic no no no 2-ChIorophenylacetic 2,4-Dichlorophenylacetic 3,4-Dichlorophenylacetic 4-Chlorophenylmercaptoacetic 3-Chlorosalicylic no no no no no * All except 2,4-D and 3-chlorosalicylic acid (Dow Chemical Co.) and 4-chloro- phenylmercaptoacetic acid (Evans Chemetics, Inc.) synthesized by R. W. Leeper, Pineapple Research Institute, Chemistry Department. t Sprays at 100 gallons per acre; chemical dissolved in dimethylformamide and applied with triethanolamine and a wetting agent. I Data of M. J. Kent, Pineapple Research Institute, Chemistry Department; 1-naphthaleneacetic acid used at 200 p. p.m. as a standard for comparison. in loss of the crop, and for this reason such compounds are not used for weed control in pineapple fields. The pineapple plant is forced to flower by the a-propionic and y-butyric analogues of 2,4-D also. How- ever, within the halogen- or alkyl-substituted phenoxyacetic acids, several of the 2,5-substituted and the 3,5-substituted compounds have some herbicidal activity, but all such compounds have thus far failed to force pineapples to flower. Results with some of these compounds and others are shown in Table 1. Pybus et al. (5) have recently re- ported on the effectiveness of certain phenylacetic acids. Of these compounds, all have had more or less activity in the split pea-stem test for growth regulator activity with the exception of 3- chlorosalicylic acid and the 3,5-substituted compounds. The latter are herbicidal apparently owing to some contact, or less general sys- temic, activity. Such materials, then, although much less effective than 2,4-D as herbicides, would merit further investigation in crops exceptionally sensitive to 2,4-D since they might be tolerated in drift amounts. Although some of them have the advantages of systemic herbicidal activity, they would be no more injurious to the crop than some nongrowth-rcgulator chemicals in a misdirected contact spray. Growth Regulators in Agriculture 805 0.3 0.6 1.2 3 6 12 6U bu j^^ Level (mg/plant) inducing 50% or more flowering active Fig. 1. Lack of correlation between activity in the split pea-stem curvattire test and induction of flowering in the pineapple. Data for 92 substituted phenoxyalkyl carboxylic acids, omitting data for 53 others inactive in the pea test which were also inactive in flower induction. The emphasis in the previous paragraphs was on toxicity, but the point should be recognized that there are qualitative differences in morphogenic responses between compounds which assay as "auxin in common in growth responses. From Figure 1 it can be readily seen that there is no particular correlation between activity in the split pea-stem curvature test and ability to induce flowering in the pineapple plant. Of the 92 variously substituted phenoxyalkyl carboxylic acids shown active in the pea test, only 33 were active in flower induction. The compounds inactive in flower induction ranged from 6 to 249 per cent of the activity of NAA in the pea test, and were tested in many cases on pineapple at rates up to a toxic level. Another example was reported some time ago by Leopold (4, p. 302) in connection with pineapple. He pointed out that the use of 806 D. P. Gowing 1-naphthaIeneacetic acid (NAA) for flower induction in this plant re- sulted in a suppression of slips.- However, p-chlorophenoxyacetic acid (PCA) induces slip production in plants forced with NAA. These two materials, although both auxins in growth responses and although both will induce flowering in the pineapple, are actually antagonistic in this other morphogenic response. When the two are applied at vari- ous rates in combination, the NAA offsets the slip-producing activity of PCA. Other instances of antagonism in morphogenic effects have been reported by the writer, e.g., that of indole-3-acetic acid for NAA, and of indole-3-acetic acid for indole-3-butyric acid in the flowering- induction response in the pineapple (2, 3). The horticulturist seek- ing a chemical to give a morphogenic response cannot afford to as- sume that if one growth regulator is active, the activity of all others will be in the same direction, and only to a greater or lesser extent. A further point which has been but little investigated is the range of morphogenic responses assignable to various levels of application, short of outright toxicity. As an example of the importance of the proper level of growth regulators to obtain a particular response, the inhibition of flowering in pineapple by NAA may be mentioned. A number of years ago, Clark and Kerns (1) reported that NAA at a concentration of 10 p. p.m. induces flowering, but suppresses flowering at 1,000 p. p.m. The induction of flowering by NAA has been a regular practice for a number of years, but the chemical inhibition of flowering on Sep Dec Mar Jun Sep Dec Mar Jun Sep Dec Mor Jun Sep I I I I 1 I I I I I I Planting Fruit Init. Harvest Vegetative Growtti I Fruit Devel. I I Planting Normal Fall Slip Planting F.I. I"!- *"•,'• H. H. H. Foil Sucker Planting Planting Fl. Fl. Fl. H.FIH.FI. H. Fl. H. H. H. >- 1 1 1 I I I I I I I I I Off-season Slip Planting Fig. 2. A sketch of the circumstances of pineapple crop production under Hawaiian conditions. Year-around harvest (H.) is desirable for cannery operations. However, several periods of fruit initiation (F. I.) in a given field are costly. (See text.) 'The slip is morphologically a fruit, but is overwhelmingly vegetative rather than fniitlike in its development and is tlie preferred planting material for succeed- ing crops. It is borne on tlie peduncle just as is the fruit, in distinction to the crown, a vegetative organ borne on the top of the fruit, and the sucker, which is a branch off the main stem. Slips and suckers normaiiv develop at the time of natural fruit initiation, and ihc crown is initiated after the florets of the inflorescence have been laid down. Groioth Regulators in Agriculture 807 pineapple plantations is not yet on a commercial scale. A moment's digression to Figure 2 will illustrate the problem. Although the crop from slips planted in the fall initiates inflorescences quite uniformly in December of the succeeding year and is harvested as a unit the following summer, to handle all the crop in this way would exceed the fruit-packing capacity of the canneries. Consequently, plantations are compelled to harvest the year around, and it is in trying to make the population in a field of fall-planted suckers, or in a field planted in an oif-season, behave as a unit that the problem arises. Commonly, in such fields, a percentage of the plants will differentiate in one month, more in another couple of months, and so forth as shown in Figure 2. Fruits and plants of different physiological ages in the same field increase costs by increasing the number of harvest periods and of harvest rounds within these periods, and they make maintenance of optimum fertilization and insecticide schedules quite impossible. Moreover, precocious fruits from small plants are often small. The development of an acceptable inhibitor of precocious flowering is therefore quite important. Figures 3 and 4 will show why the use of NAA at 1,000 p.p.m. to inhibit flowering has not become a general practice, despite the seri- ousness of the problem. Figure 3 represents an untreated 15-month- old nonfruiting pineapple plant stripped down and the leaves ar- ranged from left to right according to their position on the stem (oldest leaves at the bottom). It will be seen that as the pineapple plant grows the length of the longest leaf produced increases with age of the plants to a maximum. It will continue to hold this maximum until flowering 1201" ,i<''," 100 S 80 - 60 c o 0) 40 20 ..'" ". Leaves -2_s^,(u,'' ,«' ■HUj' i" t • r I I I 1 I - About 25 leaves Stem - from original ^ , Slip ^* ^M-l Increasing age of plant—*- Fig. 3. Lengths of leaves of nonfruiting pineapple plant 15 months after plantnig. Oldest leaves at left; each mark one leaf tip. (Leaves with broken tips omitted.) 808 D. P. Gowing 100 -^80 o £60 c Leaves -> i» " i''» « , A 'ill ,' Area of n \ ii disorganized a - 40- ,•' leaf tissue o -J 20 About 22 leaves ^^ Constricted from original .^ stem and slip NAA R j^ adventitious^ oool'nl N.AA/V^ appl'n^ XAA/v*^ roots """^"^Q | yy — - Increasing age of plant -^ Fig. 4. Representation of 15-month-old pineapple plant 5 months after treatment with NAA at 1,000 p.p.m. Some rotting of leaf bases and proliferation of adven- titious roots on basal leaf tissue at R. is induced either naturally or artificially. (The shorter leaves on the right are the immature leaves of the growing point at the stem tip). Figure 4 shows a similar plant stripped down about 5 months after application of NAA at 1,000 p.p.m. Note that from the time of appli- cation the growth of the developing leaves was severely curtailed, there was some damage to the leaf tissue, some rotting and root forma- tion on the basal white leaf tissue, a marked constriction and elonga- tion of the stem, and an unusual proliferation of roots for some dis- tance up the stem. The constriction of the stem and the evidence of disturbed growth in the leaves is eventually outgrown, and succeeding leaves attain again the previous maximum length, provided the plant is not induced to flower naturally or artificially. However, the total mass of green tissue which must contribute to the fruit has been re- duced much below that illustrated in Figure 3. Reduced plant size generally means reduced fruit size, and when a standard can size for sliced pineapple must be met, this is not acceptable. Quite apart from the commercial aspects, is the shortened leaf an indication of an induced juvenility? There is some circumstantial evidence in favor of this view. The leaves on 'Smooth Cayenne' pine- apple are normally spineless except at the tip. (This is, of course, the first tissue laid down in the growth of these monocotyledonous leaves.) When excessive levels of NAA have been used (and incidentally also when the plant resumes growth after a period of drought), some spines may be produced on the leaves just below the area of constricted growth. Parenthetically, it may be suggested that this is further evi- Growth Regulators in Agriculture 809 dence that ontogeny repeats phylogeny, because it is suspected that the 'Smooth Cayenne' plant was derived from a spiny ancestor. Two points can be made here in respect to Figure 4. One is that the quantitative differences in rate of application of NAA have quali- tatively different effects in differentiation responses. And the second point, which is suggested by this, is that other growth regulators may provide the same desirable over-all effect of inhibition of flowering without some of the undesirable (or at least nonessential) side effects of proliferation of roots, constricted stems, and spininess. We have some evidence that this is so. From these illustrations, I should like to derive the principle that a good deal of work remains yet to be done with the substituted aryl- oxyalkyl carboxylic acids and other recognized growth regulators, com- mon or uncommon. Much will be done if horticultural workers will re- fuse to take for granted that all these materials behave the same and vary only in the degree of activity. Admittedly, it is scientifically less satisfying to move into a program of testing without a well-developed hypothesis. We do have some well-developed hypotheses in the test- ing program for crop control in pineapple production, but these were formulated only after empirical testing of several hundred compounds. Some of the earlier hypotheses, as was inevitable, required modifica- tion as the body of test results increased and new evidence became available. However, the net result has been the development of in- formation useful in enlarging our conceptual framework along with its practical value. LITERATURE CITED 1. Clark, H. E., and Kerns, K. R. Control of flowering with phytohormones. Science. 95: 536,537. 1942. 2. Cowing, D. P. Some experiments on chemical induction of flowering in the pineapple. Plant Physiol. 31 (Suppl.): xxxiv. 1956. 3. . An antagonism of indolebutyric acid for indoleacetic acid. Plant Physiol. 33 (Suppl.): xx. 1958. 4. Leopold, A. C. Auxins and Plant Growth. 354 pp. Univ. Calif. Press, Berkeley. 1955. 5. Pybus, M. B., Wain, R. L., and Wightman, F. New plant growth-substances with selective herbicidal activity. Nature. 182: 1094, 1095. 1958. 6. Wain, R. L. Herbicidal selectivity through specific action of plants on com- pounds applied. Jour. Agr. Food Chem. 3: 128-130. 1955. DISCUSSION Professor Blackman: Since we are discussing tropical applications, perhaps another example might be given where existing knowledge and existing compounds can be used as a basis for a research pro- gram. I am referring to the program on the stimulation of latex flow 810 D. P. Gowing in old rubber trees which is being conducted at the Rubber Research Institute in Malaya. The new clones of rubber trees in Malaya are many times as productive as the unselected trees planted 30 to 40 years ago. There is a great need to replace the old trees as rapidly as pos- sible, but they cannot all be replaced at once because then there would be no rubber on the estate. It has been found that if esters of substi- tuted phenoxyacetic acids are applied below the tapping panel in an oil vehicle, the rubber flow is appreciably increased for as much as six months. It is relevant to the discussion of the Avena test that 2,4,5-T causes the greatest stimulation, followed by 2,4-D. Substitution in the 6 position, dimethyl substitution, or monochloro substitution, all greatly decrease the activity. The effects of other growth regulators are now being explored. Dr. Fawcett: How do 3,4-dichloro- and 2-methyl-4-chlorophenoxy- acetic acids compare with 2,4-D for the promotion of flowering? Dr. Gowing: In connection with Prof. Blackman's comments, 4- chlorophenoxyacetic acid, 2,4-D, and 2,4,5-T show about the same order of effectiveness in induction of flowering in pineapple as in stimulating the flow of latex. The 2,4,5-T is the most active of the group. Dr. Fawcett asked about the 2-methyl-4-chloro- and the 3-methyl-4- chlorophenoxyacetic acids. The 3-chloro-4-methyl compound has 249 per cent of the activity of 1-naphthaleneacetic acid in the split pea- stem curvature test but doesn't induce flowering in pineapple. How- ever, the 3-methyl-4-chloro compoiuid does induce flowering. Similarly, the 2-chloro-4-methyl compound has no activity in flower induction but has considerable activity in the split pea-stem curvature test, and the 2-methyl-4-chloro compound (MCP) is active in both tests. MCP is active in flower induction at about the same level as 2,4-D (0.6 mg. per plant for 50 per cent or more induction). The 3,4-dichloro compound induces flowering in the pineapple at about 3 mg. per plant for 50 per cent or more effectiveness. Consequently, it is excluded from com- mercial consideration as an herbicide in pineapple plantings. Dr. Sachs: In view of the multiplicity of the responses observed by Dr. Gowing, such as effects on flower initiation, herbicidal effects, etc., I am wondering about the mechanism and site of action of these sub- stances. Is there any universal point of view that can be developed? Will it be one Kingdom of Heaven or seven? I think there has been a general impression that the primary site of action of most growth regulators is in the cell walls, probably by causing them to become soft and thereby letting them extend. I don't hold to this view, or at least I don't see how one can jump from softening a cell wall to getting a nucleus to divide to getting flowers to be initiated. Growth Regulators in Agriculture 811 Dr. Gowing: "In my Father's house there are many mansions." Dr. Thimann: There are some rather striking differences between the effectiveness of growth substances in simple growth tests and their effectiveness in the production of parthenocarpic fruits. An impor- tant point to bear in mind is that in different types of tests like these, there is a very wide difference in timing. After the substance is ap- plied to the pineapple, how many weeks or months later. Dr. Gowing, is it that the flower initials appear? Dr. Gowing: It varies from 50 to 120 days. Dr. Thimann: In contrast, a growth test is usually done in 24 hrs. So one must consider that the determining factors for activity in a long time test are going to include not only activity per se, but the ability of the substance to be adsorbed or combined on other constitu- ents of the plant and its ability to be metabolized or destroyed, or at least made inactive. In considering those different phenomena that can intervene between application and finally getting the effect, we will probably get the explanation of these differences. LEONARD L. JANSEN Agricultural Research Service, USDA Beltsville, Maryland Physical-Chemical Factors of Surfactants in Relation to Their Effects on the Biological Activity of Chemicals It is not possible to state the exact manner in which wetting agents or other types of surfactants may be employed to increase the effec- tiveness of biologically active materials. In the first place, the term surfactant, which is a contraction of surface active agent, denotes any organic substance which possesses surface active properties. A great number of surfactants have appeared over the past 30 years. Primarily, the big boom started with the expansion of the soap in- dustry immediately after World War I. Development of synthetic detergents was accentuated greatly during World War II. During the past ten years there has been a tremendous boost in the number of surface active agents of all types. The variety of commercial appli- cations of surfactants is almost phenomenal. From the usage stand- point, surfactants may be classified as soaps, detergents, wetters, emul- sifiers, dispersants, spreaders, thickeners, solubilizers, etc. In this paper are presented some of the physical-chemical properties of surfactants which I believe warrant consideration in future experimentation. It also calls attention to a great bulk of information which has been brought together and summarized by Schwartz and Perry (1). This book brings up to date most of the surfactant information as it ex- isted up to 1947. Developments which occurred from 1947 to 1957 are summarized in a second volume by Schwartz, Perry, and Berch (2)- . . , We have previously examined some of the important biological applications of surfactants. Dr. Phinney has demonstrated greatly enhanced uptake of gibberellic acid from surfactant solutions. Crosby and Vlitos have found certain naturally-occurring long-chain alcohols and acids, which undoubtedly exhibit some surfactant properties, to have growth stimulatory activity. In the same manner, Stowe has ob- [813] 814 L. L. Jansen tained growth promotion from Tween 20, a polyoxyethylene sorbitan monolaurate. He has also employed surfactants in application pro- cedures to emulsify and stabilize fatty materials. Classic uses of sur- face active agents in growth-regulator and herbicide research have presumably been associated with improved wetting, sticking, or spreading qualities. They have also been used to emulsify or disperse poorly soluble or nonsoluble compounds in aqueous and/or oil systems. The majority of surfactants have a characteristic type of surface activity with respect to the energy relationships of the interfaces formed. The definite free energy relationships of these interfaces are measured in two different ways. They are referred to as surface ten- sion for the liquid-gas interface and as interfacial tension for the liquid-liquid and liquid-solid interfaces. In Figure lA are illustrated the generalized ranges of values for these free energies found for solu- tions of most sinfactants. From a value of 72 dynes/cm for pure water, surface tension falls rapidly as surfactant concentration is in- creased to approximately 0.1 per cent. At higher sinfactant concen- trations the surface tension remains more or less constant. Interfacial tension relative to surfactant concentration behaves similarly. The latter is usually measured between an aqueous and a Nujol or min- eral oil phase. Since the effective biological concentrations of sur- factants in general are greater than 0.1 per cent, one must assume that biological effectiveness is better correlated with other properties than surface energies. One point which may be significant in explaining biological effec- tiveness is the fact that actual measurements of surface and inter- facial tensions do not always coincide with theoretical values. A great many surfactants show the initial rapid drop in tension but at higher concentrations exhibit a slight increase in tension. The lowest point on the tension curve appears to correlate with the formation of col- loidal micelles and is referred to as the critical micellar concentration. Since the end of the steep drop in tension is usually found in the concentration range from 0.01 to 0.1 per cent, it seems probable that at biologically effective levels surfactants are behaving as strong col- loidal systems. Several other properties of surfactants undergo marked changes in regions corresponding to the critical micellar concentration. One of these is the relative conductivity of the solution. In Figure IB the conductivities of an homologous series of alcohol sulfonates are illus- trated. As concentration is increased, very little change in electrical conductivity is found for the C-2 to C-7 homologues. Higher members of ihc series, however, exjiibit a rapid drop in relative conductivity 400a a2 0.4 ae o.s lo CONCENTRATION (PERCENT) 0 0.£ 0.4 0.6 0.8 1.0 SQUARE ROOT OF WT. NORMALITY 03 - t 0£ ■ 0 0,2 0.4 0.6 0.8 1.0 SQUARE ROOT OF VOL. NORMALITY Fig. 1. Effects of surfactant concentration on several properties of surfactants. A. Generalized ranges of values for surface tension and interfacial tension for the ma- jority of surfactants. B. Equivalent conductances for a series of alkyl sulfonic acids (numbers indicate length of the carbon chains). C. Cation transference numbers for a series of alkyl amine hydrochlorides. D. Osmotic coefficient of sodium tetra- decylsulfate. (Modified after Schwartz and Perry (1) by permission of Interscience Publishers, Inc., New York.) 816 L. L. Jansen in the region corresponding lo the critical micellar concentration. One can conceive what tremendous effects could result from place- ment of such a solution on the charged surface of a cell wall and how these effects could be transmitted through the aqueous continuum in the cuticle and cell wall to the charged cytoplasmic skeleton. Electrophoretic and osmotic properties of surfactant solutions are also modified anomolously as concentration of the surfactant is increased. In Figure IC we see the cation transference numbers for an homologous series of alkyl amine hydrochlorides plotted against concentration of surfactant. Abrupt changes occur in cation transfer after we pass the C-8 moiety. Among higher homologues the magni- tude of the change is increased as chain-length increases to C-14, but thereafter the magnitude is lessened. The effect of concentration of a representative surfactant on the osmotic coefficient, as determined by freezing point depression, is plotted in Figure ID. The osmotic coefficient of surfactant systems is considerably less than predicted values at concentrations commonly employed in industrial and bio- logical work. In the case of the representative surfactant used in the illustration, the breakpoint occurs at a concentration of approxi- mately 0.8 per cent. One cannot comment at the present time on the importance of all of these phenomena to the behaviors of biologically active chemi- cals. They are factors, however, which conceivably could materially influence penetration, translocation, and chemical activity. The pur- pose of this presentation has been to draw attention to those factors other than surface tensions which may be influential in the biological activity of systems containing surfactants. LITERATURE CITED 1. Schwartz, A. M., and Perry, J. W. Surface Active Agents. \'ol. I. 579 pp. Inter- science Publisliers, Inc., New York. 1949. 2. , Perry, J. W., and Berch, J. Surface Active Agents and Detergents. Vol. II. 839 pp. Interscience Publishers, Inc., New York. 1958. The Next Steps JAMES BONNER California Institute of Technology The Probable Future of Auxinology The only way in which we can forecast the future of a human activity is by projecting past trends into the future. This does not allow us to predict with certainty; it only allows us to predict a probability, just as the weather forecaster predicts the weather. He says, "Well, I can measure some things today, and I can look around and see how things were yesterday, and I can see how things probably will be to- morrow if the situation develops as similar situations have developed in the past." He forecasts a probability, and as you know about weather forecasters, they are often wrong but never in doubt. And in this same spirit, tempered by a certain amount of humility, I make my own forecast of the probable future of auxinology. Let us start with the matter of auxin conferences. We've had quite a number now. The first one was held, I understand, in Paris in 1937, and it was attended by about 20 people. In 1949 a conference was held in Wisconsin and the official participants numbered about 45. At the third auxin conference at Wye in 1955, the participants numbered about 65. Today, in 1959, we have over 100 people in at- tendance. Twenty in 1937, 45 in 1949, over 100 in 1959. It is clear that the doubling time for the number of people who attend an auxin conference is about 10 years. And this enables us to predict, then, that 100 years from now, at the conference to be held in 2059, there will be approximately 100,000 people in attendance. It's a heart- warming prospect. There is another trend we can also foresee. Quite evidently the number of people who attend auxin conferences is increasing more rapidly than is the total number of people in the world. The time must therefore inevitably come when everyone, each single individual on the earth's surface, will be an auxinologist. And if we bear in [819] 820 ]. Bonner mind the relevant rates of increase of people and of auxinologists, we can determine by simple calculation that the crossover point, the time at which all people will be auxinologists, will be in the year 2229. (Don't trust the last figure.) At that time there will be approxi- mately ten billion people on the earth's surface, and they T\ill all be auxinologists. Isn't that a wonderful thought? We know further that an attendant at an auxinology confer- ence submits a paper which appears in a book, and that on the average, the individual paper contributes five pages to this book. The book that results from our conference in the year 2229 will therefore consist of fifty billion pages. If we bind the proceedings of the con- ference up in volumes, each of 1,000 pages (which is a handy size), they will constitute approximately 50 million volumes. And if we put these volumes on a library shelf, they will occupy a shelf approximately 120 miles in length. My trusted agent in the Library of Congress has studied the reading habits of scientists, and he assures me that the biologist on the average reads down the shelf of biological literature at the rate of about 12 inches a year. This means, therefore, that if an individual wishes to read the Proceedings of the Auxinology Con- ference of 2229, he must realize that the task is going to take him about one million years. This isn't such a good prospect, and in fact our forecast begins to appear rather ridiculous. It has evidently been made on too lui- sophisticated a basis. Let us therefore start afresh and base our fore- cast on an entirely different model. W^e know that all human activi- ties undergo a grand period of growth. They rise and attain a maxi- mum only to decline again. This is true of human activities, of human innovations. Innovations are innovated; they appear; they increase in number or importance; and then they disappear as they are supplanted by some new innovation. Take, for example, bows and arrows. They were invented and they increased until some large nimiber of them were present on the earth's surface, and now they're almost gone again. Or take horses on the North American continent. They were intro- duced in the year 1515. They increased, attaining substantial num- bers, and reached a maximum about the time of the first World "W^ar. Since the end of the first World War they have catastrophically de- clined in numbers until they have now almost disappeared. This same behavior is characteristic of the cx])loitation of fields of human activity. Take an oil field. We find an oil field and some people put down a well, and they start extracting the good stuff out of the ground. They get it, and other people start putting down more and more oil wells and they pump out more and more oil each year. But finally the oil gets harder to find, and you have to pump it from Probable Future of Auxinology 821 deeper and deeper. And so the rate of production of oil goes down and down and ultimately declines to zero. This has actually happened to oil fields in our country. It is an interesting fact that oil production during the exploitation of an oil field first increases with time along an S-shaped curve. It then reaches a maximum and this is followed by decline also along an S-shaped curve. The curve which describes production as a function of time is symmetrical about its maximum. Rate of production rises and falls along a symmetrical bell-shaped curve, a curve which resembles appropriately enough that which de- scribes how growth rate of the Avena coleoptile section rises and falls as we increase auxin concentration. Perhaps, then, the model that we should use for forecasting the future of auxinology is not just an un- sophisticated doubling every so often but rather that of the exploita- tion of a field. Perhaps we should think about the exploitation of a field of knowledge. And so I have investigated auxinology from this stand- point too. With what kinetics is auxin lore being extracted from nature? I have approached this question by studying how many papers are written each year on auxinological subjects, and I have found out how this number has changed with time. Now we can't just count the number of papers that are produced each year because some of them aren't very good, will never be read again, and don't really con- stitute an extraction of knowledge from nature. As measure I have used, therefore, the yearly number of auxinological papers which someone else has thought worthwhile to cite. This is a better measure. These are essentially contributions to the theory of auxinology — contributions which have influenced other workers. Now to kinetics. The first paper of modern auxinology was, of course. Professor Went's contribution published in the year 1928, so in that year there was one paper. During the next year two or three papers appeared, and in the next year four or five. The number of papers per year on auxinological subjects thereafter increased steadily and exponentially, following a nice S-shaped curve, attaining a maxi- mum in the years 1948 to 1954. And, I have found to my horror and dismay, that we are already on the declining limb of the curve; we have seen the rise and are now witnessing the decay of auxinology. We may predict that the yearly number of auxinological papers will be down to half-maximal in about the year 1965, and that the last paper on auxinology will be written about 1985. This would appear to be a rather dismal conclusion. However, it isn't actually quite so gloomy as it seems, or rather it places gloom where no gloom is really intended. I think that you will agree 822 J. Bonner that this sort of rise and fall is what we must expect in the exploitation of any and all fields of knowledge. Let me make this a point more concretely by reference to a field of knowledge that has already been thoroughly exploited and explored. About 40 years ago, biologists suddenly became aware of the importance of hydrogen ion concentra- tion. We learned that there is such a thing as pH and that we are supposed to measure it. There ensued a great gold rush of biologists to measure the pH of everything — insides and outsides of cells, dif- ferent parts of cells, the soil — everything one might imagine was pH- measured. And papers concerning the pH of objects of biological origin appeared with rapidly increasing frequency. Numbers of papers per year in this field attained a maximum and then subsequently shrank. The rise and fall of works on pH has followed the kinetics of our exploitation model. What does this mean? It doesn't mean that we're less interested in pH than we used to be. It just means that everybody know^s that there's such a thing as pH and that when you do an experiment you should measure the pH, that you've got to be careful about it and put buffers in solutions, and do all the things that biologists do to take cognizance of the fact that the pH of the solution is an important variable. W^q have essentially incorporated pH lore into biological wisdom and we use it as an everyday part of our equip- ment for being a biologist. It is therefore my prediction that by the year 1985, although the last paper specifically dedicated to auxinology will have already ap- peared in print, auxinology won't really disappear, it will just be in- corporated into the total body of classical biology in the same way that pH lore has been assimilated. When people study growth mat- ters, they'll know that there's auxin, that auxin is made in certain places, that it goes around the plant and that it makes leaves not fall off or makes cells increase in size, or makes fruits be parthenocarpic — they'll know the things that auxin does, whatever it does, and this wisdom will be used in assessing the possible existence of any new growth factor, in discussing the interaction of other growth factors, in discussing the ways in which climatic factors influence plants, etc. Auxin lore will just be a part of the background information which we have at our disposal to help us evaluate new facets of the behavior of plants. It will be a part of general plant biological wisdom. And I think that this is not too dismal a prospect. We can look forward perhaps to a decreasing interest in auxinology as a specific subject in iis own right, but we can look forward to its becoming increasingly a part of llic biological body. I forecast that this change will be at- tended by an alteration in the educational level at which we offer in- struction about auxin matters. There was a time in which peoj)le Probable Future of Auxinology 823 learned about auxin only during their postdoctoral careers. I learned about auxinology in graduate school. There are many people in at- tendance at this conference who first learned about auxinology as undergraduates. I predict that by 1985 students will learn about auxin in high school or possibly even in junior high school. It will just be everyday stuff. We have outlined one trend in auxinology: it is going to become a part of classical biology. There is another trend that I think we can foresee. We must rec- ognize the debt which plant physiology owes to the discovery of auxin and to the development of the whole field of experimental auxinology. In certain respects the development of auxinology has, I think, made plant physiologists much more into cell biologists. It has interested plant physiologists in things on a cellular level, in finding out how the auxin works and finding out what this has to do with respiration and so on. The study of auxin-controlled plant growth has resulted in the accelerated development of plant physiology along the lines of the study of plant behavior on a cellular level. This is one thing that auxinology has done for plant physiology. A second thing auxin- ology has done is that it has been responsible, perhaps more than any other single force, for making botanists aware of the existence of chemistry. Auxinology has really brought chemistry to plant physi- ology and it has taken plant physiologists to chemistry. It has had a very great effect in making plant physiology a more chemical and, I will say, a more sophisticated subject. We all know that in 1935 it was first demonstrated that it is pos- sible to make compounds synthetically which possess auxin activity and which are therefore synthetic substitutes for naturally-occurring auxin. This discovery resulted in a major gold rush in auxinology. Chemists mysteriously appeared from everywhere — I don't know where chemists appear from — there must be lots of chemists who are technologically unemployed, and who, when some new field opens up, rush in to fill the vacuum that has been created. Anyway, chemists appeared from everywhere to synthesize tens of thousands of com- pounds, which were then tested for possession or nonpossession of auxin activity. As everyone knows, some of these compounds turned out to have useful auxin activity, to be able to do things such as be herbicides or be abscission inhibitors, and so on. And so to the development of auxinology in this chemical way we owe, to a very considerable extent, the development of agricultural chemical plant physiology. And I think that we can foresee clearly that this trend will continue far into the future, to a future far more distant than I can foresee for you now. People will continue to make more and 824 /. Bonner more plant regulators. They will find compounds that tell plants to grow, or tell plants not to grow, or tell plants to make sugar, or tell them to please make amino acids. The day will inevitably come when every aspect of the activity of every kind of plant will be supervised by the giving to that plant of an appropriate chemical. The poor little plant just won't have any private life of its own at all. However, I don't really refer to this as a long-range prospect for auxinology. I think that what auxinology has done is to enunciate and develop the concept of the supervision of plant activities by the application of chemical substances and that the agricultural chemical industry which is based upon this concept is now already very much wider than the concept of auxinolog)^ itself. It started a whole new field, and a field which has grown far beyond the bounds of auxin- ology. Here, then, is another trend which we can foresee for the long- range future. When we look into the shorter-range future, I think we can get some glimpses of trends in auxinology, trends which are apparent to everyone who has attended the present conference. In the first place, it is quite apparent that some day, any decade now, we shall finally succeed in finding out how the auxin does its work. This is an immediate task of plant physiologists, and some day it will be found out. It is really rather a disgrace to the profession of plant physiology that the nature of auxin action has not yet been revealed. This sad state of affairs is due to the fact that we embarked on the study of how auxin does its work insufficiently prepared with basic knowledge. We have known too little of the constitution of plant cells and of the constitution of the cell wall, and we have been insufficiently prepared with knowledge of the basic cell biology of the plant. We have had, in fact, insufficient knowledge of the basic cell biology of any kind of creature. But now as we get this new knowledge, we may hope that we will be able to solve the problem of how the auxin does its many kinds of work which are manifested in cell elongation, bud and root inhibition, prevention of abscission, etc. And in its effort to attain a solution to the problem of how auxin works we can, I think, forecast that auxinology and indeed plant physiology generally will become what I will call, pardon the expression, more sophisticated, will become a more rigorotis science. It will become more physical, more biophysical if you will, more biochemical. Plant physiologists to come will become more enzymological because enzymology is a craft which can obviously contribute to the solution of our problems. Plant physiologists will indulge more in model making, in stochastic re- search, that is, in the making of conceptual models which can then be tested for correspondence with real life. Plant physiology will, in Probable Future of Aiixinology 825 short, progress until it resembles in its mode of operation the sciences of biochemistry and of biophysics today. So this is another trend. We know, too, that because there is such a thing as auxin, one might well look for other kinds of growth factors. That there are other kinds of growth factors is now amply attested by the fact that we know about some of them. We know about gibberellin, for ex- ample, and therefore all of the kinds of questions that have been asked about auxin can now be asked about gibberellin, too. When all of the problems of auxinology are solved, it will not mean that we have nothing to do. We can always convert ourselves to working on gibberellin, or working on the isolation of flowering hormone, or some other lesser known growth factor. There is an old saying, or if there isn't such an old saying, there ought to be one, that "Old auxin- ologists never die, they turn into gibberellinologists." So there are many things to do in the study of plant growth factors other than auxin itself. I predict, therefore, that within another 25 years certain- ly, the chemical nature and mode of action of a vast array of further plant hormones will be understood. What, now, is going to happen after plant physiologists have got- ten busy and found out about the nature of all of these other growth factors and know how all of them do their work? What will people do then? I have a suggestion. It seems to me that really the most basic biological problem that confronts us, now that the problem of the nature of genetic information and its replication has been wound up, is the problem of differentiation. We have mentioned differentia- tion many times during the course of this conference. No one has explained anything about differentiation; we just say to each other, "Oh, that's differentiation," or "This substance influences differentia- tion," or something of similar ilk. We fail to understand differentia- tion because we haven't yet really had a tool which would enable us to approach the subject in a productive experimental way. How does differentiation take place? It is my prediction that another generation from now this may well be one of the principal studies to which people who would have been auxinologists in the olden days will turn their talents. And I have a little suggestion, too, about how the approach to the study of differentiation might possibly be made. I want to make this suggestion now because perhaps some of us can be thinking about it and perhaps we can jump the gun a little and get on with the study of differentiation. It is implicit in the thinking of every biologist and has been im- plicit, too, to a small extent in our discussion at these meetings, that when cells of common cell ancestry differentiate along different path- ways and turn into different kinds of cells, these different kinds of 826 J. Bonner cells are different because they possess different kinds of enzymes. They have different enzymatic complements. They have, as it were, different enzymatic spectra. One type of cell will have lots of enzyme A and little of enzyme B, while of a second type, the converse will be true. And as a matter of fact, I believe that most students of biology now begin to be pretty well convinced that differentiation with respect to enzymatic composition is not only an accompaniment of the differen- tiation process but is very probably the cause of differentiation itself. How do cells of common descent and of common genetic consti- tution get different enzymatic complements? It is a tenet of modern biolog)', of course, that each enzyme in the cell is produced under the control of a specific gene, a gene which sits in the nucleus and says to the cell, "Please make me some of this kind of enzyme." For each enzyme there is a responsible gene. This is the central dogma of our time. We now know a little bit about how enzyme molecules are made. This is information which has accrued to cell biologists in very re- cent years. We know that enzyme molecules are made of amino acids and that the specificity of an enzyme molecule, the fact that it is this enzyme instead of some other enzyme, depends in a great measure upon the sequence of amino acids in the peptide chain of which the molecule is made. We might say that an enzyme is a message written in a 20-letter alphabet — the 20-letter alphabet of the 20 amino acids which occur in protein molecules. And we know, too, that enzyme molecules are synthesized in the cell, or even outside the cell, by spe- cial little objects which sit in the cytoplasm and do this task — objects which we in the \\'est call microsomes and which, in the East, people call microsomal nucleoprotein particles. And the evidence is pretty good that each one of these kinds of particles carries the information required to make one — and only one — kind of enzyme molecule. The newer cell biology tells us, too, that microsomal particles con- tain their information in the form of nucleic acid — information writ- ten in language of ribonucleic acid, RNA. This information is some- how transmitted to and used in the assemblage of the amino acids in the specific sequence of a specific enzyme. The microsomal particles in turn are made within the nucleus, and we don't know how this happens. W^e know only that microsomal particles are made in the nucleus, that they are made in the nucleus only in the presence of de- oxyribonucleic acid, the DNA of the genetic material. The implica- tion is iliat the message contained in the DNA of the nucleus is some- how printed off in the language of RNA, and then extruded into the cytoplasm in the form of microsomes which are responsible for the as- semblage of enzyme molecides. Probable Future of Auxinology 827 Now let us get back to differentiation. We know that every cell of the multicellular organism, so far as we can tell today (and this is backed by extensive embryological information) possesses all of the genes which are needed for the assemblage of the whole plant or the whole animal. It is quite obvious, though, that all cells do not use all of the genetic information, do not make all of the enzymes which are needed to make the whole creature. Take a pea plant for example. It has genes in it which tell the cotyledons how to make the reserve protein of pea cotyledons. But this protein does not occur in all parts of the pea plant. The genes for making reserve protein of pea cotyle- dons only do their work when they sit in the cells of a pea cotyledon and do not do their work when they sit in a cell of a root or stem or leaf or flower. Here, then, we return to differentiation. It is quite evi- dent that there is some entity within the nucleus which says to the gene either, "Please be active and make appropriate microsomes which can go out and make the enzyme for which I contain the message," or this entity, whatever it is, says to the gene, "Please be inert and do not send out your message." Differentiation consists of the sequential and properly programmed turning on and off of the genetic informa- tion of the nucleus. Perhaps part of this programming is information contained in the DNA. As one of my students once said, "Perhaps part of the information in the nucleus is information on when to use the information." But I don't know what it is that programs the nu- cleus. Perhaps one of the things that turns the genes off and on in the nucleus is auxin itself. This would indeed be jolly, but I don't believe it. But let's find out what it is that turns genes off and on during the course of development, even if it does not turn out to be auxin. In any case the study of differentiation is clearly enough one of the important tasks of the plant physiology of the future. But let us now return to auxinology, the treasury of auxin lore. What does the future hold for our chosen discipline? My principal forecast is that auxinology, as a specific science apart, will ultimately come to an end. Everything will be found out. But auxinology as a part of basic biological lore will be immortal. Supplementary Information Participants in the Conference Author Index Subject Index Participants in the Conference (Address at time of publication) •Aasheim, T0RBJORN, Botanlcal Labor- atory, University of Bergen, Bergen, Norway. Aberg, B., Institute of Plant Physiology, Royal Agricultural College, Uppsala 7, Sweden. Addicott, F. T., Department of Agron- omy, University of California, Davis, California (U.S.A.). Albert, L., American Cyanamid Co., Stamford, Connecticut (U.S.A.). Alder, E. F., Agricultural Research Center, Eli Lilly and Co., Greenfield, Indiana (U.S.A.). Andreae, W. a.. Pesticide Research In- stitute, Canada Department of Agri- culture, Research Branch, Univ. Sub Post Office, London, Ontario, Canada. Arthur, John M., 414 North Main Street, Paris, Illinois (U.S.A.). AuDUS, L. J., Botany Department, Bed- ford College, University of London, Regents Park, London, N. W. 1, England. Avery, George S., Jr., Brooklyn Botanic Garden, Brooklyn, New York (U.S.A.). Bach, M. K., The Upjohn Company, Kalamazoo, Michigan (U.S.A.). *Bak, R., National and University Insti- tute of Agriculture, Rehovot, Israel. *Baker, K. C, University of California, Los Angeles, California (U.S.A.). *Bakhsh, J. K., Botany Department, Bedford College, University of Lon- don, Regents Park, London, N.W. 1, England. Barker, G. W., Division of Tropical Research, United Fruit Co., c/o Tela Railroad Co., La Lima, Honduras, C.A. Barlow, H. W. B., East Mailing Re- search Station, near Maidstone, Kent, England. Barton, Lela V., Boyce Thompson In- stitute for Plant Research, Inc., Yon- kers. New York (U.S.A.). *Beauchesne, G., C.N.R.S., Laboratoire du Phytotron, Gif-sur-Yvette (S. et O.), France. Bennet-Clark, T. a.. Department of Botany, Kings College, 68 Half Moon Lane, London, S. E. 24, England. Bentley, Joyce A., Marine Laboratory, Scottish Home Department, Victoria Road, P.O. Box No. 101, Torry, Aber- deen, Scotland. BiTANCouRT, A. A., Divisa de Biologia Vegetal, Instituto Biologico, Sao Paulo, Brasil. S.A. Blackman, G. E., Department of Agri- culture, University of Oxford, Oxford, England. Bonner, James, Division of Biology, California Institute of Technology, Pasadena, California (U.S.A.). * Authors who did not attend. [831 ] 832 Participants in the Conference Brian, P. \V., Akcrs Research Labora- tories, Imperial Chemical Industries, Ltd., Wehvyn, England. BuKovAC, M. J., Department of Horti- culture, Michigan State University, Last Lansing, Michigan (U.S.A.). BuRSTROM, H., Botanical Laboratory, Lund, Sweden. ♦Carns, H. R., U. S. Department of Ag- riculture, Beltsville, Maryland (U.S.A.) . Carroll, Robkrt B., P.O. Box 955, Greenwich, Connecticut (U.S.A.). •Chailakhian, M. Kh., K. A. Timiriazev Institute of Plant Physiology, U.S.S.R. Academy of Sciences, Lenin Avenue 33, Moscow, U.S.S.R. Chandler, Clyde, Boyce Thompson In- stitute for Plant Research, Inc., Yon- kers. New York (U.S.A.). Chouard, p., Laboratoirc de Physi- ologic Vegetale, Sorbonne, Paris, France. Cooke, A. R., Biological Research Divi- sion, E. I. du Pont de Nemours, Inc., Wilmington, Delaware (U.S.A.). Crafts, A. S., Department of Botany, University of California, Davis, Cali- fornia (U.S.A.). Crosbv, D. C, Research Dept., Union Carbide Chemicals Co., South Charles- ton, ^^■est Virginia (U.S.A.) . Davis, D., New York Botanical Garden, Bronx Park, New York 58, New York (U.S.A.) . Deverall, B. J., Dcpt. of Plant Patliol- ogy, University of Wisconsin, Madi- son 5, \Visconsin (U.S..^.). DoRSCMNiR, K. p., Niagara Chemical Di- vision, Food Machinery and Chemical Corp., Middleport, New York (U.S.A.). •DowDiNG, L., Siiell Development Co., Modesto, California (U.S..\.). EvENARi, M., Department of Botany, Helirew University, Jerusalem, Israel. Fawceit, C. H., Agricultural Research Council Unit, Wye College, Wye, near Ashford, Kent, England. •Fellig, J., Lindc Company, Union Car- bide, Tonawanda, New York (U.S.A.). Ferri, M. G., Department de Bolanico, Universidad de Sao Paulo, Faculdailc de Filosofia, Ciencias e Letras, Caixa Postal 8.105, Sao Paulo, Brasil, S.A. Fi.EMioN, Florence, Boyce Thompson Institute for Plant Research, Inc., Yon- kers. New York (U.S.A.). Forti, G., McCollum Pratt Institute, Johns Hopkins University, Baltimore 18, Maryland (U.S.A.). Freed, V. H., Department of Chemistry, Oregon State College, Corvallis, Ore- gon (U.S.A.). Freiberg, S. R., Department of Research, United Fruit Co., Norwood, Massa- chusetts (U.S.A.). *Frost, p.. Union Carbide Research In- stitute, 32 Depot Plaza, White Plains, New York (U.S.A.). Galston, A. W., Department of Botany, Yale University, New Haven, Connec- ticut (U.S.A.). ' Goldacre, p. L., Division of Plant In- dustry, C.S.I.R.O., Canberra, Australia. Gordon, S. A., Argonne National Lab- oratory, Lemont, Illinois (U.S..\.). GowiNG, D. P., Pineapple Research In- stitute of Hawaii, Honolulu, Hawaii (U.S.A.). Hall, W. C, Department of Plant Phys- iology, Texas A. and M. College, Col- (U.S.A.). *Hallaway, Mary, Department of Bot- any, University of Oxford, Oxford, England. Hamm, p. C, Agricultural Research Laboratory, Monsanto Chemical Com- pany, St. Louis, Missouri (U.S..\.). ♦Hancock, C. R., East Mailing Research Station, near Maidstone, Kent, Eng- land. Hansch, C, Department of Botany, Po- mona College, Claremont, California (U.S.A.). Hansen, J. R., Agricultural Chemicals Division, Hercules Powder Company, Wilmington, Delaware (U.S..\.). Hayashi, T., Department of Plivsiology and Genetics, National Institute of Agricultural Sciences, Nishigahara, Kita-Ku, Tokyo, Japan. ♦Hemming, H. G., Imperial Chemical Industries, Ltd., Wehvyn, England. Henderson, J. H. M., Department of Botany, Tuskegee Institute, Tuskegee, Alabama (U.S.A.). •Herrero, F. a., Texas A. and M. Col- lege, College Station, Texas (U.S.A.). IlERRiir, R. A., Union Carbide Chemi- cals Co., Research Farm, Clayton, North Carolina (U.S.A.). Higmkin, H., Division of Biology, Cali- fornia Institute of Technology, Pasa- dena, California (U.S.A.). Participants in the Conference 833 Hill, G. D., Grasselli Chemicals Depart- ment, du Pont Experiment Station, E.I. du Pont de Nemours, Inc., Bldg. 268, Wilmington, Delaware (U.S.A.). HiLLMAN, W. S., Department of Botany, Yale University, New Haven, Connect- icut {V.S.A.). HiNMAN, R. L., Union Carbide Research Institute, 32 Depot Plaza, White Plains, New York (U.S.A.). Hitchcock, A. E., Boyce Thompson In- stitute for Plant Research, Inc., Yon- kers. New York (U.S.A.). HousLEV, S., Food Research Department, Unilever Ltd., Colworth House, Sharn- brook, Bedford, England. Jacobs, W. P., Department of Biology, Princeton University, Princeton, New Jersey (U.S.A.). Jansen, L. L., Weed Control in Crops Section, Crops Research Division, Ag- ricultural Research Service, USDA, Beltsville, Maryland (U.S.A.). Jepson, J. B., Courthauld Institute of Biochemistry, The Middlesex Hospi- tal, Medical School, London, W. I, England. Kato, J., Department of Botany, Univer- sity of California, Davis, California (U.S.A.). *Kaur, Ravindar, Yale University, New Haven, Connecticut (U.S.A.). *Kawarada, Akira, University of Tokyo, Bunkyo-Ku, Tokyo, Japan. Kefford, N., Division of Plant Industry, C.S.I.R.O., Canberra, Australia. Kessler, B., Institute of Horticulture, Unit of Plant Physiology, National and University Institute of Agricul- ture, P.O.B. 15, Beit-Dagan, Rehovot, Israel. Ring, L. J., Philadelphia College of Phar- macy and Science, Philadelphia 4, Pennsylvania (U.S.A.). Klein, R. M., The New York Botanical Garden, Bronx Park, New York 58, New York (U.S.A.). KoNiNGSBERGER, V. J., Botauisch Labora- torium en Hortus Botanicus van de Rijksuniversiteit te Utrecht, Lange Nieuwstraat 106, Utrecht, Netherlands. *Lacey, H. J., East Mailing Research Station, near Maidstone, Kent, Eng- land. *Lam, S. L., Purdue University, Lafa- yette, Indiana (U.S.A.). *Lang, Anton, California Inslitulc of Technology, Pasadena, California (U.S.A.). Larsen, p.. Botanical Laboratory, Uni- versity of Bergen, Bergen, Norway. Leaper, J. M. P., Villa Juan Jose, Puerto de Soller, Mallorca, Spain. Leasure, J. K., Dow Chemical Co., Mid- land, Michigan (U.S.A.). Lemin, a. J., The Upjohn Co., 301 Hen- rietta Street, Kalamazoo, Michigan (U.S.A.). Leopold, A. C, Department of Horticul- ture, Purdue University, Lafayette, Indiana (U.S.A.). LocKHART, J. A., Hawaii Agr. Exp. Sla., University of Hawaii, Honolulu 14, Hawaii (U.S.A.). LoHR, A. D., Naval Stores Research Di- vision, Hercules Powder Co., Wilming- ton, Delaware (U.S.A.). LoNA, F., Istituto ed Orto Botanico, Uni- versity of Parma, Parma, Italy. ♦Maclachan, G. a.. Department of Botany, University of Alberta, Edmon- ton, Canada. Marre, E., Institute of Botany, Univer- sity of Milan, Via Gius. Colombo 60, Milan, 443, Italy. Marth, p. C, USDA, Agricultural Re- search Service, Horticultural Crops Research Branch, Beltsville, Maryland (U.S.A.). Martin, A. W., National Science Foun- dation, Washington 25, D. C. (U.S.A.). Mayer, A. M., Department of Botany, Hebrew University, Jerusalem, Israel. McCallan, S. E. a., Boyce Thompson Institute for Plant Research, Inc., Yonkers, New York (U.S.A.). *McCune, D. C, Boyce Thompson In- stitute for Plant Research, Inc., Yonk- ers, New York (U.S.A.). McLane, S. R., Amchem Products, Am- bler, Pennsylvania (U.S.A.). McNew, G. L., Boyce Thompson Insti- tute for Plant Research, Inc., Yonkers, New York (U.S.A.). McRae, D. H., Agricultural Research Division, Rohm and Haas Chemical Co., Philadelphia, Pennsylvania (U.S.A.). Merritt, J. M., Plant Chemical Section, Merck & Co., Inc., Rahway, New Jer- sey (U.S.A.). 834 Participants in the Conference Miller, C. O., Department of Botany, University of Indiana, Bloomington, Indiana (U.S.A.). ♦Miller, C. S., Texas A. and M. College, College Station, Texas (U.S..\.). Miller, L. P., Boyce Thompson Insti- tute for Plant Research, Inc., Vonkers, New York (U.S.A.). MiNARiK, C. E., U.S. Army Biological \Varfare Laboratories Crops Division, Fort Detrick, Frederick, Maryland (U.S.A.). Mitchell, J. \\., USDA, Agricultural Research Service, Horticultural Crops Research Branch, Beltsville, Maryland (U.S.A.). Morel, G., Station Centrale de Physi- ologic V(^getale, Centre National de Recherches Agronomiques (route Saint-Cyr), Versailles (S. et O.), France. •MoscicKi, Z. W., National and Univer- sity Institute of Agriculture, Rehovot, Israel. MuiR, R. M., Department of Botany, University of Iowa, Iowa City, Iowa (U.S.A.). Naf, U., The Rockefeller Institute, New York, New York (U.S.A.). Nichols, R., Regional Research Centre, Imperial College of Tropical Agricul- ture, St. .Augustine, Trinidad, \\M. NiCKELL, L. C, Biological Research Di- vision, Chas. Pfizer & Co., Inc., Groton, Connecticut (U.S.A.). NiTscH, C, Laboratoire du Phytotron, Gif-sur-Yvette (S. et O.), France. NiTSCH, J. P., Laboratoire du Phytotron, Gif-sur-Yvette (S. et O.), France. •No(;uEiRA, Alexa.nora p., Divisa de Bi- ologia Vegetal, Instituto Biologico, Sao Paulo. Bnisil, S.A. OsBOR.NE, Dai'h.ne J., Agricultural Re- search Council, Unit of Experimental Agronomy, Department of Agricul- ture, University of Oxford, Oxford, England. Phinney, B. O; Department of Botany, University of California, Los Angeles 21, California (U.S.A.). PiLET, P. E., LaI)oratoire de Physiologic Vegctale, Universile de Lausanne, L;uisannc, Swil/ci land. Plaisted, p. H., Boyce Thompson Insti- tute for Plant Rcscarcli, Inc., Yonkers, New York (U.S.A.). •Poljakoff-Mavber, a.. Department of Botany, Hcljicw University, Jerusa- lem, Israel. ♦PoRTo, F., Union Carbide Research In- stitute, 32 Depot Plaza, White Plains, New York (U.S.A.). PuRVES, W. K., Department of Botany, University of California, Los Angeles, California (U.S.A.) . Rav, p. M., Department of Botany, Uni- versity of Michigan, Ann Arbor, Mich- igan (U.S.A.). *Reithel, F. J., University of Oregon, Eugene, Oregon (U.S.A.). *Remmert, L. F., Oregon State College, Corvallis, Oregon (U.S.A.). RiCKETT, H. W., The New York Botan- ical Garden, Bronx Park, New York 58, New York (U.S.A.). RoBBiNS, W. J., The New York Botan- ical Garden, Bronx Park, New York 58, New York (U.S.A.). Ruth, Jeanette, National Science Foun- dation, Washington 25, D.C. (U.S.A.). Sachs, R. M., Department of Floricul- ture and Ornamental Horticulture, University of California, Los Angeles, California (U.S.A.). Sargent, J., Herbicide Research Unit, Department of Agriculture, University of Oxford, Oxford, England. ScHULDT, P. H., Boyce Thompson Insti- tute for Plant Research, Inc., Yonkers, New York (U.S.A.). ♦Schwarz, Kaethe, Divisa de Biologia Vegetal, Instituto Biologico, Sao Paulo, Brasil, S.A. Shantz, E. M., Department of Botanv, Cornell University, Ithaca, New York (U.S.A.). Shapiro, S., Department of Biology, Brookliavcn National Laboratory, Brookhavcn, Long Island, New York (U.S.A.). *Sme\, Jane Y., Division of Biological and Medical Research, Argonne Na- tional Laboratory, Lemont, Illinois (U.S.A.). SiiGEL, S. M., Union Carbide Research Institute, 32 Depot Plaza, ^Vhite Plains, New York (U.S.A.). SiRONVAL, C, Laboratory of Plant Physi- ology, Centre de Recherches de Gor- sem, Gorsem-Saint-Trond, Belgium. Smith, Margaret S., Department of Botany, AVye College, ^Vye, near .Ash- ford, Kent, England. Steere, W. C, The New York Botan- ical Garden, Bronx Park, New York 58, New York (U.S.A.). Participants in the Conference 835 SroDOLA, F. H., USDA, Agricultural Re- search Service, Pioneering Laboratory for Microbiological Chemistry, North- ern Utilization Research R: Develop- ment Division, Peoria, Illinois (U.S.A.). Stovve, B. B., J. W. Gibbs Laboratory, Dept. of Botany, Yale University, New Haven, Connecticut (U.S.A.). SuMiKi, Y., Department of Agricultural Chemistry, University of Tokyo, Bun- kyo-Ku, Tokyo, Japan. *Takahashi, Noriko, Japan Women's University, Bunkyo-Ku, Tokyo, Japan. Teubner, F. C, Department of Horti- culture, University of Nebraska, Lin- coln, Nebraska (U.S.A.) . Thellier, M., Laboratoire de Physi- ologic Vegetale, Sorbonne, Paris, France. Thimann, K. v., Biological Laboratories, Harvard University, Cambridge, Mas- sachusetts (U.S.A.). ToLBERT, N. E., Department of Agri- cultural Chemistry, Michigan State University, East Lansing, Michigan (U.S.A.). *ToNZiG, S., University of Milan, Via Gins. Colombo 60, Milan, 443, Italy. Torrev, J. G., Biology Department, Harvard University, Cambridge, Mas- sachusetts (U.S.A.). TuKEV, H. B., Department of Horticul- ture, Michigan State University, East Lansing, Michigan (U.S.A.). TuLECKE, \W R., Boyce Thompson Insti- tute for Plant Research, Inc., Yonkers, New York (U.S.A.). Utter, L. G., Diamond Alkali Co., Re- search & Development Department, P.O. Box 348, Painesville, Ohio (U.S.A.). VAN Overbeek, J., Shell Development Co., Agricultural Laboratory, P.O. Box 3011, Modesto, California (U.S.A.). *ViLLiERS, T. A., Botany Department, Makerere College, Kampala, Uganda. Vlitos, a. J., Central Agricultural Re- search Station, Caroni Ltd., Cara pichaima, Trinidad, W.I. Wain, R. L., Agricultural Research Council Unit, Wye College, University of London, Wye, near Ashford, Kent, England. Wareing, p. F., Department of Botany, University College of Wales, Abery- stwyth, Wales. Waygood, E. R., Department of Botany, University of Manitoba, Winnipeg, Manitoba, Canada. Weinstein, L. H., Boyce Thompson In- stitute for Plant Research, Inc., Yon- kers, New York (U.S.A.). West, C. A., Department of Chemistry, University of California, Los Angeles 24, California (U.S.A.). WiGHTMAN, F., Prairie Regional Re- search Laboratory, Saskatoon, Sas- katchewan, Canada. *WiLSON, R. K., University of California, Los Angeles, California (U.S.A.). Wittwer, S. H., Department of Hor- ticulture, Michigan State University, East Lansing, Michigan (U.S.A.). Zwar, J. A., Department of Agriculture, Canberra, Australia. Author Index Aasheim, T., 43 Aberg, B., 219, 232, 448 Addicott, F. T., 559 Andreae, W. A., 92, 93, 178, 247, 643 Audus, L. J., 109, 125, 126 Bach, M. K., 273, 339 Bak, R.. 387 Baker, K. C, 559 Bakhsh, J. K., 109 Barlow. H. W. B., 127, 654, 662, 722 Barton, Lela V., 501 Beauchesne, G., 667 Bennet-Clark, T. A., 39, 55, 246, 327 Bentley, Joyce A., 25, 39, 40, 41 Bitancourt, A. A., 68, 181, 460, 777 Blackman, G. E., 231, 233, 245, 246, 588, 802, 809 Bonner, J., 247, 304, 307, 378, 443, 448, 819 Brian, P. W., 465, 469, 501, 645, 653, 655, 656. 663 Bukovac, M. J., 505 Burstrom, H., 107, 125, 245, 556, 624, 749 Cams, H. R., 559 Chailakhian, M. Kh., 531 Crafts, A. S., 339. 789. 801, 802 Crosby, D. G.. 57, 67, 68, 69, 429, 447, 448. 482 Deverall. B. J., 627 Dowding, L., 657 Evenari. M., 106, 655 Fawcett. C. H., 41, 69, 71, 94, 257. 379, 461, 810 Fellig, J., 273 Forti, G., 776 Freed, V. H., 289, 304, 305, 339, 340 Frost, P., 205 Galston. A. W., 68. 124, 338, 355, 481, 501, 611, 625, 654, 722, 775 Goldacre. P. L.. 143 Gordon. S. A.. 655 Gowing. D. P.. 272. 803, 810, 811 Hall. W. C. 751. 775, 776. 777. 778 Hallaway, Mary, 329 Hancock, C. R., 127 Hansch, C, 231, 249, 258, 431, 444 Hayashi, T., 465, 466, 579, 588 Hemming, H. G., 645 Henderson, J. H. M., 245, 455, 801 Herrero, F. A., 751 Hillman, W. S., 589 Hinnian, R. L.. 205 Hitchcock. A. E.. 775 Housley. S.. 69, 627. 642, 643 Jacobs, W. P., 397, 400 Jansen. L. L.. 428, 813 Jepson. J. B.. 93 Kato. J.. 601 Kaur. Ravindar, 355 Kawarada. A., 483, 503 KefTord, N., 40, 67 Kessler, B., 387 Lacey. H. J.. 127 Lam, S. L., 411 Lang, A., 567 Larsen, P., 43, 54, 55, 588 Leopold. A. C. 305, 411 Lockhart, J. A., 543, 557, 655 Maclachlan, G. A., 149 Marre, E., 725, 743 Mayer, A. M., 735, 749 McCune, D. C, 611 McNew, G. L., 3 Miller, C. S., 751 Morel, G., 723 Moscicki, Z. W., 387 Muir, R. M., 249, 257, 258, 431 Naf, U., 709, 722, 723 Nickell, L. G., 675 Nitsch, C, 687 Nitsch, J. P., 41. 231, 271, 377, 653, 687, 775 Nogueira, Alexandra P., 181 Osborne, Daphne J., 69. 329, 339, 443, 461 Phinney, B. O., 489, 501 Pilet, P. E., 167 PoljakofF-Mayber, A.. 735 Porto. F., 341 Purves, W. K., 589, 611, 643 Ray, P. M., 199, 258, 381, 461, 777 Reithel, F. J., 289 Remmert, L. F., 289 Robbins, W. J., 13 Sachs, R. M., 567, 653, 810 Schwarz, Kaethe, 181 Shantz, E. M., 776 Shen, Jane Y., 259 Siegel, S. M., 341, 705 Sironval, C, 521 Smith, Margaret S., 256 Stodola, F. H., 465. 468 Stowe, B. B., 67, 419, 429, 465 Sumiki, Y., 483, 503 Takahashi, N., 363 Teubner, F. G., 259, 271. 272, 777, 778 Thimann, K. V.. 40. 54, 68, 69, 91. 92, 93, 363. 377, 444, 448, 749, 773, 776, 778, 802. 811 Tolbert. N. E., 587. 655, 779 Tonzig. S., 725 Torrey, J. G.. 105 Tukev, H. B., 105 Tulecke, W. R.. 675 van Overbeek, J., 303, 304, 449, 455, 456. 458, 460, 461, 557, 657, 662 Villiers. T. A.. 95 Vlitos, A. J., 57, 67, 429, 734 Wain, R. L., 67, 71, 93, 107, 230, 231, 256. 257, 258, 272, 303, 306, 443, 456. 458. 460. 749 Wareing. P. F., 95, 105, 106, 107. 339 Waygood. E. R.. 149. 642 West. C. A.. 473, 481 Wightman, F.. 71. 91, 92, 93 Wilson, R. K., 559 Wittwer, S. H.. 259, 340, 505 [836] Subject Index AA = ascorbic acid Abscission, GA effect on, 559 Acer pseitdoplatantis, buds of, 96 Achlya, sex hormones of, 19 ACN = indole-3-acetonitrile Acrasin, 19 Acropetal transport auxin, 406 lAA, 400, 413 IBA in Helianthus, 413 Adenosine triphosphatase = ATPase, 742 Agave toiimeyana, tissue culture, 678 Age, effect of, on auxin content, 170 Aged ethylene-C'S 754, 757, 776 Ageratum mexicanum, 525 Aging in root cells by auxins, 167 Agrobacterium tiimefaciens, 181 L-alanine, 605 Algae, 25, 33 Algal extracts, chromatography of, 26 Alkylamine hydrochlorides, 815 Alkylamines as antioxidants, 345 Alkylhydrazines as antioxidants, 345 Alkyl lipides, 422 Alkyl sulfonic acids, 815 Allium cepa == onion, 345 AMe = methyl indole-3-acetate Ameristic prothallus in ferns, 712, 716 Amides of indole acids, 71 Amines as antioxidants, 345 o-Aminoacetophenones, 214 Amitrol = AT apoplastic movement in bean leaf, 792 mobility in Zebrina pendula, 796 symplastic and apoplastic movements in potato tubers, 794 uptake by barley seedling roots, 798, 799, 800 Amo- 1618^ [ (5-hydroxycarvacryl) tri- methylammonium chloride, 1-piperi- dine carboxylate] Anaerobic preconditioning, 351 Anemia factor and antheridia, 711, 714, 715 Anem.ia phyllitidis, 710 ANH2 = indole-3-acetamide Anhydrouronic ( galacturonic ) acid = AUA, 320 Anisole as antioxidant, 345 Anneau initial, activation by GA, 522 Anthesin, constituent of florigen, 524 Antheridium formation in ferns, 709 Anthraceneacetic acid, 415 Anthranilic acid, 214 Antiauxin = 2-NMSeA Antiauxins, 222, 223, 603 Antioxidants, 342, 348 grovi'th regulation, 344, 345, 346, 347, 349 interaction with peroxides, 350 Apium = celery, 507 petiole elongation, 507 Apoplastic movement of amitrol in bean leaf, 792 Apple ^ Malus Apricot trees, 387 Arabidopsis arenosa, 525 Arachidonic acid, 65 Arachis = peanut, 399, 403 L-arginine, 605 Aromatic ring, 432 Arylamines as antioxidants, 344, 345 Arylhydrazines as antioxidants, 345 Aryloxv compounds as antioxidants, 344, 345 Ascorbic acid = AA as antioxidant, 344 as inhibitor, 344 effect on concentration DHA in tissues, 731 glutathione oxidation-reduction equi- librium, 728 plasma viscosity, 727 respiration internode segments, 727 growth inhibition, 605, 726, 729 growth regulating hormone, 725 growth regulating tissue cultures, 701 translocation in plants, 726 Ascorbigen, 734 Aspartic acid, 605 Aspergillus niger filtrate, 605 Asplenioids, 710 Assay technique, 26 AT = amitrol Athyriu7n filix-mas, 709 ATPase = adenosine triphosphatase AUA= anhydrouronic (galacturonic) acid Autocatalysis, Omphalia enzyme induced, 201 Autoradiograms, 334 Auxin = IAA, 17, 18, 25, 106, 167, 223 absorption on charcoal, 305 action, 222 intracellular locale, 355 physico-chemical nature of, 451 primary mode of, new theory, 449 ortho and carboxyl groups, 449 stimulation by lipides, 419 activation, 141 activity, 52 activity indices substituted benzoic acids, 250 adaptation of pea roots to, 109 anchoring to cytoskeleton, 453 antagonist, 259 content and growth, 170 content in aging in root cells, 167 Brassica napus, 51 Glycine max, 58 leaf senescence, 329 lentil root segments, 169 Visum, 49, 50, 358 tobacco, 57, 58, 59 TJ methanol extracts iAvena), 703 Yicia faba, 49 cytoplasmic proteins, effect on, 355 definition, 490, 589 extraction from roots, 168 fat solubility, 453 [837] 838 Subject Index Auxin ( continued ) inactivation, 141, 169 by soil, 54 induced decrease in heat coagulability, 360 growth mechanism of, 307 role of respiration, 323 interaction with AA, on metabolism, 730 AA and DHA (Pisum), 731 GA, 646 mechanisms, 589, 590 metabolic basis, 611 metallic ions, 363 interconvertible, 25 Kogl's, 43 precursors in algae, 33 sparing action, 455 structure of auxins and related com- pounds, 450 synergistic action, 259, 289 GA light efFect, 611 TJ (tomato juice) on cultures Helianthus, 691 Scorzonera hispanica, 691 transport in Coleus, 404, 405 polar movement, 411 in shoots, 397 in vascular tissues, 404 rate in Helianthus and Malus. 403 relation to cell length, 401, 402 saturation, efFect in Coleiis, 406 tropisms, stimulation of, 543 Auxinology, 5, 819 Avena, 237, 240, 242 coleoptile, 44, 98, 106, 131, 138, 223, 225, 226, 307 curvature tests, 46, 48, 49, 52, 67, 97, 98 elongation, 367 extract analysis, 97 lAA induced growth deformation by applied weight, 314, 315 efFect calcium ion concentration, 312, 313 efFect potassium ion concentration, 313 pectic synthesis, 320 reorientation cellulose microfibrils, 32 relation auxin transport to cell length, 402 section, efFect AA. 726 curvature response curves, 412 leaf base section test, 660, 661 technique of test, 657, 658, 659, 660 root growth, 739 sativa straight growth, 260 straight-growth method, 26 Avocado = Persea Azaserine ^ AZS ( tumor inhibiting anti- biotic), 649, 650 AZS = azaserine (tumor inhibiting an- tibiotic ) B Bacterial contamination control by strep- tomycin, 75 Bakanae efFect, 465 Bambusa multiplex, 64 Barley fixation of C"02, 767 mobility of 2,4-D, lAA, and AT in, 797 seedling roots, uptake labeled, 798, 799, 800 Basipetal auxin transport in Arachis gynophore, 403 Helianthus of lAA, IBA. and NAA. 413 Phaseolus hypocotyl, 400, 402 BCN = -^-(indole-3-) butyronitrile Bean = Phaseolus Bean bending, 790 Bean factors = BF, 475 Bean factor I = BFI, 475, 476, 479 Bean factor II = BFII, 475, 476, 480, 490, 496. 497 Bean leaf, 792, 793 Bean stem section, 275 Beets, 782 Bellis perenitis, 525 Benzimidazole, 329, 345 Benzofuran as antioxidant, 345 Benzoic acids, substituted, 257 efFect chemical structure on growth, 249 indices of auxin and chemical activitv, 250 Benzthiophene as antioxidant, 345 Bermuda grass regrowth, 791 Beta vulgaris, 525 BF = bean factors Bile pigments as antioxidants, 345 Bioassay method of Nitsch, 58 of Pisum extract, 48 of TJ fractions, 695 Biochemical analyses of auxin, 173 Biochemical gradients in Lens roots, 172 Biochromatograms, 174 Biochromatographic analyses. 174 Biophysics of cell growth, 381 Bios, 18 in TJ. 687 Biotin, 605 Blastocholine as growth inhibitor, 699 in tomato juice (TJ), 688 Blechnoids, 710 BMe = methyl -y-(indole-3-) butyrate BNH2 = -,-(indole-3-^ butyramide Bombyx niori, 10. 12. 65 Bonding between microfibrils cell wall and matrix, 381 Brassica, 52, 94 antioxidant growth promotion in, 347 extract of, 46, 50 indole-3-propionic acid in, 94 napus, auxins in, 51 oleracea var. sabauda, extracts of, 51 rapa = turnip, 345 seed germination, 350 (2-Bromoethvl) trimethvlammonium bro- mide = TMA. EBr, 779 p-Bromophenylhydrazine antioxidant, 346 2-BrPOA, 220, 223 3-BrPOA, 220 4-BrPOA. 220. 224, 225 4-BrPOP(±), 220 Bryophyllum, 18 Bud growth, 661 Buds, growth substance and chilling, 95 Buds of Acer pseudoplatanus. 96 Butyl ester 2,4-D, 330, 331, 336 5-n-Butylpicolinic acid = fusaric acid, 466 Bz-hydroxy-o-aminoacetophenones, 214 Ca<= transport in apricot, 388, 389, 391, 392 Caffeic acid in TJ and lAA, 699, 700 Calcium as growth inhibitor, 365 interaction with EDTA, 271 translocation in apricot, 387, 389, 390, 391 Subject Index 839 Calcium ion concentration binding, 317, 379 cell wall, 310 effect on Avena coleoptile sections, 312, 313 Carbon chain length, 815 Callus formation, 277, 349 Camellia japonica, 19 Cannabis sativa, 526 Capsella biirsa-pastoris, 525 Carbazoles as antioxidants, 384 Carotenes, 770 Casein hydrolysate, 605, 701 Catabolic lattice of indole derivatives, 181, 182 Catalase, 144 Catechin, 701 Catecholase, 744 Catechol-type inhibitor, 159 Cation transference numbers, 815 Caulocaline, 422 activity in stem growth, 543 cell wall plasticity, effect on, 554 C"-carboxyl-labeled 2,4-D as auxin, 358 CCN = e-(indole-3-) capronitrile Celery = Apiiim Cell elongation driving force of, 308 in coleoptile sections, 128 Cell growth biophysics of, 381 plastic mechanism of, 352 Cell length, 401, 402 Cell volume Chlorella vulgaris increased by coumarin, 742 Cell wall Avena coleoptiles, deformation, 315, 316 deformation, interaction with lAA and Ca ions, 311 interaction with cytoplasm in growth, 323 Cellulose microfibrils, tube structure, 319, 320, 321 Chain-stopping agents, 159 l-CioH7.CH2.S.CH-(CH3)COOH = NMSeA, 222, 223 CHeCN conversion to COOH, 93 Cheiranthes cheiri, 525 Chelating agents, 363, 368 Chemistry of GA from flowering plants, 473 Chenopodium, 737 Chilling dormancy, breaking of, 98, 100 germination stimulant, 102 growth substance, effect on, 95 Chlorella pyrenoidosa bioassay of extracts of, 27 chlorophyll, photooxidation of, 527 chromatography of extracts of, 27, 30 Chlorella vulgaris growth regulation by coumarin, 742 photooxidation of chlorophyll, 527 Chloro-derivatives of phenylacetic acid, activity, 457 3-Chloro-2,6-dimethylbenzoic acids, 450 ( 2-Chloroethyl ) trimethylammonium chlo- ride = TMA.ECl, 779, 783, 784 Chlorogenic acid in TJ, 699 p-Chlorophenoxyacetic acid ^ PCA, 19, 806 Chlorophenoxyacetic acids; see also Infra- red spectra, 291 3-Chlorophenoxyisobutyric acid = 3-CIBA, 603 4-Chlorophenoxyisobutyric acid = 4-CIBA, 603 a-(p-Chlorophenoxy )isobutyric acid = PCIB, 594, 595 3-(p-Chlorophenyl)-l, 1-dimethylurea = CMU, 285 (CH3)3N+CH2CmX, 780 C0H5.O.CH2.COOH = phenoxyacetic acid Choline analogues, 780 Chromatogram Ehrlich-positive substance, 179 pigment fraction of Gossypium treated with C^'-ethylene, 766 ultraviolet decomposition products of indole-3-acetic acid, 182 Chromatograms, 79, 82, 87 Chromatographs, 85 Chromatography algae extracts, 26 amino acid fractionation, 767 indole derivatives, 185 and metabolism, 71 paper, 57, 63 radioactive tryptophan-like metabolite, 768 Chrysanthemum inhibition of stem elongation, 572, 573 dwarfing effect of choline analogues, 783 Chrysanthemum morifolium, 576 3-CIBA ^ 3-chlorophenoxyisobutyric acid 4-CIBA = 4-chlorophenoxyisobutyric acid Citrus unshiu = mandarin orange, 475, 482 Clostridium tetani, 349 2-ClPOA, 220. 227 2,3-Cl2PO-A, 221, 227 2,4-Cl2POA = dichlorophenoxyacetic acid 2,4-Cl2PO-A, 221 2,4,5-Cl3PO-A, 221 2,4,5,6-Cl^PO-A, 221 2,4,6-Cl3PO-A, 221, 227 2,5-Cl2PO-A, 221, 227 2,6-Cl2PO-A, 221 3-ClPOA, 220, 226 3,4-Cl2PO-A, 221 3,5-Cl2PO-A, 221, 227 4-ClPOA, 220, 226 4-ClPOiB, 220 4-ClPOP(±), 220 CM = coumarin CMU = 3-(p-chlorophenyl)-l, 1-dimethyl- urea CN = indole-3-nitrile C"02 fixation by barley in the light, 767 Cobalt (Co+-) as antioxidant, 349 as growth promoter, 365 synergistic action with lAA {Avena), 377 time course effect on Avena coleoptile elongation, 367 Coconut milk GA in, 683 Growth regulation of crown gall tissue tobacco, 693 lAA synergism, 693 Cofactors for the lAA-oxidase of peas, 125 Coleoptile curvature, plum extract effect on, 131 sections cell extension in, 128 transport of inhibitor through, 135 Coleus absorption of ethylene-C" by, 752 auxin movement in, 404 translocation of ethylene-C", 752 Color reactions of ai-(indole-3-) alkanecar- boxylic acids, 73 Combretum, 338 CoMe = methyl indole-3-carboxylate 840 Subject Index Competition for internal sites, 242 Composite concentration-response curves, 224 Conductances, 815 C0NH2 = indole-3-carbonaniide Cotton == Gosst/piuni Coumarin = CM and derivatives, 739 effect on movement of calcium in apri- cot, 389, 390, 391 inhibition shoot, 602 destruction, 736 effect on animals, 736 ascorbic acid oxidase, 741 catalase, 741 chromosome breakage in Allium, 736 dehydrogenases, 741 DPNH oxidase, 741 fat utilization, 741 free fatty acid liberation, 741 glucose accumulation, 741 growth and germination, 735 growth regulator in Chlorella, 742 lipase, 741 oxygen uptake, 741 peroxidase, 741 phytin breakdowm, 741 proteinase, 741 sucrose utilization, 741 volatile fatty acids, 741 growth stimulation, 736 inhibition, 736 interaction with lAA on Avena coleoptiles, 737 thiourea on germination and growth, 743 metabolism in Lactuca, 741 mitochondria, 742 phytin, 743 precursor, transcinnamic acid, 735 stimulation, 737, 738 Coumarins as antioxidants, 344 Coumarol, 742 Cresolase, 744 Cross links between polyuronide chains, 382 Croton glabellus, 678 Crown gall tissue culture, 679 Cruciferae, 51 Cucumber = Cucumis Cue wr»iis=cucumber dwarfing effect on choline analogues, 782 GA effect on stem elongation, 507, 508, 509 growth promotion by antioxidant, 346 hypocotyl elongation, 345, 351 interaction with light and auxin, 551, 552, 602 Cucurbita pepo, 551 Cupric ion chelation, 379 Cyanide inhibition of lAA oxidation, 150 Cytoplasmic proteins, 323, 355 Cytoskeleton membrane, 952 2,4-D ^ 2,4-dichlorophenoxyacetic acid Dalapon, 789, 799, 800 Dandelion = Taraxacum officinale Datura stramonium, 679 DCA ■= 2,4-dichloroanisoIe DCP = 2,4-dichlorophenol DCPIP = 2.6-dichlorophenolindophenol Deactivation phenylacctic acids, 435 Decapitation of apricot trees, 387, 389 390, 391 Deformation of Avena coleoptile, 314 315, 316 Dehydroascorbic acid = DHA, 729, 730 Dennstadtia punctilobula, 710, 711 Desoxyribonucleic acid = DNA, 5 Detoxication by formation indoleacetylas- partic acid, 179 DHA = dehydroascorbic acid Diazines as antioxidants, 345 6-Diazo-5-oxo-L-norleucine = DON, 649, 650 Dicarboxylic acid, 146 2,4-Dichloroanisole = DCA, 120, 121, 388 2,4-Dichlorobenzoic acid, 450 2,6-Dichlorobenzoic acid, 450 2,4-Dichlorophenol = DCP, 151, 155, 157 2,6-Dichlorophenol-indophenol ^ DCPIP, 186 2,4-Dichlorophenoxyacetic acid = 2,4-D absorption, 297, 790 action on crystalline enzymes, 297 concentration, 277, 278, 300, 360 cytological disturbances caused by, 245 decreasing heat coagulability in cyto- plasm of Pisum, 355 effects, 329 flowering stimulation in pineapple, 803 inhibition of monohydroascorbic acid reductase, 732 interaction with GA on Helianthus crown gall tissue, 680 Nicotiana crown gall tissue, 682 Persea crown gall tissue, 683 Pisum stem sections, 646 Vicia faba crown gall tissue, 683 lAA, disappearance of, 245 light on Pisum stem sections, 648 temperature, effect on activity of peroxidase, 299 pea root response, 115, 116, 294 retardation of leaf senescence, 329 structural formula, 450 synergism, 691 transport inhibition, 417 symplastic movement, 793, 794 Zebrina, 796 2,6-Dichlorophenoxyacetic acid, 450 2,4-Dichlorophenylacetic acid, 450 2,6-Dichlorophenylacetic acid, 450 Dicoumarol, 742 DIECA = diethyldithiocarbamate Diethyldithiocarbamate = DIECA, 729 Diethyl ether, 387 Diffusion pressure deficit = DPD, 309 1,4-Dihydro-l-naphthoic acid, 450 Dihydro-2-quinolones, 214 Dihydro-4-quinolones, 214 Dihydroxymaleic acid, 146 2,3-Diketogulonic acid, 729 p-Dimethylaminobenzaldehyde = DMBA, 45, 186 p-Dimethylaminocinnamaldehyde = DMCA, 45, 49, 50, 52 2,6-Dimethylbenzoic acid, 450 a,a-Dimethyl-IAA, 211, 212, 213 1,1-Dimethyl-hydrazine, 346 2-4-Dinitro-o-cresol, 172 2-4-Dinitrophenol, 172 2,4-Dinitrophenylhydrazine = DNPH, 186, 191, 194, 195 Dinitrophenylhydrazine, 188 Dioscorea composita, 679 Dioxindoles, 214 Diphenylether as antioxidant, 345 Diphenylurea, 605 DMBA = p-dimethylaminobenzaldehyde DMCA = p-dimethylaminocinnamalde- hyde DNA = desoxyribonucleic acid DNPH = 2,4-dinitrophenyIhydrazine Docosahexaenoic acid, 65 Subject Index 841 l-Docosanol, 61 DON = 6-diazo-5-oxo-L-norleucine Dormancy and chilling, 98 due to growth factor lack, 95 due to growth inhibitors, 95, 98 in embryo, plant, 97 in Fraxinus excelsior, 96 in peach seed, 105 in plant organs, 95 in Xanthium pennsylvanicum, 100 Dormant embryos, 99 Dosage response curve to GA, 494 Double chromatograms, 189, 190, 191 DPD = diffusion pressure deficit DPN, 347 Draba aizoides, 525 Dryopteroides, 710 Dwarf corn, 622 Dwarf plants, 616 Dwarfing effect of choline analogues, 782 effects on wheat and tomato, 781 genes in Zea mays, 489, 491 Ferulic acid of TJ, 699 Flavones of TJ, 702 Flax roots, 219, 223, 225 Florigen, 524, 532, 540 Flowering hormone, 531 Flower number stimulation in tomato, 263 Fluorescence intensity of enzymes, 298 Fluorescence in ultraviolet of a)-(indole-3-) alkanecarboxylic acids, 73 Fluorescent spots, 188 Folic acid, 605 4-FPOA, 220, 226 4-FPOP (±), 220 Fractionation of soluble proteins, 357 Fragaria vesca, 528 Fraxinus excelsior, 95, 96, 97, 98, 99, 101, 102, 103, 104 Fraxinus spaethiana, 96 Fructose-l,6-diphosphoric acid, 649 Fructose-6-phosphoric acid, 649 Fruit drops, 661 Furanocoumarins, 736 Fusaric acid = 5-M-butylpicolinic acid Fusarium heterosporum, 466 EDTA = ethylenediaminetetraacetic acid Eggplant, 782 Ehrlich-positive substance, 179 Ehrlich reacting spots, 188 Electrophilic attack on ring, 436 Electrophoresis, 185, 186, 189 Electrophoretic patterns, 619, 620, 622 Electrophoretic separation peroxidases, 618 EMBA = 4-ethyl-3-mercaptobenzoic acid Embryo chilled, growth stimulation in, 101 dormancy, 97 stimulation by TJ, 688 unchilled, growth stimulant in, 101 Enzyme breis, 630 Enzymes, 297 Epicatechin, 701 Epilobium, 787 Et = ethyl Ether soluble auxins from tobacco, 59 Ethionine, 318, 323 Ethyl = Et, 219 Ethylene-Ci* absorption and translocation, 752 anion exchange chromatogram, 760 fixation, effect of, 756 induction of leaf abscission, 776 Ethylenediaminetetraacetic acid = EDTA as chelating agent, 368, 369 interaction with calcium, 271 preparation of soluble Pisum protein, 356 stabilizer of residual pectin, 319 synergism, 370, 371, 372 Ethylene-oxide, 777 Ethyl indole-3-acetate = lAEE, 490 Ethyl indole-3-acetate = lAEt, 47 4-Ethyl-3-mercaptobenzoic acid = EMBA, 253, 254, 255 4-EtPOA, 220, 224 Euglena, 19 Euonymus japonica, 330, 331, 332, 333, 334 Extension of cress roots, 134 Extract, plum shoot, inhibitor, 127 Extraction of auxins from roots, 168 Fat solubility of auxins, 453 Fatty acids, unsaturated, 62, 65 Fern antheridium formation, 709 Ferrous iron, 365 GA = gibberellic acid; see also Gibber- ellin. GAi, GA2, GAs, GAi ^= gibberellins Gain in fresh weight coleoptile seg- ments treated with 2,4-D, 239 Gain in fresh weight etiolated stem seg- ments treated with 2,4-D, 239 Genes, dwarfing, 489 Geotropism of grass node, 327 Gerbera jamesonii, 576 Gibberella fujikuroi, 101, 468, 469, 473 Gibberellic acid = GA, 132, 172 abscission of leaf (.Gossypium), 559, 560, 561, 562 auxin activity in Pisum, 420 breaking dormancy, 101 chemical structures of As, 423 electrophoretic pattern of peroxidase activity, 622 growth regulation in buds, 661 embryos, 101 leaf growth, 660 Lemna paucipunctata, 606 Pisum, 617, 646 plasticity, 547 Rhus typhina, 634 Samolus, 567, 568, 569 shoot histogenesis, 567 in coconut milk, 683 in potatoes, 101 in resting buds, 101 in seeds, 101 inhibition of fruit drop, 661 root formation, 602 interaction with Amo-1618 in Chrysanthemum, 572, 573 auxin, metabolic basis, 611 choline, substituted, 783 3-CIBA, 605 4-CIBA, 603 CM on shoot growth, 602 coumarin on seed germination, 744 2 4-D, 646, 680, 682, 683 filtrate of Aspergillus on Phaseolus, 605 lAA, 591, 645, 657, 661 lAA and antiauxins, 605 lAA oxidase, 627, 630 842 Subject Index Gibberellic acid {continued^ K, 661 light, 649 sucrose and starvation, 650 lAA oxidase on Pisum seedlings, 606 K, 661 Ka, 605 light intensity, 647, 783 MH, inhibition of shoot growth, 602 NAA, 602, 646 Zea mays, log response in, 493 photosynthesis, 580, 581, 582, 584 preparation of, 466 retardation of leaf senescence, 329 synergism with lAA, 611, 613, 623, 651 tissue cultures, 679 Zinnia elegans, 606 Gibberellin; see also Gibberellic acid, 18 103, 422 acceleration, 531, 532 activation of, 522, 524 activity in cell multiplication, 521 morphogenesis, 543 stem elongation, 521 auxin mediation of activity, 589 biological evaluation, 505, 506, 507, 508, 509 chemical structure and activity, 503 chromatography of Phaseoliis factors, 477 derivatives and related compounds activity of, 513, 514, 515, 516 definition, 490 history, 465 long-day plants, effect on, 524 presence in dwarf Zen, 498 florigen, constituent of, 524 protein synthesis, effect on rate, 524 reversal stem growth radiation inhibi- tion, 551, 552 short-day plants, effect on, 524 spectra (infared), gibberellins and methyl esters of, 486 stimulation, 521, 522, 523 visible radiation, inactivation bv, 548 and Zen mays, 492, 494, 495, 496, 497, 498 Gingho biloba, 679, 692 Glucose-1-phosphate, 649 Glucose-6-phosphate, 649 L-glutamic acid, 605 L-glutamine, 650 Glutathione, 172, 701 Glycine, 605 Glycine niax, 58 Gordon-Weber reactions, 193, 194, 196 Gossypium = cotton, 239, 240, 242 and ethylene-C" absorption, 752 fixation, 767 translocation, 715, 767 Gossyjjiuni hirsiitnm, 560, 561 Grass node, geotropism of, 327 Group A, maize extract fractionation, 671 promotion cell division, 669, 673 Group B, maize extract auxin content, 673 promotion cell elongation, 670 Group C, maize extract, promotion cell division, 673 Guaiacol as substrate, 620, 622 Guillotine, for cutting root sections, 168 Gymnogrammoids, 710 Gynophore of Arachis basipetal auxin transport, 403 rate of transport lAA, 399 H Habituated tissues, 691, 692 H-bonds at surface cytoskeleton mem- brane, 453 HCN = ^-(indole-3-)hepanonitrile Heat coagulability, 355, 360 Helianthus = sunflower, 43 coumarin stimulation, 737, 738 crown gall tissues, 680, 682 rate of auxin transport, 403 Helianthns anmiiis, 551, 678 Heliaiithus test of TJ fractions, 695 Helianthns tuberosiis == topinambour tuber tissue synergism, 668 lAA and coconut milk, 693 lAA and TJ, 691, 693 2-Heptadecanol, 62 Herbicidal properties phenoxyalkyl carboxylic acids, 804 phenyl carboxylic acids, 804 1-Hexadecanol, 61 5-HIAA = 5-hydroxyindole-3-acetic acid Hibiscus syriacus tissue culture, 679 L-histidine, 605 Histograms of root growth, 174 of stem growth, 174 of wheat cylinder test, 80, 83, 86, 88 H/L factor, 431 H2O2 interaction with lAA, pH depend- ency, 209 8-HOQ = 8-hydroxyquinoline Hordeum vulgare, 551 Hormonal mechanism of growth inhibi- tion by light, 543 Hormone, 18 growth regulating, definition, 725 Hormones excretion of, by algae, 25 plant growth, 95 role of, on effects of radiation, 548 sex, 19 Horseradish peroxidase, 150, 152, 206 5-HTRAM = 5-hydroxytryptaniine 5-HTRPH = 5-hydroxytryptophan Humectants, in sprays, 798 Hyascyamus niger var. bierinis, 576 Hydrazines as antioxidants, 345 Aipha-hydrogen atoms, 460 Hydrogen peroxide effect on induction phase, 200 and hydrazine, 350 saturation of peroxidase, 202 Hydrolase action, 84 Hydroquinone, 154 Hydroquinone p-benzoquinone, 160 Hydroxyanthranilic acids, 214 [(5 Hydroxycarvacryl) trimethyl am- mcmium chloride, 1-piperidine car- boxylatel= Amo-1618 action of, 567 growth regulation by, 784 inhibition by, 572 5-Hydroxyindole-3-acetic acid = 5-HIAA, 181 L-hydroxyproline, 605 8-Hydroxyquinoline = 8-HOQ, 364, 368 5-Hydroxytryptamine = 5-HTRAM, 181 5-Hydroxy tryptophan = 5-HTRPH, 181 I I = inhibitor of lAA oxidase lA = indole-3-aldehyde lAA = indole-3-acetic acid lAAL = indole-3-acetaldehyde lAAld. = indole-3-acetaldehyde lAAld-NaHSOs, 45 Subject Index 843 44, 45, 47, 48, 60, 63, 67, 68, 110, 111, 113, 131, 138, 178, 179, 193, 194, 195, 214, 222, 223, 307 lAA-oxidase = indole-3-acetic acid-oxidase lAA (S.COOH), 149 -I action, 438, 439 lAEE = ethyl indole-3-acetic acid lAEt = ethyl indole-3-acetate IAN = indole-3-acetonitrile IB, 221 IBA = T;-(indole-30butyric acid Iheris amara, 521, 523, 525 ICA = indole-3-carboxylic acid ICAPA = e-(indole-3-)caproic acid IGCA = indole-3-glycolic acid IGXA = indole-3-glyoxylic IHA = ^ (indole-3-)heptanoic acid Ilex aqiiifoliiim tissue culture, 679 IN = indole Indole = IN, 172, 188, 194 derivatives catabolic lattice, 181 decomposition pathways, 181, 182 growth regulators, 57 ring, protonation, 212 Indole-3-acetaldehyde = lAAL, 181, 188, 190, 193, 194. 195 Indole-3-acetaldehyde = IAAld., 43, 44, 45, 47, 48, 49, 50, 51, 52, 54, 55, 181 Indole-3-acetamide = ANH=, 38, 47, 73, 76, 82, 93, 605 Indole-3-acetic acid = lAA, 49, 54, 55, 57, 58, 59, 90, 91, 93, 94, 97, 109, 117, 118, 120, 122, 123, 124, 144, 157, 158, 159, 163, 167, 181, 185, 187, 188, 190, 201, 208, 209, 211, 213, 246, 247, 252, 290, 305, 306, activity in Avena curvature, 412 as antioxidant, 345, 346 auxin in Pisutri, 420 cell wall deformation, 310 chelation with cupric ion, 379 destruction, 169, 170, 171, 172, 173, 175, 200, 639 effect of active and passive aspects, 322 cell wall plasticization and pectin synthesis identical reactions, 318 deposition cell wall material, 322 inhibition of Phaseolus by filtrate As- pergillus niger, 605 maintenance cell turpor, 322 plasticization cell wall, 322 pectic metabolism, 317 respiration in Avena coleoptiles, 323 xylem cell regeneration, 405 estimation of, method for, 628 formula, 450 fruit drop, stimulation, 661 and GA physiological action, 602 growth inhibition by, 254, 660, 661 growth regulation of, 62 Allium seed germination, 348 Avena coleoptile sections, 308, 312, 313, 314, 315 Brassica radicle, 348 Cucu77iis hypocotyl, 348 Lemna paucipunctata, 667 Taraxacum flower stalk, 348 inactivation, 143, 650 inhibition of GA action, 657 monohydroascorbic acid reductase, 732 oxidation, 149, 151 interaction with 3-CIBA, 605 fructose- 1,6-diphosphoric acid in Pis- um, 649 fructose-6-phosphoric acid (Pisum), 649 GA, 601, 630, 645, 646 glucose-6-phosphate (Pisum), 649 lAA oxidase, GA and light (Pisum), 630, 631 Ks, 604 K and GA (Avena), 661 light (Pisum), 647, 648, 649, 650 2-phosphoglyceric acid (Pismtti), 649 sucrose (Avena), 310 2,4,6-T, 603, 604 labeled, uptake by barley seedlings, 798 799, 800 model chemical system, 205 oxidation by lAA oxidase from Omphalia, 207 oxidizing enzymes, 145, 200 peroxidase, 150, 205 oxidation inhibition, 149 rates of oxidation, 199, 210 ratio with DCP, 155, 164 resorcinol, 155 response curvature in Avena, 412 response to Pisum root, 112 retardation by riboflavin phosphate, 152, 153 reversal of GA induced elongation sup- pressed by antiauxins, 605 synergistic response with cobalt (Avena), 377 coconut milk (Helianthus tissue), 693 EDTA, 370 GA. 611, 613, 650 TJ in Helianthus tuber tissue, 693 transport, 399, 400, 402, 414, 794 Indole-3-acetic acid-oxidase = lAA-oxidase, 109, 110, 111, 113, 118, 120, 122, 124, 143, 146, 173, 627 Indole-3-acetonitrile = ACN, 73, 76, 87 Indole-3-acetonitrile = IAN, 35, 43, 45, 47, 48, 49, 51, 52, 54, 55, 57, 58, 59, 60. 63, 90, 97 synthetic, 49 zone, 51 Indole-3-acetylaspartic acid, 38, 79, 92, 178 Indole acids, metabolism of, 71 Indole-3-acrylic acid, 94 Indole-3-aldehyde = lA, 181, 187, 190, 193, 195, 220 a)-( Indole-3- ) alkanecarbonamides, 8 1 w-(Indole-3-)alkanecarboxylic acids, 73, 77, 80, 86 w-(Indole-3-)alkanenitriles, 85, 88 Indole-3-alkanoic acids, 212 «'-(Indole-3-)butyramide = BNH2, 73, 76, 82 /3-IndoIe-3-butyric acid, 19 J-- (Indole-3-) butyric acid = IBA, 412, 414, 490 4-(Indole-3)-n-butyric acid, 210 j'-(Indole-3-)butyronitrile = BCN, 73, 76, 87 e-(Indole-3-)caproic acid = ICAPA, 73, 76, 79, 91 e-(Indole-3-)capronitrile = CCN, 73, 76, 87 Indole-3-carbonamide = C0NH2, 73, 76, 81, 82, 84 Indole-3-carboxyl acid = ICA, 73, 76, 79, 89, 90, 94 Indole-3-ethanol = tryptophol, 47 Indole-3-glycolic acid = IGCA, 181, 188, 193 Indole-3-glyoxylic = IGXA, 181, 188, 193 844 Subject hidex f(Indole-3-)heptanoic acid = IHA, 73, 76, 79 |-(Indole-3-)heptanonitrile = HCN, 73, 76, 87 5- (3 Indolemethyl)tetrazole, 450 Indolenines, 214 Indole-3-nitrile = CN, 73, 76, 85, 87 y3-(Indole-3-)propionamide = PNH-', 73, 76, 82 a-(Indole-3-)propionic acid, 227 y3-(Indole-3-)propiomc acid = IPA, 73, 76, 79. 81, 94, 211, 212, 412, 417 ;8-(Indole-3-)propionitrile = PCN, 73, 76, 87 Indole-3-pyruvic acid = IPA, 38, 94, 181 Indoles as antioxidants, 344, 345, 347 -(Indole-3-)valeramide = VNH2, 73, 76, 82, 84 5-(Indole-3-)voleric acid = IVA. 73, 76, 79 5-(Indole-3-)valeronitrile = VCN, 73, 76, 87 Indoline as antioxidant, 345 Induced decomposition of lAA, 187 Induction phase, 200 Infrared spectra of chlorophenoxyacetic acids, 292, 293 Inhibition of growth by calcium and magnesium, 365 chain-stopping agents, 159 interaction irradiation and GA, 550 reversible, 127 toxic, 127 wheat coleoptile sections, 127 Inhibitor changes due to chilling, 95 lAA oxidase activity in Pisum, 636 induction phase, extension of, 151 interaction with germination stimulant, 102 plum shoot extract, 127 transport through coleoptile sections, 135 variation in, 96 woody shoots, 127 Inhibitor of lAA oxidase = I, 637 Inhibitory principle in TJ, 693 Interaction, EDTA and calcium, 371 Interfacial tension ranges of values of surfactants, 815 Intermediate regulators, 224 Intracellular locale auxin action, 355 lodothyronines as antioxidants, 345 lonochromatogram, 185 lonogram, 186, 187 IP = isopropyl IPA^ 0-(indole-3-) propionic acid IPA = indole-3-pyruvic acid 2,IP, 4-Cl, 5-MePO-A, 221, 227 2-IPOA, 220 2,4-IjPOA, 220 3-IPOA, 220 4-IPOA, 220, 224 2-IPOP(±), 220 3-IPOP(±), 220 4-IPOP(±), 220 4-iPPOA, 220 Iris, 106 DL-iso-leucine interaction with lAA and antiauxin, 605 Isoniazid = isonicotinyl hydrazine Isonicotinyl hydrazine ^^ isoniazid as antioxidant, 345, 346, 347, 348 Isopropyl = IP, 219 2-Isopropyl-4-dimethylamino-5-methyl- phenyl-1-piperidine carboxylate meth- ylchloride, 784 IVA = 5-(indole-3-) valeric acid Jerusalem artichoke test of TJ fractions, 695 K K = kinetin K3 = 2-methyl-l, 4-dihydronaphoquinone Kalanchoe, 19 modification amino acid content by short days, 525 photooxidation of chlorophyll, 528 K and GA interaction, 661 Kenten-Mann reaction, 149, 161, 162 K, lAA, and GA interaction, 661 Kinetic phases of enzymatic lAA oxida- tion, 200 Kinetics of the induction phase, 201 Kinetin = K, 18 Kogl's auxin, 43 Ki vitamin, 423 Lactuca = lettuce acceleration of flowering by GA, 507 dwarfing effect choline analogues, 782 seed germination GA activity, 507, 512 GA-?2-alkyl esters activity, 513 growth inhibition and promoters, 106 interaction hydrogen peroxide and hydrazine, 350 phosphorus content, 743 phytin in, 742 stalk elongation by GA, 510, 511 TCA soluble fraction, 743 Lag phase, 200 Latex flow in rubber plants, 810 Leaching of dormant embryos, 99 Leaf age, ethylene-C" fixation influenced by, 756 Lemna minor, 233, 234, 240, 243 Lemna paucipunctata, 606, 607 Length increase in Pisiim, 420 Lens cnlinaris, root tips of, 167 Lens roots, biochemical gradients in, 172 Lentil, root tips of, 167 Lepidium ruderale, 521, 523, 525 Lettuce = Lactuca L-leucine interaction with lAA, 605 Light activation on O2 uptake, 163 destruction lAA by apical Pisum tis- sues, 632, 635 growth inhibition, hormonal mechan- ism of, 543 intensity, interaction with GA and TMA.ECl. 783 interaction with auxin, 552 lAA, 156 lAA and GA, 636, 647, 648 lAA, GA, and lAA oxidase, 630 lAA and DON, 649, 650 lAA and AZS, 649, 650 2,4-D, 648 NAA, 647, 648 sucrose, 647 limitation stem elongation by, 553 Lignification, 347 Lignin synthesis, 347 Liliuin longiflorum, 576 Linolenic acid, 65 Lipides, 419, 424 Localization of activity, 279, 280 Subject Index 845 Long-day grafts on short-day plants, 531 Luckwill's test, 261 Lycapersicon = tomato fruit, growth factors in (TJ), 687 GA effect on photosynthesis, 580, 581, 582. 583 interaction of light and auxin, 552 ovaries, activity of GA on growth, 507, 512 Lygodinm japonicum, 710, 711 L-lysine interaction with lAA, 605 M +M action, 438, 439 Maize = Zea mays Maize, dwarf effect of bean factors I and II, 476 effect of GA on peroxidases, 616 Maize (immature) extract auxin content, 668 factionation group A, 670, 671 group C, 671, 672 Maleic hydrazide = MH, 161, 172 growth regulation Bermuda grass regrowth, 781 shoot histogenesis, 567 shoot inhibition, Xanthinm, 573 subapical meristem, 567 interaction as cofactor in oxidation of lAA, 151 with GA on growth inhibition, 602 oxygen uptake, effect on, 157 transport Ca^^ in apricot, 388 mobility in potato tuber, 794 mobility in Zebrlna, 796 uptake by barley seedling roots, 798, 799, 800 Maleyl hydrazine as antioxidant, 345 Malic acid, 701 Malonyl hydrazine as antioxidant, 345 Malonyltryptophan, 38 Malus = apple pollen-tube extension, 133 rate of auxin transport, 403 Mandarin orange = Citrus unshiu Manganese, 149, 155, 163, 365 oxidation by redox catalyst and peroxi- dase, 162 Manganicitrate, 161 Manganipyrophosphate, 161 Manganiversene, reduction of, 160 Manganous ion as cofactor for lAA-oxi- dase of peas, 125, 145 Manganous-phenolic cofactor, 149 Manteau de Gregoire, 522 'Maryland Mammoth' tobacco, 57, 58, 59, 63, 65, 67 Mathiola incana, 576 Me ^ methyl Melitotus officinalis, 679 Melting points of w-(indole-3-)alkanecar- boxylic acid, 73 p-Menthane hydroperoxide and indole, 350 MeO = methoxy group 2-MeOPOA, 220 3-MeOPOA, 220 4-MeOPOA, 220 2-MePOA, 220, 223 3-MePOA, 220 4-MePOA, 220, 224, 225 l-MePOP(±), 220, 225 VIercapto-purines as antioxidants, 345 vieristem, subapical growth in Samoliis parviflorus, induced by GA, 567, 568 /leristeme d'attente, 524 Meristeme medullaire, 522 Mescaline, 347 Metabolic basis GA-auxin interaction, 611 Metallic ions and auxin action, 363, 365 L-methionine, 605 Methoxy group = MeO, 219 3-Methoxy-4-hydroxycinnamic acid, 172 a-Methoxyphenylacetic acid = MOPA, 417 Methyl = Me, 219 2-Methyl-l, 4-dihydronaphtoquinone = Ka, 604 Methyl ester content of pectin, 383 Methyl esters of indole acids, 71 of a>-(indole-3-)alkanecarboxylic acids, 84 Methyl-hydrazine as antioxidant, 346, 347 Methyl indole-3-acetate = AMe, 73, 76, 85 Methyl indole as antioxidant, 345 Methyl J'-(indole-3-)butyrate = BMe, 73 76, 85 Methyl indole-3-carboxylate = CoMe, 73, 76, 85 Methyl )3(indole-3-)propionate = PMe, 73, 76, 85 Methyl 3(indole-3-) valerate = VMe, 73, 76, 85 Methyl linoleate, 421, 423 Methyl oleate, 421 Methyl pyrrole, 345 MH = maleic hydrazide Microfibrils; see also Cellulose microfi- brils, 381 Mitochondria DHA effect on phosphorylation, 730 effect of coumarin, 742 phenoxy acids, 295, 296 thyroxine as an antioxidant, 349 Mitoses in meristem by GA on Samolus cell generation time, 570, 577 temperature effect on rate, 569, 570, 571 by MH, Amo-1618 and GA, 576 interaction of Amo-1618 and GA in Chrysanthennim, 573 Mn-2 (MnCb), 172 Mn-phenol-peroxidase systems, 161, 162 Model chemical system, 208 Molecule, plant regulating, design of, 6 Monodehydroascorbic acid reductase, 732 Monuron labeled, uptake barley seedling roots, 798, 799, 800 movement in potato tuber, 749 MOPA = a-methoxyphenylacetic acid Mosaic growth in cell wall, 383 Miicor spinosus, 777 N NAA = 1-naphthaleneacetic acid NalAA = sodium salt of indole-3-acetic acid NaOCl, 45 1-Naphthaleneacetic acid = NAA, 490 chelation with cupric ion, 379 curvature response in Avena, 412 formula, structural, 450 growth regulation, 806 interaction with cobalt, 366 GA in Phaseolus lateral bud growth, 602 Pisuyn stem sections, 646 light, 643, 648 PGA, 806 synergism, lack of, with EDTA, 371 transport velocity in Helianthus, 414 846 Subject Index l-Naphthalenic acid, 738 1-Naphthoic acid, 450 1-Naphthoxyacetic acid = 1-NOA, 220, 222, 227 2-Naphthoxyacetic acid = 2-NOA, 220, 222, 227, 290 curvature response curves, 412 stimulation parthenocarpy, 688 transport inhibitions, 417 1-Naphthylhydrazine as antioxidant, 346 1-Naphthylmethylphosphonous acid, 450 2-Naphthylmethylselenoacetic acid = NMSeA, 222 2-Naphthylniethylselenoacetic acid = 1- CioHT.CH2.Se-CH-(CH3).COOH = 2- NMSeA, 222, 223 a-(l-Naphthylmethylthio)propionic acid =1-NMSP, 220, 222 N-arylphthalamic acids, 259, 260 Natural factor as cofactor in oxidation of lAA, 151 Ng.E.K., 373, 374 N-HIAA = N-hydroxyindole-3-acetic acid N-hydroxyindole-3-acetic acid = N-HIAA, 181, 185, 188, 195 Ni = nitro group Nicotiana = tobacco, 57, 59, 60, 61 crown gall tissue culture, 682, 690 short-day plants, 532, 535 tissue culture, 679, 692 Nicotine-amide, 605 2-NiPOA, 220 3-NiPOA, 220, 227 4-NiPOA, 220, 224, 226 Nitriles of indole acids, metabolism of, 71 Nitro group = Ni, 219 NMSeA ^ 2-naphthylmethylselenoacetic acid 2-NMSeA = antiauxin, 220 1-NMSP ^ a-( l-naphthylmethylthio)propi- onic acid -NO:;, 219 1-NOA = 1-naphthoxy acetic acid 2-NOA = 2-naphthoxyacetic acid Nucleophilic attack on ring, 436 Oat coleoptile cylinders, preparation of, 219 -OCH:<, 219 Ochromonas viathamensis, 26 chromatogram Zone R, 34 chromatograms of extract, 29, 32, 36, 37 1-Octadecanol, 61 Omphalia flavida, 200 Onion = Allium cepa Onoclea sensibilis, 709, 710, 711, 713. 714 Onocleoids, 710 OP = osmotic pressure Opiintia monacaiitlta crown gall tissue culture, 690 special tissue culture, 679 Origin of bean stem internode, 277, 279 DL-ornithine interaction with lAA and antiauxin, 605 Oryza = rice seedlings as assay, 503 GA efPect on photosynthesis, 580, 582, 583 starch content, 586 sugar content, 585 Oscillatoria spp., 26, 27, 28 Osmotic coefficient sodium tetradecylsul- fate, 815 Osmotic pressure = OP, 308 Osmunda cinnamonea, 710 Osmunda claytoniana, 710 Oxidant-antioxidant interactions, 347 Oxidants as growth regulators, 841 resulting in lignification, 343 resulting in melanization, 343 toxicity, 343, 349 Oxidase of lAA, 144, 205 Oxidation rates of lAA, 199, 201, 210 of IBA, 210 Oxidation scheme, enzymatic of lAA, 145 Oxidized radicals = (RO.), 161 Oxindole, as antioxidant, 348 Oxindole-3-acetic acid, 213, 214 Oxindoles, 213 Oxygen action of on lAA in aqueous solution, 208 toxicity, 341, 349 uptake, 426, 742 Pantothenic acid, 605 Paper chromatography, 57, 63 Parthenocarpy stimulated by N-aryphthalamic acids, 260 tomato, 260, 265 Parthenocissiis tricuspidatus, 690, 691 Partition between ethyl acetate and water of TJ, 694 Partition coefficient of active auxins, 453 Pathways of decomposition, indole deriva- tives, 181 PCA = p-chlorophenoxyacetic acid PCIB = a-(p-chlorophenoxy)isobutyric acid PCN ^= /3-(indole-3-)propionitrile Pea curvature test of growth stimulants, 74 Pea root breis inactivation by lAA, 114 response to 2,4-D, inhibitory concentrations, 115 DCA, 120, 121 lAA, 111, 112 TIBA, 118, 119 Pea roots, adaptation to auxins, 109 Peach seed, dormancy of, 105 Peanut = Arachis Pectic carboxyl groups in cell wall, 316, 317 Pectic metabolism, 318, 322 Pectic synthesis, 318, 323 Pectin esterase, 324 Pectins of cell wall Avena coleoptile, 318, 319, 320 Pepper, 782 Perilla nankinensis and GA effect on pigment production, 525 interaction with visible radiation, 548 molar ratio chlorophyll-hematin in leaves, 526 presence in short-day plants, 532, 535, 538, 539 stimulation cell division, 521, 522 Peroxidases electrophoresis, separation, 618 electrophoretic pattern, comparison sub- strates, 620 GA effect reduction activity, 617, 618 lAA, 144, 205 Persea = avocado, 683 Persea americana, 679, 683 pH, dependence of reaction of H2O2 with lAA, 209 Phalaris canariensis, 13 Subject Index 847 Phaseolns vulgaris = bean, 274 growth regulation by coumarin, 737 GA, epicotyl elongation, 506, 509 interaction with visible radiation, 551 tissue culture, 611 peroxidases in dwarf, 616 transport in auxin, 401, 402 9,10-Phenanthroline, 369 Phenol as cofactor in oxidation of lAA, 151 Phenolase activity, 744 Phenolic cofactor = RoH, 149. 155, 161 Phenolic radical (semiquinol), 149 Phenols as antioxidants, 344 Phenothiazine as antioxidant, 344 Phenoxyacetic acid = POA, 220, 242 2,4-D, effect on Gossypium, 241 lAA, comparison of activity, 433 mitochondrial enzymes, effect on, 295, 296 para-substituents, effect of, 226 stimulation latex flow, 810 substituted absorption spectra, 295 biological activity, 295 disubstituted derivatives, activity of, 438 and molar activity, 291 and ultraviolet absorption, 294 uptake by Avena and Gossijpiinn, 242 water solubility, 295 a-(Phenoxy)-n-butyric acid = POB, 219 a-(Phenoxy)-n-caproic acid = POC, 219 Phenoxy compounds, 219 a-(Phenoxy)isobutyric acid ^ POib, 219 a-( Phenoxy )isovaleric acid = POiV, 219 a- (Phenoxy) propionic acid = POP, 219 a-(Phenoxy)-7i-valeric acid = POV, 219 Phenoxyalkylcarboxylic acids, 804 Phenylacetaldehyde, 146 Phenylacetic acids deactivation by methyl and methoxy groups, 435 model, 452 order of activity of substituents, 435 L-phenylalanine interaction with lAA, 605 Phenylcarboxylic acids, 804 Phenylethylamines as antioxidants, 345 Phenyl indole as antioxidant, 345 2-Phenylindole-3-acetic acid, 211 Phenyl pyrrole, 345 Phenylthioglycolic acids, 435 3-Phosphoglyceric acid and lAA interac- tion, 649 Phosphorus content in Lactuca seed dur- ing germination, 743 Phosphoryl oxidative uncoupler of cou- marol and dicoumarol, 742 Photooxidation of chlorophyll, 527, 528 Phototropism inhibitors, 455 Phycomyces blakesleeanus, 18 Phytin in Lactuca seeds, 742 Phytol, 61 Phytophthora, 777 Pi-electron distribution for benzoic acid derivatives, 249 Pigment content of plants, 525 Pineapple, flowering induction, 803, 804, 805, 806 Pisum = pea auxin content, 46, 48, 49, 50 growth regulation, 43, 44, 50, 51, 52, 243, 679 heat coagulability of cytoplasm, 355 interaction on growth AA, effect on internode sections, 726. 727 AA and DHA, 731 GA and lAA, 590, 592, 593, 645 on apical tissues, 632, 633 red radiation, effect, 549 reverse inhibition stem growth, 551 lAA oxidase, 632, 633 light, 632, 633 glyceride and auxin, 420, 421 method of use, 628 stem sections etiolated, 612, 613 GAi and lAA, 602 green. 613, 614, 615, 616 growth stimulants, 74 NAA, 804, 805 synergism of auxin and GA, 611, 616 Plant age, ethylene-C'* fixation influenced by, 757 Plant extracts, indole-3-acetaldehyde, 43 Plant growth hormones, 95 regulants, 3,4 regulation, expanding concepts, 13 Plant regulating molecule, design of, 6 Plant regulation, need for, 8 Plasma viscosity, effect of AA on, 727 Plastic mechanism of cell growth, 382 Plasticity of cell wall definition, 546 GA required for maintenance, 551 of Pisum, 546 visible radiation, effect of, 545 growing plant, 3 Plant tissue culture, growth regulation by GA, 678, 679 Plum extract, 131, 132, 133, 134 inhibitor, 134, 135 shoots, extract preparation, 128 PMe = methyl /3(indole-3-) propionate PNH2 = ;8-(indole-3-)propionamide ps^O , uptake by barley seedling roots, 798, 799, 800 POA = phenoxyacetic acid POB = a- (phenoxy )-n-butyric acid POB (±), 220 POC = a- (phenoxy )-ri-caproic acid POC (±), 220 pOib = a- ( phenoxy )isobutyric acid POiB, 220 pOiV = a-(phenoxy)isovaleric acid Polar group of auxin, 453, 454, 459 transport of auxins, 411 Polarity in development and regeneration, 397 Polarity of uptake, 279, 281, 282 Polypodium aureum, 710 POP = a- (phenoxy )propionic acid POP (±), 220 Portulaca oleracea, 678 Potassium as growth promoter, 365 ion concentration, 313 Potato = Solanum tuberosum POV = a- (phenoxy )-?i-valeric acid POV (±), 220 Power of movement in plants (Charles Darwin), 13, 14 L-proline, 605 ( 2,3-n-Propylene )trimethylammonium bro- mide = TMA.PBr preparation, 779 Protonating hydrogen peroxide, 213 Protonation of indole, 212, 213 Prunus serrulata, 329 Pteridine as antioxidant, 345 848 Subject Index Pteridium antheridial factor, 709, 710 Pteridium aquilinum extract of prothalli, 709 gametophytes, types of, 716 loss of sensitivity to Pteridium factor, 713 physiology of reproduction, 719 Pteridium factor in archegonium-bearing prothalli, 712, 713 Pteroids, 710 Pulularia pullulans, 134 Purine as antioxidant, 345 bases, 18 Pyridoxine, 605 Pyrimidine bases, 18 Pyrogallol as substrate, 620, 621 Pyrrole as antioxidant, 345, 348 Pyrrolidine as antioxidant, 345 Quercetrin, 701 Quinic acid, 699, 700 2-Quinolones, 214 4-Quinolones, 214 p-Quinone growth regulation by, 161 inhibition, oxidation of lAA, 154 Radiation, visible effect on internode length, 544 growth inhibition reversal by GA, 548 morphological effects, 543 Radical attack on ring, 436 Radioactive products of C"-labeled 2,4-D in bean stems, 283, 284 Radioactivity distribution in Ci*-2,4-D treated Coleus, 764 Phaseolus stems, 278, 279 Pisum cell fraction, 358 Radioautograms of C'', 752 Radiochromatograms of radioactive fractions of Coleus, 763 water-soluble fractions of Gossypiiim, 761 Radiochromatographic analyses of C'', 175 Raphanus, 552 Rb = riboflavin, 161 Rb-phosphate, 154 (Rb^Rb.2H) = redox system, 161 Reactive species, 213 Receptor in auxin response, 222 Redox catalyst = (ROH), 162 Redox system = (Rb^Rb.2H) Regeneration, acceleration by reductants, 347 Regulator-antioxidant hypothesis, 341 Resorcinol cofactor in oxidation of lAA, 151 growth regulation by, 161, 162 oxygen uptake, effect on, 157 Respiration coleoptile section, 135 germinating Lactuca seeds, 742 role in auxin induced growth, 323 Response curves in Avena curvature test, 412 Retardation, mechanism of chain-transferring agents, 160 lAA oxidation, 155 in dark and light, 164 Retarders of oxidation of lAA, 154 Retarding effect, 160 Reversible inhibition, 127 R, values of Coleus metabolites, 762 of aj-(indole-3-)alkanecarboxylic acids, 73 of phosphorylated Gossypium metabo- Ute, 762 Rhizobium leguminosarum, 726 Rhus typhina, 634 Riboflavin = Rb growth regulation by, 161, 163 non-reversal of growth inhibition by antiauxins, 605 reduced, 164 retarder of inhibition of lAA, 156, 160, 163 Ribonucleic acid = RNA, 393 Rice ^ Oryza RNA = ribonucleic acid RNase in vitro, 393 (RO. ) == oxidized radicals ROH = phenolic cofactor (ROH) = redox catalyst Root growth GA and lAA interaction, 661 on excised tomato ovaries, 688 stimulation by tomato juice, 688, 689 tomato juice and sucrose interaction, 690 Rosa sp., 678 Rosette plants, stem elongation, 567, 568 Rubus, 96 Rubus fruticosus, 691 Rudbeckia (LD plant), 532, 533, 535, 536, 537 Rumex acetosa, 678 Rumex virus tumor tissue culture, 676 Salicylyn hydrazine as antioxidant, 345 Salkowski reactions, 193, 194, 196 lAA metabolite, 178 reactive spots, 188 Salvia splendens, 522, 525, 526, 527 Samolus parviflorus, stem elongation, 567, 568 S-carboxymethyl N, N-dimethylaminodithi- ocarbamate Schema, 637 Scopeletin growth regulation, 737 retardation in oxidation of I A A, 154, 160 Scorzonera hispanica, 691, 692, 694 Seed growth-substance and chilling, 95 Selective action of 2,4-D, 233 Semiquinol (phenolic radical), 149 Serotinin as antioxidant, 346 Serotonin, 94 Short-day grafts on long-day plants, 531 Side-chains of auxin, 453 Simazin, 798, 799, 800 Sinapis alba photoperiodic induction, 526 visible radiation and GA, 551 Sinocalamus oldhami, 64 Skatole = S = Sk, 348 Skatole peroxy-radical, 149 Skoog test group C, 670 Slit pea stem curvature, 132 Sodium salt of indole-3-acetic acid = NalAA, 128, 129, 130, 132, 135. 136, 137, 138, 139 Soil conversion of Pisum extracts by, 48 treatment of auxins, 49 Subject Index 819 Solanum tuberosum = Potato, 95 tissue culture, 678 tuber disk weight increase, 366 cells permeability to water, 135 Sorbus aucuparia, 96 Specificity of model system, 210 Spectrum, 211 Spontaneous decomposition of lAA, 187 S ii^) ^ superdelocalizability, 249, 250, 251, 252, 258 Starvation of Pisum stem sections, 650 Statice sinuata, 521, 522, 523 Stem elongation in rosette plants, 567, 568 Steric factor, 431 Stilbenes as antioxidants, 344 Stimulants, growth, by acids, 65 anaerobic preconditioning, 351 S-carboxymethyl N,N — dimethylamino- dithiocarbamate, 69 Stretching of wall due to cell turgor, 381 Structural formulae of auxins, 450 Sublimation test of volatile indole deri- vatives, 187 Substituents in 4-substituted phenoxy- acetic acid, 433 Sucrose effect of lAA on progress curves, 310 effect on auxin activity of EDTA plus lAA, 370 interaction with lAA, starvation, and GA, 650 light, 647 Sugars of TJ, 702 Sunflower = Helianthus Superdelocalizability = S^ic^'^ Surface tension ranges of values of sur- factants, 815 Surfactant growth stimulation, 428 roles in sprays, 797 Surfactants, 813, 815 Survey fractional extraction scheme of radioactivity in Gossypiujn leaves ex- posed to C"-ethylene, 765 Symplastic movement of 2,4-D, 793 Synergism of cobalt plus lAA (Avena coleoptiles), 377 GA and sucrose, 591 lAA-GA, 651 Synergistic action, 743, 744 1, 2, 3, 4-T = 1, 2, 3, 4-tetrahydro-l- naphthoic acid, 450 2,4, 5-T = 2,4,5-trichlorphenoxyacetic acid 2,4,6-T = 2,4,6-trichlorophenoxyacetic acid Taraxacum = dandelion, 345, 346 Taxus sp. pollen, tissue culture, 678 tB ^ tert-butyl 4-tbPOA, 220, 224 TCA = trichloracetic acid Temperature effect on auxin transport rate, 403 interaction and TMA-ECL, 784 Termination reactions, 202 tert-butyl = tB, 219 Tetradecyl sulfate. 815 1,2,3,4-Tetrahydro-l-naphthoic acid = 1,2, 3,4-T, 450 Thiamin, 18, 605 Thiourea, 102 as germination stimulant, 100 effects, 744 interaction with coumarin, 743 DL-threonine, 605 Thymohydroquinone, 785 Thymoquinone, 785 Thyroxine, as antioxidant, 344, 345, 348 for mitochondria, 349 in growth production, 346 TIBA = 2,3,5-triiodobenzoic acid Tilia, 103 Time course effect of cobalt, 367 Tissue cultures growth stimulation by TJ crown gall tissues, 690 habituated tissues, 691 normal tissues, 692 special tissues, 692 and growth substances, 675 technique, 675, 676 response to different levels of GA, 677 TJ = tomato juice TJF = tomato juice factor TMAEBr= (2-bromoethyl) trimethylam- monium bromide = (CHs) N+CH2CH2- Br TMA. ECl = (2-chloroethyl) trimethyl- ammonium chloride TMA.PBr= (2,3-n-propylene) trimethyl- ammonium bromide Tobacco = Nicotiana Tomato = Lycopersicon Tomato flower formation, 260 fruit, 687 parthenocarpy, 265 Tomato juice = TJ active principle not an auxin, 692 growth regulation, 687 by components, 701 fractions, 694 tissue cultures, 690, 695, 702 inhibition, seed germination, 688, 689 synergism of TJ and lAA, 692, 693 Tomato juice factor = TJF, 696, 697, 698 Topinambour = Helianthus tuberosus Toxic inhibition, 127 TRAM = tryptamine Trans-cinnamic acid, 735 Translocation of C^^-ethylene in Coleus, 754 of C"-ethylene in Gossypium, 754 of urea in Gossypium, 795 Transport inhibitions, 417 NalAA, 135 velocity in Helianthus, 414 Trichloracetic acid = TCA, 743 2,3,6-Trichlorophenoxyacetic acid 2,3,6-T, 452 2,4,5-Trichlorophenoxyacetic acid = 2,4,5- T, 20, 252, 329 2,4,6,-Trichlorophenoxyacetic acid = 2,4,6- T, 318, 603, 604 2,3,5-Triiodobenzoic acid = TIBA, 110, 122, 124, 227, 243, 244 effect of varying concentration, 392 effect on activity of RNase in vitro, 393 basipetal transport of Ca^" in apricot, 389, 391 translocation of calcium, 387 growth regulation of Zinnia elegans, 606 pea root response to, 118, 119 transport inhibitions, 417 Triphenylmethane dyes as antioxidants, 344 Triticum (Wheat), 237 chromatograms, 81 coleoptile section, 123 850 Subject Index Triticum (continued) cylinder test growth stimulants, 74 histogram, 80, 83, 86, 88 leaf base test, 131, 132 leaf extracts, 150 roots, 219, 223, 225 seedlings, 780, 781 TRPH = tryptophan, 45, 93, 146, 181 Tryptamine = TRAM, 94, 181 Tryptophan = TRPH Tryptophan-like metabolite, 768 Tryptophol = indole-3-ethanol Tube structure, in cell walls, 319 Tumor inhibiting antibiotic, 649, 650 Turgor of cell, 381 Turnip = Brassica rapa Tween stimulation of auxin action, 421 Two-point attachment hypthesis, 440 of plant growth regulators to proteins, 431 Tyramine as antioxidant, 347 Tyrosine, 605 u Ultraviolet decomposition products of in- dole compounds, 182, 185, 187 Ultraviolet spectra; see also Spectra absorption spots, 188 chlorophenoxyacetic acids, 292, 294 lAA, oxidation of, 206, 207 indol-3-alkanoic acids, 212 Unsaturated hydrocarbon derivatives as antioxidants, 344 Uptake of 2,4-D, 234, 235, 236, 239 TIBA, 243 urea, 798, 799, 800 Urea translocation, 794, 795 Uredospores, 737 Vitamin B12 and non-reversal growth inhibition by antiauxins, 605 effect on Euglena, 19 Vitamin E, 605 Vitamin K, 605 Vitamins, 18 VMe = methyl 5- (indole-3-) valerate VNH2 ^ 5-(indole-3-) valeramide Volatile substances, 188 w Wall pressure = WP, 308 Water soluble growth substances, 60, 64 radioactive fraction, 758 Weigela, 507 Weight increase in potato tuber disks, 366 White's medium, 275 Woodsia obtusa, 711 Woodsioids, 710 Woody shoot inhibitor, 127 WP = wall pressure Xanthium. dormancy in, 100 leaf senescence, 329 kinetin, efFect of, 389 MH, GA, and Amo-1618, effect on in- hibition mitoses in apical meri- stem, 574 Xylem cell regeneration through auxin, 405 Yeast extract and non-reversal growth in- hibition by antiauxins, 605 DL-valine interaction with lAA, 605 Values of S/'^', 250 Vascular stem tissues and auxin trans- port, 404 VCN == §-(indole-3-)valeronitrile Verticillum alboatruni, 134 Vicia faba, 43 GA, effect on tissue culture, 681 GA and 2,4-D, interaction, 683 radiation sensitivity of stem, 547 Vigna sinensis, 678 Vinca rosea, 678 Virus tumor tissue culture, 678 Visible radiation; see also Light and growth hormones, 548 nature of, 544, 545 Zea mays = maize, 489 dwarf, 616 dwarfing genes, 489 dwarfism, 490 GA in, 489 homogenate, 620 mesocotyl, geotropism of, 327 Zea saccharata, 678 Zebrina pendiila, 796 Zinnia elegans, 606 Zone R, chromatogram of, 28 Zone X, chromatography of, 27 Zone Z, chromatograms of, 29 chromatography of, 27