BOSE INSTITUTE TRANSACTIONS VOL. V THE PHYSIOLOGY OF THE ASCENT OF SAP WORKS BY THE SAME AUTHOR 1902 1906 RESPONSE IN THE LIVING AND NON-LIVING. With 117 Illustrations, 8vo. 10s. 6d. net PLANT RESPONSE: AS A MEANS OF PHYSIOLOGICAL INVESTIGATION. With 278 Illustrations, Svo. 21s. net COMPARATIVE E L E C T RO - PHYSI- OLOGY. A PHYSICO-PHYSIOLOGICAL STUDY. With 406 lUustrations, Svo. 16s.net 1907 RESEARCHES ON IRRITABILITY OF PLANTS. With 190 Illustrations, Svo. 7s. 6d. net 1913 LIFE MOVEMENTS IN PLANTS, Vol. I. With 92 Illustrations, Svo. 15s. net 1918 LIFE MOVEMENTS IN PLANTS, Vol. II. With 128 Illustrations, Svo. 20s. net 1919 LIFE MOVEMENTS IN PLANTS, Vols. III. and IV. With 124 Illustrations, Svo. By prof. PATRICK GEDDES AN INDIAN PIONEER OF SCIENCE: LIFE AND WORK OF SIR J. C. BOSE. With Illustrations, Svo. 16s. net 1920 LONGMANS, GREEN AND CO. ON NEW YORK TORONTO BOI CALCUTTA AND MADRAS 39f THE HYSIOLOGY OF THE ASCENT OF SAP BY SIR JAGADIS CHUNDER BOSK M.A., D.SC, LL.D., F.R.S., C.S.I., CLE. DIRECTOR, BOSE RESEARCH INSTITUTE COSSIMBAZAR ENDOWMENT PUBLICATION WITH NINETY -FIVE ILLUSTRATIONS LONGMANS, GREEN AND CO. 39 TATERNOSTER ROW, LONDON, E.G. 4 NEW YORK. TORONTO BOMBAY. CALCUTTA AND MADRAS 1923 7 ^ J 3^J *.'> A/ade in Great Britain PREFACE The ascent of sap has been the most ekisive problem in Plant-physiology. The obscurity which has surrounded the subject has been in a great measure due to the lack of adequate means of detection and accurate measurement of the rate of ascent, of transpiration, of exudation, and their induced variations. Various types of automatic recorders of great sensitiveness and precision have been devised and are described in the present work, which have been of signal service in the investigations of which an account is here given. The result of these researches is to prove the existence of active pulsating cells throughout the length of the plant, in and from the absorbing root to the transpiring leaf. It is the pumping action of these cells that gives rise to the physiological conduction of sap, even in the absence of root-pressure and transpiration ; it also injects liquid into the xylem, setting up an intra-vascular pressure with the consequent mechanical transport of fluid. The situation of the active cells has been localised by means of the Electric Probe ; the cellular pulsations con- cerned in the ascent of sap have been recorded by an automatic method. The invisible changes in the interior of the plant have thus been revealed, and the effect of the changes of the environment determined from the responsive variations in the pulse-record. Other investigations are described which show that there are two distinct modes of inter-communication and inter-action between distant organs in plants : (i) the transfer of matter, and (2) the transmission of motion. The first VI PREFACE of these is brought about by the movement of sap, and the second by the excitatory nervous impulse. They give rise to two reflexes at a distance, the hydraulic reflex being antagonistic to the nervous reflex. There are, no doubt, many such reflexes corresponding to the various modes of stimulation. The complexity of the life-movements is, in fact, the expression of the combined effects of concordant and antagonistic reflexes. The ascertained facts justify the important generalisation of the unity of the physiological mechanism in plants and animals. Further investigation of the simpler life of plants may therefore be expected to lead to the solution of many intricate problems in animal life. Jt affords me much gratification to associate this work with the ' Cossimbazar Endowment,' founded for my Insti- tute by the enlightened interest taken by the Maharajah Sir Manindra Chandra Nandy, K.C.S.I., of Cossimbazar, in the advancement of research. I also take this opportunity of acknowledging the very efficient help which has been rendered to me by my research- assistants and scholars. J. C. BOSE. BosE Institute, Calcutta, January, 1922. PAGE CONTENTS CHAPTER I THE PROBLEM OF THE ASCENT OF SAP Physical and physiological theories — Inconclusive character of Stras- burger's poisoning and scalding cxperiments-^Root-pressure CHAPTER II AUTONOMOUS PULSATION Khythmic vegetable tissue — Autonomous pulsation in Desmodii.m gyraiis — ]\Iultiple response under strong stimulus — Pulsations in growth — Characteristics of pulsatory activity — ^Effect of varia- tion of internal hydrostatic pressure — Effect of maximal stimulus ■ — Effect of sub-minimal stimulus — Modification of response in sub-tonic specimens — Effect of variation of temperature on rhythmic activity- — Arrest of pulsation at the critical thermo- metric minimum — Effect of anaesthetics — Effect of dose — Action of poison — Tests for pulsatory activity — Summary CHAPTER III DETECTION AND RECORD OF THE ASCENT OF SAP Detection and record of ascent of sap — Mechanical method of Erectile Response — The Automatic Recorder for erectile response — Erectile response of Mimosa, Chrysanthemum and Itnpafiens — The Osmotic Theory — Theory of suction and root-pressure — Ascent of sap in the absence of root-pressure and transpiration — Depressed rate of ascent under increasing drought^Ascent of sap in cut stems previously exposed to air — Function of the xylem — Summary . . . , . . . .24 Vlll CONTENTS CHAPTER IV DETERMINATION OF VELOCITY OF ASCENT BY MECHANICAL RESPONSE PACE Difference of velocity of ascent in cut and rooted specimens — In- fluence of the previous history of the plant — The Duplex Method — The effect of drought — The effect of physiological anisotropy induced by stimulus — Determination of velocity in the reverse direction — Summary ...... 40 CHAPTER V EFFECT OF PHYSIOLOGICAL VARIATIONS ON ASCENT OF SAP The Potograph — Effect of physiological agents in modification of ascent — Effect of diminished internal pressure — Effect of stimulus — Modifying influence of tonic condition — Effect of variation of temperature on ascent and on growth — The critical thermo- metric minimum — Drooping of leaves during frost^Phenomenon of accommodation — Effect of anaesthetics — Effect of poison — Method of exudation — Strasburger's experiments — Summary . 51 CHAPTER VI TRANSPIRATION Physical evaporation and physiological excretion^Isolation of absorbing, conducting, and excreting organs — The Bubbling Method for measurement of transpiration— Comparison of transpiring activity of different species of plants— Ratio of transpiration from upper and lower surfaces of leaves — Deter- mination of transpiration from a single stoma — Transpiration in the absence of evaporation — The role of evaporation — Physio- logical continuity in stem and leaf — Crucial tests of physiological activity underlying transpiration — Effect of variation of tempera- ture— Effects of sub minimal and maximal stimulus — Summary 81 CHAPTER Vn VARIATION OF TRANSPIRATION UNDER PHYSIOLOGICAL CHANGE The Micro-Transpirograph — Effect of diminution of turgor on trans- piration— Effect of stimulus — Opposite effects of stimulation of upper and lower surfaces of leaf — Effect of high frequency Tesla CONTENTS IX PAGE current — Effect of electric waves — Effect of statical electric induction — Effect of thermal rays— Effect of light — Effect of red and of blue light — Effect of carbonic acid gas — Effect of ether — ■ Effect of chloroform — Summary ...... 98 CHAPTER VIII THE DIURNAL VARIATION OF TRANSPIRATION Diurnal variation of transpiration in plants with roots — Diurnal variation after removal of the root — The Radiograph — Diurnal variation of temperature and of light — Balancing evaporation against transpiration — The Differential Balance — The optimum temperature for transpiration — Summary . . 117 CHAPTER IX EXUDATION BY THE ROOT-STOCK The Recorder of Exudation — The Tilter and the Electromagnetic Writer — Composition of exuded sap — Continuous record of exudation — Effect of drought — Effect of mechanical and electrical stimulus — Effect of poison — Effect of anaesthetics — Continuity of action in root and in shoot — Activity of terminal laj'er at the cut end — Expulsion of sap by living cells — Summary . . . . . . . . . .131 CHAPTER X THE RELATION BETWEEN ROOT-PRESSURE AND EXUDATION General considerations — Diurnal periodicity of root-pressure — The recording apparatus — Relation between temperature and pressure — Diurnal variation of pressure in deciduous trees — Diurnal variation of exudation — The effect of hght — Summary . 146 CHAPTER XI DIURNAL VARIATION OF PRESSURE AND EXUDATION IN PLANTS WITH LEAVES Complexity arising from fluctuating factors of absorption and ex- cretion— Hydraulic and electric model — Diurnal variation of pressure in root stock — Effect of light — Explanation of irregular variation of pressure in trees — Diurnal variation of exudation in root stock — Positive and negative exudation — Exudation in Pithecolobiiitn—SummdiTy ....... 160 X CONTENTS CHAPTER XII THE ' WEEPING ' MANGO-TREE PACE Exudation from the Mango-tree — Chemical analysis of the exuded sap — Period of maximum pressure — Absence of exudation from hole drilled into the tree — The existence of a cavity due to dis- integration of alburnum — The lateral injection of sap by active cortex — Enhanced secretion due to local rise of temperature — Summary . . . . . . . . . .170 CHAPTER XIII EXUDATION IN PALMS The Indian Date Palm — The Palmyra Palm {Borassiis flabellifer) — The maximum quantity of exudation in a season — The total yield of sugar — Diurnal variation of exudation in Phcetiix sylvesiris — Explanation of greater exudation at night — Diurnal variation of exudation in Palmyra Palm — The action of sunlight — Absence of root-pressure — Stimulus for initiation of exudation — The magnetic analogue of polar action of cells in absorption and excretion — Summary . . . . . . . .178 CHAPTER XIV DETERMINATION OF VELOCITY OF THE ASCENT BY THE ELECTRIC METHOD Electric variation with change of turgor — Electrometric determina- tion of velocity — Galvanometric determination — Shock- effect of the hydrostatic blow — Simultaneous determination of velocity of ascent by mechanical and electrical methods — Determination of velocity by the Di-phasic method — Summary . . .194 CHAPTER XV DISCOVERY AND RECORD OF THE PULSATION OF AN INDIVIDUAL CELL The Electric Probe for detection of pulsation in the interior of the plant — Turgor and electric variation during a single pulsation — Electric pulsation of Desmodiitni — Periodic groupings in pulsations — Record of pulsation of a single cell — Cellular pulsa- tion in herbaceous plants — Pulsating cells in trees — Pulsatory activity modified under variation of temperature — Record of pulsation by Einthoven galvanometer — The period of a single pulsation — Summary ........ 206 CONTENTS XI CHAPTER XVI LOCALISATION OF PULSATING CELLS AND DETERMINATION OF WAVE-LENGTH PAGE Localisation of active layer of pulsating cells in Iinpatiens — Localisa- tion in Brassica — Amplitude of pulsation at different depths — Theory of the electric determination of wave-length — Successive electric maxima and minima — Determination of wave-length in Chrysanthemum and in Musa — Change of wave-length under physiological variation — Upsetting of the phase- difference by passage of electric current — Summary . . . . .216 CHAPTER XVII THE CELLULAR MECHANISM IN THE TRANSPORT OF SAP Initiation of pulsation — Effect of stimulus — Effect of differential hydrostatic pressure — Effect of constant electric current — Effect of variation of temperature on pulsation — Effect of anaesthetics — Effect of diminished internal pressure — Summary . . . 232 CHAPTER XVIII THE HYDRAULIC AND THE NERVOUS REFLEX Interaction between distant organs — Sachs's experiment of the growth of a branch inside a dark box — Hydraulic convection and nervous conduction — Importance of stimulus in maintenance of life-activit}^ — The Leaf a catchment-basin for reception of stimulus — Stimulation of internal cortex by transmitted ex- citation of sunlight — Antagonistic action of the hydraulic and nerve reflexes — Dual impulses under stimulus — Opposite effects of direct and indirect stimulus — Explanation of opposite geotropic responses in shoot and root — The co-ordination of nervous reflexes— -Dia-heliotropic attitude of leaves — Summ.ary 244 CHAPTER XIX GENERAL SURVEY ....... 257 INDEX . . . . . . . 273 25 ILLUSTRATIONS FIG. PAGE L. Leaves of Biophytum and of Desmodimn gyrans . . . lo 2. Record of Pulsation of Growth ..... .10 3 ,, Arrest of Pulsation of Desmodiitm under Diminished Internal Pressure . . . . . . 13 4. ,, Effect of Electric Stimulus on Pulsation of De5wzorfn/M7 14 5. ,, Effect of Stimulus of Light in Renewal and Enhance- ment of Pulsation of Desworfnrw . . . 16 6. ,, Effect of Ether and Chloroform on Pulsation of Des- modii'.in ....... 20 7. ,, Effect of Anaesthetics on Growth . . . . 20 8. ,, Effect of Poison on Pulsation of Z)e57MO<^«/7M . . 22 9. Erection of Drooping Shoot on Application of Water at cut end .......... 10. Photographs of a Potted Specimen of Impatietis before and after Irrigation ........ 26 11. Automatic Recorder for the Erectile Response of Drooping Leaf 28 12. ,, ,, for Erectile Response of Drooping Stem . 29 13. Record of Erectile Response of Leaf of Mimosa on Irrigation . 30 14. Records of Erectile Response of Drooping Leaf and Stem after Irrigation ......... 32 15. Determination of Rate of Flow of Sap in L'p and in Reverse Direction .......... 44 i6. The Potograph ......... 53 17. Record of Effect of Plasmolytic Solution in Arrest of Ascent of Sap • • 5i 18. ,, Effect of Stimulus on the Ascent of Sap . . .56 19. ,. Effect of Variation of Temperature on Ascent of Sap and on Growth . . . . . . 60 20. ,, Arrest of Pulsation of Desmodium at Critical Temperature ^linimum ..... 64 21. ,, Arrest of Ascent of Sap at the Critical Temperature 66 22. ,, Effect of Ether on Erectile Response . . . . 70 23. ,, Effect of Chloroform ...... 70 24. ,, Effect of Poison . . . . . . . 71 25. Photographs of Normal and Poisoned Wheat-seedlings . . 73 26. ,, ,, DroopingShootofC/ir}'saw///ewz2(wz after Applica- tion of Poison . . . . • • 75 26a. C hrysanihemum. Photographs of Shoots in Water and in Formal Solution ........ '77 XIV ILLUSTRATIONS FIG. 2,6b. Curves of Suction of the two Shoots of Chrysanthemum . 27. The Bubbler ........ 28. The Pitcher of Nepenthes . . . . 29. Record of JMultiple Response of Glands of Nepenthes . 30. The Micro-Transpirograph ...... 31. Record of Transpiration without and with Balance . 32. ,, Electric Stimulus on Transpiration 33. Curves Exhibiting Variation of Transpiration during Night 34. Record of Effect of Carbonic Acid on Transpiration . 35. . ,, Effect of Ether on Transpiration 36. ,, Effect of Chloroform on Transpiration 37. ,, Diurnal Variation of Transpiration . 38. Diurnal Curve of Transpiration ..... 39. Curves of Diurnal Variation of Light and of Temperature 40. The Differential Balance ...... 41. Diurnal Record of Transpiration balancing Evaporation . 42. The Tilter and Electro-magnetic Recorder of Exudation 43. Record of Exudation from the Root-stock of Ciicitrbita 44. ,, . Effect of Mechanical and Electrical Stimulus on Exudation ...... 45. Curves of Response and Recovery in Mimosa and of Exuda tion in Ciiciirbita ....... 46. Diagrammatic Representation of INIethods for Record of Exuda tion and of Pressure ...... 47. Record of Diurnal Variation of Internal Pressure in Zea Mays 48. Diurnal Record of Pressure and of Exudation in Zea Mays 49. Record of Variation of Pressure in a leafless Tree [Poinciana regia) ......... 50. Arrangement for Measurement of Exuded Sap 51. Diagram of Water and Electric Mains .... 52. Record of Diurnal Variation of Pressure in Cucurbita with Leaves 53. ,, Effect of Incident Light on Internal Pressure 54. ,, Variation of Pressure in the Rain-tree {Pithecolobiiim 55. ,, Positive and Negative Exudation in Cnc/rrfcy/a 56. ,, Positive and Negative Exudation in the Ram-tree 57. Photograph of the ' Weeping ' Mango-tree . . . 58. Record of Exudation and Pressure in the ' Weeping ' Mango tree ......... 59. Sections of the I\Iango Stem ...... 60. Phoenix sylvestris with Trunk sliced for Collection of Sap 61. Record of Exudation from the Date Palm 62. Curve of Diurnal Variation of Exudation in Phceuix sylvestri 63. ,, ,, Exudation in Palmyra Palm .... 64. The Molecular and the Cellular Model .... 65. Electrometric Record for Determination of Velocity of Ascen 66. Galvanometric Record for Determination of Velocity 67. Method of Simultaneous Determination of Velocity by Mechan ical and Electric Methods ...... 68. Simultaneous Mechanical and Electrical Records . 69. Record of Electrical Pulsation of Desmodium . 70. ,, Cellular Pulsations in Impatievs ILLUSTRATIONS XV FIG. ?!• 7'- 73- 74- 75- 76. 77- 78. 79- 80. 81. 82. 83- 84. 85- 86. 88. 89. 90. 91. 92. 93- Record of Regular Cellular Pulsations in Musa Cellular Pulsation of the Mango-tree ,, Cellular Pulsation of Ficus religiosa . ,, Cellular Pulsation in Nauclea, by Einthovcn-gal vanometer ...... The Electric Probe ....... Amplitude of Electrical Pulsation at Different Layers . Section of the Petiole of Brassica showing the Active Layer IMethod for the Determination of the Hydraulic Wave-length Record of the Determination of Wave-length in Chrysanthemum The Cyclic Record in Chrysanthemum, .... Record of the Phenomenon of Interference Determination of Wave-length in Musa Increase of Wave length by Rise of Temperature Effect of Passage of Electric Current on Variation of Phase-difference ..... Cellular Contraction under External Stimulus Enhancement of Cellular Pulsation by Electric Stimulation ...... Effect of Electric Current on Cellular Pulsation . Enhancement of Amplitude of Pulsation by Rise of Temperature ..... Effect of Chloroform on Cellular Pulsation Arrest of Cellular Pulsation under Diminished Internal Pressure . . . . . Distribution of Fibro-vascular Tissue in the Stem of Papaya Record of Dual Response in Averrhoa under Stimulus Nerve-connections of the four Sub-petioles of Mimosa with the four Quadrants of the Pulvinus . . . . . . PACE 211 213 213 214 216 217 219 224 226 226 226 227 229 235 235 237 239 240 241 247 250 253 THE ASCENT OF SAP CHAPTER I THE PROBLEM OF THE ASCENT OF SAP Physical and Physiological theories — Inconclusive character of Stras- burger's poisoning and scalding experiments — Root-pressure. Among the fundamental activities in the life of the plant are the absorption of water from the soil and the conduc- tion of the sap to all parts of the body. By them the plant obtains its inorganic food-material from the dissolved constituents of the soil, and is supplied with the water necessary to maintain its cells in that state of turgor with- out which its growth and various life-movements would become arrested. Every portion of a tall tree has to be supplied with water, which is absorbed by the root, con- ducted along the stem, and finally excreted by the leaves. Calculations have been made which show that the amount of water transpired by the leaves of a large Birch-tree may be as much as 38 kg. per day. The energy required for lifting such large quantities of water to the top of the tree must be very great, especially when, as in the giant Eucalyptus amygdalina, it attains a height approaching 450 feet (150 metres). The problem of the ascent of sap has, from the earliest days of plant-physiology, enlisted the keenest attention of numerous investigators ; but the results obtained have not yet been found to offer any wholly satisfactory solution of it. The obscurity of the subject is, in large measure, due to the presence of numerous co-operating 2 CHAP. I. PROBLEM OF ASCENT OF SAP agencies of but secondary importance ; the inquirer is very apt to be led into the error of confining his attention to one or other of these, thus missing the essential factor in the problem. There is a voluminous literature on the different theories proposed in explanation of the ascent of sap, the enumeration of which here is out of the question : I must content myself with mentioning only some of the more important of them. They may be roughly classified as physical or physiological. According to the first, living cells take no part in the process. The second or physiological theory assumes, on the other hand, that the transport of water is fundamentally due to the activity of living cells, the movement being promoted secondarily by physical agencies. Of the physical forces that have been invoked, obviously neither capillarity nor atmospheric pressure can offer any explanation of the phenomenon. There is a mainly physical theory, due to Dixon and Joly, and to Askenasy (1895), that has received more support than any other, according to which the ascent is brought about by the transpiration from the leaves. The fluid in the mesophyll-cells of the leaves becomes concentrated by evaporation. x\n osmotic attraction is thus set up in the leaves, and the suction thereby exerted is supposed to be transmitted back to the roots through cohering columns of water in the wood- vascular tissue. This theory labours under various difficul- ties. To begin with, it is inconceivable that slow osmotic action could produce a sufficiently rapid current of water. For I show, in the chapters on the subject, that the velocity of ascent may become more than 20 metres an hour, even in the complete absence of transpiration. There is, more- over, no conclusive proof that, under actual conditions, the water-columns within the plant could possess the necessary tensile strength : for the cavities of the wood- vessels and tracheides contain air-bubbles which must impair their cohesion. Ewart (1905) has shown that, in THEORIES OF ASCENT OF SAP 3 order to maintain the transport of water, a pressure- column iive or seven times as great as the height of the tree would be necessary. He insists that the osmotic attraction developed in the parenchymatous cells of the leaf could not possibly exert so great a force. Turning next to the supposition that living cells may be instrumental in producing water-movement, Scliwendener assumes ' that the requisite energy is furnished in some as yet unexplained fashion by the living elements of the wood, thereby confessing his adherence to the views previously formulated by VVestermaier, God- lewski, and Janse, who all maintain that the ascent of sap is a vital and not a purely physical process.' Godlewski postulated a periodic variation in osmotic pressure, during which the osmotically active substance is alternately broken down and built up afresh ; he was, however, ' un- able to prove this hypothesis. Hence no discussion of his theoretical conclusions is necessary, nor of those of Janse and Westermaier as to the way in which living cells may act in raising water. ' ^ Strasburger (1891-1893), on the other hand, en- deavoured to disprove the physiological theory by his experiments in poisoning and scalding trees. He showed that solutions of copper sulphate and of picric acid, in spite of their poisonous character, ascended to the top of the tree. He also killed portions of the stem by heat, and yet the uppei living and leafy portions were found to remain turgid for a few days. My experiments on the subject will be found in Chapter V ; they lead to a con- clusion diametrically opposite to that of Strasburger. Strasburger's views have met with strong criticism from Pfeffer (1892) and from Ursprung (1904-5). Ursprung thinks that the living cells of the stem may, in some way, maintain the vessels in a favourable condition for conduction of water, or be instrumental in the ascent ^ Physiological Plant A-natoniy, Haberlandt, English translation, 1914, p. 321. 4 CHAP. I. PROBLEM OF ASCENT OF SAP of sap. In support of this he carried out a series of experi- ments in which lengths of petioles and stems of plants were killed by the action of high or low temperatures, or by poisonous solu+ions. He found that by killing por- tions of the stem, the wilting of the leaves above the dead area took place in the course of two to nineteen days, and that the greater the length of the stem that was killed, the earlier was the resulting wilting of the leaves. It has been objected that the wilting of the leaves may not be due to the death of the intervening tissue, but to secondary reactions. Boehm believed that the wilting was brought about by the plugging of the vessels with mucilage. Dixon regards it as being caused by the intro- duction of poisonous or plasmolysing substances from the dead tissue. None of these various theories has been found to be completely satisfactory, as Pfeffer,^ in summarising them, points out : ' How and by what means the water is so rapidly trans- ferred even' to the summits of the tallest trees has not yet been satisfactorily explained, it has unfortunately not even been determined whether the aid of living cells is quite unnecessary.' "^ The experimental methods generally employed by observers labour under the disadvantage that long periods of time are required, which must necessarily introduce many complications. The wilting of the leaves, more- over, is a very crude index for the detection of induced physiological change. The ideal method would lie, not in the employment of average statistics, but in the quick measurement of the change in the rate of ascent of sap caused by some physiological variation. Such a method for the record of the ascent of sap would make it possible to subject the process to various crucial tests, which would decide once for all whether it is physical or 1 Pfeffer, Physiology oj Plants, vol. i. p. 226. *'^^^ 2 Pfeffer, ibid., p. 220. ROOT-PRESSURE 5 physiological. I describe in the succeeding chapters several appliances of great sensitiveness which I have been able to devise for the purpose. Returning to the physiological theory, it should be borne in mind that a vague assumption of protoplasmic activity is not a sufficient explanation of the phenomenon of the propulsion of sap in plants. It is necessary further to determine the character of the cellular activity underlying the ascent, how that activity is initiated, and by what means a definitely directed transport of sap is maintained. As regards the last point, no satisfactory explanation has been offered. Still greater difficulties and complica- tions are introduced when we take into account other phenomena connected with the ascent of sap, such as the root-pressure, the occurrence of positive and of negative pressure, and the relation between the root-pressure and ' bleeding ' of injured plants. The root-pressure is sup- posed to force the water up and thus to help in the ascent of sap. But when this pressure is most needed, as during the rapid ascent of water to meet active transpiration by the leaves, it disappears or becomes nega- tive. The internal pressure of the tree is also subject to changes which appear to be erratic. Finally, the pheno- menon of ' bleeding ' is supposed to be due to root-pressure. No definite relation is however found to exist between the pressure and the exudation at the cut surface ; the Palms, in fact, exhibit vigorous exudation in the complete absence of any root-pressure. It is thus seen how necessary it is to arrive at a compre- hensive theory which will explain not only the ascent of sap but also other phenomena associated with it, which are quite inexplicable in the existing state of our knowledge. My object in the present work is to attempt to formulate such a comprehensive theory, based upon experimental evidence. Reference may be made to a long course of investigation which I undertook (1904-1906) on the subject of the ascent 6 CHAP. I. PROBLEiM OF ASCENT OF SAP of sap.^ It was shown that the transport of water is main- tained by physiological action, and that it is not the mere presence of living cells, hut their rhythmic or pulsating activity, which maintains the ascent of sap. Very little definite information has hitherto been available as regards the characteristics of the rhythmic vegetable tissues. A detailed account of investigations on the subject will be found in the works just referred to: but I give in the next chapter a brief statement of the characteristics which distinguish the pulsating from the ordinary tissue, for these criteria will afterwards be em- ployed in proof of the pulsatory character of the tissue concerned in the ascent of sap. 1 Plant Response (1906) and Comparative Electro-physiology , (1907) : Longmans and Green. CHAPTER II AUTONOMOUS PULSATION Rhythmic vegetable tissue — Autonomous pulsation in Desmodium gyrans — Multiple response under strong stimulus — Pulsations in growth — Characteristics of pulsatory activity — Effect of variation of internal hydrostatic pressure — Effect of maximal stimulus — Effect of sub-minimal stimulus — IModification of response in sub tonic speci- mens— Effect of variation of temperature on rhythmic activity — Arrest of pulsation at the critical thermometric minimum^ — Effect of anaesthetics — Effect of dose — Action of poison — Tests for pulsatory activity — Summary. Before describing the characteristics of pulsating tissues in plants, it will be of interest to form a mental picture of the physiological mechanism in the propulsion of sap. I have, in my previous works,^ shown the fundamental similarity of response in plant and in animal tissues. There is in fact no physiological action in the animal which is not to be found also in the plant. This being so, it may be instructive to refer to the means by which one-directioned propulsion of fluids is maintained by animal tissues. Let us take the instance of a multiciliated tissue ; here the cilium at one end gives, as it were, a signal which is followed serially by the rest, the multiple activity being continued for a long time. It is clear that if such a multi- ciliated tissue took the form of a hollow tube, the ciliated surface inwards, and if the tube were filled with water, then, owing to this peculiarity of the multiple-responding cilia, water would be driven in one direction. In the cir- culation of blood in animals, it is the sinus which gives ^ Plaiit Response (1906) ; Comparative Electro-physiology (1907) ; Irritability of Plants (1912) ; Life-Movements of Plants (1919-20) : Long- mans and Green. 8 CHAP. II. AUTONOMOUS PULSATION the signal, and the rhythmic contraction of the heart proceeds towards the ventricle ; the pumping action thus initiated determines the uni-directional flow of blood. Have we any proof that plant-cells are possessed of a similar rhythmic activity ? The detection of this in a single cell is surrounded with many difficulties. Micro- scopical examination, even if practicable, would show little or no effect : for, assuming the diameter of a cell to be of the order of 0-05 mm., its contraction or expansion would not cause any change of more than ten per cent. ; the variation of length would, theiefore, be something like o -005 mm. The period of a single pulsation of a plant is comparatively slow, being of the order of a minute or so. The problem then is the detection of a rate of change in length of the order of 0-00002 mm. per second, which is beyond the power of a microscope.^ Rhythmic Tissues Fortunately we have other means for the detection of rhythmic activity in plants, specially in pulvinated organs. The most striking example of this is found in the lateral leaflets of the Telegraph Plant, Desmodiitm gyrans. The cells of the lower and upper halves of the pulvinule execute alternate contractions : the result of the contraction of the more excitable lower half is a quick down-movement of the leaflet ; while the lower half is in the phase of recovery, the less excitable upper half undergoes contraction with a resulting slow up-movement. The period of a com- plete pulsation varies according to circumstances from a minute to four minutes or so. These pulsatory movements take place without any immediate external stimulus and arc therefore described as ' spontaneous ' or self-originated ; such spontaneous pulsation of the vegetable tissue exhibits ^ It is, however, possible to detect ultra-microscopic movements b}- a special electric method wliich will be described in a subsequent chapter. RHYTHMIC TISSUES 9 all the characteristics of the spontaneous movement of the animal heart.^ There are other plants which exhibit multiple pulsa- tion under special conditions. An example of this is furnished by the leaflets of BiopJiytmn sensitivum, which are normally in a state of quiescence. Multiple responses of the leaflets are, however, evoked by the application of a strong stimulus, the persistence of the pulsatory activity being dependent on the intensity and duration of the stimulus. // is thus seen that under normal conditions certain tissues, like those of Desmodium, exhibit very pronounced pulsatory activity. In other words, rhythmic activity is strongly developed in certain tissues, while it is but feebly developed in others. The former maintain their rhythmic activity under normal conditions ; whereas intense stimu- lation is required to arouse the latter. Further, there is no strict line of demarcation between the phenomena of multiple and of autonomous response. In very favourable circumstances for absorption of excess energy from without, Biophytum becomes an automatically responding plant like Desmodium. Conversely, under un- favourable conditions, that is to say, when the sum-total of its energy is below par, an automatically responding plant like Desmodium ceases to exhibit any pulsations : but the leaflets, now at standstill, will, like those of Biophytum,, give multiple response under strong stimulus. With regard to the uni-directioned propulsion of fluid in the plant, it has been pointed out that in the animal it is determined by the propagation of excitatory waves. Such a propagation of excitatory waves in vegetable tissues is exhibited in a very striking manner by Biophytum. If we apply a drop of strong salt solution at the inner end of the petiole, repeated excitations will be found to be 1 The pulsatory activity of tissues has been variously described as spontaneous, rhythmic or autonomous, and I use these terms in that sense. 10 CHAP. II. AUTONOMOUS PULSATION propagated from the point of irritation, in strict sequence, from each pair of leaflets outwards to the next (fig. i). Fig. I. Leaves of Biophytum (left) and of Desmodmm gyrans Application of salt at s gives rise to multiple excitation in the leaflet of Biophytum. The lateral leaflets of Desmodtum gyrans (right) execute autonomous pulsations. Autonomous activity of a pulsatory nature is also well marked in growing organs/ as is demonstrated in the records obtained by means of the High Magnification Fig. 2. Record of Pulsation of Growth taken with the High Magnification Crescograph Crescograph. The growth-pulsations consist of a series of alternate expansions and contractions, the latter being the smaller of the two ; the resultant growth in length is the difference between the elongations and the contractions (fig. 2). Sometimes the growth-activity alternates on 1 Irritability of Plants, p. 288. MODIFICATIONS OF PULSATORY ACTIVITY II two sides of the organ as the result of lateral pulsations, just as the up-and-down oscillation of the leaflet of Des- modium is produced by the alternate activity of the upper and lower sides of its motile organ. In previous investigations of these two typical instances of pulsatory activity it has been ascertained that it can be modified in very definite directions by variations of the physiological conditions : a brief summary of these in- vestigations is given in the following pages. If now the ascent of sap be found to be similarly affected by the same physiological variations, it may reasonably be concluded that it too is essentially a phenomenon of pulsatory activity. The following table summarises what has been determined for the movements of Desmodium and for the rate of giowth, as well as what may be anticipated for the ascent of sap. Table I. — Different Modes of Response to Induced \ariation OF Autonomous Activity „ , . J-. Induced enhancement of 1 Induced depression of Pulsatory activity g^^i^i^y g^ti^f^ty Pulsation of Desmo- Enhanced frequency and .Diminished rate or dium ' ampHtude, or both ; arrest Movement of growth Increased rate of growth Ditto Ascent of sap \ Enhanced rate of move- Ditto ment of sap i 1 The Characteristic Modifications of Pulsatory Activity under Physiological Variations The effects of physiological variation will be considered in the following order : (i) the effect of variation of internal hydrostatic pressure ; (2) the effect of external stimulus of sub-minimal and of maximal intensity ; (3) the modifying influence of the tonic condition ; (4) the effect of variation of temperature ; {5) the determination of the critical point of thermometric minimum for the arrest of response ; (6) the effect of anaesthetics, and (7) the effect of poison. 12 CHAP. II. AUTONOMOUS PULSATION (i) The Effect of Variation of Internal Hydrostatic Pressure A certain amount of internal pressure is necessary for the initiation and maintenance of rhythmic activity. This is seen in the renewal of the pulsation in the quiescent heart of the snail when the intracardiac pressure is increased. When a plant is subjected to drought, the turgor and the internal hydrostatic pressure become diminished. A diminution may also be produced artificially by the plas- molytic withdrawal of water. Conversely, an increase of internal hydrostatic pressure may be produced by fixing the cut end of the stem or of the petiole in the short arm of an U tube, and applying hydrostatic pressure by a water column in the longer arm of the tube. Desmodium Pulsation. — When water is withheld from Desmodium, the leaflet ceases to pulsate, the activity being renewed on irrigation. The arrested pulsation of a de- tached leaflet ma}' also be revived by the application of hydrostatic pressure. The pulsatory activity is thus dependent on the internal pressure. The converse is demonstrated by the plasmolytic withdrawal of water inducing an arrest of the normal pulsation. A solution of KNO3 applied at the cut end of the petiole bearing the pulsating leaflet, induces a continuous diminution of the amplitude of pulsation culminating in an arrest. Restoration of the normal pressure by substitution of water renews the pulsation (fig. 3). Growth. — Parallel effects are seen in the phenomenon of growth. Growth becomes arrested under drought and is renewed after irrigation. Partial drought diminishes the rate of growth ; application of warm water at the root increases the turgor of the plant and enhances the rate of growth. A plasmolytic solution, on the other hand, diminishes the rate. Thus in a series of experiments with a growing specimen of the flower-stalk of Zephyranthes, the normal growth-rate under partial drought was 0-04 /i MODIFICATIONS OF PULSATORY ACTIVITY 13 per second. On irrigation with warm water the rate was enhanced to 0-20 /x ; after this temporary increase the steady growth settled down to 0-08 /x. On application Fig. 3. Arrest of Pulsation of Desmodinni Leaflet due to Diminished Internal Hydrostatic Pressure induced by KNO3 Solution applied at Arrow ; Subsequent Revival on Substitution of \\'ater at Inverted Arrow of KNO3 solution to the root, the rate of growth was found to be diminished to 0-03 yu, per second, or to a third of the previous rate. Table II. — Effect of Variation of Internal Hydrostatic Pressure on Growth [Zephyranthes) Condition of experiment Rate of Growth Dry soil ..... After application of warm water . Steady growth after one hour After application of KNO3 solution i 0-04 ;u per second ' 0-20 ,, I o-o8 I 0-03 (2) The Effect of External Stimulus In vigorous specimens, all modes of maximal stimula- tion induce a diminution of turgor, a contraction, and a decrease of pulsating activity. These may be regarded as the normal responses of the plant to stimulus. 14 CHAP. II. AUTONOMOUS PULSATION Desmodium Pulsation. — The inhibiting action of stimulus on the pulsation is seen in the record (fig. 4) of the effect of electric stimulus of moderate intensity. The pulsation is seen to become arrested. On the stoppage of stimulus, the after-effect is often found to be an enhancement above the normal. Growth. — Various stimuli, mechanical, electric, or photic, retard the normal rate of growth. This retardation in- creases with the intensity and duration of the stimulus, and Fig. 4. Effect of Electric Stimulus on Pulsation of Desmodium gyrans Note inhibition as the direct and enhancement as the after-effect of stimulus applied at s. culminates in an arrest of growth. Thus under electric stimulation, the normal rate of growth in a specimen was found depressed from o • 30 /i- to o • 09 //. per second. Stronger stimulus induced an arrest. Under the action of light, the rate of growth in a second specimen was found to be diminished from 0-47 /* to o-io fi per second. Stronger intensity of light induced an arrest of growth. The above results are obtained with maximum stimulus ; sub-minimal stimulus, however, is often found to induce an effect which is opposite to that of the maximal, that is to say, an enhancement of activity. .MODIFICATIONS OF PULSATORY ACTIVITY 1 5 (3) Modifying Effect of Tonic Condition I will now refer briefly to certain very unexpected results obtained in the course of my investigations on the response of vegetable tissues to external stimulus. It was found that the normal sign of response is liable to modifica- tion, the variation being definitely related to the physio- logical condition of the tissue, which may be at or below par. These two conditions will be designated as the normal and the suh-tonic. This difference in the initial condition of the tissue, though outwardly indistinguishable, is revealed through characteristic changes in the response to a testing stimulus. • The generalisation arrived at in regard to the charac- teristics of response in the two conditions is that, the response of a suh-tonic tissue is of opposite sign to that of the normal. This applies to all tissues, ordinary or rhythmic. As an illustration, the pulvinus of Mimosa normally responds to stimulus by contraction and the resulting fall of the leaf. But if the plant be kept in dark- ness or in other unfavourable conditions, its physiological tone falls below par, and the sign of response undergoes a reversal ; the pulvinus now responds to the same stimu- lus by expansion, and consequent erection of the leaf. Successive stimulations, however, improve the tonic condition, with the result that the abnormal response is gradually converted to the normal.^ As regards the autonomous rhythmic tissues, their activity declines or becomes finally arrested with increasing sub-tonicity. This condition may be artificially induced by keeping the whole plant or a cut specimen under unfavour- able conditions. The rhythmic activity manifested in pulsation or in growth may thus be made to undergo a continuous decline culminating in arrest. Just as the response of Mimosa in a sub-tonic condition exhibits a response of opposite sign to the normal, so a 1 Cf. Life-Movements of Plmits, vol. i. p. 221. l6 CHAP. II. AUTONOMOUS PULSATION rhythmic tissue also exhibits this reversal in sign of response when it is in a sub-tonic condition ; that is to say, that a stimulus which inhibits the activity in a normal specimen, renews or enhances the activity in a specimen which is in a condition of sub-tonicity. Desmodiiim Pulsation. — If a cut specimen of Desmodinni be kept in the dark, the amplitude of pulsation of the leaflets is greatly reduced in the course of about eight hours, and comes to a total stop in the course of eighteen hours. If we now apply the stimulus of an electric shock, the pulsa- tory activity is found to be revived, the persistence depend- FiG. 5. Effect of Stimulus in renewing Pulsation of Desmodium gyrans, originally at standstill Successive exposures to light for five, ten and forty-five minutes. A portion of record is omitted. ing on the intensity and duration of stimulation. Similar effects are produced by the application of the stimulus of light. Thus the application of strong light for five minutes gave rise to a single pulsation in a leaflet previously at a standstill. The next application of light of the same intensity was for ten minutes, and this gave rise to four pulsations — two during and two after application (fig. 5). Light was next applied for forty-five minutes, and the pulsatory activity persisted for nearly an hour after the cessation of exposure. These results show that the spon- taneous pulsation, so called, is not self-originated, but is really due to an antecedent external stimulus. The per- sistence of autonomous activity is thus dependent on the amount of stimulation to which the plant had previously MODIFICATIONS OF PULSATORY ACTIVITY 1 7 been subjected : the energy supplied by the environment becomes, as it were, kitent in the plant, increasing its power of work. Growth. — Diametrically opposite effects of stimulus on normal and on subtonic specimens are also met with in the phenomenon of growth. Thus while the effect of stimulus on a normal specimen is a retardation, its effect on a sub-tonic specimen is an enhancement of the rate of growth. The following table gives the quantitative results of the effect of stimulus on the growth of sub-tonic specimens. Table III. — ^Acceleration of Growth under Stimulus in Sub-tonic Specimens Specimens Stimulus Rate of Growth Wheat-seedling Scirpus Kysoor Previous After electric stimulation Previous After 5 minutes exposure to light 0-05 ^i per second 0-I2 ,, 0-30 ^ per second 0-40 ,, In the sub-tonic Wheat-seedling, stimulus enhanced the rate two and a half timics. In 5. Kysoor also stimulus enhanced the rate of growth by more than thirty per cent. It is thus seen that the effect of stimulus is modified by the physiological condition of the tissue. (4) The Effect of Variation of Temperature If we take the cardiac muscle of the animal as an example of rhythmic tissue, it is found that a rise of tem- perature quickens the pulsation ; lowering of temperature, on the other hand, slows it down. Desmodium Pulsation. — A rise of temperature induces an enhanced frequency of pulsation. Thus in a particular series of experiments it was found that, during a period of twelve minutes, there were four pulsations at 28° C, which increased to six at 31° C, and to ten pulsations at 34° C. Growth.— The effect of variation of temperature on i8 CHAP. II. AUTONOMOUS PULSATION growth is similar to the above. It is found that a rise of temperature enhances the rate of growth up to an optimum point which varies in different species of plants. The follow- ing table shows that the rate was continuously increased from 0-03 /i to 0-92 /x as the temperature was raised from 26° C. to the optimum temperature of 34° C. When the temperature was raised one degree above this optimum point, the rate of growth underwent a decline to 0 -84 yu,. Table IV. — The Rate of Growth at Various Temperatures Temperature Rate of Growth Temperature Rate of Growth 26° c. ■ 27° ,, 28°,, 29°,, 30°,, 0-03 /u per second 0-I2 ,, o-i6 ,, 0-22 ,, 0-32 31° c. 32° ,, 33°,, 34°,, 35°,, 0-45 /x per second 0 ■ 60 , , o-8o 0-92 ,, 0-84 ,, (5) The Critical Point of Thermometric Minimum Lowering of temperature slows down the pulsation of Desmodiiim leaflet till at a critical point it becomes arrested. This arrest is however not permanent, since a revival takes place as soon as the temperature is raised above the critical point. As I give in a subsequent chapter records of the arrest and revival of the pulsatory activity below and above the critical point, I need only state here that in Desmodium the pulsation generall}^ becomes arrested at or about 17° C. Lowering of temperature likewise induces a diminution of the rate of growth till, at a critical temperature, growth becomes arrested. This critical point varies in different species of plants ; but in several tropical plants examined, it was found to be about 22° C. (6) The Effect of Anaesthetics Anaesthetics when given in large doses act as poisons, causing the death of the plant. With regard to the action MODIFICATIONS OF PULSATORY ACTIVITY I9 of poisonous agents in general, the amount of the dose is of importance ; the striking general result which I have ob- tained in this connection is, that while a poisonous solution of moderate strength arrests or abolishes all life-activity, a small dose enhances it. Opposite effects are thus pro- duced below and above the critical dose. With strong poisons the range of safety is very narrow ; with less toxic agents, however, the range is wider, and by regulating the dose it is not difficult to produce either a stimulating or a toxic effect. Ether is less toxic than chloroform, and it is easy to obtain with it the stimulating effect of a small dose. Though the application of chloroform is apt to prove fatal to the plant, yet even here we can obtain the opposite effects of small and large doses without great difficulty. For when a large quantity of chloroform is applied, the plant absorbs it slowly ; the preliminary effect is therefore the same as that of a small dose in the enhancement of activity. Long-continued application, however, brings about the toxic effect. While all modes of rhythmic activity are enhanced by the application of small doses of anaesthetics, continued application produces arrest of activity and ultimate death, as is illustrated in the following records of Desniodiuni pulsations and of growth. Desmodium Pulsation. — Beginning with the effect of ether, the leaflet used was in a slightly depressed state, so the introduction of dilute ether-vapour into the plant- chamber induced an enhancement of activity (fig. 6, a). Continued application arrested the pulsation ; but the arrested activity could be revived by substituting fresh air for the ether-vapour. The effect of chloroform is shown in the record (fig. 6, h). The leaflet in this case was in a state of standstill ; the preliminary stimulating effect is seen in the renewal of the arrested pulsation. The continued action of chloroform caused arrest, and the death of the plant as seen in the spasmodic contraction and the resulting down-movement. 20 CHAP. II. AUTONOMOUS PULSATION Growth. — I have carried out numerous experiments on the effect of anaesthetics on the growth of various organs. Fig. 6 (a) Effect of small dose of ether in enhancing the pulsation of Desmodium gyrans (b) Effect of chloroform ; note the preliminary enhancement, followed by depression, arrest, and death as indicated by the spasmodic contractile movement downwards. The results obtained are similar in all cases. The specimens were placed in a closed chamber with an opening for the Fig. 7. Effect of Anaesthetics on Growth (a) Enhancement under small dose of ether ; (b) preliminary enhancement followed by spasmodic death-contraction, under the action of chloroform. passage of the connecting link by which the plant was attached to the High Magnification Crescograph. The MODIFICATIONS OF PULSATORY ACTIVITY 21 magnification employed was about looo times, tlie successive dots in the records being at intervals of fifteen seconds. The anaesthetic vapour was intro- duced into the chamber by means of an inlet pipe. The record was taken on a moving plate, and the first part of the curve indicates by its slope the normal rate of growth. Application of chloroform produced a preliminary enhancement of growth, seen in the sudden erection of the curve. Continued application induced an arrest, as seen at the turning point of the curve. This is the critical point, for further application of the anaesthetic produced a sudden spasmodic contraction giving rise to the reversal of the curve (fig. 7, b). The apex of the curve demarcates life from death. After this reversal, spots of discoloration appeared in the plant ; these spread very rapidly and the specimen became wilted as a consequence of death. (7) The Effect of Poison In regard to the action of different poisons, it must be remembered that a certain substance may prove very toxic to one plant and not so much so to another. Plants may also become accommodated to the action of a poison. Growth. — Poisons retard or abolish growth. Thus in a particular experiment, the application of one per cent, solution of copper sulphate depressed the rate of growth from the normal 0-45 /^ to 0-13 /^ per second. Prolonged application of the poison killed the plant. Desmodium Pulsation. — A poisonous solution of potas- sium cyanide was applied directly to the pulvinule of the leaflet ; this caused a complete arrest of pulsation in the course of seven minutes (fig. 8, upper record). In another experiment the poison was applied at a distance, namely at the cut end of the petiole which carried the pulsating leaflet. In this case the arrest of pulsation took place much later, i.e. after thirty-eight minutes, the 22 CHAP. II. AUTONOMOUS PULSATION delay being due to the time taken by the poison to ascend through the intervening distance (tig. 8, lower record). This experiment also demonstrates that a poisonous solution can pass through a killed tissue, owing to the suctional activity of the cells higher up, a fact that bears Fig. 8. Effect of Poison on the Pulsation of Leaflet of Desmodium In the upper record the poison was appHed directly on the pulvi- nule. The lower record exhibits the effect of application of poison at the cut end of the petiole, the arrest taking place much later. The gap in the lower record represents an interval of twenty-four minutes. upon Strasburger's experiments already mentioned. The matter is discussed in Chapter V. The physiological characteristics of pulsatory activity have now been described, as ascertained in fulh^ investigated instances of plant-movement. It now remains to deter- mine, by the application of similar methods, whether or not the ascent of sap responds in an essentially similar manner. SUMMARY 23 Summary The following have been shown to be the .physiological characteristics of pulsating tissues. i. Pulsatory activity is depressed or arrested under diminished internal pressure ; ii. Normal pulsation is inhibited by the action of strong stimulus, the after-effect of which may be an enhancement of activity ; iii. Sub-minimal stimulus enhances autonomous pulsa- tion ; iv. The response of a sub-tonic tissue is opposite to that of the normal ; that is to say, stimulus revives the arrested, and enhances the enfeebled, activity ; V. Rise of temperature up to an optimum enhances and fall of temperature depresses, rhythmic activity ; vi. Pulsation is arrested at a critical point which is the temperature minimum ; arrested pulsation is revived when the temperature is raised above this critical point ; vii. A small dose of an anaesthetic induces an enhance- ment of activity ; viii. Pulsation is arrested under the continued action of a large dose of the anaesthetic ; ix. Rhythmic activity is permanently abolished by the action of poisons. CHAPTER III DETECTION AND RECORD OF THE ASCENT OF SAP Detection and record of ascent of sap — Mechanical Method of Erectile Response — The Automatic Recorder — Erectile response of Mimosa. Chrysanthemum and Impatiens — The Osmotic Theory — Theory of suction and root-pressure — Ascent of sap in absence of root-pressure and transpiration — Depressed rate of ascent under increasing drought — Ascent of sap in cut stems previously exposed to air — Function of the xylem — Summary. In the study of the ascent of sap great difficulty is en- countered in the measurement of the rate of fiow and its induced variations. The withering of leaves, as stated before, is a very crude and unreliable test ; some more exact method is essential. Though the direct observation of the movement of sap inside the plant is practically impossible, yet we may • detect and measure some of the effects induced by it. In electric measurements we arc unable to see the passage of electricity, but are nevertheless able to detect and measure the current by its various effects, such as the production of heat, the directive action on the magnetic needle, the movement of a string across the magnetic field, the chemical effect, and so on. It is thus possible to construct different types of galvanoscopes or galvanometers possessing various degrees of sensibility. Similarly, by taking advantage of the effects produced by the conduction of flow of sap in a plant, we should be able to construct various instruments for the detection or measurement of its ascent. I have, in fact, devised two different methods for this purpose, namely, those of mechanical and of electrical response. In the present chapter 1 describe in detail the principle and construction of the automatic Mechanical Recorder, reserving the descrip- tion of the Electrical Recorder for a subsequent chapter. METHOD OF MECHANICAL RESPONSE ^D The Method of Mechanical Response The principle of the method will be understood from the following experiments. A cut specimen of ChrysaHthemitm coronarium is subjected to drought, when the plant doubles over, the leaves shrink and appear crumpled up and dried ; Fig. 9. Full Erection of the cut Shoot with Drooping Leaves on Application of Water at the Cut End. (Chrysanthemum) Photographs showing the difference before (right) and after irrigation (left). in fact the plant seems to be dead. But irrigation brings about a marvellous transformation through the ascent of sap ; the original turgor is restored, the bent stem straightens up and the withered leaves spread out in their original vigour. This is shown in the photograph reproduced (fig. 9), in which complete recovery took place in a time as short as fifteen minutes. I also reproduce 26 CHAP. III. DETECTION AND RECORD photographs of a potted Impatiens subjected to drought. The rate of ascent of sap here is much slower than in Chrysanthemum ; a partial recovery occurred in the course of two hours, complete recovery being attained after four hours (fig. lo). In nature the plant experiences great fluctuations Fig. io. Photographs of a Potted Specimen of Impatiens The first shows the effect of drought, the second exhibits partial recovery two hours after irrigation, and the third shows full recovery after four hours. in its state of turgor. Thus in Bengal there was no rain for six months from October last. The temperature in April had risen to 40° C. or 104° F., so that the plants were suffer- ing from excessive drought when the rains came down in the middle of April. There was thus great variation as regards the available source of supply of water ; and we shall presently have occasion to discuss the manner in which variable conditions of drought affect the ascent of sap. Potted plants are similarly subjected to periodic variation : on watering the plant, the stem and the METHOD OF ERECTILE RESPONSE 27 leaves become turgid : after one or two days the loss by transpiration from the leaves will reach a point when it will be greater than the supply of water through the ascent of sap, the result being a slight drooping of the leaves. Confining our attention to a particular leaf, we find that fresh irrigation causes an erection of the leaf to the horizontal outspread position. This erectile movement docs not take place immediately after irrigation ; a cer- tain time is required for the ascending sap to reach the leaf-joint so as to increase its turgor and thus cause the responsive erectile movement of the leaf. I designate this time-interval as the latent period. In the case just mentioned, the leaf is the responding organ : but the bent portion of the drooping stem itself may be employed as the responder ; for after irrigation, the ascending sap, reaching the bend in the stem, will cause it to straighten. We have thus two means of detecting the ascent of sap, namely, the erectile response of the drooping leaf, and the erectile movement of the drooping stem. These are so slight that the course of erection from its initiation to uniform movement cannot be made out by mere eye- observation. ]\foreover, there remains the important element of the time-relations of the response. For the fulfilment of our requirements, it is therefore necessary to devise special apparatus giving automatic records. The Automatic Recorder for Erectile Response The responsive movement induced by the ascent of sap is recorded by the apparatus (fig. ii). The indicating leaf is attached by a thread to a magnif\'ing lever made of fine glass fibre ; the lever itself is mounted on jewel bearings. The magnification may thus be raised from five to a hundred times. The bent tip of the long arm of the lever inscribes the erectile response on a smoked glass plate, kept oscillating to and fro by means of clock-work. 28 CHAP. III. DETECTION AND RECORD This oscillating device offers the double advantage of eliminating any friction of the recording lever against the glass plate, and of securing the accurate time-relations of the curve of response. Adjustment is made so that the Fig. II. Automatic Recorder for the Erectile Response of Drooping Leaf Leaves b and c are attached to two recording levers, B to the upper and c to the lower. The sap reaches b before reaching c ; hence the earlier response recorded by the upper lever. The clock-work for the oscillation and lateral movement of the plate is not shown in the figure. oscillation of the plate takes place once in fifteen seconds ; the distance between successive dots therefore represents a definite interval of time. The plant with the slightly drooping leaf is suitably clamped and mounted on a stand, the clamping being just sufficient to prevent slipping ; too great a compres- sion would, obviously, retard the ascent. The lower end of the plant is cut and water applied to it. We shall METHOD OF ERECTILE RESPONSE 29 presently lincl how the curve of response enables us to determine the characteristics of the ascent of sap and its induced variations. For the accurate determination of the velocity of ascent, two different levers are employed, as seen in the illustra- tion ; the first being attached to the lower, and the second to the upper leaf, one being vertically over the other. The advantage of this Duplex Method will be described later. The record of the drooping stem is obtained by supporting it by means of a clamp a little below the point where it begins to bend, this bent portion being the responder. Water is supplied at the cut end of the stem, and the erectile response recorded in the usual manner. The arrange- ment for taking record of the response of an intact plant with root is shown in fig. 12 ; the pot containing the plant is placed inside a larger vessel, v, into which water is poured for irri- gating the plant . The Oscillating Recorder illustrated here is of a more compact type than the one previously described. In illustration of the method described above, I will first describe an experiment with a potted specimen of Mimosa piidica. The plant was in a condition of a slight drought, and the responding leaf was exhibiting a slow and a continuous fall, due to diminishing turgor of the pul- vinus. We know that a sudden diminution of turgor takes place under the action of stimulus which causes a quick Fig. 12. Automatic Recorder for Erectile Response of Drooping Stem c, clockwork ; v, outer vessel ; s, screw adjustment for rais- ing or lowering the plant. 30 .CHAP. III. DETECTION AND RECORD fall of the leaf ; in the present case the gradual diminution of turgor due to increasing drought caused a slow movement of fall. This is seen in the first part of the curve (fig. 13). On irrigation at the vertical line, the fall of the leaf became arrested and then reversed to an erectile move- ment. This took place in the course of thirty seconds, which Fig. 13. Erectile Response of Leaf of Mimosa on Irrigation Down-curve shows gradual fall of leaf under drought. Irrigation at the vertical line induced erectile movement. Application of ice-cold water at arrow arrested the movement in the course of fifteen minutes. The gap in the record represents an interval of ten minutes. (Successive dots at intervals of fifteen seconds.) is the time taken by the ascending sap to reach the pulvinus from the absorbing root. On the attainment of a uniform rate of erectile movement, cold water was applied to the root, at the point in the record marked with an arrow. This brought about an arrest of the erectile movement in the course of about fifteen minutes. We have already seen that the application of cold induces a diminu- tion of pulsatory activity ; the arrest of the erectile response in this experiment is thus attributable to a retardation METHOD OF ERECTILE RESPONSE 3I of the ascent of sap induced b}^ physiological depression of rhythmic cells. The method of experiment with Mimosa described above, though of much theoretical interest, labours under certain disadvantages. First, it may be supposed that the results are peculiar to ' sensitive ' plants ; secondly, the very great sensitiveness of the pulvinus demands special precaution against accidental disturbance. 1 therefore prefer to employ the leaves of ordinary plants as indicators of +he ascent of sap. Most of the investigations described below have been carried out with Chrysanthemum and Impatiens. Chrysanthemum m.ay be gi'own in Calcutta from December to ]\Iarch, Impatiens is available during the rest of the year. In the records of different cut specimens given in fig. 14, the distance intervening between the cut end of the stem and the responding organ is the same in all, namely 15 cm. The response of the leaf of Chrysanthemum in the following experiments is shown in fig. 14, a, that of the drooping stem in 14, b. They are seen to be very similar, the cause of this resemblance being that in the two experiments the leaf and the stem function merely as indicators of the ascent of sap. In the record of the erectile response of the stem (fig. 14, b), it is seen to take place after the third dot, that is to say, forty-five seconds after the application of water to the cut end of the stem ; as the intervening distance for the transport of sap was 15 cm., the velocity of ascent was 200 mm. per minute. The record (fig. 14, c) was obtained with a drooping stem of Impatiens ; the erectile response occurred 2 '5 minutes after irrigation, the velocity of ascent being 66 mm. per minute, or less than a third of the velocity in Chrysanthe- mum. The curve attained an uniform slope in the course of six minutes after the application of water, and this uniformity was maintained for a considerable length of time, in fact so long as the bent portion of the stem did 32 CHAP. III. DETECTION AND RECORD not become too erect. For securing an uniform curve, the drooping stem should make an angle of about 5° below the horizon. Uniform slope of curve indicates uniform rate of the ascent of sap ; enhancement of the rate under stimu- lating agents is demonstrated by a sudden erection of the curve, also by wider spacings between the successive dots. Induced depression, on the other hand, is indicated by the Fig. 14. Records of Erectile Response of Drooping Leaf and Stem after Irrigation (a) Erectile response of ' varnished ' specimen of Chrysanthemum with leaf as an indicator (see p. 36). (b) Erectile response of drooping stem of Chrysanthemum. (c) Erectile response of drooping stem of Impatiens. \d) Erectile response of C hrysanthemum stem the cut end of which had been exposed to air (see p. 37). flattening of the curve, and by the closeness of the succes- sive dots. Having secured accurate methods for the determination of the rate of ascent of the sap, I defer to a subsequent chapter the study in detail of the effect of physiological agents upon it. For the present I will describe certain important experiments which will show definitely that the generally accepted theory of the ascent of sap is quite untenable in its essential details. THE OSMOTIC THEORY 33 The Osmotic Theory The first stage in the process, the passage of water from the soil into the plant, is described as follows by a well-known author ^ : ' The cells of the root-epidermis absorb water osmotically from the soil. The water ab- sorbed by the epidermis is transferred to the centre of the root since the cell-sap is in a state of greater concentration there than it is in the epidermis, and it will continue to be so transferred until a similar osmotic pressure prevails throughout all the cells of the transverse section. Water in the same way will pass osmotically into segments of young vessels while these are still in an embryonic state and possessed of normal cell-contents. When, however, a segment fuses with the next older segment, an immediate dilution of its osmotically active cell-sap must take place since it is essentially water that is found in adult vessels. The question then comes to be, how can water be abstracted from the cell-sap of a parenchymatous cell and transferred to the lumen of a vessel ; one would expect the precisely converse process to take place.' There is thus a barrier between the parenchymatous cell and the xylem which cannot be crossed by osmotic action. A different explanation has to be found for the transfer of water to and from the xylem according to different circum- stances. This is afforded by the theory of cellular pulsa- tion according to which the liquid is injected by the living cells into the wood- vascular tissue. Pulsatory activity is dependent, as we have seen, on the internal hydrostatic pressure and the resulting state of turgor. The difference of hydrostatic pressure between two points will be one of the factors in determining the direction of the propulsion of sap from a place of higher to a place of lower potential, from the more to the less active region. The sap-movement 1 Jost, Plant Physiology, English translation, p. 49. 34 CHAP. III. DETECTION AND RECORD will thus follow the ' turgor-gradient,' tending to equalise the difference of turgor in different parts of the plant. Theory of Suction and Root-Pressure Passing now to the consideration of the further move- ment of water in the stem, some idea of the prevalent view will be obtained from the following quotation : ' It is certain that the water is not merely driven upwards from the root, or base of the stem by the root-pressure acting like a force-pump, but that the removal of water from the conducting channels exerts a force transmitted backwards as far as the absorbing organs, causing in these a corresponding entry of water.' ^ The motive power is thus assumed to be the root- pressure, supplemented by the backwardly transmitted negative pressure caused by transpiration from the leaves. Confining our attention to the latter, it would follow that the greater the partial vacuum produced by transpiration the greater would be the backwardly transmitted suctional force and the corresponding enhancement of the rate of ascent. As regards the channel of conduction of water, it is considered as certain that the xylem alone subserves this function ; the well-known ' ringing experiment ' is supposed to offer conclusive proof : ' In order to break the con- tinuity (of the cortex) two circular incisions are made round the stem right into the wood and the intervening ring of tissue removed. If this " ringing " be not done too extensively, and if due care be taken that the stem does not become dried -up or rotten at the region of ringing, the leafy crown will remain fresh for a long time, and the transport of water will not be interrupted to any appreciable extent by the ringing. We may conclude therefore that the conduction of water is effected by the wood.' ^ This 1 Pfeffer, Plant Physiology, English translation, p. 208. - Jost, Plant Physiology, English translation, p. 48. THEORY OF SUCTION AND ROOT-PRESSURE 35 experiment is by no means conclusive, since the injection of water into the xylem (see Chapter XII) by the active cortex below would carry the water through the short stretch of the woody tissue fiom which the cortex had been removed. Another argument adduced in support of this view is the supposed abolition of ascent of sap in a stem when its cut end has been exposed for a short time to the air : ' When a stem has been cut across, air is drawn into the opened tracheae and the tracheids, owing to the internal negative pressure, and hence the absorption of water is rendered more difficult. In herbaceous plants the lessened rate at which the water is then absorbed is sufficient to cause a pronounced flaccidity even when the cut stem is immediately placed in water.' ^ Thus, according to the generally accepted theory, the ascent is mainly due (i) to the root-pressure together with the internal negative pressure and backward suction due to transpiration ; it follows from that theory (2) that the greater the condition of drought caused by transpiration, the quicker should be the rate of ascent ; and (3) that the ascent should be stopped by previous exposure of the cut end of the stem, the vessels being choked with injected air. The theory of cellular pulsation asserts, on the other hand, that (i) the ascent is due to the independent activity of living cells which extend throughout the length of the plant, hence neither root-pressure nor transpiration is essential to the process ; (2) the propulsion being due to cellular activity, which is enhanced under increased internal pressure, the rate of ascent should be diminished under condition of drought ; and that (3) it is not the dead vessels, but the living tissue which takes an active part in the conduction ; hence the previous exposure of the cut end of a stem to air should not cause a stoppage of the ascent. I proceed to describe experiments which prove (i) that the ascent may take place at a vigorous rate in the complete ^ Pfefier, Plant Physiology, English translation, p. 231. 36 CHAP. III. DETECTION AND RECORD absence of root-pressure and transpiration, (2) that the rate of ascent is diminished under increasing drought, and (3) that the exposure of the cut end of the stem to air does not abolish the conduction of water in the stem. Ascent of Sap in the Complete Absence of Root- Pressure and of Transpiration I took a specimen of Chrysanthemum which had been subjected to incipient drought : its root and all but a single indicating leaf were removed. The stem and this single leaf were coated with vaseline for the complete elimination of transpiration. The specimen was duly mounted, and a fresh cut made at the lower end of the stem, to which water was applied by raising a beaker of water from below. It will be seen (Fig. 14, a) that the erectile response of the indicating leaf took place two dots, that is 30 seconds, after the application of water at the cut end ; the intervening length was 15 cm., and the velocity of ascent was thus 300 mm. a minute, or 18 metres per hour. In certain other instances the velocity was found to be as high as 70 metres per hour. It is obvious that this high rate of conduction could not possibly be due to slow osmotic action. Moreover, in the experiment just described, there was no root-pressure to propel the water, nor any transpiration to suck it. Hence it follows that it is the cellular activity throughout the length of the stem which causes the propulsion of sap. Depressed Rate of Ascent under Increasing Drought Experiments on the effect of increasing drought in depressing the rate of ascent will be given in full detail in the next chapter : I here give a summary of some of the results. In the stem of Chrysaiithemum, the average rate of ascent under moderate drought was found to be ASCENT OF SAP IN CUT STEMS 37 230 mm. per minute. This was depressed under excessive drought to 18 mm. per minute, or to about one thirteenth. In Impatiens, the average rate in cut stems was 70 mm. ; under excessive drought this was depressed to 7-5 mm. per minute, or to about a tenth. Ascent of Sap in Cut Stems previously exposed to Air For this experiment, a shoot of Chrysantliemum subjected to drought was taken and its cut end was exposed to air for more than half an hour. The xylem-vessels would then be filled with air under atmospheric pressure which would block the channels. I also removed all the leaves except the solitary indicator, and smeared the stem and the leaf with vaseline, thus producing a complete abolition of transpira- tion. There could now be no backwardly transmitted suctional force, nor was there any channel for conduction through the xylem, now choked with air. According to the current theory, there should be a complete abolition of the ascent of sap under the particular circumstances described above. According to the pulsatory theory, however, there should be no such abolition ; the pulsations of the semi- dried cells at the cut end would, it is true, be arrested ; but this arrest would not be permanent. For after the absorp- tion of water there would be a slow revival of activity ; the record would thus show a prolonged latent period followed by an ascending curve less erect than that of plants in which drought was not so pronounced. The record given in fig. 14, d shows that an ascent of sap did take place along the stem in which the cut end had been previously exposed to air, and from which the transpiring leaves had been removed. The response-record, moreover, shows the characteristics which were expected. The latent period is prolonged to eight minutes, and the relatively slow rate of ascent is found in the gentle slope of the curve of the erectile movement. 38 CHAP. III. DETECTION .\Nn RECORD Thus by two independent tests we arrive at an identical conclusion that the ascent of sap takes place not by physical transference along the dead xylem, but along the living cells by means of their pulsating activity. Other experiments will be described in a subsequent chapter which will offer independent proof of the underlying physiological action in the transport of sap. Function of the Xylem The experiments described above prove that the xylem is not essential for the ascent of the sap. I have been able by an electric method to localise the tissue which by its pulsatory activity maintains the ascent of sap (see Chapters XIV, XV). This is the cortex which abuts on the fibro- vascular tissue. In dicotyledonous stems there is thus a cylindrical sheath, which subserves the purpose of rapid conduction of sap. The inactive xylem- vessels are situated very near the active cortex, within a fraction of a millimetre or so ; hence it is easy for the active cortex to force the sap laterally into the xylem during the phase of contraction. The xylem may, therefore, be regarded as a reservoir, water being pumped into or withdrawn from it according to the different circumstances. It will also be shown in the chapters referred to above that the fundamental mechanism in the ascent of sap is the same in herbaceous plants and in tall trees. Additional means, however, become increasingly necessary to meet the excessive demand for water in trees during active trans- piration. In herbaceous plants the distance of the soil- water is not too great : but in tall trees it is necessary to have a near source of supply of water, a ' soil-extension,' as it were, in the shape of conduit-pipes filled with water. These conduit-pipes are the young xylem-vessels (alburnum) for mechanical transference of water during the emergency of active transpiration from the leaves. Physical forces alone, such as capillarity or the cohesive power of water- SUMMARY 39 columns, cannot raise water to any great height. There is, however, no such limit in the case of propulsion of sap by the physiological action of living cells. When transpiration is feeble, the normal ascent along the cortex supplies every portion of the tree with water. The leaves become turgid, and the xylem filled with sap. During active transpiration, however, the physiological conduction is not sufficient to meet the demand, and water is withdrawn from the xylem-reservoir. Two factors are thus brought into operation : the physiological conduction by and along the active cortex, and physical transference along the xylem. SUMMARY Drooping leaf and drooping stem become erected in consequence of restoration of normal turgor by the ascent of sap after irrigation. The automatic record of the erectile response gives an indication of the rate of ascent of sap. According to the generally accepted theory, the ascent of sap is due to root-pressure and suction exerted by tran- spiring leaves, the rate of ascent increasing with increased drought and transpiration. Conduction is supposed to take place exclusively through the xylem ; if this were so, the ascent would be stopped by exposure of the cut end of the stem, since the vessels would then be choked by the injected air. In disproof of these views the following facts have been experimentally demonstrated. The ascent of sap takes place with great rapidity in complete absence of root-pressure and transpiration from leaves. The rate of ascent is decreased under increasing drought. The ascent of sap persists in stems whose cut ends have been previously exposed to air. The above experiments prove (i) that the xylem is not essential for conduction, (2) that neither transpiration nor root-pressure is essential, and (3) that there are channels other than the xylem for the ascent of sap. CHAPTER IV DETERMINATION OF VELOCITY OF ASCENT BY MECHANICAL RESPONSE Difference of velocity of ascent in cut and rooted specimens — Influence of the previous history of the plant — The Duplex Method — The effect of drought — The effect of physiological anisotropy induced by stimulus — Determination of velocity in the reverse direction — Summary. In the last chapter two methods for obtaining records of the rate of ascent of sap were described. The results ob- tained by the application of these methods with the highest degree of accuracy will now be given, dealing with such questions as whether the velocity is the same in intact plants with roots and in cut stems ; the effect of increasing drought on velocity ; the effect of stimulus on the rate of ascent ; the effect of physiological anisotropy in inducing differences of velocity of ascent on the two sides of an organ ; and, finally, the velocity of movement of sap in a direction opposite to the normal. In the determination of the velocity in different speci- mens, I was at first greatly puzzled by the widely divergent values obtained, which ranged from 0-3 mm. to about 700 mm. per minute. A long course of investigation enabled me, however, to detect the causes of this divergence : I found (i) that the velocity was not the same in different species of plants, the velocity in Chrysanthemum, for example, being higher than in Impatiens ; (2) that the velocity was higher in thick than in thin specimens ; (3) that it depended on the temperature, a rise of temperature up to an optimum enhancing the rate of ascent ; (4) that the velocity was higher in a cut stem than in a specimen with roots ; (5) that INFLUENCE OF PREVIOUS HISTORY 41 the rate of ascent is modified by the previous history of the plant ; (6) that it is affected by the condition of drought to which it had been subjected ; (7) and that it is modified by the action of stimuhis, the after-effect of which may be persistent. With regard to the velocity of ascent in specimens with roots, the following table gives results which I obtained with potted specimens of Impatiens in a condition of moderate drought : Table V.^ — ■^'ELOCITY of Ascent of Sap in Specimens of Impatiens with Roots, 30° C. Specimen Length Time Velocity per minute I 2 3 4 5 82 mm. 90 ,, 90 ,, 100 ,, 100 ,, 9 minutes 10 ,, 9 10 ,, 10 ,, 9 mm. 9 ,, 10 „ 10 ,, 10 „ The velocity in Impatiens in a ' moderate ' condition and at a temperature of 30° C. is thus found to be of the order of 10 mm. per minute. This velocity is lower than in cut specimens of Impatiens, which was found to be about 60 mm. per minute. The reason of this difference is found in the fact, already stated, that in specimens with roots, the fine root-hairs offer great resistance to the entrance of water. The Influence of the Previous History of the Plant In normal specimens of Impatiens, i.e., those subjected to moderate drought, the velocity of the water-transport has been shown to be of the order of 10 mm. per minute. In a particular specimen, however, the result was found to deviate greatly from the normal. The latent period was very short, which meant a great enhancement of the rate of transport of water, which was independently exhibited 42 CHAP. IV. DETERMINATION OF VELOCITY OF ASCENT by the marked steepness of the curve of erectile response. In attempting to discover the cause of the anomaly, I found that the specimen had been warmed artificially to hasten the drooping of the stem, and this must have necessarily raised the temperature of the soil. The specimen, however, had been kept in the experimental room for several hours before the commencement of the experiment, by which time the temperature had returned to the normal. It thus appeared that the warming of the soil had stimulated the roots, the after-effect of which persisted even after return to the normal temperature. In order to put this surmise to experimental test, I took three batches of similar plants which drooped to the same extent from drought. The first batch was kept as the control, the temperature of the soil of the second batch was raised, while that of the third was lowered. For producing vari- ation of temperature of the soil, two boxes were made with circular openings, through which the conical pots were let down, so as to close the opening, the pots being exposed to the air of the chamber. The temperature inside one of the boxes was raised by an electric heating coil, and that of the other lowered by fragments of ice placed at the bottom. The soil in the two sets was thus brought to about io° C. above and below the normal. It should be noticed that it was not the plant as a whole, but the roots embedded in the soil, that were subjected to the action of variation of temperature. After this the pots were kept in the experimental room for several hours till they attained the normal temperature, as indicated by a thermometer imbedded in the soil. Records were next taken after irrigation with water at the ordinary temperature, which revealed in a striking manner the dif- ference in their past history. The control batch gave the characteristic records which have been previously described, the average velocity of the transport of water being about 10 mm. per minute. The batch whose roots had been stimulated several hours before by warmth, now gave records which exhibited a very short latent period and a THE DUPLEX METHOD 43 velocity which was more than ten times the normal. The batch whose roots had previously been cooled exhibited no response for a considerable length of time, sometimes not even for hours. The velocity in such cases was about 0'3 mm. per minute, or one-thirtieth the normal. It is thus seen how profoundly the activity of the ascent of sap is modified by the previous history of the plant. We shall see later how the depression of irritability of the root affects other activities of the plant. The Duplex Method It is necessary here to explain certain difficulties which are encountered in the accurate determination of the velocity of ascent. These arise (i) from the loss of time due to the physiological inertia of the responding leaf or cut portion of the stem, (2) from the loss of time required for absorption of water by the root, (3) from the difficulty of measuring the intervening distance from the root, since its exact position with its numerous side-branches is very indefinite. These difficulties are, however, eliminated by the Duplex Method of record, where two indicating leaves, situated vertically one over the other, give successive responses to the arrival of water at the two points (see fig. 11). The response of the lower leaf indicates the moment of the arrival of the ascending sap at that leaf ; the delay in the response of the second gives the time-interval for the ascent of sap from one leaf to the other. The physiological inertia of the two leaves being about the same, this source of error is eliminated by taking the difference of the two latent periods. The question of the distance of the root does not arise at all in this method of determination. We have, however, to bear in mind the characteristics of the leaf -arrangement on the stem. The leaves in Chrysan- themum are arranged in a spiral, so that the fifth leaf is situated vertically above the first. A particular ascending fibro-vascular bundle, moreover, gives off lateral branches 44 CHAP. IV. DETERMINATION OF VELOCITY OF ASCENT to the first, the fifth, and the ninth leaf counted in order from below ; the vertically situated leaves are thus connected with each other. Another important fact is, as stated previously, that it is the cortex abutting on the fibro- vascular strand which is mainly concerned in the ascent of Fig. 15. Diagram for Determination of Rate of Flow of Sap in Up and in Reversed Direction In the left diagram there is a vertical slit in the stem shown by dotted line ; a piece of mica is inserted in this slit. Water applied at the slanting cut surface x travels upward from A to B to c, after which it crosses over to d, and follows the reverse course d, e, f. Diagram to the right shows reverse direction of flow by irrigation of the leaves. A is the normal direction of the ascent, and d the reversed direction of flow. sap. It thus follows that a particular cortical strand of tissue goes straight up, supplying the sap to the first, the fifth, and the ninth leaf. If the root of an intact plant or the cut end of the stem be irrigated, we can observe the successive erection of the vertical row of the leaves. A, B, C, or F, E, D, on the two opposite sides, and thus determine the velocity THE EFFECT OF DROUGHT 45 of ascent between A and B or between B and C and so on (fig. 15). I find that the velocity is approximately constant in the middle portion of the stem, which is neither too old nor too young. This will be seen in the results of the follow- ing experiment on the determination of the velocity in a specimen of Chrysanthemum which had been subjected to drought. The distance between a and b was 97 mm. and the time-interval between the successive responses was forty-five seconds. The velocity was therefore 130 mm. per minute. The distance between the leaves b and c was 62 mm., the time-interval thirty seconds, and the velocity 124 mm. per minute. The above results show that this differential method enables us to determine the velocity with great accuracy. The Effect of Drought The following experiments demonstrate that the velocity is decreased under increasing drought. The results given in the accompanying tables may be regarded as typical of the effect of slight and of excessive drought on velocity. The specimens employed were cut stems of Chrysanthemum, and of Impatiens. Table VI. — Showing the Effect of Slight and Excessive Drought on the Velocity of Ascent ChrysantJiemuin Moderate drought Excessive drought x. Distance Time in | Velocity per in mm. ■ seconds minute j^ Distance in mm. Time in seconds Velocity per minute 1 no 30 220 mm. i i lo 2 100 30 200 ,, 2 120 3 100 22 270 ,, ; 3 105 1 330 420 360 20 mm. Mean velocity = 230 mm. per min. Mean velocity =18 mm. per min. 46 CHAP. IV. DETERMINATION OF VELOCITY OF ASCENT Impatiens Moderate drought Excessive drought No. I 2 Distance in mm. Time in seconds Velocity per minute No. I 2 Distance in mm. Time in seconds Velocity per minute no 100 90 90 73 mm. 67 „ 120 no 900 930 8 mm. 7 „ Mean velocity = 70 mm. per min. Mean velocity = 7-5 mm. per min. From the above tables we find that in the case of Chrysan- themum the velocity was decreased under excessive drought from the average value of 230 mm. to 18 mm. per minute, and in Impatiens from 70 mm. to 7*5 mm. per minute. These results prove conclusively that the velocity of ascent becomes decreased under increasing drought. The Effect of Physiological Anisotropy induced by Stimulus I will next describe certain unexpected results which I obtained in the determination of the velocity. It is natural to expect that the velocity of ascent along the different flanks of the same stem would be the same. But in the determination of the velocity on the opposite sides of the stem of Chrysanthemum I found that though it was the same in a certain number of cases, it was widely different in others. Further investigation showed that the velocity was more or less uniform on all sides of specimens which had been grown in situations where the sunlight did not fall directly on the plant. In other specimens, of which the side facing south had been exposed to the action of sunlight, the north side being protected from it, though there was no visible difference in the two sides of the plant, yet an impressed physiological difference became revealed by the different speeds with which the sap ascended the two sides. I give in the following tables certain typical cases out of a large number. THE EFFECT OF PHYSIOLOGICAL ANISOTROPY 47 Table VII. — Showing the Difference in the Velocity of Ascent ON the Sunny and the Shaded Sides of Chrysanthemum Under slight drought Sunny side Shaded side j^ ' Distance Time in in mm. seconds Velocity per minute 103 mm. 157 ,. 187 „ 168 ,, No. I 3 4 Distance in mm. Time in seconds Velocity per minute 1 155 2 196 3 125 4 224 1 90 95 40 80 270 280 335 140 40 25 50 20 325 mm. 072 „ 474 ,. 420 ,, I Under excessive drought Sunny side Shaded side j^ Distance Time in in mm. 1 seconds Velocity per minute No. I 2 3 4 Distance in mm. Time in seconds Velocity per minute I 2 3 4 100 150 160 60 340 480 660 250 18 mm. 19 ,, 14 .. 14 .. 1- 125 i 130 130 1 150 156 180 170 170 58 mm. 52 ,, 44 ., 60 „ It has thus been shov^^n that, everything else being the same, (i) the velocity in a fully drooping specimen is, as previously shown, lower than in a semi-drooping specimen, and (2) the velocity of ascent in the shaded side is markedly higher than in the sunny side. This refers to the middle portion of the stem where the velocity is uniform. This latter result also proves that osmotic action could not be the determining factor in the ascent of sap. For Arrhenius has shown that the osmotic pressure in a plant is lower when growing in shade than in the open.^ The explanation of the lower velocity in the side stimulated by light has been arrived at by the experiments which will be described in detail in the next chapter ; it is that stimulus 1 Arrhenius, N. K. vet Akad, Nobel Inst., 5, No. 15, 1-20. 48 CHAP. IV. DETERMINATION OF VELOCITY OF ASCENT in general induces a diminution of velocity of ascent, and that this diminished velocity persists as an after-eftect of strong and long-continued stimulation. Determination of Velocity in the Reverse Direction In a plant subjected to drought, the root-cells cease to function from the absence of water-supply. The resultant diminution of turgor and hydrostatic pressure thus arrests the rhythmic activity underlying the ascent. If now, in a plant subjected to drought, water be applied to the top of the stem, it will be found that the direction of the flow of sap will be reversed, i.e. from above downwards. The following experiment demonstrates this in a striking manner. A drooping stem had all the leaves cut off except the terminal one. A beaker of water was raised so that the leaf was immersed in it (fig. 15). The result was that in a very short time the bent stem became erected, so that the leaf was lifted out of the water, the leaf having acted as an absorbent organ. There next arises the question as to the relative rates of the flow of sap in the normal up direction and in the reversed down direction. This determination I have been able to carry out with great accuracy by the following arrangement (fig. 15). We make a vertical slit dividing the stem to a certain height into two halves, and place a piece of mica between the two ; the slit is carried above the right leaf c, and i cm. below the left leaf d. A specimen was chosen which had been uniformly exposed to the light from the sky and not to one-sided sunlight. The normal conducting power was thus the same on all sides. The lower end of the stem has a slanting cut, so that, by partly immersing the end, only the right half of the stem was supplied with water. Owing to the physio- logical interruption by interposition of the piece of mica, the movement of sap on irrigation was from A to b, and then to c, causing successive erection of the drooping DETERMINATION OF VELOCITY IN REVERSE DIRECTION 49 leaves. After this the sap had to ascend i cm. and cross over to the left through a distance of about 4 mm., which was the diameter of the stem ; it then caused erection of the leaf d. After this the flow of sap was reversed in a downward direction, and the successive erections of the leaves took place in a reverse order, d, e, f. The respective time intervals enable us to determine the velocity of movement in an upward, in a transverse, and in a down- ward direction. The specimen, it should be remembered, was under considerable drought and the general rate of the flow of the sap was therefore slow. Table VIII. — -Giving the Rate of Ascent and the Rate of Reversed Flow downwards Distance Time interval Velocity per minute Ascent Reversed flow . AB 80 mm. BC 80 ,, de 75 mm. EF 75 ,, 245 sec. 240 „ 18 mm. 20 ,, 1620 sec. 1500 ,, 2-8 mm. 3-0 ,. The average rate of ascent was thus found to be 19 mm. per minute, and the rate of flow in a dov\mward direction to be 2*9 mm. The period required for the transverse conduction through 4 mm. was five and a half minutes, or at a rate of 0*7 mm. per minute. Thus, representing the slowest rate of transverse conduction by i, the rate of reverse flow downwards would be 4, and the normal ascent rate 27. Summary The velocity of ascent may be determined with the highest degree of accuracy by the Duplex Method, in which the latent periods of the absorbing and responding organs are eliminated. The velocity in a cut stem is higher than in an intact 50 CHAP. IV. DETERMINATION OF VELOCITY OF ASCENT plant with roots ; the difference is due to great resistance offered by the fine root-hairs to the entrance of water. The velocity of ascent is modified by the plant's previous history as regards the favourable or unfavourable con- ditions to which it had been subjected. The effect of excessive drought is to lower the rate of ascent of sap. Sunlight, acting as a stimulus, retards the rate of ascent : the after-effect of this stimulus is persistent ; anisotropy is thus induced between the sun-exposed and the shaded side of the same stem. The velocity in the shaded side is higher than that in the sun-exposed side. The velocity of flow of sap in the downward direction is about eight times slower than in the normal upward direction. The rate of transverse conduction is about 27 times slower than that of normal ascent. CHAPTER V THE EFFECT OF PHYSIOLOGICAL VARIATIONS ON THE ASCENT OF SAP The Potograph — Effect of physiological agents in modification of ascent- Effect of diminished internal pressure — Effect of stimulus — Modify- ing influence of tonic condition — Effect of variation of temperature on ascent and on growth — The critical thermometric minimum — Drooping of leaves during frost — Phenomenon of accommodation — Effect of anaesthetics — Effect of poison — Method of exudation — Strasburger's experiments — Summary. The various crucial tests discriminating the pulsatory activity of living tissues have been given in Chapter II. These are, the effects of diminished pressure, of the action of stimulus, of the modifying influence of tonic condition on response, of variation of temperature, of the critical thermometric minimum, of small and large doses of anaesthetics and of poisons. In the present chapter will be considered the application of these physiological tests to the ascent of sap, not only in intact specimens with roots, but also in cut stems. In addition to the two reliable and sensitive methods for the investigation of the velocity of the ascent of sap and its induced variations, namely, those of the response of drooping leaf and of drooping stem, a third method, the method of the Potograph, is now introduced as an indepen- dent and confirmatory test. The experiments were carried out, unless stated to the contrary, with cut stems. The Potograph The rate of water-movement may be indirectly deduced from the successive readings of the index of a potometer. 52 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION The results obtained with the apparatus in general use are not, however, free from error. The readings again are necessarily discontinuous, and important phases of the induced changes are thereby missed. In order to determine any variation of water-movement, a laborious process of construction of curves from the given data is necessary. It is therefore impossible to obtain any im- mediate indication of the normal rate of suction and its induced variations. For overcoming these drawbacks I devised the Poto- graph, by which the curve is directly obtained ; the inspection of the curve is sufficient to afford all the in- formation as to the normal rate of water-movement and the direct and the after-effects of external agents on that movement. The apparatus consists of (i) an arrangement by which the immersed part of the specimen may be readily subjected to the action of different excitatory or depressing agents ; (2) a potometric tube by which the normal rate of suction and its variation is observed ; and (3) a contrivance by means of which the excursion of the water- index and its time-relation become recorded. For this last, I employ two different methods : the first is automatic, in which the image of the opaque index is thrown upon a photographic plate allowed to fall at an uniform rate by means of a clockwork. The second is a much simpler device, that of following the movement of the index with a recording pen resting on a drum, round which is wound the paper for record ; the drum is kept revolving by a clockwork at a known and adjustable speed. When the excursion of water is followed in the way described, a curve is obtained the ordinate of which represents the quantity of water sucked up, and the abscissa the time. The slope of the curve gives the rate of water-movement ; so long as this is uniform the slope remains constant. If any stimulating agent increases the rate, there is an immediate flexure in the curve, which becomes steeper. A depressing THE POTOGRAPH 53 agent lessens the slope of the curve ; the arrest of water- movement is indicated by a horizontal record.^ The new type of the Potograph is shown in fig. i6 ; it possesses several advantages over its predecessor, one of which is facility of regulating the temperature of water. The stopcock s allows the introduction of water or other solutions into the vessel. For this purpose the Fig. 1 6. The Potograph The suction of water bv the plant is recorded by following the excursion of the water-index in the capillary tube with the recording pen, which traces the curve on the drum d, kept revolving by the clock c. s, stopcock for the introduction of water; Si for exit; So connects the capillary tube with the plant-vessel. stopcock Si, for exit of water, is opened and s^, in con- nection with the capillary tube, closed. After the intro- duction of water or a solution, s and Sj are closed and s^ opened. A thermometer inserted into the water-vessel indicates the temperature. The spiral of platinum wire for electric heating is placed at the bottom of the vessel, care being taken that the rootlets do not come in contact with the heating coil. Electric connections are made 1 For greater detail, as also for the more sensitive Method of Balance, cf. Plant-Response and Electro- Physiology. 54 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION with the coil by means of the electrodes e and e'. The current from a battery of cells is regulated by a rheostat, and the rise of temperature in the water-vessel may thus be adjusted without any difficulty. In order to secure accurate results it is necessary that the temperature of water in the vessel should remain constant. For ordinary experiments in which the effect of variation of temperature is not required, the temperature of the vessel does not in practice vary from the temperature of the room. But when we wish to study the physiological effect of variations of temperature, complications arise from the gain or loss of heat by the water in the vessel. This is reduced to a minimum by enclosing the plant-vessel in an insulating cover of thick felt, or b}' placing it inside a box filled with mica-dust. It is also easy to construct a correction-curve for the particular apparatus. The error introduced in neglecting this correction is, however, less than 2 per cent. Having described the different methods for obtaining the record, we may now enter upon the detailed study of the effects of physiological changes in inducing variation of the normal rate of the ascent of sap. Effect of Diminished Internal Pressure Diminished internal pressure may be produced by the action of drought or by plasmolysis. These were shown to induce a depression or arrest of the pulsation of Desmodium gyrans and of growth (p. 12). A condition of drought diminishes or arrests the ascent of sap. This is not solely due to the absence of water for transmission, but also to the depression of the pulsatory activity of the cells. Thus in a series of experiments carried out with the cut stem of Chrysanthemum, the conducting power of the specimens subjected to excessive drought was found to be depressed to as much as one- thirteenth the normal rate (p. 45). THE EFFECT OF STIMULUS 55 The application of a plasmolytic solution of KNO3 diminishes or arrests the ascent ; thus a dilute solution of KNO3 applied at the cut end of the stem of Impaticns arrested its erectile response ; a stronger solution applied to a different specimen induced not only an arrest but an actual reversal, that is to say, a drooping movement (Fig. 17, a, b). The effect of plasmolytic solution in diminishing the rate of suction was also determined by the independent method of the Potograph. The normal rate of suction of a cut stem of Croton was 36 c.mm. per Fig. 17. The Effect of Plasmolytic KXOg Solution in Arrest of Ascent of Sap Irrigation at vertical line induced normal erectile movement. Application of KNO3 solution at arrow arrested the response. The left figure shows the effect of strong, the right figure the effect of dilute, solution of KNO3. minute ; application of dilute KNO3 solution reduced it to 17 c.mm. per minute. The Effect of Stimulus It has been shown that in normal specimens the effect of stimulus is to depress or inhibit pulsatory activity, whether in Desmodiuw or in growing organs. Jn experi- menting on the effect of stimulus on the ascent of sap, a cut stem of Impatiens was taken, into which two pins had been thrust at a distance of two centimetres from each other, these serving as electrodes for the passage of induction- 56 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION shocks. After attainment of an uniform erectile response, strong electric shock was applied (at arrow, fig. 18, a). This is seen to have induced an arrest of ascent in the course of fifteen seconds ; the arrest persisted for a considerable length of time. Fig. 18. Effect of Stimulus on the Ascent of Sap in Normal and Sub-tonic Specimens {a) Efiect of strong stimulus applied at arrow in arresting ascent. (b) Effect of stimulus of moderate intensity inducing arrest with subsequent recovery. (c) Effect of stimulus on sub-tonic specimen in which ascent was at a standstill. Stimulus of moderate intensity initiated ascent for a short time ; stronger stimulus at s' produced persistent ascent. In another experiment, the induction-shock applied was only moderate. This gave rise to a temporary arrest, followed by recovery alter three minutes. Results similar to those obtained with electric stimulus were also obtained with other modes of stimulation, such as that of light. The stimulus of sunlight has already MODIFYING INFLUENCE OF TONIC CONDITION 57 been shown to induce a persistent diminution ot the rate of ascent (p. 47). Modifying Influence of Tonic Condition The effect of stimulus on a sub-tonic specimen has been shown to induce renewal or enhancement of pulsation in Desmodinm, or an enhanced rate of growth in growing organs (p. 15). The effect of stimulus on a sub-tonic tissue is thus diametrically opposite to that on the normal. Similarly, the ascent of sap in sub-tonic specimens is enhanced under the action of stimulus. This is seen in the record of a sub-tonic specimen of Impatiens. The sub-tonicity of the specimen is evidenced by its inability to suck up water even after irrigation, the record remaining horizontal. Application of electric stimulus of moderate intensity at s induced a transient renewal of the ascent ; stimulus of stronger intensity applied at s' induced a renewal which persisted for a considerable length of time (fig. 18, c). I also studied the effect of stimulus on sub-tonic speci- mens of plants with roots by the potographic method. The plants were mounted on the recording Potograph and afterwards placed in a dark room, till the normal suction was nearly abolished. This occurred in Zea Mays after twenty-four hours ; but in Impatiens the arrest did not take place till after several days. In Zea Mays the rate of suction declined to 0-24 c.cm. per minute. Electric stimulation of a definite intensity and duration was now applied to the lower end of the plant, one electrode of the induction-coil being dipped in the water-vessel, and the other applied to the stem 2 cm. above the root. The effect of the first stimulation was to enhance the rate from o -24 to o-6o c.cm. per minute, that is to say, it more than doubled the rate. The second increased it to 0-85, and the third raised it still higher to i-o c.cm. per minute, which was the climax, for the fourth stimulation induced a decline. 58 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION as in normal specimens. Effects similar to the above were also obtained with Impatiens. Table IX. — The Effect of Electric Stimulus on the Ascent OF Sap in Sub-tonic Specimens Specimens Stimulation Rate of ascent of sap Zea Mays -| Impatiens ■ \ Normal First stimulation Second ,, Third Fourth „ 0-24 cubic cm. per minute o-6o ,, „ 0-85 0-40 ,, ,, Normal 0-30 cubic cm. per minute First stimulation 0-78 ,, ,, Second ,, 0-26 ,, ,, Effect of Variation of Temperature Rise of temperature has been shown to enhance the autonomous activities of Desmodmm pulsation and of growth ; fall of temperature, on the other hand, causes a depression (p. 17). Variation of temperature also induces similar effects in the ascent of sap. The investigation was carried out by two different methods, first by the Erectile Response of drooping stems and second by the method of the Potograph. The specimens were Impatiens with and without roots. I will first describe the results obtained by the Erectile Method with a rooted specimen. Record was first taken of the actual rate, showing that the plant was exhibiting a continuous drooping, as seen in the down curve (fig. 19, a) ; watering the plant, at the vertical line, with water at normal temperature, arrested the drooping and brought on the erectile response. Cold water was next applied at c ; this caused a flattening of the curve indicating the relative depression of the rate. Warm water was next EFFECT OF VARIATION OF TEMPERATURE 59 applied at h, with the result of a great enhancement of the rate of erection, and therefore of ascent, as shown by the erect curve and the increased distance between the successive dots. The record given by a magnifying lever labours under the defect that an arc is described, on account of which the flexure caused by the variation of the rate of ascent is not so pronounced towards the end as in the middle of the curve. The defect arising from the curvature in the record may, however, be eliminated ; it is least pronounced in the middle part of the record through a length of about 5 cm. We take the record on a stationary plate, after adjusting the lever slightly below the middle ; the plate is next moved sideways through about i cm., and the next record is commenced at the same level as the first. This is accom- plished by lowering the plant, the stand on wliich it is placed being provided with a rack and pinion. Successive records of the effect of different temperatures are thus obtained in the middle part of the plate. In fig. 19, 6, are given successive records showing the effect of variation of temperature on the ascent of sap, the temperature rising from 30° to 35° C. and falling once more to normal 30° C. It will be seen that it took fifteen minutes to cover a distance of 40 mm. at the beginning of the experiment, and sixteen minutes for covering the same distance, at 30° C, after completing the cycle of temperature-variation ; the two determinations for 30° C. are practically the same, the average period for the same length of the record being 15-5 minutes. At 35° C. the same distance was described in eight minutes. Hence the rate of ascent of sap at 35° is about 1-9 times that at 30° C. I also reproduce, for comparison, a record of growth taken on a stationary plate at normal temperature, under cold, and under warmth (fig. 19, c). Method of the Potograph. — We determine first the normal rate of suction of a cut stem of Impatiens at 6o CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION 30° C, which is the temperature of the room in summer. Warm or cold water is then introduced into the plant- vessel, and the record obtained shows the effect of rise or fall of temperature on the rate of suction. In studying the effect of rise of temperature we introduce warm water, say, at 35° C, or the rise is effected by means of the electric Fig. 19. Effect of Variation of Temperature on the Ascent of Sap and on Growth (a) Record of erectile response on a moving plate. Cold water applied at c arrested the ascent, while application of warm water at H enhanced it. {b) Effect of cyclic variation of temperature of 30°, 35°, and 30° C. on the ascent of sap ; record taken on a stationary plate. Depressed rate is indicated by the closeness of successive dots ; enhanced rate indicated by wider spacings of the dots. (c) Effect of variation of temperature on the rate of growth taken on a stationary plate by the Crescograph. n, normal rate, c the depressed rate under cold, and h the enhanced rate under warmth. heating coil. After the attainment of the steady condition, record is taken of the resulting rate of suction. Correction for thermometric effect.- — In carrying out experiments on variation of temperature we have to apply a correction for the thermometric effect. It is to be re- membered that the vessel of the potometer acts as the bulb of a thermometer. When the water in the vessel is at a higher temperature than that of the room, there is a loss of heat through conduction and radiation. The loss can EFFECT OF VARIATION' OF TEMPERATURE 6 1 be greatly reduced by a non-conducting cover. Again, other things being equal, the rate of loss of heat and of fall of temperature will be greater the greater the difference between the temperature of water in the vessel and that of the surrounding temperature. With a difference of one or two degrees, the rate of loss will be very slight. During the fall of temperature the water in the vessel will contract, and the index will show this by a movement which is in the same direction as that of the suction by the plant. For obtaining the absolute rate of suction we have, therefore, to apply a correction, which is to be subtracted from the observed rate. When the water in the vessel is at a lower temperature than that of the room there is a gain of heat and a consequent expulsive movement of the water-index, which is in a direction opposite to that of suction. The actual suction will be greater than what is observed, and we have to add a correction for the true rate. It is therefore necessary to obtain a correction-curve for different tempera- tures applicable for the particular apparatus. Experimental method of obtaining the correction-curve. — For determining this correction, a glass stopper closes the aperture through which the lower part of the plant is inserted into the vessel. The water of the vessel is raised 5 degrees above the temperature of the room, this being the maximum rise generally employed in the experiments. Observations are commenced after the attainment of a steady condition. The thermometer inside the vessel shows the rate of fall of temperature ; and the movement of the water-index the rate of contraction of the water due to the fall of tempera- ture. It was found that, under the conditions of the experi- ment, the temperature fell from 35 -5° C. to 34-5° C. in the course of forty minutes, and the total contraction of the index was 98 mm. ; the average rate of contraction is therefore 2 -4 mm. per minute for the mean temperature of 35° C. The average rate of suction of Impatiens at 35° is, on the other hand, about 135 mm. per minute. The correction for the apparatus at a temperature 5° C. above that of the 62 CHAP. V, THE EFFECT OF PHYSIOLOGICAL VARIATION room is thus i-8 per cent. For temperature five degrees below that of the room the correction is of the same order but of positive sign. For smaller differences of temperature the correction is negligible. In studying the effects of change of temperature on the ascent of sap, a rising temperature can be kept under better control by the electric heating of the platinum coil immersed in the vessel than by pouring in hot water. Lowering of temperature is effected by the introduction of cold water. The surrounding temperature in Calcutta varies from about 22° C. in winter to nearly 40° C. in summer. Effect of variation of temperature. — The experimental plant employed was Impatiens ; the temperature of the water at the cut end of the stem was first lowered and the record taken at 25° C. ; it was next raised to 30° C, and afterwards to 35° C. The record obtained gives the move- ment of the index in mm. per minute. The absolute quantitj^ of water in cubic mm. sucked up by the plant is found by multiplying the rate of movement of the index by a constant, which for the capillary tube used was 0-24. The following table gives the rate of suction at different temperatures. Table X. — -The Kate of Suction at Different Temperatures Temperature Rate of movement of index Absolute rate of suction per minute per minute 25° C. ] 38 mm. 8-7 cubic mm. 30° C. [ 81 mm. I9'4 ,, ,, 35° C. i 150 mm. 36*0 ,, ,, Effect of cyclic variation of temperature. — An investiga- tion was next carried out on the effect of cyclic variation of temperature, that is to say, of the determination of the rate of suction at different temperatures for both thermal ascent and descent. Observations were made as the temperature EFFECT OF VARIATION OF TEMPERATURE 63 rose successively from 25° to 30° and then to 35° C. ; the temperature was next varied in a reverse direction from 35° to 30° and afterwards to 25° C. The two sets of results did not at first exhibit any close agreement. Further investigation showed that this was due to the fact that sufficient time had not been allowed for physiological adjustment to the changed conditions. In studying the effect of a given temperature, the specimen should be subjected to it for at least twenty minutes before taking the record. With this precaution the record of a cyclic change is extraordinarily consistent. The following table gives the results of the observations. Table XI. — The Effect of Cyclic \'ariation of Temperature. (Capillary Constant 0-24) Temperature rising Rate of suction per minute Temperature falling . ^^'^ ^5^^^^°" "" 25° c. 30° c. 35° C. 36 mm. 71 mm. 120 mm. 35° C. 120 mm. 30° C. 69 mm. 25° C. i 36 mm. 1 It will be seen that the suctional activity is enhanced during rise and depressed during fall of temperature, and that the rate of suction for a given temperature during the ascent and descent is practically identical. Comparison of the rate of Ascent and of Growth at di§ event temperatures. — It will be instructive to compare the effect of variation of temperature on the two autonomous activities of the ascent of sap and of growth, at about the medium temperature between 30° and 35° C. By the Potograph we found the rates of ascent at the two temperatures to be in the ratio of 81 : 150 or as i : 1-85. By the method of Erectile Response also the ratio of the rates of ascent for the same difference of temperature was seen to be 1:1-9. The rates of growth (Table IV, p. 18) at the two temperatures were found to be o -32 /x and o •8<^ /x, the ratio 64 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION being as i : 2-6. The induced variations in the activity of ascent and of growth may, therefore, be taken to he of the same order. The Critical Temperature Minimum I have explained (p, i8) that the autonomous pulsation of Desmodium leaflet becomes arrested at a temperature below the critical. This is shown in the record (fig. 20) , in Fig. 20. Record showing the Critical Temperature for the Arrest of Pulsation of Desmodium Leaflet The arrest took place at i7°C. and revival at i8°C. which the arrest in a summer specimen took place at 17° C, the pulsation being revived at 18° C. The plant becomes accustomed to a lower temperature in winter, when the critical temperature is 11° C. The mean critical point for the leaflet of Desmodium may therefore be taken as 14° C. When the temperature is lowered, the arrest of pulsation persists so long as the temperature remains at or below the critical point ; rise of temperature above this point revives the pulsation. The same living tissue may thus exist in two different conditions, namely, an inactive and an active state, which can be made to alternate by merely lowering CRITICAL TEMPERATURE-MINIMUM 65 or raising the temperature below or above the critical point. No proof of physiological activity could be more direct or convincing than the alternate arrest and revival induced by this definite physiological variation. As regards growth, the minimum temperature for arrest in Scirpus Kysoor I find to be 22° C. ; the arrested growth becomes feebly revived when the temperature is raised by 1° C, the rate of growth being now 0*02 yu. per second. The critical temperature for arrest of growth in S. Kysoor is thus found to be about 8 degrees higher than the mean critical point for the Desmodium leaflet. This shows that the growing cells are more sensitive to the adverse influence of low temperature than the fully grown cells in the pulvinule of Desmodium. It is thus seen that lowering of temperature below the critical point arrests the rhythmic activity of the Desmodium leaflet and of growth, a rise of temperature above that point causing a revival. Hence, a crucial proof in demonstratio'ii that the maintenance of the ascent of sap is effected by the rhythmic activity of living cells would be afforded by the alternate arrest and renewal of the ascent by temperature- variations below and above the critical point. In endeavouring to ascertain whether or not the ascent of sap is arrested at a critical temperature, I used the method of Erectile Response for the determination in specimens with roots. The Potographic method was employed, more especially for ascertaining whether or not the critical tem- perature was the same or different for the stem and the root of the same plant. Method of Erectile Response. — I obtained the erectUe response of Impatiens after irrigation with water at the normal temperature of 30° C. ; on the attainment of the steady rate, water at 10° C. was applied to the root ; this was found to cause an arrest of the ascent, as indicated by the stoppage of the erectile movement. The temperature was next allowed to rise slowly, and when the thermometer buried in the soil indicated 23° C, there was a slow 66 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION up-movement indicating resumption of the ascent of sap (fig. 21, a). The critical temperature for arrest was there- fore below 23° C, or at 22° C. Watering the soil does not produce a quick variation Fig. 21. Record of Erectile Response showing Critical Tempera- ture for Arrest of Ascent of Sap (a) First portion of the curve shows normal rate of erection under irrigation with water at 30° C. Application of water at 10° C. caused arrest in the course of fourteen minutes, after which the plant exhibited drooping. As the temperature rose to 23° C. there was a resumption of ascent, as seen in the erectile movement. (6) The plant was placed with roots in cold water, which caused an arrest of suction and consequent drooping. When the temperature of water rose to 23° C. the ascent of sap and the erectile movement were resumed. (Impatiens.) of temperature. It would be better, were it possible, to place the root in water and adjust the temperature. But a difficulty would arise in the washing of the soil from the roots, and the fixing of the plant in a vessel of water. By the time this had been accomplished the root would have CRITICAL TEiMPEKATURE-MINIMUM 67 absorbed enough water to cause a more or less complete erection of the stem, after which it would be impossible to obtain any further record of any erectile movement. It now occurred to me that the difficulty could be overcome by washing the roots with water at a low temperature, say, at 10° C, and placing them in a vessel of water at 15° C. As this temperature is below the critical point, the rhythmic activity would be arrested, with the resulting arrest of absorption. This surmise proved to be amply justified, and nothing could be more surprising than the fact that the plant previously under drought, with its roots greedy for absorp- tion of water, was unable to absorb even when immersed in water. In fact the record (fig. 21, b) shows that the plant continued to exhibit a drooping movement, as if the roots were buried in a very dry soil. The temperature of the water in the vessel was now allowed to rise ; the drooping movement was arrested, and the reverse erectile movement commenced at 23° C. The critical point for arrest is again found to be at or about 22° C. Drooping of leaves during frost. — The experiments described above offer a very satisfactory explanation of the drooping of the leaves which is observed during frost, and the recovery when the plant is brought into a warmer atmosphere. The ascent of sap, as we found, becomes arrested below the critical temperature, which is lower in cold climates than in the tropics. The temperature during frost would, in most cases, prove to be below the critical point ; hence the drooping of the leaves is due to the arrest of the ascent of sap. The suctional activity is restored by the higher temperature, and by the renewal of the ascent of sap and the restoration of turgor, the leaves regain their normal condition. The Potographic method. — I first tried to find out if suction by a cut stem of Impatiens was arrested at a suffi- ciently low temperature. The stem was mounted in the apparatus, the vessel being fiUed with water at 12° C. ; after this the temperature was allowed to rise gradually. 68 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION It was found that no suction was recorded at the low temperature ; but as the temperature of water in the vessel rose to i6° C. there was an initiation of suction. The critical point of suction is thus about 15° C. Suction could be renewed or arrested repeatedly by variation of temperature above and below the critical point. Phenomenon of accommodation. — While repeating the above experiment, I became aware of the very interesting phenomenon of accommodation by which the plant adjusts itself to changing external conditions. The critical point for the arrest of suction obtained from the first experiment was 15° C. ; repeating it a second time gave 14° C. for the arrest. A third repetition gave 13° C. as the critical point ; this was found to be the lowest obtained with this specimen, as further repetition did not exhibit any variation. The average critical point for the cut stem of Impatiens is thus 14° C, which is also the average critical point for the Desmodium leaflet. This coincidence is certainly very remarkable. The critical point for Impatiens with roots was next determined. The experiment was commenced with the temperature of water at 14° C, when suction was found to be completely arrested ; it was found to be feebly renewed at 23° C. ; the critical point is therefore below this, i.e., 22° C. A second experiment gave an identical result. This is a remarkable confirmation of the result obtained by the method of Erectile Response which has already been described. Thus by the independent methods of the Erectile and of the Potographic Response, we arrive at the same value for the critical point for the activity of the root, which is 22° C. The critical point for the cut stem is 14° C, or 8 degrees lower. The root is therefore more sensitive than the stem to the adverse effect of lowering of temperature. It is very remarkable that in certain tropical plants the critical point, 22° C, for the suctional activity of the root should be the same as the critical point for growth. This THE EFFECT OF ANAESTHETICS 69 may be due to the fact that it is tlie growing portions of the root which are more actively concerned in the absorp- tion of water ; or it may be that the irritability of the root is, in general, greater than that of the stem, on which account its activit\' is arrested at a relatively higher temperature. A rhythmic tissue may thus be alternately rendered active and inactive, above and below a critical temperature. Above the critical point, rhythmic activity is exhibited by the pulsation of the Dcsmodium leaflet and in the move- ment of growtli : below the critical point, in the inactive state, these manifestations become arrested. Since varia- tions of temperature above and below the critical point induce a similar alternation of states of activity and in- activity in the movement of the sap, the conclusion is inevitable that the movement is effected by a rhythmic tissue. The Effect of Anaesthetics We found that a small dose of ether induced an enhance- ment of pulsation in the Desmodium leaflet, and an enhance- ment of the rate of growth. Chloroform gave a preliminary enhancement followed by a decline and arrest (p. 19). The effect of anaesthetics on the ascent of sap is precisely similar, as will be seen in the following experiments. After the attainment of the uniform erectile response, dilute ether was applied at the cut end of a stem of Chrysanthemum ; this is seen to induce a great enhancement of response, which continued for a considerable length of time (fig. 22, a). A similar effect was induced when the anarsthetic was applied to the root (fig. 22, b). Application of a dilute solution of chloroform causes an extraordinary increase of the rate of ascent as the immediate effect. The record (fig. 23, b) gives us a striking example of the immediate stimulation caused by the appli- cation of chloroform, the activity being enhanced more than fifteen-fold, as seen in the 'jumps' of successive dots 70 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION inscribed by the lever on the oscillating plate. Continued action of chloroform causes depression and arrest . The experiments described above prove that the action of anaesthetics on the various rhythmic activities is in every way uniform. A small dose of the anaesthetic enhances the pulsation of Desmodium leaflet, the rate of growth, Fig. 22 Fig. 23 Fig. 22. Effect of Ether in Enhancement of the Erectile Response (a) Effect of appHcation at the cut end of the stem. (i) Effect of apphcation at the root. [Chrysanthemum.) Fig. 23. Effect of Chloroform on Erectile Response (a) Cut stem of Chrysanthemum.. (b) Drooping stem of Impatiens. Note sudden enhancement as the immediate effect. and the rate of the ascent of sap. A strong dose, on the other hand, stops pulsation, growth, and the ascent of sap. The Effect of Poison It has been pointed out (p. 21) that rhythmic activity is abolished, in the leaflet of Desmodium and in growing THE EFFECT OF POISON 7I organs, by the action of poisons. I now give the results of experiments made in order to ascertain whether or not the ascent of sap is similarly affected. I employed two different methods for the demonstration of the arrest of the ascent of sap by poison, the method of Erectile Response and the method of Exudation. It should, however, be borne in mind that certain poisons are more toxic for a given plant than for others. Moreover, Fig. 24. Effect of Poison on the Ascent L, Effect of dilute solution of formaldehyde on the response of a drooping leaf of Chrysanthemum, l', the effect of stronger solution in inducing quick arrest. a, The normal erectile response of drooping stem of Impatiens ; b, c, and d, the effects of increasing strengths of solutions in retardation and ultimate arrest of ascent. (See text.) plants often exhibit a certain amount of accommodation to poisonous agents. The Method of Erectile Response. — After the attainment of an uniform rate of erectile response in water with a droop- ing leaf of Chrysanthemum, a dilute solution of formalde- hyde was applied at the cut end of the stem. This induced at first an arrest of ascent, followed by a feeble attempt at recovery ; but the arrest soon became permanent. In a second experiment a stronger solution was applied. This caused a quick arrest (fig. 24, l, l'). 72 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION Another series of experiments was carried out with cut shoots of Impatiens. In fig. 24, a, is given the erectile record of a drooping stem, when the cut end was placed in a vessel of water. The erectile movement is seen to take place with great rapidity. After the commencement of the normal erectile response, i per cent., 1-5 per cent., and 2 per cent, solutions of formaldehyde were applied to different specimens. The records b, c, and d exhibit the effects of increasing strengths of solution in inducing increasing retardation of ascent, culminating in an arrest. The Method of Exudation. — In the following experiments I employed specimens of seedlings of Wheat with roots. The exudation of water at the tips of the seedlings of various Graminece is a visible indication of the activity of the ascent of sap. The experiments to be presently described were carried out with more than 100 different seedlings, and the results obtained were, without a single exception, in perfect agreement with each other. The mode of pro- cedure was as follows : the apparatus has two trenches ; one of these was filled with water and the second with i per cent, solution of poisonous agents like potassium cyanide or sodium arsenite. A dilute solution of poison was used, as the object was to paralyse the plant and thus arrest its activity : too strong a dose would have caused immediate death and wilting of the plant. Each row of seedlings was placed with their roots in water and in the solution of the poison respectively. The specimens were placed under a glass cover, and in the course of a few hours it was found that while drops were being exuded vigorously by the seedlings with their roots in water, not a single drop was found at the tips of the poisoned plants (fig. 25). These experiments were repeated many times with the same result. The exudation of drops of water is not the only mode of expression of spontaneous activity. This is also exhibited in active growth, and nothing could be more striking than the simultaneous arrest of exudation and of growth under THE EFFECT OF POISON 73 the action of poison in the same seedhngs. The experi- ments described below were carried out on three groups, each group consisting of six seedlings, so selected that for every specimen of a given length in one group there were two of l'"iG. 25. Photographs of Normal and Poisoned Wheat Seedlings Note exudation of water-drops and active growth in the former (front row), and the absence of exudation and growth in the latter. the same length in the other two groups. The first group had their roots placed in water ; the second, in dilute solution of potassium cyanide ; the third, in dilute solution of arsenious acid. The following tabular statement gives the results in the three groups, the observations being continued for forty-eight hours. 74 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION Table XII. — The Effect of Irrigation with Water and Poisonous Solutions on Growth and on Exudation No. Original length Length after forty-eight hours 1 Group I Group II (water) (KCN) 1 1 Group III (arsenious acid) I 3 4 5 6 40 mm. 42 mm. 43 mm. 45 mm. 45 mm. 46 mm. 1 100 mm. 1 43 mm. 100 mm. 43 mm. 102 mm. 44 mm. 107 mm. ' 47 mm. 108 mm. 46 mm. 109 mm. 47 mm. 44 mm. 45 mm. 46 mm. 48 mm. 47 mm. 50 mm. Exudation of water was copious and growth active in Group I, but completely arrested in Groups II and III. It will be noted that while the normal specimens became more than doubled in length by growth, the poisoned speci- mens showed practically no growth. These latter drooped and died in the course of a few days. The difference between the normal and poisoned specimens will be seen in the photograph of the apparatus containing two rows of four seedlings each, originally all of the same length. The seedlings with roots in water are seen exhibiting vigorous growth, and with exuded water trickling down the side. The seedlings in the second row are seen to be in a state of arrested growth and with no exudation (fig. 25). As regards the effect of poison on erectile response of drooping stems, nothing could be more striking than the photographs reproduced below of the effect of formalde- hyde solution in the arrest of ascent of sap in drooping shoots of Chrysanthemum (fig. 26). In a previous illustration (fig. 9) it is shown how the stem with its cut end in water becomes fully erected, with its leaves outspread in a turgid condition, in so short a time as fifteen minutes. In the present case, however, the cut stem in formaldehyde solu- tion persisted in the drooping condition ; so presumably the ascent of sap was completely abolished. The specimen never recovered, but exhibited even greater drooping after THE EFFECT OF POISON 75 eight hours ; subsequently it died from the effect of the poison and became decomposed. These results give conclusive evidence that poisons affect the ascent of sap just as they do the movements of the Desmoditim leaflet and the process of growth ; it may therefore be inferred that, like them, the ascent of sap is dependent upon the activity of living cells. The experiments described above on the effect of poison in the arrest of ascent of sap have an important bearing upon Strasburger's results, already referred to (p. 22). The Fig. 26. Photographs of Drooping cut shoot of Chrysanthemum placed in solution of Formaldehyde, which caused increased drooping, instead of full erection by ascent of water as in fig- 9 erroneous inferences drawn from them have had the most disastrous effect on the advance of investigation of this subject, as will be seen from the following extract : ' Owing to the researches of Strasburger, all vital theories have re- ceived a severe blow, if indeed they have not been directly disproved. Further, no positive evidence has been advanced in support of these theories, and one accepted them because purely physical explanation appeared to be inadequate.'* Now, no evidence could be more direct and convincing in support of the physiological theory than the continued arrest of ascent in a drooping stem with its cut end in a poisonous solution, and the renewal of ascent in a similar ^ Jost, Plant Physiology , English translation, p. 75. 76 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION specimen with its cut end in v/ater. The results of a com- plementary experiment described below will be found to be even more convincing. In this, we take two vigorous specimens, a and b, with their cut ends in water. They are in every respect similar to each other, the rate of suction in a being i-i c.c, that in b, i-o c.c. per hour, a and b were then placed in two similar test-tubes, one filled with water and the other with a 10 per cent, solution of formalde- hyde ; a layer of oil was spread over the surface of the two Hquids to prevent evaporation. The two test-tubes being previously calibrated (making allowance for the volume of the immersed stem), the rates of subsidence of the liquids will show the rate of suction and the ascent of sap in the two cases. The experiments were carried out inside the laboratory before a window. It was a rainy day, and the variation of temperature during the five hours of the experi- ment was slight. The specimen b, which was as erect and outspread as a, being placed in the poisonous solution, exhibited a collapse of the first pair of leaves in the course of five minutes, similar effects being produced in others in sequence from below upwards. The rise of poison could be followed by the discoloration ; the stem also collapsed, and the plant became a huddled mass of dying tissue (fig. 26A). The difference in the rate of suction observed in the two cases offers the most striking and conclusive proof of the activity of living cells in the ascent of sap. In specimen A the rates of suction and ascent were practically uniform throughout the five hours of the experiment. The suction continued unabated for several days in succession. In con- trast with this is the rapid fall of the rate in b due to gradual rise of poison, which put the successive zones of the living stem out of operation. The normal rate of i-o c.c. fell to 0-6 c.c. one hour after the action of the poison ; after two hours it was reduced to 0-35 c.c, after three hours to 0-2 c.c, and after four hours to o-i c.c. Suction was completely abolished after five hours. The difference in the two cases THE EFFECT OF POISON 77 is exhibited in a striking manner by the two curves given in fig. 26B. It may be thought that the effect of poison in woody trees might be different from that in the herbaceous stem of Chrysanthemum. The next experiment was, therefore, undertaken with two similar shoots of a Mango-tree, each bearing a rosette of eleven leaves. The average rate of suction of the specimen maintained with its cut end in water Fig. 26A. Photographs of two Shoots of Chrysanthemum, originally erect : the one to the left with cut end in Water, and the other in Formaldehyde Solution was 0 -g c.c. per hour ; the normal rate of the other specimen was I -I c.c. After treatment with the poisonous solution, the rates at successive hours were 0-7 c.c, o- 4 c.c, 0-21 c.c, and o • 12 c.c The suction was practically abolished after five hours. But to return to the consideration of Strasburger's experiments on the ascent of poisonous solutions in the trunks of trees. They do not afford conclusive evidence that the ascent of sap is independent of living cells : for it is only reasonable to attribute the ascent, in his experi- y8 CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION ments, to the suctional activity of the living tissues beyond and above the poisoned region. That liquid, even poison- ous liquid, can travel through dead tissue is proved by the experiment with Desmodium, described on p. 22, as also by others. The killed tissue of the poisoned stem becomes passive ; but so long as the parts above remain alive, it continues to exhibit suction, which is completely Fig. 26b. Curves of Suction of a in Water, and b in Poisonous Solution. Ordinate represents Quantity of Water sucked, and the Abscissa, the Time Note that the slope of the curve a remains unchanged for days, indicating continuous suction (the portion of the curve after 2 p.m. has been omitted), while the curve b shows con- tinuous diminution and final abolition of suction. abolished only when the stem is killed throughout its entire length. It would take a long time to kill a large tree by applying poison to the root or to the cut surface of the trunk . Incidental reference may be made to Strasburger's scalding experiments. The account of the following experiment will be of interest. The root of a plant was killed by boiling water. On return to the normal tem- THE EFFECT OF POISON 79 perature, the suction of water, instead of being arrested, was found to be enhanced even above the normal. This result does not in any way invalidate the physiological theory ; for it was only the portion of the plant killed by boiling water which lost its activity, whereas that of the unkilled portion above remained unaffected. The greater rate of suction after killing of the root is due to the fact that instead of the extremely attenuated channels of the root- hairs through which water was previously absorbed, there was now substituted the broad-sectioned stem, the dead mass of the roots acting as a piece of moist cloth for supply- ing water to the living tissue. The experimental evidence condensed in the following table proves that, under certain physiological changes, the ascent of sap undergoes variations which are identical with those of other forms of rhythmic activity, and justifies the conclusion that it is, like them, a rhythmic pheno- menon carried on by essentially the same mechanism — that of living pulsating cells. Table XIII. Showing the Effect of Physiological Changes on all Forms of Rhythmic Activity Physiological variation Induced effect of rhythmic activity Pulsation of Desmodium Activity of growth Ascent of sap Diminished pressure Arrest Arrest Arrest Efltect of sub-tonicity ,, ,, i» Effect of stimulus on Normal specimens . Inhibition Inhibition Inhibition Subtonic ,, Renewal Renewal Renewal Effect of poison Arrest Arrest Arrest Effect of anaesthetics SmaU dose . Enhancement Enhancement Enhancement Large dose . Arrest Arrest Arrest Rise of temperature . Enhanced Enhanced Enhanced frequency growth ascent Fall „ Diminished Diminished Diminished frequency growth ascent Critical temperature Mature organs About 14° C. About 14° C. Growing organs About 22° C. About 22° C. 8o CHAP. V. THE EFFECT OF PHYSIOLOGICAL VARIATION Summary Diminished internal hydrostatic pressure induces a depression of the ascent of sap. In sub-tonic specimens stimulus causes an enhancement of the rate of ascent ; in normal specimens it induces a retardation or arrest of ascent. Rise of temperature enhances the rate of ascent, while fall of temperature depresses it. The ascent is arrested at a temperature below the critical point ; an identical tissue can thus be made a con- ductor or non-conductor of the sap by raiding or lowering the temperature above or below the critical point. In the tropics the critical temperature-minimum is higher than in colder climates. The following relates to the critical point in several tropical plants : — The critical point of the fully grown pulvinule of Desmodium is about 14° C, which is the same as the critical point of ascent of sap in the cut stem of many plants : in growing organs, growth-activity is arrested even at the relatively high temperature of 22° C. : absorption and the ascent of sap in rooted plants are also arrested at about 22° C. The effect of small doses of anaesthetics is to enhance the rhythmic activity of Desmodium leaflet, of growth, and of the ascent of sap. Large doses induce an arrest. Poisons arrest the ascent of sap in specimens with roots and also in cut shoots. These experimental results prove conclusively that it is the pulsatory activity of living cells which maintains the ascent of sap in the plant. CHAPTER VI TRANSPIRATION Physical evaporation and physiological excretion — Isolation of absorbing, conducting, and excreting organs — The Bubbling Method for measure- ment of transpiration — Comparison of transpiring activity of different species of plants — Ratio of transpiration from upper and lower surfaces of leaves — Determination of transpiration from a single stoma — Transpiration in the absence of evaporation — The role of evaporation — Physiological continuity in stem and leaf — Crucial tests of physio- logical activity underlying transpiration — Effect of variation of temperature — Effects of sub-minimal and maximal stimulus — Summary. The study of the ascent of the sap in the stem has shown that it is effected by an independent activity of its own : that it takes place in the absence of a root to propel the sap or of leaves to suck it (p. 36). The direction of propa- gation is determined, as we have seen (p. 34), by the turgor- gradient, from the more turgid to the less turgid region of the plant. We have found that the activity of the root is not specifically different from that of the shoot, for the modifi- cation of the ascent of sap under physiological variation is essentially similar in cut stems and in intact plants with roots. We have arrived at the conclusion that the various manifestations of the ascent are brought about by the co-operative activity of living cells throughout the length of the plant, the absorbing root and the conducting stem. It now remains to study in detail the excretion of water at the upper end of the plant by the transpiring leaves. This is important, inasmuch as the state of turgor, internal pressure, exudation, and various other phenomena connected with the ascent of sap, are deter- 82 CHAP. VI. TRANSPIRATION mined by the relative gain and loss of water by absorption and by transpiration. The problem is highly complex, since the different organs of absorption, of conduction, and of excretion are subjected to different conditions and to diverse modes of stimulation. The root buried in the soil is to a great extent protected from the fluctuating changes in the environment. It may nevertheless exhibit a diurnal periodicity ; though whether or not such periodicity exists is not definitely known, and it would be necessary to undertake an investigation on the subject. I have observed that exposure of the stem to the action of light has the remarkable effect of checking the rate of conduction (p. 47). Sunlight, which by its heating effect enhances the transpiration from the leaves, may thus exert an inhibitory action on the conduction of water in the stem. Finally, the transpiring leaves are sub- jected to the numerous fluctuating changes of the environ- ment, to variations of humidity, to mechanical disturbances caused by the wind, to variation of temperature, and to the alternating action of light and darkness. The combined effects of these varying influences, which act unequally upon the three regions of the plant, are thus seen to be very numerous, and the complications which thus arise may well appear baffling. But the difficulty in the solution of such intricate problems will by no means be lessened by the employment of mere verbal phrases, nor by any argument of a teleological character which offers no real explanation of the underlying physio- logical mechanism. Nor can any scientific advance be made by the unjustifiable employment of physico-chemical processes in explanation of phenomena which are beyond their scope. There has hardly been any recent contribu- tion to plant-physiology so important as that of the inves- tigation of osmotic action. But it would be a distortion of truth if it were to be assumed that the extremely slow osmotic action could give rise to a velocity of ascent of sap which may be as high as 70 metres per hour ; or that ISOLATION OF INDIVIDUAL ORGANS 8^, J the quick pulsatory movements of certain plant-organs is brought about by the alternate and spontaneous manu- facture and destruction of osmotically active substances. The problem of the ascent of sap and its diverse mani- festations, though highly complex, is not insoluble. By isolation and separate investigation of the individual factors it will be possible to remove the obscurity which surrounds it. It is obvious how necessary it is to isolate an individual organ for the study of its characteristics. Neglect of this has often led to conclusions which are quite unjustifiable ; thus from the observation that the application of dilute acids at the root induces a reduction in transpiration, it has been concluded that this agent has a retarding effect on transpiration itself. This in- ference is not logical, since the effect might as well have been due to an induced variation in the absorptive power of the root, or in the conducting power of the stem. It is more likely that the chemical agent affected all the different activities of absorption, conduction, and tran- spiration alike. The difficulty attending the separate investigation of the activities of the three regions of the plant arises principally from the absence of exact methods and suitable apparatus for investigation : but I have endeavoured to overcome it by the invention of various instruments of precision which will be presently described. Complete isolation of the different regions from each other is im- possible, since, as will be shown, a physiological continuity exists throughout the plant. Approximate isolation may, however, be secured. Experiments have already been described on the ascent of sap in isolated stems from which the root and transpiring leaves had been removed. The root may be isolated by cutting the root-stock close to the ground and studying its activity by observing the rate of exudation from the cut end of the stock. Finally, the trans- piring activity may be studied by taking a single leaf with the cut end of its short petiole immersed in water. 84 CHAP. VI. TRANSPIRATION The current view of transpiration is that it is mainly a phenomenon of physical evaporation. Thus, to quote Pfeffer : ' Transpiration is influenced by the same external conditions as the evaporation of water in general. . . . Transient and rapid changes, such as the movements of the stomata, serve to modify the transpiration according to the conditions existing at the moment, and thus exercise a certain regulatory control. . , . The actual evaporation of water is a purely physical phenomenon, dependent in a plant, as in a dead body, upon the physical properties of the body in question.' ^ The object of the following experiments is to ascertain whether the giving up of water by the transpiring leaf is essentially a physical process, as Pfeffer suggests in the above quotation, or a physiological process of excretion effected by the pulsatory activity of living cells. In carrying out this investigation on transpiration it was necessary to devise a sensitive apparatus by which the rate of normal excretion and its induced variations could be rapidly determined with a high degree of accuracy, which will now be described. The Bubbling Method A moderate-sized transpiring leaf is mounted water- tight in a graduated vessel with a side-tube containing a drop of non-adhesive oil to act as a valve. This prevents evaporation from the side-branch, and also serves as a means of counting the air-bubbles that enter the vessel from the outside (fig. 27). Transpiration, by removing a certain quantity of water from the vessel, causes a slight vacuum ; the normal pressure is, however, immediately restored by a bubble of air, which enters the vessel by lifting up the oil-valve. The drop of oil falls back and the valve is closed once more, and the process is repeated time after time. 1 Pfeffer, Plant Physiology, English translation, pp. 236, 240. THE BUBBLING METHOD 85 Each bubble thus indicates the removal of a definite quantity of water by transpiration. This Bubbling ]\Iethod has proved to be a very accurate and valuable means of investigation. Under constant external conditions the interval between successive bubbles is extremely regu- lar. Thus transpiration from a full-grown leaf of Naiiclca, kept in a room temperature of 30° C, caused the appearance of successive bubbles at exact intervals of ten seconds, without any variation. The specimen was next removed to a cooler room with a temperature of 28° C. The bubbling period now be- came slowed down to twelve seconds, and this remained constant for the next hour. It will presently be shown that this depression in the rate of excretion is due not to physical but to physio- logical variation. Other experiments will be de- scribed which will show that definite physiological changes are attended by induced variation in the ex- cretion, as detected in the change in the bubbling period ; that is to say, a stimulatory agent quickens the rate of bubbling ; depressors, on the other hand, cause a slowing down of the rate. The rate of bubbling observed in a given specimen depends (i) on the sensibility of the apparatus, (2) on the transpiring activity of the species of Fig. 27. The Bubbler Note the drop of oil at the bend which acts as a valve. 86 CHAP. VI. TRANSPIRATION the plant, (3) on the size of the leaf and (4) on its physio- logical condition. The rate of bubbling, on account of the above circumstances, exhibits a wide variation in different specimens. But in one and the same specimen a great uniformity of excretion is observed under constant external conditions. The absolute rate of transpiration may easily be deter- mined by finding the constant of the apparatus. For this purpose we ascertain, by a sensitive balance, the difference of the weight of the Bubbler containing the leaf at the beginning and at the end of an hour. This difference represents the loss of weight by transpiration. A count having already been taken of the number of bubbles in the course of the hour, each bubble represents the loss of a definite quantity of water by transpiration. Thus, in a particular experiment, the leaf of Thtinhergia grandt/Iora lost o-i68 gram of water in the course of an hour, during which 60 bubbles were counted. The loss per bubble was thus 0-0028 grm. With a still more sensitive apparatus it is possible to detect a loss of one mgrm. Determination of the loss of water by weighing requires a long time, whereas the Bubbling Method enables us to obtain an immediate indication of the absolute rate of transpiration and its induced variations. In less vigorous specimens of leaves the average rate is found to be constant, though the intervals between successive bubbles vary slightly above and below the mean interval. Thus in a particular leaf of Thunbergia the successive bubbles occurred at intervals of 29, 31, 30, 30, and 29 seconds. In order to find the relative transpiring activity of different species of plants, I took leaves the area of which was nearly the same. I thus found that the rate of trans- piration of Thunbergia was half that of Nauclea. In Crassu- lacese the transpiration is very feeble ; in Bryophyllum calycinum the activity of excretion was found to be one- fifth that of Nauclea. I also determined the evaporation RATIO OF THE TWO LEAF-SURFACES d>y of water from a free surface, and the transpiration from an approximately equal area of leaf. Representing the evaporation as loo, transpiration from Nauclea was 45, from Thunhergia 20, and from Bryophyllum calycinimi it was 9. Ratio of Transpiration from the Upper and Lower Surfaces of Leaves Many leaves bear stomata only on the lower surface, on account of which transpiration at that surface is relatively greater. This may be qualitatively found by the use of cobalt paper, which becomes reddened earlier at the lower surface. To obtain quantitative results, Garreau employed the laborious method of cementing the leaf in two bell- jars containing vessels of calcium chloride. The relative increase in weight of the two vessels of calcium chloride gives the amount of water transpired respectively by the upper and the lower surfaces of the leaf. The following method is more direct and simple ; the results moreover are very accurate. The cut end of the petiole of the leaf is fixed air-tight in the apparatus (fig. 27), which is then placed on a sensitive balance, and the loss of weight deter- mined for, say, fifteen minutes ; this is the total transpira- tion, T, for both the upper and the lower surfaces. The upper surface of the leaf is then smeared with freshly boiled vaseline, which prevents transpiration from the upper surface ; the loss of weight in fifteen minutes now indicates the transpiration, l, of the lower surface only. The leaf is now smeared on the lower surface as well ; the loss of weight should now be zero ; if any loss occurs in this condition, it must be due to some unavoidable leakage ; in practice this is found to be negligible. Having found the total transpiration t, and l, the transpiration from the lower surface, t— L gives the transpiration from the upper surface. Representing the total transpiration, T, by 100, we thus obtain the percentage of transpiration 88 CHAP. VI. TRANSPIRATION at the upper and lower surfaces respectively. The ex- periment is next repeated with a fresh specimen, but this time the lower surface is smeared with vaseline, which gives u, the transpiration from the upper surface : t— u is then the transpiration from the lower surface. The results of the first experiments were not found to be very consistent ; this was traced to impurities in the vaseline, which contained traces of moisture. The difficulty was overcome by boiling the vaseline before applying it to the leaf. With this precaution the results were found to be highly satisfactory The following is a summary of the results. 1. Leaf of Nauclca (small size) : Rate of total transpiration per minute . 0-000382 grm. Upper surface . . . . -23 per cent. Lower surface . . . . . 76 ,, ,, Leakage . . . . . . i ,, ,, Total . .100 2. Leaf of Nauclea (large size) : Rate of total transpiration per minute . o' 000681 grm. Upper surface ..... 21 -5 per cent. Lower surface . . . . • 77'o >, >> Leakage . . . . . • r '5 ,, ,, Total . .100 The leakage is thus seen to be negligible ; the average ratio of transpiration from lower and upper surfaces of the leaf of Nauclea is thus 76-6:22, or about 3-5:1. In Thunhergia the ratio is about 4:1. Determination of Transpiration from a Single Stoma It may be of interest to obtain an approximate idea of the transpiration from an individual stoma at a temperature of 30° C. By means of a standardised Bubbler, the loss of transpiration from both the surfaces of a particular leaf of Nauclea was found to be 600 mgrm. per hour. The transpiration from the lower surface of the Nauclea leaf DETERMINATION FROM SINGLE STOMA 89 is, as we have seen, 4';^ part of the total, and was therefore 467 mgrm. per hour. Portions of the epidermis taken from different parts of the lower surface of the leaf gave the average number of stomata to be 820 per sq. mm. The area of the leaf was 20,800 sq. mm. ; hence the total number of the transpiring stomata was approximately 17 millions. Transpiration from an individual stoma was thus about 0*000028 mgr. per hour. In investigating the induced changes of transpiration, it is not necessary to determine the absolute rate, but only the relative variation. Thus in an experiment on the effect of change of temperature, the normal rate of transpiration was one bubble per ten seconds, or one-tenth of a bubble per second. In order to avoid fractions, it is better to take an hour for the unit of time. The hourly rate of transpiration at 30° C. was thus 360 bubbles, and it was depressed to 300 bubbles per hour at 28° C. The rela- tive change in the transpiring activity induced by slight cooling is thus in the proportion of 360 : 300, As the in- vestigation on the induced variations of activity of trans- piration is carried out with an identical specimen and with the same bubbler, it is sufficient to determine the ratio of the normal rate to that of the changed rate. The trans- piratory activity will therefore he relatively measured by the number of bubbles per hour. Transpiration in the Absence of Evaporation It has already been mentioned that the generally accepted view of transpiration is that it is essentially a phenomenon of physical evaporation. I am, however, able to describe several decisive experiments which prove that excretion from leaves takes place even in the absence of evaporation, thus affording a conclusive proof that transpiration is an active physiological process. The first experiment of the series was carried out in the Mayapuri laboratory in the hill-station of Darjeeling 90 CHAP. VI. TRANSPIRATION during continuous downpour of rain on the break of the monsoon. The air was surcharged with moisture. The tem- perature indoors was i6° C, and the transpiring activity of a single leaf of Hydrangea was found to be 40 bubbles. Water was sprayed on both the upper and lower surfaces of the leaf ; this did not arrest the transpiration ; the bubbling persisted at the rate of 24 per hour. In a second experiment the transpiring activity during a heavy downpour was 38, the leaf being placed outside, but protected from the rain. The transpiration persisted, after exposure to the rain, at the same rate for half an hour, after which it was lowered to 20. The next experiment is still more decisive ; evaporation from the leaf was prevented by thickly coating both the upper and the lower surfaces of the leaf with freshly boiled vaseline. It is true that the leaf under this abnormal condition is deprived of the supply of oxygen on which the various activities of its life depend. If it be a case of active secretion, this will induce only a depression of the rate, but not arrest. If, on the other hand, transpiration is dependent on evaporation, the fact will be demonstrated by the immediate arrest of transpiration. Experiments on these lines were carried out with various leaves. Thus in a vigorous leaf of Nanclea the normal rate of transpiration was 180 bubbles per hour ; after smearing both the surfaces with vaseline the bubbling was found to persist ; after an hour the rate was 139 ; at the eighth hour the excretion was still persistent, the rate being 30 bubbles per hour. The results of other experiments were similar, the only difference being that in less vigorous specimens the decline of the rate of bubbling was more rapid than in the above case. It may now be asked : what happened to the excreted water ? Examination of the smeared leaves showed that the excreted water became collected in small patches under the film of vaseline, which prevented its escape into the atmosphere. This was specially marked on the lower PHYSIOLOGICAL CONTINUITY IN STEM AND LEAF 9I surface of the leaf which bore the greater number of stomata. The experiments described above prove conclusively that the excretion from leaves is an active process independent of evaporation. I shall, in order to avoid ambiguity, use the term trans- piration only in the sense of active excretion in the further discussion of the subject. The Role of Evaporation Though evaporation is not essential for excretion, it is important in the removal of the excreted water, and thus in maintaining a state of diminished turgor in the transpir- ing region. We have seen that the flow of sap is determined by the turgor-gradient, and this difference could not be permanently maintained unless evaporation quickly re- moved the excreted water, and caused a partial drought at the upper end of the plant. The reason for the gradual diminution of excretion in the vaselined leaf is the accu- mulation of water which could find no vent for escape, and which tended to produce a flow of sap in the reverse direction. Physiological Continuity in Stem and Leaf The ascent of sap involves physiological continuity throughout the plant. The existence of this continuity is demonstrated in the fact, already described (p. 48), that, in a plant subjected to drought, the leaf on being supplied with water exhibits absorption, thereby pro- ducing a reversal in the direction of the flow of sap. The terminal leaf in the above case functions as a root, that is to say, the organ which excretes is also capable of absorption. Further experiments are given below which prove the existence of this continuity in the leaf. Just as it has 92 CHAP. VI. TRANSPIRATION already been shown that the root and the shoot are affected ahke by definite physiological changes, so now it will be shown that the petiole, the midrib, and the lamina respond in an identical manner. The Effect of Variations of Temperature The effect of raising the temperature of the water at the cut end of the stem, in enhancing the rate of ascent, has already been described, as also the converse effect of a fall of temperature (p. 58). I have now to describe experiments on the effect of thermal variation on trans- piration, (i) when the lamina is subjected to a change of temperature, and (2) when the distant petiole is subjected to thermal variation, the lamina itself being kept at a constant temperature. As regards the effect of change of temperature on the lamina, it may be remembered that the transference of a leaf from a warm to a cold room was found to be attended by depression of the rate of transpiration (p. 85). The experiment on the effect of variation of the temper- ature of the petiole was carried out as follows. The cut end of the petiole of a leaf of Naiiclea was mounted, with a thermometer, in a metallic tube. This tube was placed inside a larger vessel, which could be filled with water at different temperatures. I first produced a gradual lowering of temperature from 29° C, which was the temperature of the room, to 10° C. It is to be understood that the petiole alone was subjected to the lowering of the temper- ature, the lamina being maintained at the temperature of 29° C. After completion of the series of observations, the temperature inside the tube was allowed to return to the room-temperature, and afterwards raised from 29° C. to 32° C. The following table shows the effect on transpiration of cyclic variation of temperature of the petiole. EFFECT OF ELECTRICAL STIMULATION 93 Table XIV. — Effect of Variation of Temperature of the Petiole ON Transpiration Temperature falling Transpiring activity 29° C. 23° c. 18° C. 219 124 82 Temperature rising 29-5° C. 30-5° C. 32° C. Transpiring activity 275 300 360 It will be seen that during the fall of temperature of the petiole the transpiration underwent a decrease, while during the rise the activity was enhanced, in spite of the fact that the transpiring lamina itself was maintained at an uniform temperature. Effect of Electrical Stimulation One of the crucial tests of the rhythmic nature of a tissue is its reaction to stimulus : sub-minimal stimulus inducing an acceleration of activity, maximal stimulus retarding or inhibiting it (p. 14). This test was applied to the various parts of the leaf in the following experiments. Stimulation of lamina. — The electrical stimAilus has the special advantage that it can be easily graduated from sub-minimal or from maximal intensity. Electrical connections were made at two diagonal points on the lamina of Nauclea, which was stimulated by feeble induction shocks lasting for a minute. The normal transpiring activity was 116 ; but after feeble stimulation it was increased to 150, the enhancement being 29 per cent. The normal activity was restored in the course of twenty minutes. The electrical stimulus was now gradually increased. This induced a diminution of activity from the normal 116 to 87, or by 25 per cent. Further increase of intensity of stimulus induced a continuous diminution of activity, culminating in an arrest. Similar effects were also obtained with the leaves of Thunhergia. Stimulation of the midrib. — I next applied stimulus to the midrib of the leaf ; this induced results similar to 94 CHAP. VI. TRANSPIRATION those of the stimulation of the lamina. Under feeble stimulation the transpiring activity was enhanced from the normal 150 to 190, i.e., an enhancement of 27 per cent. Recovery took place after fifteen minutes ; strong stimulation now depressed the activity by 67 per cent. Stimulation of the petiole. — Electrical stimulus was applied to the petiole of another leaf of Nauclea. Feeble stimulus was found to enhance transpiration by 34 per cent. ; stimulus of stronger intensity induced a depression by 44 per cent. The foregoing experiments show that the tissues of the transpiring leaf, both petiole and lamina, give the reactions characteristic of rhythmic tissue to electrical stimuli, sub- minimal or maximal. We are now in a position to trace a complete picture of the physiological mechanism of the ascent of sap throughout the length of the plant. The pulsatory activity is initiated in the root, effecting the absorption of water from the soil. Similar activity in the cortex pumps the water up through the stem from cell to cell, and also injects water into the vascular tissue of the xylem (p. 175), along which it is physically transferred. The water conveyed by physio- logical conduction and physical convection reaches the leaf, where it is distributed along the veins and their numerous ramifications, and is eventually excreted or transpired into the intercellular spaces, whence it finds exit to the atmosphere outside by evaporation. Physiological continuity as regards the ascent of sap thus exists in the plant, and any distinction of a specific activity of the root, of the shoot, or of the leaf is not merely arbitrary but highly misleading. For the excretion at the upper end results from the additive activities in all the regions of the plant. Brief reference may here be made to other modes of excretion, by glands, by water-pores, and from wounded surfaces. No hard and fast line can, however, be drawn in these different manifestations. Excretions in general EXCRETION BY GLANDS 95 result from the additive action of cells throughout the length of the plant. The possession of a specially active Fig. ;8. The Pitcher of Nepenthes, and Transverse Section showing the Glandular Structure terminal layer may, however, render the excretion by an organ more or less independent of the rest. Such a specially active layer is found in the glands of the pitcher of Nepenthes (fig. 28). The pronounced rhyth- mic activit}^ of the layer is shown by the multiple electric responses ex- hibited by it (fig. 29). Excretion can therefore take place in the pitcher even in a condition of partial drought. Further, active ex- cretion may take place even in the absence of glandular organs, for we shall find that Palms, which possess no glandular organs, exhibit active excretion even Fig. 29. Multiple Response given by the Glandular Tissue of the Nepenthes 96 CHAP. VI. TRANSPIRATION in the absence of root-pressure. Excretion by the nectaries is no doubt helped by the osmotic withdrawal of liquid by the concentrated sugar-solution outside ; but the first excretion must have occurred without this adventitious aid. Similarly, evaporation from the leaves promotes the maintenance of the turgor-gradient in the plant, by which the uniformity of transpiration is secured. Summary The activity of transpiration can be accurately deter- mined by the Bubbling Method. Taking the evaporation from a given surface of water as 100, the transpiration from an equal leaf-surface of Nauclea is 45, of Thiinhergia 20, and of Bryophyllum calycinum g. Accurate determination can be made of the relative transpiration from the upper and the lower surfaces of the leaf by the Bubbling Method. In Nauclea the ratio is 1:3-5; ill Thunhergia it is i : 4. On the lower surface of an average-sized leaf of Nauclea there are 17 millions of stomata ; at a temperature of 30° C. the rate of transpiration from an individual stoma is 0*000028 mgrm. per hour. Transpiration persists for a length of time even after the smearing of both the surfaces of the leaf with vaseline. It is thus an active process not essentially dependent on evaporation. Transpiration is appropriately modified under physio- logical variations. Application of feeble electrical stimulus to the lamina enhances transpiration, while strong stimulus retards or arrests it. The effects described are also produced when the midrib, or the petiole, is stimulated instead of the lamina. When the lamina is subjected to a rise of tempera- ture, its transpiring activity is enhanced : the same result is obtained on raising the temperature of the distant petiole, SUMMARY 97 and lowering of the temperature of the petiole induces a depression in the rate of transpiration. These experiments prove that transpiration is a physiological process carried on by rhythmic tissue forming part of a rhythmic system continuous through- out the plant for the absorption and distribution of water. CHAPTER VII VARIATION OF TRANSPIRATION UNDER PHYSIOLOGICAL CHANGE The Micro -Transpirograph — Effect of diminution of turgor on transpira- tion— Effect of. stimulus — Opposite effects of stimulation of upper and lower surfaces of leaf — Effect of high frequency Tesla-current — Effect of electric waves — Effect of statical electric induction — Effect of thermal rays — Effect of light — Effect of red and of blue light — Effect of carbonic acid — Effect of ether and of chloroform — Summary. In the previous chapter we found that transpiration is a phenomenon of active excretion ; it was also shown that it undergoes responsive variations under changes of temperature and under the action of electric stimulus. We shall in the present chapter consider the response of the excreting leaf to various further tests of its pulsatory activity. These are : (i) the effect of diminished internal pressure ; (2) the effect of diverse modes of stimulation ; (3) the effect of light ; and (4) the action of anaesthetics. In addition an account of the action of thermal rays, of electric waves, of high frequency Tesla-current, and of statical electrical induction will also be given. Detailed explanation has already been given of the very reliable and sensitive Method of Bubbling for the deter- mination of induced variations of transpiration. As it cannot, however, be made to record the induced variation, it was necessary to devise a second method, which would be automatic and would inscribe a record of the effects induced. This has been secured by the Micro-Transpirograph, whose automatic records afford all the necessary information as regards the normal rate of transpiration and its induced variations. THE MICRO-TRANSPIROGRAPH 99 The Micro-Transpirograph In fig. 30 a reproduction is given of the photograph of a moderately sensitive apparatus. An U-tube filled with water has a float, F, on one side and the transpiring leaf on the other. The free water surface bears a certain thickness of oil to prevent evaporation, the cut end of the short stem bearing the leaves (or the petiole of a single leaf) being im- mersed in the water below the oil. The float is attached Fig. 30. The Micro-Transpirogra^)h The transpiring leaf on one side of an U-tube, and a iioat, F, on the other. The descent of the iioat caused by transpiration from the leaves is recorded by a writing-lever on a smoked oscillating plate of glass. For obtaining balance, the vessel v is raised or lowered by rack and pinion r. s^, stop-cock by the manipulation of which the writer may be adjusted at any position on the recording plate. to a recording lever which inscribes the record on a smoked glass plate kept oscillating by a clock-work. For continu- ous record to exhibit diurnal periodicity, the sensitiveness may be considerably reduced by making the diameter of the U-tube large and reducing the magnification of the recording lever. For researches on the effect of various external agents on the rate of transpiration, the sensitive- ness may be exalted to any extent desired (i) by selecting an U-tube with a narrow diameter, and (2) by increasing lOO CHAP. VII. VARIATION OF TRANSPIRATION the magnification produced by the recording lever. For ordinary purposes a magnification of 50 times by a single lever is quite sufficient. But this magnification can be greatly increased, and it is thus possible to record the loss of a milligram of water from the transpiring leaf. The greatest difficulty encountered in practice is that of the sticking of the float against the side of the U-tube, arising from unequal capillary action at the opposite sides. This may be obviated by making the tube in which the float moves perfectly vertical and preventing rotation of the float. The first is secured by a levelling arrangement of the tube, not shown in the figure. The float is attached to one arm of the lever which moves in a vertical plane ; jewel-bearings reduce its friction to a minimum. The float itself is made of a hollow aluminium tube which is very accurately turned. These precautions remove all difficulties in the perfect working of the apparatus. Subsidence of the float, caused by loss of water by transpiration, gives a record on the smoked glass plate, kept oscillating at intervals of twenty or thirty seconds according to different requirements. The record is taken on a moving plate, and the slope of the curve gives an indication of the rate of transpiration. The effect of any physiological variation is seen in the change of the slope of the curve, or in the widening or shortening of the intervals between the successive dots, the former indicating the enhancement, and the latter the depression of the rate. There is, however, a far more sensitive method available which enables us to detect not only the immediate but also the after-effect of the external agent. This is the Method of Balance, in which the level of the float at the beginning is maintained constant, the rate of loss by transpiration being exactly compensated by an equal rate of supply. Under these circumstances the record becomes horizontal. The balance is easily secured by the supply of water from the vessel v, the rate of which is roughly adjusted by the stop-cock, the finer adjustment being produced by a slight raising or lowering of the vessel by means of a rack THE MICRO-TRANSPIROGRAPH lOI and pinion. The normal rate is shown in the up-curve in the first part of the record of transpiration given in fig. 31 ; after the estabhshnient of the balance, the record is seen to become horizontal. The leaves were next subjected to a saturated atmosphere by holding a hollow vessel coated with moist blotting-paper over them. The effect of the reduced transpiration is seen in the imme- diate upsetting of the balance downwards. After the removal of the cylinder the balance became re-established as seen in the record, which again became horizontal. Had the after-effect been one of enhancement, the balance would have been upset in the op- posite direction with an upward movement. The horizontality of the record thus de- notes recovery to the normal rate. We have now two independent means, namely, the methods of Bubbling and of the Micro-Tran- spirograph, for investigating the effect of external agents on transpiration. In some of the following cases both these methods were employed, the results of which will be found to be in perfect agreement with each other. Most of the experiments described below were carried out with a single leaf, with the cut end of the petiole in water. Fig. 31. The Record of Transpiration, N without, and M with Balance, which is upset downwards by subjecting Leaf for a Short Time to Saturated Atmosphere Note re-estabhshment of balance, shown by record becoming horizontal, on restora- tion to original atmospheric condition. 102 CHAP. VII. VARIATION OF TRANSPIRATION Effect of Diminution of Turgor This condition may be artificially induced by a plasmo- lytic agent. The Bubbling Method was employed. The experiment was made with a leaf of Nauclca ; the cut end of the petiole was placed in water, and the normal rate of transpiration wcs determined. On substituting a 2 per cent, solution of glycerin for the water, the effect was found to be a depression of transpiration. The normal activity, 120, of the leaf was reduced to 85 in the course of fifteen minutes. Transpiration persisted at the lower rate during the whole time of the experiment, which lasted for two hours. The Effect of Stimulus We shall next study the effect of various modes of stimulation on transpiration. These are, the action of electric stimulus, which could be easily varied from sub- minimal to maximal ; the effect of mechanical friction ; and the action of light. Electrical Stimulus. — The effect of this, as determined by the Bubbling Method, has already been described in the last chapter ; it was shown that while feeble stimulus induced an enhancement, strong stimulus gave rise to the opposite effect of retardation and arrest. These results are confirmed by the Method of the Balanced Transpirograph. In this a record was first taken after securing exact balance, as shown in the horizontal record. An electric shock of feeble mtensity was next applied to the lamina, which is seen to upset the balance in an upward direction, indicating an enhancement of activity (fig. 32, e). The restoration of normal activity is seen to have taken place after a certain interval of time, as indicated by the record becoming horizontal. The effect of a strong stimulus applied at e is seen in the next record (fig. 32, f) ; the balance is upset downwards. EFFECT OF STIMULUS I03 which indicates a depression of transpiration. After a certain interval of time the record is seen to liave become horizontal, indicating the restoration of normal rate, and then there was an up-movement of the curve followed by a per- manent horizontal record. The result is significant, showing that while stimulus depresses activity, its after-effect may be an enhancement of activity. This was also found to be the case in the record of Desmodium pulsation (see fig. 4). The Fig. 32. Effect of Electric Stimulus on Transpiration (e) Enhancement of transpiration under feeble stimulus. (e) Depression induced by strong stimulus. Note the en- hancement as the after-effect, indicated by the subsequent up-curve. opposite effects of small and of large doses of chemical agents may be regarded as a phenomenon analogous to the above ; a small dose being equivalent to a feeble, and a large dose to an intense, stimulus. Mechanical Stimulus. — Electric stimulation acts diffusely on the lamina, and it is therefore impossible to apply it locally on the upper or on the under side of the leaf. Mechanical stimulation, however, labours under no such difficulty, and the upper or the lower side may thus be stimulated, one surface at a time. This brought out a very interesting difference in the two responses, as will be seen in the following experiments. 104 CHAP, VII. VARIATION OF TRANSPIRATION Responses to stimulation of the upper and the lower surfaces. — For this purpose different leaves of Thunbergia were taken and the surfaces stimulated by rubbing them with a brush. The experiments were carried out in the following order. The normal activity of transpiration was first determined, after which the upper surface was stimu- lated, causing an increase in the rate of transpiration. The rate was once more determined after an interval of fifteen minutes, by which time it had returned almost to the original value ; this I will designate as the second normal. The lower side was next stimulated, and a dimin- ution was the effect. It was invariably found that while the stimulation of the upper surface induced an enhance- ment of transpiration, that of the lower surface caused a diminution of the rate. The following are typical results obtained with three different specimens : Table XV. — Variation of Transpiration by Mechanical Stimulation OF Upper and Lower Surfaces of the Leaf {Thunbergia) 1 Number i Condition of experiment Transpiring activity Percentage of variation Normal ..... , Stimulation of upper surface Second normal .... Stimulation of lower surface 1 35 50 38 20 + 43 -47 II. Normal ..... Stimulation of upper surface Second normal .... Stimulation of lower surface 36 48 40 22 + 34 -45 + 45 -61 III. Normal ' 88 Stimulation of upper surface . 128 Second normal . . . . 91 Stimulation of lower surface . 35 Effec 40 Effec 51 ,t of stimulation of upper surface ei per cent. ,t of stimulation of lower surface d per cent. ihanced trans epressed trans piration by pi ration by EFFECT OF HIGH FREQUENCY TESLA-CURREXT I05 The important result obtained from the above experi- ments is that, under moderate stimulation, the upper and the lower sides of the leaf exhibit responses of opposite sign. The following considerations may possibly offer an explanation of this difference : 1. The transpiration of the lower surface is the more effective, the excretion from this surface being about four times greater than that from the upper. Now friction of the lower causes a direct, and of the upper, an indirect, stimulation of the more irritable and effective lower surface. It will be shown (Chapter XVIII) that direct and indirect stimulation often give rise to responses of opposite sign. 2. In a dorsi- ventral organ, the lower side is, generally speaking, more irritable than the upper. Thus in the pulvinus of Mimosa the lower side I find to be eighty times the more excitable. There is reason to believe that in the lamina also the excitability is greater on the lower side. Moreover, it has already been demonstrated (p, 93) that while a sub-minimal stimulus induces an enhancement of transpiration, a maximal stimulus retards it. Now an identical stimulus which is sub-minimal for the less excitable upper side of the leaf may prove to be maximal for the more excitable lower side. Hence it is probable that the friction of the upper surface acted as a sub-minimal stimulus, enhancing transpiration, whilst the friction of the under surface acted as a maximal stimulus, retarding it. Effect of High Frequency Tesla-Current If one terminal of a Tesla-coil be connected with a plate of metal, the latter becomes charged with oscillatory current several hundred thousand times per second. The space round the plate now becomes the field of alternating lines of electric force. When a leaf is placed in this field, its transpiring activity undergoes a definite variation, as will be seen in the following experiment. A leaf of Thunbergia was placed at a distance of 15 cm. from the plate of metal in I06 CHAP. VII. VARIATION OF TRANSPIRATION connection with the Tesla-coil. The normal activity was 29 ; this was depressed to 22 after exposure to the field of alternating current, the variation being — 24 per cent. On the stoppage of the action of the coil, the leaf recovered its normal rate after an interval of twenty minutes. Effect of Electric Induction A plate of metal was held at a distance of 15 cm. above the leaf and parallel to it. The plate was charged by a Wimshurst-machine, alternately with positive and negative electrification. In both these instances the rate of trans- piration was found to be increased. Thus in a leaf of Thimbergia the normal rate of 38 was enhanced to 54 under electric induction, the increase being 30 per cent. This effect takes place when the plate is held parallel to the leaf so that the lines of induction are perpendicular to the leaf. There is, however, no change in transpiration when the surfaces of the plate and the leaf are perpendicular to each other, that is to say, when the lines of induction are parallel to the surface of the leaf. In electric culture, the high tension net-work no doubt exerts a statical induction and thus enhances the normal transpiration on which the supply of inorganic food-material to the plant depends. But if the high-tension current were alternating, then the transpiration would undergo a diminution ; and the result would be the differential effect of statical induction and of alternating field of electric force. This latter effect may be eliminated by the use of a valve by which an uni-directional current can be maintained. Effect of Electric Waves I have shown elsewhere that Hertzian Waves of sufficient intensity diminish the rate of growth. ^ They have a parallel effect in the depression of the rate of transpiration. 1 Lije-Movements in Plants, vol. i. EFFECT OF THERMAL RADIATION AND FIGHT I07 Thus the normal rate of transph^ation in a leaf of TJiunbergia was 76 ; the effect of Hertzian Waves of short length acting for a minute was to induce an immediate diminution to 68 ; this continued for the next five minutes, by which time it reached the value of 62, the diminution being 18 per cent. After this the leaf exhibited a gradual recovery, and in the course of twenty minutes it attained the normal rate of 76. Effect of Thermal Radiation Thermal radiation was produced by means of an electric heating-iron, the heat-rays being allowed to impinge on the leaf. The normal activity of the experimental leaf of Nauclea was 63, and the immediate effect was an enhance- ment of the rate of transpiration to 80 ; this increase con- tinued for ten minutes after the cessation of radiation, the activity being enhanced to 109, or an increase of 73 per cent. The increase is due to the rise of temperature, which has a very pronounced effect in enhancing transpiration. The increase of transpiration under ordinary light is to some extent due to the presence of heat-rays. Effect of Light It is supposed that light enhances transpiration, either directly by widening the opening of the stomata or in some other way at present little understood. That the widening of the opening of the stomata under light plays but a subor- dinate part in enhancing transpiration is made probable by the following considerations : (i) Bonnier and Mangin found that exposure to light enhanced transpiration in Fungi ; the effect of light is thus independent of the presence of stomata. (2) In ordinary leaves the transpiration is modified by the action of light on the upper surface which bears no stomata. (3) The enhancement of transpiration by slight rise of I08 CHAP. VII. VARIATION OF TRANSPIRATION temperature is more pronounced than that induced by exposure to Hght. The increase may therefore be more or less due to the heat-rays present in the Hght (4) Light does not always induce an enhancement of transpiration ; under definite conditions it causes a diminution. Action of Light on the Widening of the Stomata and on Transpiration The following experiments were undertaken to find the relative variation of transpiration due to the widening of the stomata under light. A leaf of Thunhergia placed in a dark corner of the room at constant temperature at 30° C. exhibited a transpiring activity of 38. It was next brought near the window and subjected to the action of the stronger diffused light of the sky. This should, accord- ing to the generally accepted view, enhance the trans- piration by the widening of the stomata under light. The temperature might have been slightly higher near the window, though a sensitive thermometer did not indicate any difference. But in spite of the greater illumination and the possible rise of temperature, the leaf exhibited a diminished rate of transpiration which was lowered from the normal 38 to 29, a diminution, that is to say, of 23 per cent. The theory that the widening of the stomata en- hances transpiration is thus seen to fail in this particular case, which shows that transpiration is actually diminished under light. In a second experiment I made continuous observation of the transpiring activity of two different leaves of Nauclea, A and B, from 6 p.m. to 6 am. next morning. The results obtained were very similar (fig. 33) ; it will therefore be sufficient to give a detailed account of the variation of transpiration in b. The fall of temperature from 6 p.m. to midnight was fairly uniform, from 32 -8° C. to 29 -8° C, or a fall of half a degree per hour. The transpiring activity WIDENING OF THE STOMATA 109 declined from 116 to 71 -5. It will be noted that the trans- piration was still considerable, though the leaf had been subjected to continuous darkness for several hours. The subsequent changes were practically determined by variation Fig. 33. Curves exhibiting Variation of Transpiration at Xight in two Leaves, a and b Note sudden depression of transpiration by a fall of half a degree in temperature at midnight, and an enhancement of trans- piration by a rise of one-third of a degree after 2.30 a.m. Light early in the morning caused a transient enhancement of transpiration. of temperature ; there was a sudden fall of temperature of half a degree after midnight to which the leaf responded by a diminution of transpiration from 71-5 to 55, i.e., a depression of 23 per cent. At 2.30 a.m. there was a rise of temperature of one-third of a degree, and the trans- no CHAP. VII. VARIATION OF TRANSPIRATION piration exhibited an enhancement of lo per cent. Light began to appear at 5 a.m. ; this induced a transient rise, which subsided after a time, and the curves of temperature and of transpiration subsequently followed a parallel course. The experiments described above show (i) that the effect of rise of temperature on transpiration is far more pronounced than that of light ; (2) that the heating effect of light may often account for increased transpiration ; and (3) that li^ht sometimes induces a dimimition of trans- piration instead of an enhancement. Light as Stimulus The pulsation of Desmodium is arrested by exposure to strong light, which also retards or arrests growth. We have seen, moreover, that while moderate stimulus retards growth, a feeble stimulus enhances it. The growth of a less excitable sub-tonic tissue exhibits acceleration under an intensity of light which in a more excitable tissue would induce retardation (p. 17). As regards the relative effectiveness of various rays in retarding growth, it is known that while the more refrangible rays of the spectrum, blue and violet, are highly effective, the red rays at the opposite end of the spectrum arc less effective or ineffective. Bearing these facts in mind, 1 undertook the following experiments with the object of ascertaining whether or not the process of transpiration responds to the action of light of different wave-length in the same manner as the process of growth. Light-filter for Red and Blue-violet Light In order lo determine the relative effect of the less refrangible red at one end of the spectrum, and of the more refrangible blue-violet rays at the other, I at EFFECT OF RED AND BLUE-VIOLET RAYS III first employed a bichromate of potash solution as a light-filter to separate the former, and an ammoniated solution of copper sulphate for the latter. The results I obtained with these filtered lights were often found to be anomalous : in consequence, as I discovered later, of the impure character of the light, due to the overlapping of the spectra. The light transmitted through the bichro- mate solution contains red, orange, yellow and green, while that transmitted through copper sulphate includes green, blue and violet rays. I therefore made special light-filters with coloured glasses which gave red in the region of Fraun- hofer's lines B and C, the wave-lengths being from 680 to 600 /Li/i ; and blue- violet light in the region of Fraunhofer's line G, the wave-lengths being from 460 to 380 //.yu,. I thus obtained two spectral lights widely separated from each other. The source of light was an incandescent electric lamp of 200 candle-power. This was used in preference to the arc-lamp, the light from which cannot be maintained constant, which radiates a very large amount of heat-rays. The incandescent lamp was placed inside the lantern and a slightly divergent beam was allowed to fall on the leaf for a definite length of time by manipulating a shutter. The interposition of the colour-screen and a parallel-sided glass trough filled with alum-solution eliminated the heat- rays. The room was kept in perfect darkness. The Effect of Red and Blue-violet Rays on Transpiration The mode of procedure in the following experiments was : (i) the observation of the normal rate in darkness ; (2) observation of the effect of exposure to blue-violet light for five minutes ; (3) observation of recovery which was practically complete in the course of twenty minutes ; (4) the effect of exposure to red light for five minutes ; (5) the observation of recovery to normal after twenty 112 CHAP. VII. VARIATION OF TRANSPIRATION minutes ; (6) the effect of exposure to blue-violet light for the second time. A complete cycle of operations was thus carried out with a particular leaf. In other cases the order of pro- cedure was reversed, that is to say, the observation was taken first with red, and afterwards with blue-violet light. The following is the detailed account of a typical experi- ment. The normal transpiring activity of a leaf of Thun- bergia in the dark was found to be 45. On exposure of the more sensitive lower surface for five minutes to blue- violet light, the rate of transpiration ttnderwent a diminution to 36, or a variation of — 20 per cent. On the cessation of exposure, the normal rate of 45 was restored in the course of twenty minutes. Red light was next applied for five minutes, and this caused an enhancement of rate to 59, or a variation 0/31 per cent. After an interval of twenty minutes, recovery was nearly complete, the rate being 42. Exposure to blue-violet light was resumed for five minutes ; this caused a depression to 33, i.e., a variation of ~2i per cent. ; this is practically the same as the variation of — 20 per cent, obtained at the beginning of the series. The table on p. 113 gives the results obtained with three other specimens of Thunbergia and one specimen of Nauclea. In all the cases given above, it was invariably found that the blue- violet light, which is effective in inducing a retardation of growth, also caused depression in trans- piration ; red light, which is ineffective in retarding growth, acted like a sub-minimal stimulus, inducing an enhancement of the rate of transpiration. These results show that the average depression under blue-violet light was about — 36 per cent., the mean acceleration under red light being about + 68 per cent. I also experimented on the effect of stimulation of the less excitable upper surface of the leaf : curiously enough, this gave responses similar to those obtained by mechanical stimulation of the two sides, but reversed. In this experi- EFFECT OF CARBONIC ACID 113 ment, the blue-violet light acting on the upper side caused an acceleration of transpiration, while the red induced a depression. The effects produced were, however, feeble compared to those induced by the stimulation of the more excitable lower side : the increase of transpiration caused by blue-violet light was +4*5 per cent, of the normal, the depression by red being 9 per cent. Table XVI. — Showing the Variation of Transpiration under Red and Blue-violet Light for Exposures of Five Minutes Specimen Light Transpiring activity Percentage of variation -h 75 per cent. — 29 ,, ' I. Thunbergia Normal Red light Blue-violet 24 42 17 2. Thunbergia Normal Red light Blue-violet M 31 25 -!- 50 per cent. — 27 ., 3. Tliiinbcrgia 1 " Normal Red light Blue-violet 82 iKi 38 ,, We study next the effect of various anaesthetics on transpiration ; of these carbonic acid gas ma\' be taken as the mildest ; chloroform, on the other hand, as the most intense and toxic after long application. The Effect of Carbonic Acid A broad inverted funnel was made to enclose the trans- piring leaf. After measuring the normal rate of transpira- tion of a leaf of Nauclea, a glass jar containing carbonic acid was emptied over the funnel. This increased the 114 CHAP. VII. VARIATION OF TRANSPIRATION transpiration almost three-fold, from the normal 28 to 70, after the application of the gas. Continued application of the gas, however, caused a slight depression compared with the normal. In another specimen of the Nauclea leaf, the first effect was an enhancement from the normal 106 to 133 ; continued action of the gas induced a depressed activity of 100. I may here describe the remarkable effect of moderately high temperature in modification of the above effect. The result described above was in winter, in January of the present year. The experiment was Fig. 35 Effect of Carbonic Acid Gas in Enhancing the Rate of Transpiration Fig. 35. Effect of Ether ; Preliminary Enhancement followed by Depression repeated in May, when the room temperature was 32° C. The preliminary effect of enhancement was now practically absent, and the rate of transpiration became depressed from the beginning under the action of the gas. Thus in a given experiment with Nauclea the normal rate of 200 was depressed to 163 in the course of a few minutes. The enhanced transpiration under carbonic acid is shown in a record (fig. 34) of the balanced Transpirograph, in which the balance is upset in an upward direction. The Effect of Ether-Vapour The vapour of ether was applied in a manner similar to the above. The immediate effect was an enhancement EFFECT OF CHLOROFORM 115 of transpiration as seen in the upset of the balance upwards, followed by depression under the continued action of the anaesthetic as seen in the down-curve (fig. 35). The Effect of Chloroform Finally, I studied the effect of chloroform-vapour on the transpiring lamina. This is seen to induce a great depression of the rate, as indicated by the upsetting of Fig. 36. Effect of External and Internal Application of Chloro- form in depressing Transpiration the balance downwards (fig. 36). In another experiment, dilute chloroform was applied to the cut end of the petiole by a side-tube. This internal application also caused a great depression of transpiration, as seen in the right-hand record. The results of applications of the anaesthetic to the lamina and to the petiole are thus identical. Summary Transpiration and its induced variations can be auto- matically recorded by the Micro-Transpirograph. Plasmolytic agents applied at the cut end of the petiole induce a diminution of transpiration of the leaf. Il6 CHAP. VII. VARIATION OF TRANSPIRATION Application of feeble electric stimulus to the lamina causes an enhancement, while stronger stimulus induces a depression or arrest of transpiration ; the after-effect of a moderately strong stimulus is often an enhancement of transpiration above the normal. Mechanical stimulus of moderate intensity applied to the upper surface of the leaf induces an enhancement of the rate of transpiration ; the same stimulus applied to the more sensitive lower surface causes a depression of the rate. The activity of transpiration is depressed when the leaf is placed in a field of rapidly alternating electric force. Electric waves also induce depression. Statical electric induction, both positive and negative, enhances the rate of transpiration. Rise of temperature of the leaf caused by thermal radiation enhances the rate. The effect of light is complex on account of the presence of two antagonistic factors : of heat-rays which enhance transpiration, and of light-rays which, acting as a stimulus, retard it. The rays at the two extreme ends of the spectrum affect transpiration in opposite ways. The action of blue- violet light on the under side of the leaf causes diminution ; that of the red rays causes enhancement. The action ol light on the upper surface of the leaf induces an effect opposite to that of its action on the lower surface. Carbonic acid induces an enhancement of transpiration ; long-continued action induces a depression. Ether induces a preliminary enhancement followed by depression. Chloroform depresses the transpiring activity of the lamina. The same effect is induced by application of dilute chloroform to the cut end of the petiole. The above experiments offer independent proof that transpiration is, essentially, not a physical but an active physiological process. CHAPTER VIII THE DIURNAL VARIATION OF TRANSPIRATION Diurnal variation of transpiration in plants with roots — Diurnal varia- tion after removal of the root — The Radiograph — Diurnal variation of temperature and of light — Balancing evaporation against trans- piration— The Differential Balance — The optimum-temperature for transpiration — Summary. Having studied the effect of physiological variations on transpiration, we may attempt to ascertain if the rate of transpiration undergoes a daily variation. Should this prove to be the case, it will be necessary to determine the external changes to which this variation is due. For the purposes of this investigation it is necessary to obtain a continuous record of the transpiration for twenty-four hours. The self-recording Transpirograph, already described, may be used for this purpose, taking the precaution of reducing its sensitiveness to a considerable extent, for the record would otherwise go off the plate. The sensitiveness may be diminished to any extent desired by increasing the diameter of the tube at the opposite ends of which the plant and the recording float are adjusted. The apparatus was fixed in a place free from vibration, the leaves being exposed to the light of the sky, but not to direct sunlight. Two separate records with two specimens of Chrysanthemum were taken simultaneously on the same plate, which was allowed to fall down vertically at an uniform rate by means of a clockwork. It was not necessary to move the plate laterally for obtaining the time-record, since this was secured by the dots in the record produced by the oscihation of the recording plate at definite intervals of half an hour. The distance between Il8 CHAP. VIII. DIURNAL VARIATION OF TRANSPIRATION the successive dots then affords a striking indication of the relative rapidity of transpiration at different periods of the day. The general similarity of the two records affords strong evidence that the result is not accidental, but is due to similar physiological changes in both. Diurnal Variation of Transpiration in Plants with Roots The record for twenty-four hours obtained with Chrysanthemum with root is given in fig. 37. The vertical • • • • 1 1 • • 10.A.M. 1P.M. ^RM. * i 10 P.M. i . . . . 10 AM. Fig. 37. The Record of Diurnal Variation of Transpiration {Chrysanthemum) record is reproduced as horizontal for convenience of in- spection. The successive dots, as already stated, are at intervals of half an hour, and the enhanced rate of trans- piration is seen in the widening of the spacings. The upper record represents the transpiration from 10 a.m. to 4 P.M., i.e., for six hours, and the lower record from 4 p.m. to 10 A.M. next morning, that is to say, for eighteen hours. The distance covered is the same in the two records, hence the average transpiration between 10 a.m. and 4 p.m. was three times quicker than that between 4 p.m. and 10 a.m. We also find that the maximum transpiration occurred at 2 P.M., which was also the thermal noon, or the period of temperature maximum ; the minimum transpiration coincided with the temperature minimum between 4 and AFTER REMOVAL OF THE ROOT II9 5 A.M. The rate was I -7 c.c. per hour at 6 A.M. ; it increased with the rise of temperature; at midday it was 9-8 c.c.; the maximum temperature at or about 2 p.m. was also the period for maximum transpiration, which was 12-9 c.c. After this the temperature fell and the rate of transpiration also declined; at 4 p.m. it had fallen to 9-8 c.c; the minimum transpiration of i c.c. was attained at or about 5 a.m. The maximum transpiration at thermal noon was thus about thirteen times that at thermal dawn. Diurnal Variation after Removal of the Root The record was continued for the next twenty-four hours, but now after the removal of the root. We observe a diurnal variation similar to that in the last experiment, but with a general enhancement of the rate. Thus the ratio of the maximum transpiration of the plant with the root and without it was 12 -9: 22 -3. The ratio at the minimum temperature at 5 a.m. was 1:1-5. The trans- piration of the shoot was thus increased to about i -7 times after the removal of the root. The table on p. 121 gives the rates of transpiration for twenty-four hours of the plant with and without root. The curves in fig. 38 also show the relative rise and fall of the rates in the two cases ; the lower curve with the root, and the upper curve after the removal of it. The rate of transpiration at every hour of the day and night was relatively greater after the removal of the root. What, now, is the cause of this difference in transpiratory activity ? The answer to this question will also solve the difficulty concerning the disparity between the amount of water absorbed by the root and the power of the stem to conduct and the leaves to transpire it. That this is regarded as anomalous, is clear from the following quotation : ' We may imagine that the water was forced up by root-pressure. . . . But the question at once rises as to 120 CHAP. VIII. DIUKNAL VARIATION OF TRANSPIRATION whether the amount of water supphed by the root is approxi- mately enougll to replace what is lost by transpiration. Some experiments carried out by Sachs (1873) are worthy of consideration on this latter point. He compared the amount of sap given off in a definite time from the root of a herbaceous plant, with the amount sucked up by the shoot, whose cut end had been submerged. A root-stock Fk;. 38. The Diurnal Curve of Transpiration The lower is the record of Chrysanthemum with root ; the upper, of the shoot without root. of Nicotiana latissima gave off about 16 c.cm. of sap in five days, but its shoot absorbed 200 c.cm. A similar disparity was exhibited in other cases also. Further, it is very improbable that the secretory capacity of the root is sufficient of itself to compensate for loss of water due to transpiration.' ^ The apparent anomaly arises from the erroneous supposition that transpiration from leaves is merely a phenomenon of physical evaporation. It has been shown, ' Jost, Playii Physiology, FInglish translation, p. 63. AFTER REMOVAL OF THE ROOT 121 however, that it is effected by the activity of living cells, not only of the terminal layers from which excretion actually takes place, but also of a system of cells extending throughout the plant from the absorbing root to the ex- creting leaf. There is thus a co-ordinated physiological mechanism such that each region of the plant controls and is controlled ])y the rest. Examples of this have already been described, where drought, by depressing the cellular activity, affected not only the absorption by the root, but also the conduction of sap through the stem and the trans- piration from the leaves. Thus an increased absorption in one region affects the rate of ascent or of excretion in a different region. The root-hairs by their attenuated channels offer great resistance to the inflow of water into the plant. Consequently the removal of the root accounts for the enhancement of the quantity of water absorbed, as in Sachs's experiment, and of the transpiration from the leaves, as seen in Table XVIL ; for the root (on which the supposed root- pressure depends), far from increasing the rate of ascent, actually impedes the flow of sap. T.'^BLE XVII. — Showing the Absolute Rate of Transpiration for 24 Hours in Chyysanthemum, with and without Root Rate of transpiration Rate of transpiration Time Time With root Without root With root Without root 6 A.M. I -7 C.C. I -g C.C. 6 P.M. 7-1 C.C. 10-3 CO. 7 „ 2-3 ,, 2-7 ,, 7 „ 5-9 ,, 8-5 „ 8 „ 3-4 „ 4-2 ,, 8 ,, 4-8 „ 6-4 „ 9 ,, 4-6 ,. 5-8 „ 9 •,, 3-8 „ 4-9 „ ID ,, 6-0 ,, 8-6 ,, 10 I, 3-0 ,, 3-6 „ II ,, 7-7 ,, 12-2 ,, II ,, 2-4 ,, 2-8 „ Noon 9-8 „ 15-6 „ Midnight 2 -o ,, 2-5 ,? I P.M. II-8 ,, 20-2 ,, I A.M. 1-7 „ 2-4 „ 2 ,, 12-9 ,, 22-3 ,, 2 ,, 1-5 „ 2-3 „ .3 ,, I2-0 ,, 20-5 ,, 3 ,, 1-4 „ 2-2 ,, 4 ,, 9-8 „ 17-8 „ 4 ., I -2 ,, 1-8 „ .5 „ 8-5 „ 14-2 „ 5 „ I -o ,, 1-5 „ 0 ,, 1 7-1 „ IO-3 „ 6 ,, 1-2 , , 1-6 ,, 122 CHAP. VIII. DIURNAL VARIATION OF TRANSPIRATION The Radiograph We shall next attempt to determine the cause of the diurnal periodicity of transpiration. We have here two variables, namely, the daily variation of temperature and the recurrent change of light and darkness. The daily variation of temperature was recorded by a thermograph placed near the transpiring leaf : but there was, unfortu- nately, no apparatus available for the continuous record of the variation of light. This difficulty was, however, overcome by an apparatus which I have devised for the automatic record of variations in the intensity of light. ^ Selenium exhibits a diminution of resistance under light, and the increasing deflection given by a galvanometer in circuit with a battery of voltaic cells gives an indication of the increasing intensity of light. But the continuous passage of a current gives rise to a counter electromotive force, and the selenium-cell then becomes unreliable. For overcoming this difficulty, the photo-electric cell was placed in the fourth arm of a Wheatstone-bridge, so that there was no deflection in the galvanometer under condition of balance in darkness. A vertical tube projects over the selenium-cell, which is closed with an electro-magnetic shutter. The battery of cells is normally cut off from the circuit, which is closed at definite intervals by a clockwork which actuates three keys in succession. By these means the battery is closed, the shutter of the selenium-cell is opened, and the deflected index of the galvanometer is recorded on a moving piece of paper. The keys are then automatically opened, and the process repeated at definite intervals of time, which may be varied from five minutes to an hour. I reproduce curves of diurnal variation of temperature and of light taken in March (fig. 39), which will give a general idea of the hourly changes. In summer the light 1 The detailed account of the Radiograph will be given in vol. iii. of the Transactions, Bose Institute. THE RADIOGRAPH 123 appears about an hour earlier, and disappears an hour later ; in winter the day is shorter by about two hours. The temperature-curve shows a steep rise, attaining its maximum at the thermal noon, about 2 p.m. ; the fall is more gradual, and the minimum temperature is attained between 5 and 6 a.m. The above is true in settled weather Fig. 39. The Curves of Diurnal Variation of Light and of Temperature conditions ; in unsettled weather there are fluctuations in the diurnal curve. As regards light, there is a very steep rise in intensity during the forenoon, the maximum being attained at noon ; the thermal noon, as already stated, is later by about two hours. There is a steady decline in intensity from noon to 4 P.M., after which the fall is abrupt till the total dis- appearance of light after 6 p.m. Comparing the transpiration-curve with those of light and temperature, we find that the effect of light is practi- cally negligible. The maximum light is at noon, but the 124 CHAP. VIII. DIURNAL VARIATION OF TRANSPIRATION maximum transpiration occurs two hours later, at the thermal noon. The similarity of the curves of transpira- tion and of temperature is, moreover, very striking. This proves that the diurnal variation of transpiration is mainly due to the periodic change of temperature. The question next arises as to whether the increase of transpiration with the rise of temperature is simply due to increased evaporation, or whether a physiological element also enters into the" problem : for rise of temperature en- hances not only evaporation but physiological activity also. Hence it becomes necessary to distinguish the effect of one factor from that of the other by some discriminating test. Evaporation no doubt helps indirectly in the uni-directioned ascent of sap by maintaining the turgor-gradient. But the independent factor of physiological activity in trans- piration may exhibit a course which is not exactly parallel to that of evaporation. The Differential Balance It is possible to discriminate between evaporation and transpiration by balancing one against the other. This is done by placing the transpiring leaf on one pan of a balance,, say the right, an equivalent area of water-surface being placed on the left pan for evaporation. It has been shown (p. 87) that the equivalent area of water is about one-fifth that of the leaf-surface of Thunbergia. But for our present purpose it is necessary to obtain not an approxi- mate but a very exact balance. This is secured by the Surface-Variator which will be presently described. By this adjustment the loss by evaporation from the water- surface in the one pan is made to balance exactly the loss by transpiration from the leaf placed in the other pan. If transpiration be a simple phenomenon of evapora- tion, then the loss at the two pans of the balance will be equally affected by variations of temperature, and the index of the balance once adjusted to zero will always THE DIFFERENTIAL BALANCE 125 remain there. But the index is found to be upset in one direction or the opposite according to the variation of Fig. 40. The Differential Balance The Surface Variator, c, seen on the left pan of the balance. A vertical wire from c passes through a guide-hole in the bent piece of metal, p, the upper pan containing shots for trans- ference to c. The right pan contains the transpiring leaf l, mounted in a vessel with a side-tube with an oil-valve, g, plate for recording movement of index r of the Differential Balance. On the right-hand smoked glass is recorded the variation of temperature. temperature. This proves that transpiration is not the same as evaporation, but that an additional physiological factor is involved in the process. The Differential Balance is shown in fig. 40. On 126 CHAP. VIII. DIURNAL VARIATION OF TRANSPIRATION account of the various sizes of the leaves and their different physiological condition, it is not easy to obtain an exact balance between evaporation and transpiration. This difficulty has been overcome by the device of the Surface Variator, which consists of an inverted cone floating in a beaker of water : the area of the evaporating surface may be continuously varied by varying the depth of immer- sion of the inverted cone used as a sinker. The cone is sunk by the addition of small shots. After obtaining a balance of weights on the two sides of the balance, the shots, which are kept on the small upper pan, are trans- ferred to the sinker. The balancing thus remains the same, the change produced being only in the area of the evaporative surface of water. The exact balancing of transpiration against evaporation at any definite tempera- ture may be secured by this contrivance without any difficulty. The long index records any variation in the balance throughout twenty-four hours. The record is taken on a small glass plate which oscillates to and fro at intervals of half an hour. The record of diurnal variation of temperature is also taken on a second plate ; for this the usual metallic thermometer is employed. For pre- vention of disturbance of the index by air-currents, it is necessary to place a cover over the apparatus. For this a glass cover is not suitable, since it interferes with the free evaporation and transpiration ; fine netting was therefore substituted for glass in the cover. The whole apparatus was placed in a large greenhouse. The record of the differential result of transpiration balancing evaporation is given in fig. 41. The trans- piration from a leaf of Thunbergia was exactly balanced at 5 P.M. The index did not, however, remain in the zero position, but drifted to the left till next morning, indicating a depression of transpiration compared with evaporation. Comparison with the thermographic record brought out the interesting fact that this relative depression of trans- piration occurred during the fall of temperature. When THE DIFFERENTIAL BALANCE 127 the temperature began to rise next morning, the index began to return to zero. As the temperature rose further, the balance became displaced once more, but this time to '••.. "-