Alden Springer CRAFTS, born June 25, 1897, at Fort
Collins, Colorado. A.B. University of California 1927; Ph.D.
Univ. of California 1930. National Research Council Fellow
1930-31 at Cornell University. Asst. Botanist, Calif. Agri.
Expt. Station, Davis, 1931-36; Asst. Botanist and Asst. Prof,
of Botany, Univ. of Calif, at Davis 1936-39; Associate Botanist
and Assoc. Prof, of Botany 1939-46; Botanist and Prof, of

Botany 1946 . Guggenheim Fellow July 1 to December 31,

1938; Research Fellow in Biology, Harvard University
July 1 to December 31, 1938; Fellow AAAS 1940-—. Mem-
ber A.A.A.S.; Bot. Soc. Amer. ; Amer. Soc. Plant Physiol.;
Calif. Bot. Soc; Amer. Soc. Range Management. Sect'y
Western Sect. Amer. Soc. Plant Physiol. 1937-39, Vice Chair-
man 1939-40, Chairman 1940-41. Visiting Professor, Puerto
Rico Agri. Expt. Sta. July 1, 1947-June 30, 1948. Honorary
Societies, Sigma Xi, Phi Sigma, Alpha Zeta, Phi Beta Kappa,
Gamma Alpha.

Herbert Bashford CURRIER, born Oct. 16, 1911 at Rich-
wood, Ohio. B.S. Ohio State University 1932, M.S. Utah
State Agric. College 1938, Ph.D. Univ. of California 1943.
Teaching Asst. in Botany, Utah State Agric. College 1937-38;
Teaching Asst. in Botany 1938-39, Associate in Botany 1939-
42, College of Agric, Univ. of California, Davis; Research
Chemist, Basic Vegetable Products Co., Vacaville, Calif. 1942-
46; Asst. Botanist, Calif. Agric. Expt. Sta. and Asst. Prof, of

Botany, College of Agriculture, Univ. of Calif. 1946 .

Member A.A.A.S., Bot. Soc. Amer., Amer. Soc. Plant Physiol.,

Sec'y Western Sect., Amer. Soc. Plant Physiol. 1948 .

Honorary societies: Sigma Xi.

Clifford Ralph STOCKING, born June 22, 1913 at River-
side, California. B.S. Univ. of California 1937, M.S. Univ.
of California 1939, Ph.D. University of California 1943. Teach-
ing Asst. in Botany, Dept. of Botany, Univ. of Calif. 1938-40;
Associate in Botany, College of Agric, Univ. of Calif., Davis,
1940-42; Food Chemist, Puccinelli Packing Co., Turlock, Calif.
1942-45; Asst. Botanist and Asst. Prof, of Botany, College of
Agric, Univ. of Calif., Davis, 1946-. Member A.A.A.S., Bot.
Soc. Amer., Amer. Soc. Plant Physiol. Honorary Societies:
Alpha Zeta, Phi Beta Kappa, Sigma Xi.

• A NEW SERIES OF PLANT SCIENCE BOOKS •

edited by Frans Verdoorn
Volume XXI

WATER

in the

PHYSIOLOGY

of

PLANTS

Plate illustrating Hales' "Experiments, whereby to find out the force with
which trees imbibe moisture." — From his "Vegetable Staticks" (ed. 3, 1738).

WATER

IN THE

PHYSIOLOGY

OF

PLA:NrT5

BY

A. S. CRAFT5

Professor of Botany

University of California,

Davis, California

H. B. Currier and C. R. Stocking

Assistant Professor of Botany
University of California

Assistant Professor of Botany
University of California

1949

WALT HAM. MASS., U.S.A.

i

JruolisJieo by me (^nronica .Dotanica Company

First published MGMXLIX

By the Chronica Botanica Company

of Waltham, Mass., U. S. A.

Copyright, 1949, by the Chronica Botanica Co.

All rights reserved, including the right to reproduce

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Made and printed in the U. S. A.
Designed by Frans Verdoorn

PREF ACE

In writing a modern scientific monograph, the writers must choose be-
tween a detailed treatment of the Hterature and a brief exposition of the
facts and principles of the subject. In this volume, we have attempted to
cite most of the relevant literature; in Chapter 1, the broader reviews;
within the subsequent chapters, the more specialized papers. From these,
we have repeated the minimum of material essential to a connected discourse
on the subject, feeling that interested students will refer back to the original
papers for details.

Thus, whereas this is intended as a reference work, it is hoped that its
reading will not be so tedious as to preclude its use in the classroom. If
any part seems needlessly long, it is because we deem the material of particu-
lar concern to certain workers in the field, or because it pertains to our own
research. For this, our only excuse is that one usually reports most accu-
rately that subject in which he is personally interested.

One further choice that must be made in treating so broad a subject is
that of vocabulary. One could use the loose and rather indefinite termi-
nology of two or three decades ago; or he may employ the more accurate
wording of the modern plant physiologist; or he might adopt the highly
technical language of the physical chemist. Again we have compromised.
This volume has been written for use by present day botanists and plant
physiologists ; hence, we have used their language. At the same time, we
have cited several recent treatises that employ, almost entirely, the vocabu-
lary of physical chemistry. We realize the need for more accuracy in the
definition and use of technical terms ; also the desirability of a universal
language for all scientists. On the other hand, the writing of a monograph
for a certain group in a language with which they are not familiar is futile.

If the language of physical chemistry is to be adopted by physiologists,
and most modern students will agree that it should be, two changes must be
made. First, physical chemistry must become a required course for all
undergraduates in physiology, and its terminology should be introduced
and used relatively early in their careers. Second, physical chemists must
develop a deeper appreciation for certain fields of physiology which, up to
the present, have been somewhat or even totally neglected. Although many
of the living systems with which the physiologist deals may be so complex
as to offer little hope for thermodynamical analysis by present methods,
their tremendous importance with respect to the well being of man, both
through his own physiology, as well as that of the plants and animals upon
which he depends for food and clothing, demands constant and sympathetic
study. And the exact vocabulary of physical chemistry must be adapted to
these complex functions if it is to be of use in their study. Though the
thermodynamicist may be content to treat only the initial and final states
of a system, the physiologist is interested in every mechanical detail of its

Crafts et al. — viii — Water in Plants

operation, and his studies must deal with the description as well as the
mathematical aspects of its functioning. Ultimately, all these problems must
submit to analysis in terms of the molecular mechanics of the systems in-
volved.

With regard to the above problem, we hope that this volume will serve
two purposes. For plant workers, we trust that it will develop an apprecia-
tion for the exact analytical methods of the physical sciences. Much of the
progress in the field of water relations has resulted from use of such methods.
For the physical chemist, on the other hand, we hope to have presented a
challenge to broaden, if need be, his view of the physical universe to en-
compass those many living systems upon which his very existence depends.
There should be some common ground where these two may meet to work
out their common problems for the mutual benefit of all. And if such co-
operation between fundamental and applied scientists will lead to a warmer
feeling of appreciation by both for their common interests, much good will
have been done. The field of plant water relations presents many problems
upon which such cooperative effort may be profitably spent. We hope that
our volume may present these problems in such a way that many workers
will be stimulated in their study.

In conclusion, we would like to express our appreciation to our many
friends who have contributed to the writing of this monograph. Through
conferences, discussions, and, in some instances, almost daily contacts, they
have helped clarify some of the intricate problems involved in plant water
relations. For reading parts of the manuscript we are especially indebted
to Dr. L. E. Davis ; for use of manuscripts, which at the time of writing
were not yet published, we thank Dr. B. S. Meyer and Mr. T. C. Broyer ;
for counsel and advice in the development of the concepts of osmosis and
osmotic pressure we express our appreciation to Drs. N. E. Edlefsen,
F. A. Brooks, Max Kleiber, H. A. Young, H. G. Reiber and R. B.
Dean. Translations of many foreign papers were kindly put at our dis-
posal by Dr. F. J. Veihmeyer and Mr. T. C. Broyer. For encourage-
ment throughout the preparation of the manuscript and aid in its final or-
ganization we are grateful to Dr. W. W. Robbins.

This book has been produced during troubled times. Because publication has
been delayed, many current papers are not cited in the text. Some of these we are
reporting here in an effort to bring our Bibliography up-to-date.

Workers on the properties of liquids and solutions are in general agreement that
the unusual behavior of water results from coordination of its molecules by hydrogen
bonding. Assuming a bonding energy of 4.25 K cal. per mole, Taft and Sisler (1947)
calculate that, of the energy absorbed upon heating, 11 per cent is utilized in breaking
bonds during melting of ice, 16 per cent is used raising the temperature from the
melting to the boiling point, and "JZ per cent goes in the vaporization process.
Weissler (1949), using the velocity of sound at different temperatures to determine
coordination, concludes that water undergoes a decrease in association of about 7.2
per cent between 0°C. and 100° C. Sound waves will detect molecular aggregates that
are stable for 10"*^ seconds. A previous value of 13.2 per cent was found using Raman
spectrum analysis. The difference is due to the fact that the latter method detects
aggregates that are stable for only lO"'^'* seconds.

In contrast to Taft and Sisler, Searcy (1949) calculates a value of 6.4 ± 0.5
K cal. for the H-bond energy in water. He concludes that repulsive as well as at-
tractive forces contribute to the dipole energy.

Using a new formula to determine an index of association in liquids, Parshad
(1947) has calculated values between 240 and 325 for a series of non-polar compounds;

Water in Plants — ix — Preface

values of 403 to 745 for some common associated organic liquids ; and a value of 5010
for water. The theory that liquids contain points of abnormal coordination (holes)
within their structure is receiving continued support and many calculations are being
made on the activation energy required for movement of molecules from position to
position (flow). Studying thermal conductivity of liquids, Palmer (1948) finds that
in associated liquids H-bonds assist in the collisional transfer of heat. This is brought
about by (1) causing orientation of the molecules in the direction of flow, and (2) as-
sisting transfer by rupture of bonds at the high-temperature end and reforming them
at the low-temperature end. If the entropy of vaporization can be considered a measure
of H-bonding, then in water 80 per cent of heat transfer is by this mechanism.

As the picture of water structure becomes clearer, many of the anomalous proper-
ties of solutions are being elucidated. Swinging from early assumptions, based on
studies of dilute solutions, that all solutions are perfect and that apparent deviations
result from association or compound formation, physical chemists went to the other
extreme and proposed that all solutes are completely ionized with abnormal behavior
resulting from the interaction of force fields (Debye and Huckel). Now a middle
course wherein ionic and molecular interaction as well as chemical bonds are given due
consideration seems best. With the introduction of spectrum analysis distinctions can
be made and the type of bonding identified. Gradually the relations between the
thermodynamic properties and the molecular structure of solutions are being studied,
and concentrated as well as dilute solutions are being given consideration (Redlich,
1949). The literature on the properties of solutions is too large to consider here and
the reader is referred to the Journal of Chemical Physics, the Journal of Physical
Chemistry and Chemical Reviews as fertile sources.

Coordination of water is reflected in the high internal pressure or cohesional force
which this compound exhibits. Temperley (1947) has shown that liquid water can
support tensions up to 60 atmospheres in glass tubes. Using air-free water in Berthelot
tubes Scott et a7. (1948) could develop a tension of only 32 atm. Though these values
differ appreciably, the latter is sufficient to explain the flow of sap to the tops of the
tallest trees. Hydration phenomena have also received attention. For example, Simha
and RowEN (1948) conclude that, in systems containing moist cellulose, silk, and wool,
behavior at low moisture levels can be explained in terms of adsorption theory ; at
high moisture levels the systems can be analysed in terms of a theory of polymer-liquid
mixtures ; in between the two extremes a transition occurs.

Although osmotic pressure measurements continue to serve in the determination
of the molecular weight of large molecules, little change has occurred in the theoretical
aspects. BuRSTROM (1948) has attempted an evaluation of the significance of turgor
of the living cell. Departing from the classical definition "turgor is the pressure acting
from inside the cell on the cell wall" (p. 58, last line), Burstrom by devious analysis
arrives at the expression T = O — E where T = turgor pressure; O = osmotic
value of the cell sap ; and E = osmotic value of the external medium. To us this T
is simply a net or corrected osmotic pressure. It is shown as such in Burstrom's Fig.
2 (page 61). It could equal our turgor pressure only when the cell is in equilibrium
with the external solution of concentration E. As in several previous cases (Crafts,
1943), we feel that this redefinition of terms is not justified. It leads to such con-
fused statements as — "T increases as the cell loses water — " (page 62, lines 1 and 2),
"turgor — expresses a pressure realized in the cell — ," "This deduction of turgor pres-
sure— exactly shows the pressure actually exerted from within the cell on the cell
wall" (p. 63), and "The turgor pressure must decrease when a cell absorbs water and
the wall pressure increases" (p. 64).

This dilemma seems to arise from a failure to appreciate that (in our symbols)
when OP = DPD, T = 0, when OP = TP, DPD = 0, and at all other states OP
= DPD + TP. While these relations are most easily examined at equilibrium, a
state of flux does not invalidate them. And the definitions which they imply are
simple, clear, and in accord with classical considerations of osmotic pressure.

In the field of cell water relations a number of valuable contributions have ap-
peared. In a general discussion of swelling and shrinking (Trans. Faraday Soc,
1946) water-holding forces in biological systems received critical consideration. The
nature of vacuolation of protoplasm following certain types of swelling is made more
clear by Faure-Fremiet. Seifriz presents convincing arguments for a distinction
between osmotic pressure and imbibition, and discusses swelling and shrinking phe-
nomena in the light of known protoplasmic structure.

In a subsequent general discussion on interaction of water and porous materials
(Faraday Soc. Discussion, 1948), Bennet-Clark restates his belief that water secre-

Grafts et al. — x — Water in Plants

tion from the cytoplasm into the vacuole of the plant cell actually occurs. In consider-
ing Levitt's (1947) claim that secretion values reported are thermodynamically un-
sound, Bennet-Clark casts doubt on the validity of the value used by Levitt to repre-
sent water permeability in calculation of water velocity.

LuNDEGARDH further identifies an active water mechanism in roots with the ab-
sorption of salts from the culture medium. As in previous work, he assumes that
"anion respiration" provides the energy.

Recent findings have added to our understanding of auxin-induced water uptake
by cells. In an important paper Levitt (1948) offers convincing evidence that auxin-
enhanced water absorption by potato discs is not active water uptake, as suggested by
several investigators, but rather a result of altered plasticity of the cell wall. The
manner in which auxin may increase wall plasticity is not understood but Kelly
(1947) has shown that it is an aerobic process in Avena coleoptile segments. Audus
has critically reviewed the problem of cell elongation (Biol. Rev., 1949).

Studies on water secretion and transport by Nitella cells have been extended by
Osterhout (1949). That the pumping mechanism demonstrated in Nitella under
somewhat arbitrary conditions could operate in certain kinds of cells seems validated
by a theoretical consideration by Franck and Mayer (1947) of an osmotic diffusion
pump.

The absorption of water by root systems is being given detailed consideration by
Kramer, whose book on the subject is in press. Frey-Wyssling (1941) has demon-
strated guttation under conditions of high water absorption and low transpiration in
several woody species, and Daniel (1949) observed xylem exudation from excised
roots of several coniferous species. Studies on the effect of certain salts on increasing
root pressure and tissue resistance to infiltration in sunflower, tomato, and dahlia were
made by Volk (1944).

Hagan (1949), by measuring water intake by excised root systems of sunflowers
growing in moisture-deficient soil, has verified the autonomic diurnal cycle of root
activity studied by Grossenbacher (1938). The diurnal cycles continued for about
6 days but were terminated if the roots were aerated with N2 or CO2. Similar cycles
observed in roots washed free of soil indicate that the activity is a function of the root
and not of its environment. Such roots in water exuded water during the day and
withdrew it during the night. Hagan attributes this activity to changing hydration of
the cytoplasm.

Mitscherlich (1947) has pointed out that water requirements of plants follow
the law of plant growth. Water requirements are high for plants having short vegeta-
tive periods and they decrease with increasing nutrient supply. More specific data
on the effects of potassium and calcium on the water relations of plants were given
by Woestmann (1942). Potassium induced water storage, water uptake, and trans-
piration, probably through its effect of promoting hydration of the protoplasm. Calcium
diminished protoplasmic hydration and consequently lowered protoplasmic permeability
and hence water absorption. Water storage and transpiration were reduced. Plants
deficient in nitrogen showed decreased transpiration, a failure of guard cells to func-
tion normally, and an increase in cold resistance and protoplasmic viscosity (Gessner
and Schumann, 1948).

Wadleigh (1945) has made a new approach to the problem of plant-soil water
relations. He devised a method for integrating the variables that account for the total
soil-moisture stress against which the plant absorbs water. When leaf elongation of
cotton plants was expressed empirically as a second degree function of soil-moisture
stress, values close to IS atmospheres for the stress inhibiting leaf growth were calcu-
lated (Wadleigh and Gauch, 1948). Additional data indicating that under good
conditions of soil aeration and fertility the rate of vegetative growth increases with
increasing soil moisture within the range from near the permanent wilting percentage
to near soil saturation have been presented by Haynes (1948). He concludes that
corn plants subjected to daily periods of temporary wilting produce small vegetative
growth when under humid conditions. On the other hand, Wilson (1948&) has
shown that tomato stem tips can maintain their highly hydrated state at the expense
of older mature tissues. Thus they can increase steadily in length throughout the
day and night even when the plant as a whole passes through a diurnal cycle with the
water balance on the negative side during the day.

Morton and Watson (1948) conclude that the rate of leaf production by apical
meristem is unaffected by water supply. The number and size of cells per leaf in-
creased with increasing water supply. Net assimilation was greatly reduced by water
deficit.

Water in Plants — xi — Preface

Reviewing plant-water relations, Tageeva (1946) found that wheat plants have
reduced water content as they mature ; upper leaves dehydrate more than lower ones.
Nevertheless, the upper leaves have a greater rate of photosynthesis than lower ones.
Low water supply results in an increase in soluble carbohydrates leading to decrease
in photosynthesis. Eaton and Ergle (1948) find that in certain plants such as cotton,
water deficit may depress carbohydrate utilization to a greater extent than it does
photosynthesis leading to an increased sugar content.

In the discussion mentioned above (Faraday Soc. Discussion, 1948) papers by
VAN DEN HoNERT, LEWIS, FoGG, and Crafts deal with water and food movement in
plants and the relations of leaf cell walls and leaf surfaces. Van den Honert, con-
sidering the role of living root cells, xylem conductors, living leaf cells and the
"gaseous part" (intercellular spaces, stomata, and air layer around the leaf) concludes
that the greatest resistance to water movement is the "gaseous part." He agrees
with Gradmann's (1928) views that this phase may exert limiting influence on water
transport.

Lewis presents further data on the hydrophobic nature of the outer walls of leaf
mesophyll, while Fogg pictures the effects of varying water conditions of underlying
tissues on the nature of the leaf surface.

Crafts, in an analysis of the physical nature of sieve-tube protoplasm, and sieve
tube walls, concludes that the mature, functioning sieve tube is a highly specialized
structure. The optical nature of the walls and their high water content suggest that
they have a loose open structure. The degradation of the sieve-tube cytoplasm at-
tending maturation of these elements results in a loss of their semipermeability. It
is concluded that this also denotes an open mesh structure. It is suggested that the
open structure of sieve-tube walls and cytoplasm constitute specialization favoring a
mass flow of the assimilate stream. Went and Hull (1949) present evidence for a
temperature coefficient of less than 1 for both rate and intensity of sugar transport in
the tomato plant.

The perplexing problem of stomatal behavior was reviewed critically by Wilson
(1948a). Theories proposing that stomatal action can be accounted for by changes in
permeability, changes in enzyme activity, or variation in colloidal hydration were not
considered adequate as an explanation for the observed effects of light, temperature,
and humidity on stomatal aperture.

Literature cited: — Audus, L. J. 1949: Biol. Rev. 24: 51-93. — Burstrom, H. 1948: Physiol.
Plant. 1: 57-64.— Crafts, A. S. 1943: Chronica Bot. 7: 386-8.— Daniel, T. W. 1949: Plant
Physiol. 24: 327-30. — Eaton, F. M., and D. R. Ergle 1948: Plant Physiol. 23: 169-87. — Fara-
day Soc. Discussions No. 3, 1948: 1-293. — Faraday Soc. Trans. 1946, 42B: 1-304. — Franck, J.,
and J. E. Mayer 1947: Archives of Biochem. 14: 293-313. — Frey-Wyssling, A. 1941: Ber.
Schweiz. bot. Gas. 51: 321. — Gessner, F., and M. Schumann 1948: Z. Naturforsch. 3b: 36-41. —
Gradmann, H. 1928: Jahrb. wiss. Bot. 69: 1-100. — Grossenbacher, K. A. 1938: Plant Physiol.
13: 669-76. — Hagan, R. M. 1949: Plant Physiol. 24: 441-454. — Haynes, J. L. 1948: Jour. Amer.
Soc. Agron. 40: 385-95. — Kelly, S. 1947: Amer. Jour. Bot. 34: 521-6. — Levitt, J. 1947: Plant
Physiol. 22: 514-25. — Levitt, J. 1948: Plant Physiol. 23: 505-15. — Mitscherlich, E. A. 1947:
Z. Pflanzenernahr., Dungung u. Bodenk. 38: 202-15. — Morton, A. G., and D. J. Watson 1948:
Ann. Bot. 12: 281-310. — Osterhout, W. J. V. 1949: Jour. Gen. Physiol. 32: 553-66. — Palmer,
G. 1948: Ind. Eng. Chem. 40: 89-92. — Parshad, R. 1947: Jour. Phys. Chem. 15: 761-2. — Red-
LicH, O. 1949: Chem. Rev. 44: 1-5. — Scott, A. F., D. P. Shoemaker, K. N. Tanner, and J. G.
Wendel 1948: Jour. Chem. Phys. 16: 495-502. — Searcy, A. W. 1949: Jour. Chem. Phys. 17:
210-11. — Simha, R., and J. W. Rowen 1948: Jour. Amer. Chem. Soc. 70: 1663-5. — Taft, R. W.
and H. H. Sisler 1947: Jour. Chem. Educ. 24: 175-81. — Tageeva. S. V. 1946: Trudy Inst.
Fiziol. Rastenii un K. A. Timiryazeva 4, No. 1: 161-75 and 176-92. — Temperley, H. N. V. 1947:
Proc. Phys. Soc. (London) 59: 199-208. — Volk, A. 1944: Bodenk. u. Pflanzenernahr. 34: 190-204.
— Wadleigh, C. H. 1945: Soil Sci. 61: 225-38. — Wadleigh, C. H., and H. G. Gauch 1948:
Plant Physiol. 23: 485-95. — Weissler, A. 1947: Jour. Chem. Phys. IS: 210-11. — Went, F. W.,
and H. M. Hull 1949: Plant Physiol. 24: 505-26. — Wilson, C. C. 1948o: Plant Physiol. 23:
5-37. — Wilson, C. C. 1948b: Plant Physiol. 23: 156-7. — Woestmann, E. 1942: Jahrb. wiss.
Bot. 90: 335-81.

July 1949 The Authors

'^j LIBRAR ,

'" '' V MASS. / '>.

ACKNOWLEDGEMENTS

Grateful acknowledgement is made for permission to reproduce certain copy-
righted materials. Our Table 12 comes from Haldane's paper in the Biochemical
Journal ; Figure 41 and Table 3 and a part of Table 4 are from Hober, Physical
Chemistry of Cells and Tissues, courtesy of the Blakiston Co., Philadelphia; Figures
22-27 are from Strugger's Praktikum der Zell- und Gewebephysiologie, Gebriider
Borntraeger, Berlin. The latter also permitted reproduction of (our) Figures 33 and
36, and Tables 21, 22 and 24, appearing in the periodicals Jahrb. f. wiss. Bot. and
Protoplasma. The Canadian Journal of Research has permitted reproduction of our
Figure 10 from a paper by Babbitt. Our Table 17 is taken from a paper by Morse,
published by the Carnegie Institution of Washington ; to the Faraday Society we are
indebted for Table 10 and a portion of Table 4, from papers in the Transactions by
London and Porter respectively; Tables 8, 11, and 13 are reproduced from a paper
by Frazer and Myrick, courtesy The Journal of the American Chemical Society. By
permission of the Alacmillan Co., New York and London, we have copied (our)
Figure 50 from Fogg's paper in the periodical Nature, and Table 36 from Dixon's
monograph, Transpiration and the Ascent of Sap in Plants. The New Phytologist and
Dr. T. A. Bennet-Clark have allowed reproduction of Fig. 42 and Tables 30 and 34.
Figure 12 comes from Hildebrand's Solubility, Reinhold Publishing Co., New York ;
Figure 40 from a paper by Osterhout and Murray, by permission of the Rockefeller
Institute for Medical Research, New York; Tables 14, 15, and 16 from Berkeley,
Hartley and Burton in the Transactions of the Royal Society ; Table 45 after Leick
in Abderhalden's series on biological methods, published by Urban and Schwarzen-
berg, Berlin. Figures 11, 19, and 49 are copied from Meyer and Anderson's Plant
Physiology, copyright Van Nostrand Co., Inc., New York.

Thanks are also due the following for authorization to quote from original publi-
cations: Dr. Matilda M. Brooks; The Faraday Society (Transactions); Journal
of Experimental Biology; Longmans, Green and Co., Inc., New York (Findley, Os-
motic Pressure) ; Reinhold Publishing Co., New York (Hildebrand, Solubility of
Non-Electrolytes) ; The Ronald Press Co., New York (Adolph, Physiological Regu-
lations) ; John Wiley & Sons, Inc., New York (Gortner, Outlines of Biochemistry,
2nd Ed.; Bull, Physical Biochemistry; Heuser, Cellulose Chemistry) ; Williams and
Wilkins Co., Baltimore (Chemical Reviews).

Where other materials not listed above are used in the text, the source is indicated.
Unfortunately the authors were unable to contact a few authors and publishers. To
all whose published data were found useful the writers wish to express their gratitude.

The Chronica Botanica Co., International Plant Science Publishers

*A New Series of Plant Science Books': —

I.MacDougal: Tree Growth (revised ed. in prep.)
2. Grant: Wood Pulp (first ed. out of print, revised

ed., published abroad, available at $8.50)
3.Darrah: Principles of Paleobotany (revised edition

in preparation)
4.Pfeiffer: Experimentelle Cytologie (being re-
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CONTENTS

Preface vii

Acknowledgements xiii

Contents xv

List of Text Illustrations xviii

List of Tables xx

Chapter I
INTRODUCTION 1

Chapter II

STRUCTURE OF WATER: — Introduction — Structure —
Early Theories — The Hydrols — The Faraday Symposium — Later
Work on the Hydrol Theory — "Normal" and "Polar" Liquids —
Molecular Concentration — Modern Studies — X-Ray Analysis —
The Second Faraday Symposium — Current Research — Summary
OF Water Structure 3

Chapter III

PROPERTIES OF SOLUTIONS: — Introduction — The In-
terrelation OF THE Properties of Solutions — The Forces Between
Atoms and Molecules — Molecular Interaction and Water Bind-
ing— Hydration — Mobility of Ions — Summary 18

Chapter IV

OSMOSIS AND OSMOTIC PRESSURE: — Introduction —
Refinement of the Osmotic Pressure Law — Causes for Non-Ideal
Behavior of Solutions — Summary 32

Chapter V

THE MECHANISM OF OSMOSIS: — Introduction (Osmosis

— Osmotic pressure — Diffusion — Diffusion pressure — Diffusion pres-
sure deficit — Turgor — Turgor pressure) — The Kinetic Basis of
Osmosis — Osmosis in Liquid Systems — The Mechanics of Osmosis

— The "Solvent" and "Solute" Pressure Theories — Calculation
BY Indirect Methods — Evaluation of Diffusion Pressure — The
Diffusion Pressure of the Solvent — Summary 43

6660;^'

Crafts et al. — xvi — Water in Plants

Chapter VI

WATER AS A PLANT COMPONENT — INTRACELLU-
LAR DISTRIBUTION OF WATER: — Introduction — Anatomy
OF Cells — Water Content — Binding Forces — Water of Cell
Walls — Protoplasmic Water — Water in Vacuoles — ^ Methods
FOR Determining Water Partition — Equilibrium Between Phases

— Summary 59

Chapter VII

THE OSMOTIC QUANTITIES OF PLANT CELLS: — A

Description of the Quantities and of their Interrelations — Mem-
branes AND Permeability — Permeability to Solutes — Permeabil-
ity TO Water — Permeability of Cell Walls — Osmotic Pressure.
Methods of Measuring Osmotic Pressure in Plants. The Plas-
molytic Method — Limitations of the Plasmolytic Method — Plas-
MOLYTic Method, Experimental Procedure — Further Uses of Plas-
molysis — The Plasmometric Method — > Cryoscopy — E;cpression
of Sap — The Cryoscope — Physical Principles — Freezing-Point
Determination — Vapor Pressure Methods — Osmometric Methods

— Other Methods for Osmotic Pressure — The Magnitude and
Variation of Osmotic Pressure in Plants — Diffusion Pressure
Deficit. Methods of Measuring DPD — The Cell Method — Strip
OR Simplified Method — The Weight Method — Other Methods
FOR DPD — The Magnitude and Fluctuation of DPD Values —
Turgor of Plant Cells — Measurement of Turgor — Intercellular
Movement of Water — Summary 7Z

Chapter VIII

ACTIVE CELL WATER RELATIONS: — Introduction —
Evidence from Plasmolytic and Cryoscopic Measurements — Inter-
pretation OF Sap Expression Data — Plasmolytic Behavior of Cells

— Cryoscopy of Tissues and their Equilibrium Bathing Solutions

— Auxin and Water Uptake — Measurement of Diffusion Pres-
sure Deficits — Evidence from Studies of Frost and Drought Re-
sistance— Evidence from Animal Physiology — Relation to
Solute Accumulation — Relation to Other Plant Functions —
Mechanisms — Summary Ill

Chapter IX

UPTAKE AND MOVEMENT OF WATER IN PLANTS: —

Introduction — Water Absorption — Active Absorption — Passive
Absorption — Methods of Estimating the Availability of Water —
Water Movement — Cohesion Theory — Cohesion and Adhesion —
Liquid Continuity in the Xylem — Dye Penetration — Freezing
of Stems — Transpiration Experiments — Direct Observation of
Xylem — Tensions Developed in the Xylem, Direct Measurements

— Potometer Experiments — "Suction Tension" Measurements —
Newer Metpiods of Estimating Tensions in Xylem — Rate of Water
Movement Through Plants — Quantity of Material Moved and
Area of Transport — Indicators (Salts — Dyes — Radioactive tracers

— Heat) — Variations in the Rate of Sap Flow — Water Balance
and Redistribution — Translocation of Solutes — Summary . 148

Water in Plants

xvii

Contents

Chapter X

WATER LOSS AND WATER RETENTION: — Introduction

— Laws of Evaporation — Evaporation from a Free Water Surface

— Evaporation from Tubes — Factors Affecting Evaporation from
Free Water Surfaces of Uniform Shape and Area (Solutes — Tem-
perature— Dryness of the air — Atmospheric pressure — Wind velocity)

— Transpiration — Methods of Measuring Transpiration — Ex-
pression OF Results — Cuticular Transpiration — Fluctuations in
the Rate of Transpiration — Internal Factors Affecting Trans-
piration — Wall Structure — Physical Resistance to Water Move-
ment— Osmotic Properties — Internal Surface — Stomatal Num-
ber AND Distribution — Methods of Measuring Stomatal Openings

— Genetic Factors — Stomatal Regulation of Transpiration —
Effect of Mineral Nutrients on Transpiration — Influence of
Disease, Sprays, Dusts, and Waxes on Transpiration Rate — Phys-
iological Effects of Water Deficits — Subaqueous Transpiration

— Conclusions — Summary 179

Bibliography 209

Author Index 228

Subject Index 233

i-jH^Wat^

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LIST OF TEXT ILLUSTRATIONS

Figure 1. — Trihydrol molecule as pictured by Sutherland 4

Figure 2. — The tetrahedral water molecule of Pennycuick 8

Figure 3. — The polar water chain 9

Figure 4. — The hexagonal ring structure of water, comparable with the crystal

structure of ice 9

Figure 5. — Diagram indicating the self-ionization of a water chain 10

Figure 6. — Electronic distribution in the simple water molecule 11

Figure 7. — The crystal lattice of ice as pictured by Bernal and Fowler ... 12
Figure 8. — Diagrammatic illustration of the shift from the hexagonal lattice of ice

to a closer packed structure for water 15

Figure 9. — Typical vapor pressure-water content curves for sand and clay showing

the values of the permanent wilting percentage and field capacity for each . . 23
Figure 10. — Curves showing the relation between moisture content and relative

humidity of the atmosphere surrounding cotton, and spruce wood 24

Figure 11. — Apparatus for demonstrating differential diffusion of gases ... 45
Figure 12. — Diagram showing the relations between attractive and repulsive forces

between molecules 47

Figure 13. — Diagram showing the relations of partial and total vapor pressures of

solutions according to Raoult's Law 48

Figure 14. — Apparatus useful in analysing problems of osmosis 49

Figure 15. — Relations among osmotic pressure, diffusion pressure deficit, and turgor

pressure for an ideal osmometer 50

Figure 16. — Calculated and observed values of osmotic pressures of cane sugar

solutions ^'*

Figure 17. — Several types of parenchyma cells found in plants 60

Figure 18. — Protein structure pictured by Frey-Wyssling 67

Figure 19. — A diagrammatic presentation of the relations among osmotic pressure,

diffusion pressure deficit, and turgor pressure as applied to plant cells . . . 73

Figure 20. — Three forms of TP-volume curves for plant cells 74

Figure 21. — The relation between concentration of plasmolyzing solution and degree

of plasmolysis 86

Figure 22. — Plasmolysis types as illustrated by Strugger 87

Figure 23. — Onion epidermal cell showing cap plasmolysis 88

Figure 24. — Tonoplast plasmolysis of onion epidermis 88

Figure 25. — Vacuole contraction in a cell of the fruit flesh of Ligustrum vulgare 89
Figure 26. — Vacuole contraction in an onion epidermal cell having the vacuole strong-
ly stained with neutral red 89

Figure 27. — Vacuole contraction as shown by leaf cells of Elodea canadensis after

staining with neutral red 90

Figure 28. — Press cylinder outfit used to express small amounts of sap from plant

tissue 91

Figure 29. — Construction of cryoscopic apparatus 92

Figure 30. — Calibration curve for the apparatus shown in figure 29 94

Figure 31. — Freezing curve constructed according to recommendations of Mair,

Glasgow, and Rossini 95

Figure 32. — The Barger Halket method for determining the osmotic pressure of

plant sap 96

Figure ZZ. — Osmotic pressure determination by the method of Ursprung and

Blum 96

Figure 34 A. — Diffusion pressure deficit values determined by the volume method 102
Figure 34 B. — Diffusion pressure deficit values determined by the weight method 103
Figure 35. — Diagrammatic illustration of water movement along a gradient of

DPD 109

Figure 36. — Osmotic pressure values of leaf cells of Bergenia cordifolia . . .114
Figure 37. — Seasonal variations in the dry matter content, inexpressible water, and

water expressed as sap after different treatment of leaves of Pinus rigida . . 121

Water in Plants — xix — Illustrations

Figure 38. — The effect of 0.2 M sucrose, 0.2 M sucrose plus 10 mgm. of indole-
3-acetic acid per liter, these concentrations of sucrose and auxin plus 10"^ M.
KCl, these concentrations of sucrose and auxin plus 10"* M. K fumarate, on

changes in the wet weight of aerated potato slices 128

Figure 39. — Effect of auxins on water uptake of potato tissue under sterile and non-
sterile conditions 130

Figure 40. — Apparatus for demonstrating "anaphoresis" 141

Figure 41. — Diagrammatic representation of two membranes, one permeable to cations

only, the other, anion permeable 142

Figure 42. — Electroosmotic flow according to Bennet-Clark and Bexon . . .143
Figure 43. — DPD gradient from the root up the stem of Arthrophytum haloxylon 164
Figure 44. — Diurnal fluctuation in transpiration and in DPD at different heights on

the stem of Arthrophytum haloxylon 165

Figure 45. — DPD measurements on the petioles of squash plants 166

Figure 46. — DPD values of solutions in the petioles of a squash plant in the

laboratory 167

Figure 47. — Arrangement of heating element and thermocouples used by Huber and

Schmidt for measuring slow rates of movement of sap streams 170

Figure 48. — Diurnal variation in rate of sap movement in Larix, Picea, and Fagus 171
Figure 49. — Potometer with growing plant for study of water absorption and loss 186
Figure 50. — Diurnal fluctuation in the wettability of leaf cuticle as shown by change

in contact angle of water drops 188

Figure 51. — DPD of the atmosphere at 20°C as related to relative humidity . . 189
Figure 52. — Relation between atmospheric temperature and relative humidity for an

atmosphere containing .004 pounds of water per pound of dry air 190

Figure 53. — Diurnal fluctuations in DPD of the atmosphere at a station in Wash-
ington, D.C., during a 10-day period in June 1932 191

Figure 54. — Diurnal fluctuations in water loss from rooted Coleus leaves under con-
trolled conditions 192

Figure 55. — The effect of DPD of Chlorella cells upon photosynthesis .... 201
Figure 56. — Experimental conditions for demonstrating subaqueous transpiration 204

Plate illustrating Hales' "Experiments, whereby to find out the force
with which trees imbibe moisture." — From his "Vegetable Staticks"
(ed. 3, 1738) iv

Dutrochet's apparatus "pour mesurer la force de I'endosmose." — Plate
1 from his "Memoires pour servir a I'Histoire Anatomique et Physi-
ologique des Vegetaux et des Animaux" (1837) xxii

Pfeffer's original apparatus to determine the osmotic pressure of so-
lutions.— From his "Osmotischen Untersuchungen" (1877) . . 242

Vignettes on pages xvii, xxi and 227 and 232
from Hales' "Vegetable Staticks".

LIST OF TABLES

Table 1. — Some thermal properties of water and several elements and compounds 4
Table 2. — Physical properties of water and some other common liquids ... 4

Table 3. — Characteristics of molecular interaction 20

Table 4. — Contributions of the three constituents of the van der Waals forces to

intermolecular forces in the liquid state 21

Table 5. — Osmotic pressures of sucrose solutions at constant temperature . . 32

Table 6. — Influence of temperature on osmotic pressure 32

Table 7. — Comparison of osmotic and gas pressures 33

Table 8. — Observed and calculated osmotic pressure values for sucrose solutions at

30° C 34

Table 9. — Osmotic pressure of cane-sugar solutions at 20°C; observed and cal-
culated values 35

Table 10. — Calculated hydration numbers for sucrose solutions at various tempera-
tures and concentrations 36

Table 11. — Osmotic pressure of solutions of cane sugar at 30°C 37

Table 12. — Osmotic pressure of sucrose solutions at 0°C 37

Table 13. — Observed and calculated osmotic pressure values for sucrose solutions

at 30°C 38

Table 14. — Observed and calculated values of the osmotic pressure of sucrose

solutions 3S

Table 15. — Observed and calculated values of the osmotic pressure of a-methyl

glucoside solutions 39

Table 16. — Observed and calculated values of the osmotic pressure of calcium

ferrocyanide solutions at O'C 39

Table 17. — Volume of weight-normal solutions of cane sugar at 0°C . . . . 47
Table 18. — Relation between relative humidity, vapor pressure, and diffusion pres-
sure deficit of atmospheric moisture at 20°C 56

Table 19. — Water content, in per cent of fresh weight, of some common food

materials 62

Table 20. — Walls of the phloem of potato stolon expressed as percentage of total

cross section of the phloem 65

Table 21. — Measurement of adhesion pressure in Beta vulgaris root 84

Table 22. — Longitudinal strips of beetroot (Futterriibe) 14 cm. long, soaked for

various times in water, then overnight in 0.5M KNO3 84

Table 23. — Effect of water saturation on Og values 84

Table 24. — Isotonic coefficients of various salts calculated on a weight molar basis 86

Table 25. — Change in volume of potato tuber tissue cylinders immersed 8 hours in

sucrose solutions of varying concentration at 20°-22°C. Values are based on

normal volume 98

Table 26. — Changes in surface dimensions of epidermal strips from Sedum nicaense

leaves. Values are arbitrary units 98

Table 27. — Osmotic pressure of various tissues determined plasmolytically, expressed

as mols of KNO3 per liter of solution 100

Table 28. — Effect of varying soil moisture supply upon the DPD of plant tissues 106
Table 29. — Osmotic quantities in the hypocotyl of Helianthus annuus seedlings,

determined at normal volume 106

Table 30. — Cryoscopic and plasmolytic measurement of osmotic pressure in vari-
ous plants 112

Table 31. — Description of beets employed in PCD determinations, and periods dur-
ing which tests were made 115

Table 32. — Summarized results of plasmolytic and cryoscopic tests on various lots

of beets 116

Table 33. — Results of deplasmolysis and replasmolysis tests on beetroot tissue.

Atmospheres at 22.5°C 116

Table 34. — Comparison of osmotic pressure values determined plasmolytically and
those determined cryoscopically on various fractions of expressed leaf sap . .118

Water in Plants

XXI

List of Tables

Table 35. — Efifect of killing tissue on the osmotic pressure of expressed sap . .119
Table 36. — Comparison of saps expressed from living and killed tissue .... 120
Table 37. — Osmotic pressure of sap expressed from living vs. killed (by heat)

leaves 120

Table 38. — Osmotic pressure of sap expressed from living as against dead (frozen)
tissue, comparing tvi^o types of beets. Values in atmospheres at 22.5 °C . . . 122

Table 39. — Plasmolysis of beet cells in their own sap 123

Table 40. — Osmotic pressure determinations of tissue cylinders and of their equi-
librium bathing solutions. Atmospheres at 22.5°C 126

Table 41. — Cryoscopy of sap expressed from several marine algae and of the sur-
rounding sea water. Atmospheres at 20° C 127

Table 42. — Osmotic pressures of cell sap from cotton seeds of three ages before and

after immersion in sucrose solutions 132

Table 43. — Freezing points of living tissue, dead tissue, and expressed sap. At-
mospheres at 22.S°C 134

Table 44. — Summary of investigations on rate of sap movement in plants . . . 169
Table 45. — Transpiration of Solidago virga aurea in milligrams per gram fresh

weight per minute. Cut surface renewed hourly 185

Table 46. — Ratios between internally exposed cell walls and external leaf surface 195

Table 47. — Water requirement in Zca as affected by nutrition 198

Table 48. — The effect of decrease in water content on the amylolytic activity and

starch dissolution of sunflower leaves kept in the dark for 18 hours 202

Table 49. — Influence of water absorption on meiosis in Oenothera 203

PLi

F.2.

r.4.

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—

A 1
a.

B
h

J)ej-,rut^ -par Z.'/^l^nc . Grave par JuSrai.w 'J^-riieu .

Dutrochet's apparatus "pour mesurer la force de Tendosmose." — Plate 1 ]rom^ his "Memoires
pour servir a I'Histoire Anatomique et Physiologique des Vegetaux et des Animaux" (1837).

Chapter I
INTRODUCTION

The relation of water to plant cells is at the same time one of the oldest
and one of the most modern of physiological problems. Malpighi, Hales,
Knight, and a host of early plant scientists recognized this important field
of study. Survey of modern literature proves that water relations still
claim the attention of leading plant physiologists. Viewpoints change;
methods improve; still the underlying principles of water absorption and
utilization challenge the researcher. Almost all plant functions involve
water relations in some form or other; provision of adequate water for
maximum growth of plants is the basis of successful agriculture in many
parts of the world.

Writing a concise monograph on water relations is a difficult task for it
involves selecting from an immense literature material relevant to modern
concepts and problems. To be of value, furthermore, such a monograph
should contain original work by the authors or it should involve synthesis
of new concepts from previous publications. Availability of modern ab-
stracting and reviewing agencies renders almost useless a mere compilation
or uncritical review of published articles.

This volume aims to attain both of the above objectives ; it also attempts
to aid students in becoming familiar with contemporary literature in the
field. A balanced treatment of the subject involves a description of the
structure of water and aqueous solutions, a review of the concepts of the
mechanism of osmosis, consideration of the water relations of individual
cells, and finally, analysis of the functions of absorption, movement, reten-
tion, and loss of water by organized plants. It is obvious that space cannot
be devoted to a detailed historical review. Mainly as an aid to students, the
following introduction to source materials is included.

Works of Malpighi (1675), Hales (1738), and Knight (1801) are of historical
interest only. Ideas in their time were confused by failure to clearly distinguish be-
tween the water relations of plants and animals.

Discovery of the microscope stimulated interest in plant structure. The earlier
works on conducting systems are thoroughly covered by Strasburger (1891) and
Haberlandt (1914). Meanwhile Dutrochet (1827), Pfeffer (1877), van't Hoff
(1887 and 1888), and de Vries (1918) laid the foundations for a physical analysis of
cell water relations through their studies on osmosis and osmotic pressure. Work by
Berkeley and Hartley (1906), Morse (1914), Frazer and Myrick (1916), and
Berkeley, Hartley, and Burton (1919) provides a quantitative basis for such analysis.

Theoretical treatment of osmotic systems by Lewis (1908), Haldane (1918),
Bancroft and Davis (1928), and others has materially broadened our concepts while
reviews by Findlay (1919), Meyer (1938), and Ursprung (1938) have marked
progress in this field. Of treatments in physico-chemical texts, those of Washburn
(1921), Lewis and Randall (1923), Glasstone (1940), and Getman and Daniels
(1943) are noteworthy.

Application of physical principles to the problems of water conduction in trees led
to the cohesion theories of Dixon (1914) and Renner (1915). Measurements by
MacDougal (1926) have largely substantiated the theoretical considerations involved
as have biophysical studies by Bode (1923).

The general field of plant water relations has been covered by the works of Living-
ston (1903),_Maximov (1929a), and Walter (1931fo). Ursprung (1938) has pre-
sented a detailed review of most of the early work including his own and that of his
collaborators. Ecological aspects of water relations are stressed by Maximov (1929a),
Walter (1931&), and Montemaktini (1943). Applications of the principles of soil

Grafts et al. — 2 — Water in Plants

physics and plant physiology to problems of water utilization by plants have been made
by Briggs and Shantz (1912) and Maximov (1929o) ; and more recently by Veth-
MEYER and Hendrickson and their associates (see papers from 1927 to 1946). The
relations of soil salinity to plant growth have been investigated at the U.S.D.A. Salinity
Laboratory. This and other works on soil salinity have been reviewed by Magistad
(1945).

Thermodynamic treatments of plant and soil water relations are given by Edlefsen
and Anderson (1943) and Broyer (1946), and also by Stern (1933).

Specialized aspects of water relations involve the binding of water by cellular con-
stituents including cellulose, proteins, and the living protoplasm. Hydration of cellu-
lose is treated by Stamm (1936), Heuser (1944), and Wise (1944). Protein hydra-
tion is discussed by Gortner (1938), Schmidt (1938), and Sponsler, Bath, and
Ellis (1940). Sponsler (1940) and Frey-Wyssling (1938) have studied hydration
phenomena of the living protoplasm.

Problems of permeability are reviewed periodically in the Annual Review of
Physiology. In 1945 this topic was covered by S. C. Brooks. Work on the uptake
and exchange of solutes is described by Hoagland (1944) and Kjjogh (1946). The-
ories of membrane permeability are extensively treated in the works of Brooks and
Brooks (1941), Davson and Danielli (1943), and Hober (1945).

Osmotic quantities of cells and their interrelations have received detailed attention
by Thoday (1918), Hofler (1920), Beck (1928), and Meyer (1938, 1945). Cryo-
scopic determination of osmotic pressure values has been stressed by Walter (1931a)
and his associates. Ursprung (1938) has compiled much of the work on the plasmoly-
tic method. Harris (1934) has accumulated an abundance of data on freezing point
lowering of plant saps, particularly as related to distribution of the plants.

Bennet-Clark, Greenwood, and Barker (1936) have renewed the interest in ac-
tive cell water relations. Papers by Bennet-Clark and Bexon (1940, 1943, 1946),
Mason and Phillis (1939), van Overbeek (1942, 1944), and Lyon (1942) treat
various aspects of the same subject.

Water relations are intimately involved in frost and drought resistance. Reviews
by Levitt (1941) and Scarth (1944) report work on frost resistance. Maximov
(1929a, 1929&, 1941), Tumanov (1929), Vassiliev and Vassiliev (1936), Martin
(1930), and Aamodt and Johnston (1936) have covered drought resistance.

The role of water in the physiology of plant cells has been studied in detail in re-
cent years. Kuster (1935), Ursprung (1938), and Guilliermond (1941) have pro-
vided monographs covering this subject, while Brauner (1932) and Strugger (1935)
describe methods for studying the physiology of cells. The AAAS' monograph "The
Cell and Protoplasm" (1940) covers many aspects of cell physiology as does also the
monograph of the American Society of Plant Physiologists, "The Structure of Proto-
plasm" edited by Seifriz (1942).

Finally, Reed (1942) gives a historical view of plant water relations with addi-
tional reference material ; two recent books covering the field of physical biochemistry
by Bull (1943) and Hober (1945) provide a wealth of information for the student
of cell physiology.

Chapter II
STRUCTURE OF WATER

Introduction : — Most cells originate in a highly aqueous medium ; they
use water in growth and other vital processes ; in most instances, they con-
sist largely of water throughout their life span. Logically there should be
an intimate relation between the functions of cells and the properties of
water and dilute aqueous solutions. A study of plant water relations im-
presses one that this is true.

Water enters into the composition of carbohydrates, proteins, and other
compounds making up the walls and protoplasts of plant cells ; it is com-
bined with various colloidal constituents of cells as water of hydration ; it is
involved in many metabolic processes in the plant ; and it exists as a liquid
in the solutions occupying the vacuoles of living cells and the lumina of
conducting elements. Due to the many forces tending to reduce the activity
of water molecules, it may be difficult to distinguish between the various
states in which water exists within the plant.

Combining the elements hydrogen and oxygen, water in its simplest
form corresponds to the formula H2O. Many more complicated forms
have been proposed to explain its various properties. Most of these in-
volve association or polymerization.

Water boils at 100° C. and freezes at 0° C. when under one atmosphere
pressure; it is liquid throughout the temperature range at which plants
thrive. Many plants can survive freezing temperatures ; certain spores can
stand boiling; photosynthesis and transpiration may take place through a
range from near freezing to the thermal death temperature around 40° to
50° C. Many plant functions, however, find their optimum within the
range from approximately 20° to 30° C. The occurrence over a large por-
tion of the earth's surface of temperatures within these narrow limits de-
pends, among other things, upon the large latent heats of vaporization and
freezing of water, and upon its heat capacity and conductivity. Hender-
son (1924), discussing the fitness of the environment, points out the many
unusual properties of water, most of which apparently contribute in some
way to the well-being of plants. It is interesting to note the exceedingly
narrow range of temperature and moisture within which our important
economic plants thrive, and to point out the role played by water in main-
taining these conditions.

Structure: — Water has long been recognized as a compound of unusual
character. Tables 1 and 2 list some of the physical properties of common
elements and liquids. Water stands out, having a very high heat of vapori-
zation and a fairly high heat of fusion. (For further data of this type, see
Bernal and Fowler, 1933, Table III). Of the compounds listed, water
has the greatest surface tension, internal pressure, and dielectric constant.
These latter properties indicate the interatomic forces present in the mole-
cule.

Formed from hydrogen which boils at — 253° C. under atmospheric
pressure, and oxygen which boils at — 180° C, water boils at 100° C. If
normal in its properties water should boil at a very low temperature vari-
ously estimated at from — 65° C. to — 100° C. Its freezing point should

Crafts et al.

4 —

Water in Plants

Table 1. — Some thermal properties of water and several elements and compounds:

Substances

Heat capacity per gram
formula weight

Calories Temp. ° C.

Heat of fusion
calories per gram

Calories Temp. ° C.

Heat of vaporization
calories per gram

Calories Temp. ° C.

Hydrogen 3.4 0

Oxygen 3.5 0

Nitrogen 3.5 0

Sulfur 5.3 0

Water, liquid 18.1 0

Water, solid 9.1 0

Carbon dioxide 8.7 0

Ammonia 8.8 0

Hydrogen sulfide .... 4.6 10

Sulfur dioxide 15.4 10

15.0

6.1

10.4

80.0

45.4
108.0

—219.0

—210.0

119.0

0

— 56.2

— 75.0

108.0
50.9
47.6

66.3
539.0

87.0
327.0
132.0

95.0

—252.8

—182.9

—195.5

444.6

100.0

— 60.0

— 33.4

— 61.4

— 10.0

Table 2. — Physical properties of water and some other common liquids: —

Substances

Surface tension
Dynes/cm. Temp.

Internal pressure
Atmospheres

Dielectric constant
E Temp. ° C.

Water 75.6 0

Mercury 47.6 20

Carbon disulfide 35.3 0

Benzene 31.6 0

Carbon tetrachloride 29.0 0

Hexane 20.5 0

Ethyl alcohol 24.0 0

16,400
13,050
5,400
4,050
3,640
2,020
7,200

81.000

17

2.600

0

1.002

100

1.003

110

1.874

20

1.006

100

be around — 100° C. to — 150° C. In comparison HgS, containing sulfur
that boils at 444° C, is a gas boiling around — 61° C.

H
H-6-

H
-6-H

V

Fig. 1. — Trihydrol molecule as pictured by Suth-
erland (1900).

Other anomalous properties of water include its minimum specific heat
at 37.5° C, its maximum density at 4.0° C, and its great expansion upon
solidification. This latter property has played an important part in all at-
tempted explanations of the structure of water.

Early Theories: — The earliest concepts of the liquid structure of water pictured
it as composed of spheroid molecules, heterogeneously arranged, and having the un-
ordered motion of a very dense gas. As early as 1884, however, Whiting visualized
liquid water as a solution of ice particles and Raoult (1885) pictured association of
water molecules into groups of four. Vernon (1891) explained the maximum density
of water by association of the molecules from (H20)2 above 4° C. to (H20)4 below
that temperature. In 1892 R5ntgen independently proposed that liquid water is made
up of a saturated solution of ice in some other form of water. He pictured ice mole-
cules as complex, but less dense than water; when ice melts the volume is decreased.

Chapter I — 5 — Introduction

As water is warmed, however, thermal expansion increases the volume. Rontgen ex-
plained the maximum density as a combination of these two effects.

The compressibility of a normal liquid decreases with temperature. Rontgen from
the association theory suggested that pressure would decrease the number of ice mole-
cules ; hence there should be a point of minimum compressibility of water. Such a
point occurs at about 50° C. He explained the increase of the thermal coefficient of
expansion at pressures around 3,000 atmospheres by the effect of pressure in breaking
up ice molecules. He suggested that the point of maximum density would occur at
lower temperatures under pressure and that the freezing point would also be lowered.
This has proved true. The anomalous decrease in viscosity with increasing pressure
he explained on the assumption that the simpler water molecules had a lower viscosity
than ice molecules.

The Hydrols: — In 1900 Sutherland proposed that water vapor is H2O (hydrol),
ice pure (H20)3 (trihydrol), and liquid water a mixture of (H20)2 (dihydrol) and
(H20)3 in proportions dependent upon the temperature. Figure 1 shows the latter
molecules as pictured by Sutherland. At 0° C. the fraction of (H20)3 in water was
calculated to be 0.375 ; at 20° C, 0.321 ; at 40° C, 0.284 ; at 60° C, 0.255 ; at 80° C,
0.234; and at 100° C, 0.203. At the critical temperature water was supposed to be
composed of nearly pure hydrol. At 4° C. Sutherland supposed it to be approxi-
mately Vs (H20)3 and Yi (H20)2.

Sutherland concluded that the latent heat of fusion of ice is mostly a latent heat
of dissociation of trihydrol into dihydrol, partly masked by heat of solution of trihydrol
in dihydrol ; and that the latent heat of vaporization also includes the heat of dissocia-
tion of the dihydrol and trihydrol of water into the hydrol of steam. The specific heat
of water is not an ordinary specific heat but includes heat of dissociation. Pressure
dissociates trihydrol ; at temperatures between 0° and 100° C. trihydrol is completely
dissociated at pressures about 2300 atmospheres. Since pressure causes dissociation,
tension should bring about association ; at temperatures below 40° the surface layer
of water he thought to be largely trihydrol.

Sutherland explains the polymerization of hydrol on a tetrad oxygen valence, the
assumption being that three H2O molecules of trihydrol are bound into a triangular
grouping by the extra oxygen bonds. The sharp melting point of ice depends upon
molecular resonance resulting in the breaking of these bonds.

Armstrong, et al. (1908) proposed the possible existence of water isomers of differ-
ent structure, the hydrones. Active water molecules, hydrone (HOH) or hydronol

/H
(H20(' /-^■^T ), take part in chemical reactions whereas inactive molecules formed by

association are closed and hence chemically inert. Such molecules he diagrammed
thus:

Dihydrone Trihydrone Tetrahydrone

H2O = OH2 H2O — OH2 H2O — OH2

\ / II

O H2O — OH2
H2

Dissociation takes place constantly and is conditioned by temperature and the pres-
ence of solutes. (H20)n^n H2O.

Solution of HCl would produce the following molecules :

H20/S HCL H2O: CIH

\'-' \OH

The first two he considered active, the third inert so long as it remained closed. Dilu-
tion should bring about increase in the active forms.

The Faraday Symposium : — The constitution of water engaged the attention of the
Faraday Society in their symposium of 1910. The association theory predominated and
most papers concerned estimation or measurement of the degree of association under
different conditions. Guye (1910) calculated the association factor assuming that
association occurs in both the vapor and liquid states. His values were 80° C, 1.90;
100° C, 1.86; 120° C, 1.82. These compared well with values derived from surface
tension measurements. Much of the interest in association grew out of work on molec-
ular weight determination by physical measurements such as surface tension, cohesion,
etc. Guye, however, emphasized the chemical nature of association and used the term
polymerization more often than association.

Grafts et al. — 6 — Water in Plants

BousFiELD and Lowry (1910) from studies on solution volume proposed that liquid
water is a ternary mixture of hydrol, dihydrol, and trihydrol. Cooling water results
in production of polymerized ice molecules ; heating causes dissociation yielding more
steam molecules (hydrol). E^ch change results in increase in volume, this increase
being superposed upon the expansion or contraction caused by change in temperature.
They showed that the solution volume (change in volume upon addition of a solute)
goes through a maximum between 0° and 100° C. when salts having a strong affinity
for water are dissolved.

According to Bousfield and Lowry, this phenomenon depends upon the presence
of steam molecules at temperatures above the maximum, and ice molecules at tempera-
tures below, both of which are destroyed by the addition of the hydrate-forming solute.
The regions of minimum volume at temperatures below and above this point of maxi-
mum solution volume represent points of maximum density for the combined water
(water of hydration) and a density value of 1.24 for the water of crystallization of the
sulfates of certain divalent metals is quoted as an example of the contraction due to
intermolecular forces. Such reasoning brings the solution-volume phenomenon into
agreement with the theory used to explain the maximum density of water at 4° C.

Concerning the valence forces of trihydrol, Sutherland (1910) diagrammed to
scale the structure of the molecule and calculated from atomic diameters its dimensions.
He arrived at a density value of 0.986 whereas that of ice at 0° C. is 0.917. Since the
discrepancy is only about 8 per cent, the method suggests that some such grouping
may be involved in the formation of ice crystals. Suthejrland maintained that water
is a binary system, hydrol, as such, being present in water in quantities too small to
detect. Water of crystallization, he suggested, was solid hydrol having a mean density
of 1.31.

In a discussion of ionization, Sutherland (1910) stated "ionization of all electro-
lytic solutions at all strengths is complete" and explained the apparent lack of ioniza-
tion of so-called weak electrolytes on the basis of mobility of ions. He pointed out the
remarkable change in volume occurring when metals of the lithium family combine with
halogens, but did not agree with either the theory of partial ionization or the hydration
theory to explain the retention of this altered volume by the ions in solution. The
mutual energy of the ion and solvent, representing both attractive and repulsive forces,
are concerned ; ionic mobility is dependent upon dielectric capacity ; undoubtedly forces
between solute and solvent are involved.

Sutherland attempted an explanation of the unusually high mobilities of hydrogen

and hydroxyl ions; he calculated the heat of fusion of water of crystallization to be

1.8 K. cal. per mol ; the heat of vaporization is 5.0 K. cal. This totals 6.8 K. cal. for

the change from hydrol of vapor to the solid hydrol of crystallization, evidence of a

profound change in the internal electrical energy of the hydrol brought about by the

proximity of the electrical fields around the molecules of salt. Nernst (1910) showed

that the diflferences in the specific heats of steam, water, and ice could be explained by

the relation

2H2O = (H20)2 + 2.5 K. cal. (1)

Professor Walker (1910) summarized the principal conclusions of the symposium
with the statement "I should think, as a result of this discussion, one will soon find,
even in the textbooks, that whilst ice is trihydrol, and steam monohydrol, liquid water
is mostly dihydrol with some trihydrol in it near the freezing point, and a little mono-
hydrol near the boiling point."

Later Work on the Hydrol Theory: — Bousfield (1914) has proposed that the
vapor pressure of water has an intimate connection with the proportion of steam mole-
cules present, and that the addition of solutes reduces the numbers of both ice and steam
molecules in water. This explains the reduction of vapor pressure by solutes. In
1917 he attempted to reconcile the osmotic properties of solutions with the structure
of water. He attributed osmotic pressure to the thermal agitation of the water vapor
molecules, and explained the depression of the vapor pressure and of the freezing point
as resulting from a shift in the equilibrium conditions of the liquid water. This con-
trasts with the view of van't Hoff, Arrhenius, and many subsequent workers on the
osmotic relations of solutions.

Cryoscopic measurements, used primarily to determine molecular weights, indicated
that water in solutions was associated. Studies on specific heats of crystalline hydrates
also confirmed this view. Studies on the structure of ice have given association fac-
tors of from 3 to 23 while data from vapor density are controversial, some indicating
association, others none. A compilation of estimates of water association is given by
Barnes and Jahn (1934, Table A).

Chapter II — 7 — Structure of Water

Studies on solution volume, viscosity, surface tension, compressibility, and other
properties of solutions have yielded a variety of evidence on the structure of water.
Richards and Palitzsch (1919) interpret their results as indicating that trihydrol is
bulky tending to dissociate under the influence of solutes. Pagliani (1920), Rabino-
vicH (1922) and Richards and Chadwell (1925) consider polymerization as one
factor in explaining viscosity and volume changes upon compression of solutions.
Bancroft and Gould (1934) studying the effect of the Hofmeister series on boiling
points, adsorption by gelatin, electromotive forces of cells, solubility of gases, and
heats of neutralization of acids, conclude that the two factors determining the order of
the series are 1) displacement of the water equilibrium and 2) selective adsorption.
With gelatin displacement of the water equilibrium is the more important ; with al-
bumin selective adsorption predominates. They conclude that, under ordinary condi-
tions, liquid water is a mixture of trihydrol, dihydrol, and monohydrol coexisting in
reversible equilibrium.

According to this interpretation, chloride, bromide, thiocyanate, and iodide ions
tend to change dihydrol into monohydrol, and have no specific effect on trihydrol be-
yond that caused by the above mentioned shift in equilibrium. They concluded that
nitrate ion tends to convert trihydrol into monohydrol ; that the sulfate ion tends to
convert trihydrol and monohydrol into dihydrol ; that the electrolytic solution pressure
of hydrogen is greater in dihydrol than in monohydrol ; that of oxygen is less. Sulfur
dioxide tends to depolymerize water. Trihydrol tends to promote fluorescence, mono-
hydrol to check it. Changes in viscosity of water and salt solutions with changing
pressure fit the concept of displacement of the water equilibrium by pressure and by
salts. Since the Debye-Hiickel theory postulates interaction of the ions as the only
disturbing factor in the behavior of solutions, Bancroft and Gould consider that it
must break down at all concentrations at which displacement of the water equilibrium
is a disturbing factor.

Rao (1934), from Raman spectrum studies concluded that water vapor consists of
single H2O molecules, water is predominately dihydrol and ice trihydrol. Though
ice contains no monohydrol, liquid water at 0° C. to 98° C. contains varying propor-
tions of all three molecular types. The shifting values that he presents fit well into
the picture of shifting coordination with temperature presented by more modern studies.

"Normal" and "Polar" Liquids : — Starting with the properties of
pure water, and proceeding to studies on aqueous solutions, the investiga-
tions of physical chemists on the structure of water have thus led many
to the belief that the anomalous behavior of water and solutions can largely
be explained on the basis of association. On the other hand, studies involv-
ing many other Hquids and proceeding from a consideration of the prop-
erties of ideal solutions as described by the laws of van't Hoff, Raoult,
and Henry, give a different view. Though often attributing anomalous
behavior to association of liquids, this latter type of study emphasizes
intermolecular forces, stressing the properties of both solute and solvent
as they relate to the behavior of the solvent. As noted by Hildebrand
(1924, pp. 84-85) objections to assuming association as explaining devia-
tions from Raoult's law apply not so much to the concept as to the arbi-
trary choice of definite polymers. Chemists have long distinguished two
classes of liquids : one, the "normal liquids" having low dielectric constants,
surface tensions, heats of vaporization, etc. ; the other "polar Hquids" hav-
ing high values for these constants. Normal liquids are poor solvents for
electrolytes, polar liquids good solvents. The former include parafiins,
benzene, carbon tetrachloride, bromine, and carbon disulfide ; the latter
water, alcohol, ammonia, sulfuric acid, and acetone. Salts melt to form
polar liquids.

The fundamental distinction between the normal and the polar or asso-
ciated liquids lies in the greater symmetry of the fields of force surround-
ing the molecules of the former, the field surrounding a polar molecule
being unsymmetrical. The polar molecules have great mutual attractions
resulting in greater cohesions, internal pressures, surface tensions, and

Grafts et al.

8

Water in Plants

heats of vaporization. They tend to "squeeze out" non-polar or sHghtly
polar molecules resulting in strong deviations from Raoult's law and low
solvent power.

This concept differs from that of the previous group in that it postu-
lates not definite polymers (dihydrol, trihydrol, etc.) but a liquid mass
that tends, by intermolecular attractive forces, to become one large
"group" molecule, as Langmuir has termed it. Such a mass resembles in
a way the crystal of sodium chloride in which the identity of any single
molecule of NaCl has completely disappeared.

If the forces between molecules of one type are great, these molecules
will tend to associate, and solubility of compounds of the other type will be
low. This is illustrated by the low solubility of paraffins, benzene, etc., in
water. If the attractive forces between polar and non-polar molecules are
great, negative deviations from Raoult's law will occur and the molecules
will tend to unite to form compounds.

Fig. 2.— The tetrahedral water molecule of Pennycuick
(1928). The oxygen nucleus with its two helium electrons is
omitted.

This concept eliminates the distinction between chemical and physical
attractive forces. All intermolecular forces are electric in nature, as con-
ceived by Sutherland as early as 1902.

Molecular Concentration: — In 1929, Longinescu attempted to reconcile the com-
plexities of liquid association under the concept of molecular concentration. Asso-
ciated liquids differ from normal ones in the degree of accumulation or crowding of
the molecules into a given volume. The molecular concentration C^ equals 1000 times
the density divided by the molecular weight. Thus the molar concentration of ether is
9.6; of ecu, 10.4; of toluene, 9.4; of benzene, 11.2; of water, the largest of all, 55.5.
For 350 compounds molar concentration values lie between 55.5 for water and
0.09 for tristearin. All organic liquids that are considered associated have values near
10. In 16 cases cited by Longinescu the association factors determined by various

workers are compared with the value —^ and the agreement is very close.

By this concept there is no molecular complexity ; only the number of simple mole-
cules in the unit volume varies; when this number exceeds a certain value, experi-

mentally determined by the formula

lOOd

W

here d = density, and m = molecular

Chapter II

— 9 —

Structure of Water

weight, the state of molecular accumulation occurs. As van der Waals explained the
difference in compressibility of gases by molecular attraction, Longinescu explains
molecular association in liquids by the internal compression of molecules. Thus he
restates Avogadro's law, "Equal volumes of fluids, and possibly solids, at the same
temperature and under the same external pressure, contain numbers of simple mole-
cules proportional to the internal pressure." Since Avogadro's law expresses the
molecular weight in the gaseous state by the equation 28.9d = m, Longinescu's ex-
pression for molecular weight in the liquid state would be lOOd = m. This would give
liquid water a molecular weight of 100 and a degree of molecular concentration of 5.5.
Though this concept is useful in consideration of molecular weight, Longinescu made
no attempt to explain such anomalies as the maximum density, minimum specific heat,
and expansion upon freezing of water.

Modern Studies: — In 1928 Pennycuick, pointing out that oxygen
may display a valence of four and hydrogen of two, attributed many of the
anomalous properties of water to the activity of these auxiliary valence
forces. Assuming a tetrahedral structure for the water molecule as shown
in Figure 2, Pennycuick stated that water can attach itself to other mole-
cules either through its own negative electron pairs or through its positive
hydrogen nuclei.

OH
H

OH

• •

=Q^H

0:H

• •

H

H

H

:0:H

• •

H

0:H

H

+

Fig. 3. — The polar water chain. (From Pennycuick, 1928).

The molecule being small (cf. Longinescu above) with 4 active aux-
iliary points of attack, its great activity is not surprising.

To satisfy the auxiliary valences of oxygen and hydrogen, and to ex-
plain association Pennycuick proposed that polar chains may be formed
as in Figure 3.

^- -/y ,n:^ ■ '^

■O-
H

Fig. 4. — The hexagonal ring structure of water,
comparable with the crystal structure of ice. (From
Pennycuick, 1928).

Furthermore these may close to form hexagonal rings as in Figure 4.

Crafts et al. —10— Water in Plants

This structure supports the view, obtained from x-ray analysis of ice,
that each oxygen atom is surrounded by four atoms of hydrogen and that
each hydrogen atom lies symmetrically between two atoms of oxygen. A
clear picture of such structure can only be obtained from a three dimen-
sional view of the crystal lattice model. Such a view shows that the term
ice molecule has no meaning, the atom being the only real unit of structure.
Pennycuick pictures the hexagonal units in ice fabricated into a con-
tinuous lattice structure ; the primary and secondary valences are of equal
strength, making the whole structure symmetrical.

In liquid water HOH chains would be irregular and distorted because
of tetrahedral structure; if the ends of a chain combine and neutralize each
other, the resulting ring would be relatively inactive {cf. Figure 4), Such
rings should play important parts in the structure of water and ice, and the
tetrahedral form of the water molecule led Pennycuick to conclude that
such rings in water must contain six oxygen nuclei.

In contrast with the crystal lattice structure of the solid, with zero re-
sultant field around each atom, in the liquid the molecule preserves its in-
dividuality and the system lacks stable structure. With definite resultant
molecular fields aggregation would occur, the whole liquid forming a three-
dimensional, continually-changing network with linkages of varying
strength in which a single linkage could exist only momentarily. Every
normal liquid must possess this type of association. Non-polar or normal
liquids, having symmetrical arrangement of valence forces, would not form
definite polymers, all molecules being constantly in a state of flux. Mole-
cules of polar or associated liquids, having asymmetric fields, would tend to
combine to form stable groups, and these {i.e., Figure 4) would behave
like solute molecules moving as independent units through the remainder
of the liquid which is normal in its behavior. This view of Pennycuick
differs from the older one that water is a mixture of mono-, di-, and tri-
hydrol, each having a statistically average existence expressible as a con-
stant value.

HO-H:0:HO:=^

• • • • • •

H H H

H:0:H

• •

H

++

• • • •

H H

Fig. 5. — Diagram indicating the self-ionization of a water chain. (From Penny-
cuick, 1928).

If water is a mixture of relatively stable hexagonal molecules in a nor-
mal solvent, it may represent an intermediate step between vapor and the
hexagonal crystal structure of ice. The auxiliary fields of the molecules
are presumably strong enough to build as far as the (HOH)^ ring but the
molecular energies are sufficiently strong to prevent coordination into a
lattice. The fraction of water polymerized would vary with temperature
and Pennycuick considered the values of Richards and his co-workers
as most probable, i.e., about 28 per cent as polyhydrol at 20° C.

In terms of the above suggestions, Pennycuick proposed that when
ice melts there is a loosening of the structure and a breaking of the rings,
resulting in a contraction of volume or an increase in the density of the
liquid. On the other hand, the increase of molecular energy with rise in

Chapter II

11

Structure of Water

temperature brings about wider spacing of the attractive centers. The
minimum volume shown at 4° C. results from alternate predominance of
these effects. The latent heat of fusion is not unexpectedly high ; it repre-
sents only the separation and not the destruction of ring structure. Evapora-
tion breaks all the intermolecular bonds ; as high as three-fifths of the latent
heat of vaporization has been ascribed to the dissociation of the complex
molecules. Water is unusual in its solvent power because the solute is held
in solution by its auxiliary valence forces that the solvent is able to neu-
tralize. Since a solute will first be attacked by the more active unassociated
molecules, the effect of solution is to reduce the number of such molecules
with a corresponding shift in equilibrium and a decrease in association. The
lowering of the temperature of the maximum density of water and the
decrease in compressibility upon addition of a solute may be explained on
this basis. The predominance of compounds containing six and twelve
molecules of water of crystallization results from incorporation of hexa-
hydrol in crystals. The self -ionization of water is explained by occasional
breaks as indicated in Figure 5.

The high dielectric constant of water is pictured as resulting from the
short-time displacement of a hydrogen nucleus under the influence of the
field due to a lone oxygen electron pair as rupture of a polar chain occurs.
The abnormal velocities of hydrogen and hydroxyl ions are related to the
possible addition of water at the active end and loss at the inactive end of
each ion under the influence of an applied E.M.F. Thus by applying the
principles of molecular structure and crystal structure to water, many of
the abnormal or anomalous properties may be explained. He fails to ex-
plain the reason for polymers that would act as solute molecules. The
formation of such stable groups in contrast to the tendency toward lattice
formation is difficult to visualize.

Fig. 6. — Electronic distribution in the simple water mole-
cule. (From Bernal and Fowler, 1933).

X-Ray Analysis : — Analysis by x-rays has become an invaluable tool
in the hands of the scientist. It has been used in the attack on the struc-
ture of water. Chadwell (1927) points out the discrepancies in the

Crafts et al.

12

Water in Plants

earlier interpretations of such studies. Ice apparently has lattice structure
but workers disagree on the details. Pennycuick accepts the interpreta-
tion of Bragg that each oxygen atom is surrounded by four atoms of hy-
drogen and that each hydrogen atom lies symmetrically between two oxy-
gen atoms. Assuming a tetrahedral structure for the water molecule and
a hexagonal ring structure for association of water molecules, a lattice
structure may be visualized.

Bernal and Fowler (1933) discuss in detail the structure of ice and
water from the standpoint of infra-red and Raman absorption spectra and
x-ray diffraction. They see no reason to assume that the molecules of
liquid water differ from those of steam except for small mutual deforma-
tions. They picture the electronic distribution in the simple water molecule
as shown in Figure 6.

Fig. 7. — The crystal lattice of ice as pictured by Bernal and Fowler (1933).

This shows the oxygen atom at the center with two hydrogens in a hori-
zontal plane with the HOH angle 103°-106°, very near the tetrahedral
angle. The two charges Px represent the negative charges in the vertical
plane at right angles to the HOH plane. The net electronic density distri-
bution resembles a tetrahedron with two corners of positive and two of
negative charge. The radius of the molecule is taken to be 1.4A.

Analysing the x-ray scattering curve for liquid water, Bernal and
Fowler arrive at a fundamental grouping of five molecules, one in the
center surrounded by four others to form a tetrahedron. This structure,
however, is not flexible enough to cover all requirements imposed by experi-
mental results. Studying the distribution functions for the neighbors around
a water molecule they conclude that water may have three chief forms
of arrangement : water I, tridymite, ice-like, present to a degree below 4° C. ;
water II, quartz-like, predominating at ordinary temperatures ; water III,
close packed, ideal liquid, ammonia-like, predominating at high tempera-
tures below the critical point at 374° C. Close packed in the above sense
refers to a uniformity of distribution in which deviations from the average
spacing are at a minimum. These forms pass continuously into each other
with change in temperature. The liquid is homogeneous at all tempera-
tures but the average mutual arrangements of the molecules resemble
water I, II, and /// in more or less degree.

Chapter II — 13 — Structure of Water

The immediate effect of breaking down the relatively empty ice-like
structure of water I would be a decrease in volume to water II (maximum
density) followed by an increase of a more normal type to water III where
the increase in spacing due to thermal agitation would more than com-
pensate for the change from the quartz-like to the close-packed structure.
The fact that several forms of ice occur under different pressures and that
these vary in density confirms the x-ray evidence that the structure of water
is not a close-packed one. Trihydrol, dihydrol, and hydrol, as visualized
by the earlier workers have no direct structural analogy with water I, II,
and ///. Bernal and Fowler explain, by means of geometrical internal
structure of the liquid, physical properties which the hydrol theory at-
tempted to explain in terms of hypothetical molecules. Ice, they conclude,
has a crystalline structure in which the H nuclei and hence the orientation
of the molecules are fixed. Every molecule is surrounded by four others
in a tetrahedron with the H nuclei opposite two of the neighbors and one
of the H nuclei of each of the remaining neighbors lying opposite a nega-
tive corner of the original molecule. This forms the simplest possible
regular physical structure. It is illustrated in Figure 7.

Pauling (1939) differs from Bernal and Fowler stating that if the
water molecules in ice are oriented in a definite way so that a unique con-
figuration could be assigned to the crystal there should be no entropy. The
fact that ice retains appreciable entropy even at very low temperature in-
dicates that the molecules retain some freedom for motion and that the
crystals can exist in a number of configurations. This is further borne
out by the fact that above about 200° K. the dielectric constant of ice is of
the same order of magnitude as that of water.

Bernal and Fowler conclude that the magnitudes of the dielectric
constants of water and ice can be explained by molecular rotation, but only
if the majority of the water molecules are not entirely free to respond by
orientation to the electric field. They propose that its unique properties
are due not only to its dipole character, but even more to the geometrical
structure which is the simplest that can form extended four-coordinated
networks. Studying ionic solutions they postulate that the strongly polariz-
ing ions H*, Li*, Na"^, all divalent and trivalent positive ions, and OH" and
F- are hydrated ; whereas NH4+, Rb*, Cs"^, and most negative ions are not.
The degree of ionic hydration, they conclude, depends mainly on the ionic
radius, and is the same in solutions as in crystals. In view of their dipole
character it seems to us that all ions should be hydrated, the effects of
NH4"*', Rb*, Cs*, and most negative ions upon the activity of water being
relatively less than the effects of those listed above as being strongly hy-
drated.

Concerning the high mobility of hydrogen and hydroxyl ions, Bernal
and Fowler arrive at a new theory. Assuming H* to exist in solutions as
{OU-^y, they suggest that a proton may jump from one water molecule to
another along the coordinated network when favorable configurations oc-
cur. Similarly for OH" a proton under the influence of an electric field
may pass from H2O to the (OH)- and the systems can separate again
with the ion and neutral system interchanged. Thus with models built al-
most entirely from absorption and diffraction data, Bernal and Fowler
have postulated a structure for water that accounts for many of its unusual
properties. They picture water as an organized system of irregular, four-
coordinated molecular groups exhibiting three different intermolecular ar-

Grafts et al. — 14 — Water in Plants

rangements in the liquid form. Properties are explained on the basis mainly
of internal geometrical structure.

Katzoff (1934) made x-ray studies on water and arrived at a mole-
cule having essentially the same electron distribution as postulated by Ber-
NAL and Fowler (Figure 5). He also agreed on the arrangement of
molecules in the liquid where each molecule has four others around it. He
found no evidence for the quartz-like structure (water II) nor for the
degree of close packing described by Bernal and Fowler in water at high
temperatures. If in ice each oxygen is tetrahedrally surrounded by four
others with the hydrogens on or near the center lines between adjacent
oxygens, then water appears to have a broken down ice structure with less
regularity of arrangement but still maintaining the tetrahedral order char-
acteristic of ice. Hot water also maintains the fundamental four-coordina-
tion but has more randomness of arrangement than that of cold.

The Second Faraday Symposium: — A symposium of the Faraday
Society on the structure and molecular forces in pure liquids and solu-
tions, in 1936, brought together many workers in this field and accumu-
lated in one publication (Trans. Faraday Soc. Vol. 33) the current views.
Kendall (1937) commented on the changing opinion regarding the na-
ture of liquids. The hard and fast distinction between the solid, liquid,
and gaseous states has largely disappeared. Dififerences do exist; the
close packing of molecules in the liquid state renders intermolecular forces
much greater than those between gaseous molecules. In contrast to the
older belief that such forces were essentially similar in nature (ordinary
van der Waals attractions or valence forces) the modern view involves the
stresses caused by dipole structure, the coulomb forces in electrolytes, and
the metallic cohesive forces in metals. Association into definite groups
(polyhydrol concept) has been largely abandoned, most scientists subscrib-
ing to the view of Langmuir that a liquid consists of one loose molecule.

If the solute in a solution were in the gaseous state the heat of solution
of a gas would be zero, those for a liquid and solid respectively should ap-
proximate the heats of vaporization and sublimation. This is not the case.
In the ideal case, the heat of solution of a gas is equal to its heat of con-
densation, the heat of solution of a liquid is zero, and the heat of solution
of a solid is equal to its heat of fusion. In other words, any substance dis-
solved in a liquid, itself assumes the liquid state.

Ideal solutions are rare; solutions deviating from Raoult's law have
been studied in detail and their behavior explained on the basis of the mag-
nitude of forces between solute and solvent molecules. Whereas most
positive deviations were at one time explained in terms of association of the
solvent, they are now considered to result from differences in internal pres-
sure. Negative deviations which Longinescu (1929) states have been
"timidly assumed" to be non-electrolytic dissociation, Kendall explains as
resulting from molecular attractions that, in the extreme case, result in com-
pound formation. This is in agreement with Hildebrand (1924).

The simple concept of intermolecular forces exemplified by van der
Waals force was expanded by London (1937) to account for orientation,
induction, and dispersion effects and MacLeod (1937) proposed a com-
pressional effect to cover the forces of molecules under high pressure.

Bernal (1937) postulated points of abnormal coordination in liquids
to explain their properties. His theory pictures the liquid as like an or-
dered solid containing a number of holes. Water, he concluded, is a liquid

Chapter II

— 15 —

Structure of Water

whose molecules, though tetrahedral in structure, behave as spheres with
directed intermolecular forces. The low (fourfold) coordination results
from hydrogen bonds that affect both viscosity and thermal properties.

HiLDEBRAND ( 1937) Stated that the term "association" under which all
departures from normal behavior of liquids have been lumped must now
be subdivided into association arising from the interaction of dipoles, and
that due to the formation of definite chemical bonds. To hydrogen bonds
he assigned great importance. Substances containing hydroxyl, carboxyl,
or amino groups show a type of association markedly different from that of
most other dipoles. These bonds are of particular interest to biochemists
because of their existence in proteins.

Current Research: — Morgan and Warren (1938) by carefully con-
ducted x-ray diffraction analysis of water showed that, whereas water in the
neighborhood of freezing temperature has open tetrahedral structure as in

ice, at higher temperatures the tetrahedral
bonding becomes less sharply defined. In
ice the intermolecular separation of neighbor-
ing molecules is 2.76A ; of next nearest
neighbors it is 4.5A. In liquid water this
distance for nearest neighbors is 2.90A ; for
next nearest neighbors it is 4.5A or slightly
more. Because the density of water at 0° C.
is greater than that of ice, Morgan and
Warren believe that the increased density
is due, not to a crowding in of next nearest
neighbors, but to a filling in between the first
and second neighbors, in other words a loss
of the highly regular pattern of the ice lat-
tice and substitution of a closer packed struc-
ture. They picture this as a shift from the
hexagonal lattice of ice to a quadrangular
pattern having a density 27 per cent greater
as indicated in Figure 8. The line indicat-
ing the next nearest neighbor distance of
4.5A is strong in their diffraction picture at 1.5° C. ; it is less distinct at
13° C, and still less at 30° C, and disappears at 62° C. This adds weight
to their conclusion that the structure of water changes with rise in tempera-
ture through this range. Although the average number of molecules per
unit space (density) is greater, the coordination may decrease; from an
average value of 4 at 0° C. the coordination number decreases approaching
a value of zero in the vapor.

The concept expressed by Bernal (1937) that a liquid possesses points
of abnormal coordination that act as "holes" into which neighboring mole-
cules may "flow" has been elaborated by later workers on liquids (Altar
1937). In their theory of liquid structure Hirschfelder, Stevenson, and
Eyring (1937) picture a mechanism of viscous flow involving the forma-
tion of double molecules that rotate into new positions and then separate.
The ability of such double molecules to rotate depends upon empty spaces
or holes into which the molecules pass during rotation and the activation
energy of viscous flow they consider to be needed for the formation of
these holes. Since the activation energy of normal liquids is around y^
to 14 the heat of vaporization they assume the required hole to be ^ to

Fig. 8. — Diagrammatic illustra-
tion of the shift from the hexagonal
lattice of ice to a closer packed
structure for water. Redrawn from
Morgan and Warren (1938).

Grafts et al. — 16 — Water in Plants

% the space occupied by the molecule since it requires the same energy
to make a hole of molecular size against the forces of cohesion as to
vaporize a molecule. A property peculiar to liquids is that such holes are
shared communally by all molecules. For a thermodynamic consideration
of viscous flow of electrolytic solutions see Harned and Owen (1943,
p. 67).

In their theory of the viscosity of liquids as a function of temperature
and pressure, Ewell and Eyring (1937) state that the unusually high
viscosity of water is brought about by the hydrogen bond structure. When
a molecule in such a liquid flows, it must not only break van der Waals
and dipole forces, but hydrogen bonds as well. As water is warmed the
number of these hydrogen bonds that must be broken decreases rapidly
and this is reflected in a decreasing coordination number. Assuming that
the activation energy for cavity formation in the liquid is Ye the molec-
ular vaporization energy, they calculate the following coordination num-
bers for water :

Temperature °C.

Coordination number

0

2.68

50

1.44

100

1.04

ISO ■

0.60

Stewart (1939), on the other hand, considering the mean coordination
of large numbers of molecules, finds that the coordination of water in-
creases with the shift of structure from the hexagonal, four coordinate lat-
tice of ice to the more closely packed structure of water. He emphasizes
the extremely labile character of the liquid and points out that the presence
of ions or rising temperature does not contract the existing structure of the
fluid but tends to shift the coordination and increase the closeness of pack-
ing. The views of Stewart and the previously cited workers may be
brought into agreement if one considers the difference between the actual
coordination of a single molecule at a particular moment and the average
coordination of a large group over an indefinite time. With increasing
temperature the number of actual bonds per molecule at a given time will
decrease ; on the other hand, because of the increasing thermal agitation the
possibilities for contacts between different molecules increase immensely
and the tendency toward bonding is greater. This is particularly true dur-
ing the shift from the stable structure of ice to the labile structure of liquid
water ; the increased closeness of packing still furthers this tendency. In a
later paper, Stewart (1943) states that increase in temperature (0° to
4° C.) breaks hydrogen bonds and alters the structure of water, decreasing
its molal volume. Ions in aqueous solution break hydrogen bonds and alter
water structure, decreasing its molal volume ; they consequently increase
the pure temperature expansion of the solvent. He uses the phrase
"smeared out" to describe the relation of liquid to crystal structure of
water (1944). He concludes that water should be visualized as having
open tetrahedral structure and that the true aqueous solution develops a
new liquid structure in which both solvent and solute participate. Finbak
and Viervoll (1943) picture the structure of water as a three-dimensional
network of pliable branched chains of tetrahedrons with the corners linked
together in such a way that rotations about the lines between centers of
neighboring tetrahedrons may occur.

Eley (1944) points out that for water the energy required to produce
a cavity is small at low temperature and increases until it reaches 80° C,

Chapter II — 17 — Structure of Water

at which point it equals the energy of solution of a gas. This explains the
decrease in solubility of gas in water with increasing temperature. Eley
relates this effect to the polarization of the water molecules around the
cavity. Since the energy required to make a cavity of molecular size equals
the internal energy of evaporation, for one mol at room temperature it
would be about 10 K cal. The energy of polarization for a molecule the
size of water is shown to be about 9 K cal. per mol. The difference, 1 K
cal. per mol, represents the net energy required to form cavities within the
liquid equivalent to one mole volume.

Since the polarization of molecules around a cavity would disorganize
the open tetrahedral structure of water, the formation of cavities would
contribute to a close-packed structure. With increasing temperature, how-
ever, the energy supplied by polarization would decrease due to the in-
creasing rotational freedom from normal thermal effects. Consequently,
the net energy required to produce cavities would increase. It seems pos-
sible also that increased energy might be needed to form holes in the close
packed structure of liquid water. This would agree with the conclusion of
Morgan and Warren (1938) concerning the shift in structure between
1.5° C. and 83° C

Eley postulates further that of all the cavities in water only certain ones
are available to neutral molecules, whereas ions and water molecules are
interchangeable in the lattice structure. Polarization of water about a cav-
ity occupied by an inert gas is limited to the immediate neighborhood ; about
an ion the force field extends through several molecular shells tending to
increase the regularity of molecular configuration. For compilation of the
physical and chemical properties of the water see the monograph by Dorsey
(1940). Detailed reviews of the structure of water as it relates to biologi-
cal problems are given by Barnes and Jahn (1934) and Blanchard
(1940).

Summary of Water Structure : — This review of work on the structure of water
indicates that, far from being a simple mass of spherical molecules, randomly arranged,
and independently agitated by thermal energy, water is composed of polar molecules
coordinately arranged in some sort of lattice-like network and bound by a number of
intermolecular forces such as dipole attractions, London forces, and hydrogen bonds.
As ice, water apparently assumes a normal four-coordinated hexagonal structure with
12 HoO molecules per unit cell. Although variously assumed to have three-fold as-
sociation, hexagonal ring structure, and a four-coordinated hexagonal lattice (the lat-
ter seems best substantiated by modern methods of analysis), the important point is
that a regularity of structure compatible with geometrical arrangement of molecules
and intermolecular forces predominates in water in the solid form.

As liquid, water apparently has abnormal coordination and several forms may
occur. These forms apparently coexist and the shift from one to another depends
largely upon temperature and the presence of solutes. Though liquid water forms a
continuum with sufficient order to give an x-ray diffraction pattern, the existence of
any one molecular configuration is only of statistical significance, the total structure
being in continual flux.

Recent theories of liquid structure postulate points of abnormal coordination that
act as cavities or holes. Such holes are shared communally by all molecules of the
liquid, and viscous flow involves formation of double molecules that rotate into new
positions within the holes. With rise in temperature a shift in structure from the hexag-
onal ring lattice to a closer-packed quadrilateral structure apparently occurs but co-
ordination of the individual molecules decreases.

The dipole character of water molecules and their tendency toward hydrogen bond-
ing are important in their role as solvent. In the formation of solutions the participa-
tion of both solute and solvent in the structure should be considered.

As water passes into the vapor state, intermolecular distances increase immensely,
forces between molecules diminish, and, it seems agreed, coordinate structure gives way
to the chaos of the typical gaseous state.

Chapter III
PROPERTIES OF SOLUTIONS

Introduction : — Studies on the structure of water have tended to single
out the water molecule as being most influential in molding the molecular
architecture of aqueous solutions ; where solutes have been considered, the
primary emphasis has been on the effects of solute molecules upon the ar-
rangement and properties of water molecules.

The Interrelation of the Properties of Solutions : — Studies on solu-
tions have tended to center about the effects of solute molecules upon such
properties of the solution as osmotic pressure, thermal properties, vapor
pressure, and the like. Most of the calculations have involved an equation
of state, that is, an equation expressing the relations between pressure,
volume, concentration, temperature, and similar physical characteristics.
Starting from the concept of an ideal solution, they have attempted to recon-
cile deviations from ideality with known or predicted properties of the
solute and solvent.

The first comprehensive theory of dilute solutions was founded by
van't Hoff (1887, 1888). Acquainted by the Dutch botanist de Vries
with the osmotic experiments of Pfeffer, he formulated an equation re-
lating osmotic pressure with the concentration of a solution. The expres-
sion is

P„V = nRT (i)

where P^ = osmotic pressure of the solution,
V = volume of the solution,
n = number of mols of solute,
R = the molar gas constant, and
T = absolute temperature.

Proving that for dilute solutions the value of the constant R was identi-
cal with that of the gas equation, van't Hoff arrived at the important
generalization that the osmotic pressure of a dilute solution is equal to the
pressure that the molecules of the solute would exercise if they existed in
a gaseous state in the volume occupied by the solution. As pointed out by
FiNDLAY (1919), the unique value of this finding lies in the fact that the
concept of osmotic pressure and the use of semi-permeable membranes
allows the properties of solutions to be treated like those of gases, particu-
larly the quantitative relation between colligative properties and concen-
tration.

Though his law has been misused, van't Hoff realized that it applied
only to very dilute solutions. This is apparent from the expression given
because, in it, osmotic pressure varies as the ratio of mols of solute per
volume of solution, whereas in an exact law it should depend upon the
ratio of solute to solvent molecules and the forces between them. The
above law provides no correction for the effect of the solute molecules upon
the properties of the solvent or vice versa.

Chapter III —19— Solutions

The ideal equation for one mol of gas is usually given in the form

PgV = RT (2)

where Pg = the pressure of the gas against the walls of the container,
V = the volume of the gas,

R = a proportionality constant commonly termed the molar gas constant, and
T = absolute temperature.

Though useful for gases at high temperature and low pressure this law,
because it neglects attractive forces between molecules and provides no cor-
rection for the space occupied by the gas molecules, is invaUd for gases at
low temperatures and high pressures. Thermodynamically it is based on the
assumption that gas molecules are elastic spheres having no attractive
forces and occupying no space.

To correct the weaknesses of this law van der Waals derived the
equation

(Pg + ^) (V-b) = RT (i)

in which

-^is a measure of the attractive forces of the gas molecules or the co-
hesive forces as applied to liquids. V-b is the free space unoccupied by
molecules. The van der Waals constants a and b have been widely used
in dealing with the properties of non-ideal gases and liquids.

Many other refinements to the gas laws have been offered. Emphasis
here is on the fact that just as the ideal gas law finds an analogy in van't
Hoff's law of osmotic pressure, the van der Waals equation, and subsequent
improved equations have been applied to the relations of solutions.

Meanwhile Raoult had proposed a law relating the vapor pressure of
a solution to the mol fraction of the solvent or solute in it.

p = p°Xl = p° (1— X2) (4)

where p = the vapor pressure of the solution,

p° = the vapor pressure of the pure solvent,
XI = the mol fraction of the solvent, and
X2 = the mol fraction of the solute.

Lewis (1908) recognized the validity of Raoult's law as applied to
both dilute and concentrated solutions and Hildebrand (1924) has pointed
out its wide applicability to studies on solutions. Ideal solutions may be
defined as those that obey Raoult's law at all temperatures and pressures.
Only liquids having equal changes in pressure with temperature at constant
volume will obey Raoult's law. Such liquids mix without heat of dilution
or change in volume.

From the above considerations it is possible to formulate a thermo-
dynamic equation of state to cover all liquids.

(w).-(w)v-

where P = pressure on the liquid,

E = total energy of the liquid,
V = volume of the liquid, and
T = absolute temperature.

Hildebrand (1936) gives the following empirical expression for the
members of this equation :

Grafts et al. — 20 — Water in Plants

(6)

Internal Attractive Repulsive Thermal External

Pressure Pressure Pressure Pressure Pressure

a = the constant of the van der Waals equation,
V = the molal volume of the liquid,
c = a constant, and

r^;; measures an intermolecular force of attraction between the molecules of a liquid.

The difficulty often experienced in using such an equation is in evaluat-
ing the energy factor E from data on osmotic pressure, or thermal prop-
erties. HiLDEBRAND and others have determined values for the internal
pressure of liquids by measuring changes of pressure with temperature at

constant volume / ^ \ and substituting these in equation (5).
\ ^T )v

The Forces Between Atoms and Molecules: — The properties of
solutions depend ultimately upon the molecules and the electric forces
within and between them. The ideal gas law assumed that there was no
attraction between molecules but experiment proved that at low tempera-
tures and high pressures attractive forces existed.

Deviations from ideality in solutions are even greater than in gases.
Much of the modern theory of solutions deals with analysis of the forces
between atoms and molecules.

The electrons within a molecule are in constant motion. The average
positions can be so designated that the potential at a distance from a mole-
cule due to its charge can be calculated. If e represents charge and r dis-
tance, the field strength of a charge varies as -^ according to Coulomb's
law. This field strength is zero for a neutral atom. Atomic ions such as
Na"" or CI" will have fields varying as - . More complex neutral mole-
cules or ions in general have potentials varying as -7^ ' -p ' etc. The term -^

represents the field strength of a dipole. The term ^ represents a qua-

drupole moment, important when the effective dipole is small.

By similar treatment it is possible to calculate the force upon a second
particle located at a distance from the first. An ion in a field of the type
mentioned above experiences a force of translation ; a neutral molecule with
an electrical moment does so only if the field is non-uniform. Characteris-
tics of the above forces of interaction are summarized in Table 3, num-
bers 1, 2, and 5.

Table 3. — Characteristics of molecular interaction
(front Bateman, in Hober, 1945) : —

Type Attractive force

number Interacting particles proportional to

1 Ion — Ion e, e', • r"^

2 Ion — Permanent dipole =p u' e, • r"^

3a Permanent dipoles coaxial u u' • r"*

3b Permanent dipoles at right angles o

4 Ion — Induced dipole a ei^ • r"^

5 Permanent dipole — Induced dipole a u^ • r"''^

6 Transient dipole — Transient induced dipole a^ • r"^

Ci, e'l denote ionic charge. a denotes polarizability.

u, u' denote dipole moment. r denotes distance between centers.

Chapter III

— 21

Solutions

In addition to the forces described above, dipoles and higher poles ex-
perience a torque making certain orientations more stabile than others. For
this reason the electric axis in the field of an ion tends to orient toward the
ion. In the field of a second dipole the axes tend to become parallel. These
forces are disturbed by thermal agitation so that complete orientation is pre-
vented under most conditions but the tendency tov^ard alignment is re-
flected in an attractive force termed the orientation efifect.

When two molecules approach each other until their electronic charges
encounter the fields of their nuclei there are strong attractive forces be-
tween them. As they come even closer together their electronic clouds no
longer screen their nuclear charges ; the nuclei repel each other by the
electrostatic Coulomb forces. The strength of primary chemical bonds
cannot be accounted for by the attractive forces mentioned above at the
equilibrium point but the polarization effect does explain some types of
residual force.

Even spherically symmetrical molecules are polarized in an external
field and the forces between the inducing particle and the induced dipole are
shown in numbers 4 and 5 of Table 3. Spherically symmetrical molecules
with a zero average field may show temporary asymmetry and hence give
rise to a fluctuating field. These transient fields induce transient electric
moments producing the dispersion forces listed in number 6 of Table 3.

Table 4. — Contributions of the three constituents of the van der Waals forces to inter-
molecular forces of the liquid state {selected data front Hober (1945) and

London (1937) : —

h'

0

0

S

Compound

>

<

u

_!
0

.J

<

^-. w
2 2

X S

2

w

S 0

>•

h

5X

0

OS 0
w >

I-

1—1

0 ®.
« 0

CD
0
'"^ (—1

. to

0
§ Si

S

M

.a

Q

Ph

W

0

)-H

P

CO

32.7

1.50

39.7

0.12

1.99

14.3

0.0034

0.057

67.5

HI

0.38

5.40

12.0

0.35

1.68

382.0

HBr

37.5

4.51

44.1

0.78

3.58

13.3

6.2

4.05

176.0

HCl

30.8

3.72

40.7

L03

2.63

13.7

18.6

5.40

105.0

NHs

24.5

4.22

36.9

1.50

2.21

16.0

84.0

10.00

93.0

H2O

18.0

5.53

30.4

1.84

1.48

18.0

190.0

10.00

47.0

Table 4 presents values for the contributions of the orientation, induc-
tion, and dispersion effects to the van der Waals forces between molecules
for six well known compounds. Common values for the van der Waals
constants a and b are also included.

Although the details of atomic interaction cannot be treated here it
seems well established that under certain conditions, as atoms or molecules
come together there is a tendency toward an equilibrium distribution of
outer shell electrons in the internuclear region that constitutes the "electron
sharing" postulated by electronic theories of valence. These forces are
responsible for all attractions between non-ionic particles exemplified by
numbers 3, 5, and 6 of Table 3.

A detailed analysis of the general theory of molecular forces is given
by London (1937). The above treatment follows closely that of Bateman
in Hober (1945).

The development of the modern views of intermolecular forces has
come through many stages. Early chemists speaking in terms of chemical

Grafts et al. — 22 — Water in Plants

affinity drew a sharp distinction between primary and secondary or residual
affinities. They failed to realize that chemical affinity has dimensions of
both quantity and intensity. Organic chemists recognized coordination
groups or spheres within which valence forces became identical and they
recognized conjugation as a means of satisfying unsaturated valence force.
It was not however until the discovery of the electron and the demonstra-
tion of its relation to valence that a clear picture of chemical affinity became
available. In 1916 Lewis presented his electronic theory of valence postu-
lating complete transfer of electrons to explain the formation of ionic, or
valence bonds, and mutual sharing of electrons to explain covalence.

Examples of these types of bonding are given in the following re-
actions.

Ionic: :Na: • + -Ci: ^ :Na: :Ci: (7)

H
Covalent : 4 U ■ + ■ C ■ ^ U -.C-.U (8)

H

Covalences may be of two types, normal and coordinated. In the nor-
mal type (equation 8), both of the reacting atoms contribute an electron
to complete the octet and thus form a stable bond ; in the coordinate type
both of the electrons are supplied by one of the atoms, which results in a
somewhat weaker bond. These two types of covalent linkage may be gen-
eralized in the following way :

Normal covalent: A- +B_»A:B (9)

Coordinate covalent : A : -|- B — > A : B (10)

The hydrogen bond, a somewhat different type of linkage, which is
partly ionic and partly covalent in character, is of great significance in
biology. This is found only between atoms of high electronegativity {i.e.,
F, O, N, and CI) and the strength of the bond is determined by the degree
of electronegativity and the ionic radii of the groups joined. Hydrogen
bonds are involved in many biological systems. As explained they are re-
sponsible for coordination of water in the liquid and solid states. They
are responsibe for formation of chelate rings as in the following dimer of
formic acid vapor.

0 •

• • H

0

/

\

H -

- C

c

\

/

0

H ■ •

• -0

H (11)

They account for the hydration of colloids such as cellulose and pro-
teins. And they form the bridges responsible for the 3-dimensional archi-
tecture of many bio-colloids, possibly of the living protoplasm. For a de-
tailed treatment of chemical affinity see Remick (1943).

In aqueous solutions in which the biologist is interested practically
every form of bond known to the chemist may be involved. The nature of
the forces of coordination between water molecules has been pointed out
in Chapter II. The values of 1.04 at 100° C. and 0.60 at 150° C. (page 16)
given by Ewell and Eyring indicate the coordination of water vapor.
At 0° C. the number is variously estimated from 2.68 (page 16) to 4.

Water molecules are pictured as small spheres having two positive and
two negative residual valence charges. The separation of these charges
gives the molecule dipole character and water has a dipole moment of 1.85.

Chapter III

23

Solutions

The force of attraction between water molecules is equal to the product of
their dipole moments divided by the fourth power of the distance between
centers. This is great enough to bring about a quasi-crystal lattice as men-
tioned in the previous chapter. The energy of the O-H-O bond of water
is assigned a value of 4.5 K cal. Electronic theories of valence have in-
dicated that all types of bonds result from mutual potential energies of
atomic nuclei and their associated electrons.

Bateman also considers van der Waals forces as active in the deter-
mination of the molecular volume of liquids. They are brought into cal-
culations to explain the discrepancies between the observed and calculated
values of the osmotic pressures of concentrated solutions, possibly because,
here too, they afifect the partial molal volume of the solvent. London
(1942) has treated systems containing long-range van der Waals forces
that may be of biological significance. Such long-range forces acting in
conjugated chains may account for the elasticity of rubber. In hydro-
philic systems these forces may even be visualized as affecting the properties

0 Z 4 ^ 6 10 /Z Af J6, /6 20 22 Jif 26 2S^

Gram J water per wo gram^ of soil

Fig. 9. — Typical vapor pressure-water content curves for sand and clay-
showing the values of the permanent wilting percentage (PWP) and field
capacity for each. (From Veihmeyer and Edlefsen, 1936).

of whole molecules and molecular aggregates. Eley (1944) discusses the
effects of ions upon the heat capacity of water from the standpoint of hydra-
tion shells and long-range structure. Thus we perceive how, starting with
nuclear atoms, and utilizing the directive forces of electrons, it is possible
to build a picture of morphological organization conditioned largely by the
geometry of molecules and restriction of their random motion by bonding
forces. And dominating this picture, as it applies to biological systems,

Crafts et al.

24 —

Water in Plants

are those influences that determine the behavior of molecules in the liquid
state.

Molecular Interaction and Water Binding: — From the foregoing
discussion it seems evident that the nature of aqueous solutions is definitely-
conditioned by the forces exerted by water molecules among themselves
and upon molecules of the solute. In simple one salt solutions the force
relations may be analysed without too much difficulty but as the solutions
become complex a satisfactory analysis is impossible. The problem is
made difficult not alone by the complexity of the relations between solvent
molecules and between solvent and solute molecules but by interactions be-
tween solute and solute both as these relate to the solvent and to the state
of aggregation of the solute. Although a number of types of forces are
exerted between the molecules of a solution as illustrated by Table 3,
these forces are all electrical in nature and are so integrated that within a
single system they form a smooth series. This is reflected in the form of
the vapor pressure : water content curves for soils and cellulose as illus-
trated by Figures 9 and 10.

/oo

t

20

y

^

/

/

/

/

X

/

/
/
/

/

/ /

.04- .06 . IZ ./6 ,10 .24 .26 iZ

Moisture content

Fig. 10. — Curves showing the relation between moisture content
and relative humidity of tlie atmosphere surrounding cotton (solid
line), and spruce wood (broken line). Redrawn from Babbitt
(1942), figure 3.

In biological systems molecular structures of great size and complexity
are often encountered. Much research has concerned the forces binding
water to molecularly dissolved solutes and to colloids. Studies on colloidal
hydration have given rise to a concept of water adsorption termed for con-
venience, "bound water" about which much controversy and confusion has
centered. The many definitions given including "water — in living tissues
in a state different from that of water as we know it in bulk" (Gortner,
1938), "adsorbed water," "oriented water molecules," "non-solvent water,"
"non-freezable water," and the like, all indicate that water in the bound
state deviates in physical or chemical properties from water in an ideal solu-
tion. Gortner states, "Heats of hydration, dielectric constant measure-
ments, and the contraction of the system colloid-water — all indicate that
the H2O molecules in bound water are more closely packed and are prob-
ably specifically oriented in relationship to each other possibly in a more
or less true crystal lattice which is more densely packed than the crystal

Chapter III —25— Solutions

lattice of ice." Out of these definitions has grown the idea that in colloidal
systems water may exist in two different states, bound water and free water.

Modern studies have changed this concept. Just as the idea of definite
water polymers has been displaced by the newer picture of coordinated
water molecules making up a continuum of quasi-lattice properties, so has
the concept of bound water been gradually broadened to the extent that all
inter- or intra-molecular forces affecting the activity of water molecules
are given consideration. For this broader concept the term hydration is
commonly used. From one viewpoint it seems that there is no such thing
as free water ; all water molecules in the liquid state have restricted mo-
tion because of their own intermolecular bonding. The restrictions and
the forces causing them may vary in intensity because of the variety of
possible types of hydration complexes ; the water molecule, however, does
not lose its chemical identity by reason of the forces with which it is held.
Although Bernal and Fowler postulate three types of water, they state
that they pass continuously into each other with change in temperature.
To deny the binding of water molecules would require a complete neglect
of those secondary valence forces that, through hydrogen bonding, account
for so many of the unusual properties of water. To adhere to the sharp
distinction between bound and free water, on the other hand, would neces-
sitate drawing a clear line between the behavior of such valence forces
toward crystalloidal, and colloidal substances, a line that is not indicated in
physico-chemical studies of solutions. That Gortner was fully aware of
the nature of these forces is evidenced by his statement, "We may con-
clude therefore that the forces which bind water on the surface of the
lyophilic colloids are of the same nature as the forces which cause the as-
sociation of water in bulk and which immobilize water molecules in the
ice crystal lattice. However, there is evidence that these forces on a sur-
face or at an interface may be of greater magnitude than the forces of asso-
ciation of water molecule for water molecule or the forces which tend to
arrange water molecules in the ordinary ice crystal lattice. Therefore at
least a part of the molecules of the bound water film may be expected to
have an activity which is less than the activity of the H2O molecule in the
ordinary ice lattice" (1938, p. 304).

It should be pointed out here that these forces show no unique response
to colloids ; they are the forces that cause hydration of ions and molecules
as well as micelles and hence cause the major deviations from ideality of
aqueous solutions.

It is often implied that bound water bears a numerical relationship to the
colloid {i.e., so many grams of water per gram of colloid) and that this
relationship is constant at different concentrations of the solution. If we
can believe the vapor pressure : water content curves this is not the case.
These curves show that as the water content of a solution decreases the
intensity of the binding force per water molecule increases. Therefore, if
equilibrium exists between the solute and the solvent, it seems evident that
the amount of water bonded to the colloid shifts continuously with water
content, there being much loosely held water at high water contents. As the
water content decreases, the remaining water is more tightly held.

For example, in the hydration of cellulose it has been postulated that,
between the dry and saturated conditions, three different mechanisms are
responsible for holding water. At low water contents the water molecules
are pictured as being held as monomolecular layers, probably by hydrogen
bonds. At intermediate contents the layers become polymolecular with a

Grafts et al. — 26 — Water in Plants

diminished intensity of bonding force per molecule of water. As the water
content approaches saturation capillary condensation is suggested to ac-
count for the loosely held water. However, these three mechanisms are
so closely integrated that the vapor pressure : water content curves are
smooth. Babbitt (1942) shows such a curve in his Figure 3. The rela-
tion of binding energy to vapor pressure in terms of relative humidity is
shown in his Figure 7.

GoRTNER (1938) has reviewed the literature on bound water and has
described thirteen methods for measuring it. Though results vary, all
methods show appreciable quantities of water that fail to obey the laws of
dilute solutions. Deviations from laws of concentrated solutions are not
always so great.

Any molecular species which dissolves in water, when added to a water
mass, tends by means of its own valence forces to satisfy those of the
water. When added, not only the water molecules in the immediate vicinity
but statistically all water molecules in the system are affected, as evidenced
by a change in vapor pressure and a corresponding lowering of activity.
With continued addition of solute there results a smooth and progressive
reduction in activity of the water. Provided there is no change in state,
the initial addition of solute to the solvent results in reduced activity also
of the solute. The case of a single undissociated solute is relatively simple
to analyse. With dissociation of the solute molecules three activities are
involved, and with the addition of two or more solutes a high degree of
complexity occurs with the possibility pointed out by Chandler (1941),
that sufficient decrease in two or more activities may actually allow increase
in a third without disturbing the thermodynamic balance. This phenomenon
may possibly explain negative values for bound water that are found oc-
casionally.

As one deals with more complex situations involving both colloidal
and crystalloidal solutes, accurate determination of the activities of all
constituents becomes impossible. Hence many of the measurements on
so-called "bound" water do not necessarly prove its existence but simply
reflect complex intermolecular reactions that are incapable of analysis. On
the other hand, there are many examples of the hydration of colloids in
which small but measurable amounts of water are held by intense binding
forces. The use of the term "bound" to describe such water is very con-
venient.

Hydration: — The relations of the solvent to the solute in aqueous
solutions has received much study and theories of hydration enter into most
considerations of the properties of solutions. In Armstrong's (1908)
theory of the polymeric isomerism of water the presence of solutes is sup-
posed to affect materially the dissociative change

(HoOni^nHoO {12)

When a compound such as HCl dissolves, complexes are assumed to be
formed as follows:

H H

HoO , HCl , and H2O : CIH

\ \

CI OH

The first two are active, the last inactive so long as it remains closed.

Chapter III

27

Solutions

Dissolved molecules may be converted into inactive closed groups :

H H

/ \

RX + XR ±^ RXXR + 2H2O

\ /

OH HO

(iJ)

Dilution would increase the number of active molecules of the type

OH H

HCi J whereas in concentrated solutions the hydrolyzed solute HoQ

\

H

\

CI

would increase.

Most of the earlier workers on osmotic properties of solutions postu-
lated hydration of the sucrose molecules to explain the fact that observed
values of osmotic pressure were higher than calculated values. Calcula-
tions were based on the assumption that a hexahydrate existed in solution
(FiNDLAY, 1919) ; Haldane (1918) postulated a pentahydrate ; for the
highest concentrations lower hydration values were used (Findlay, Tables
XXV and XXXI).

Pennycuick (1928) pictures the reaction between H2O and HCl as
follows :

HCl + HoO ^ H":C1;

H:0:-
H

(i^)

This compound would react with ammonia in this manner

H

NHs + HCI-H20 ^ H : N ;

H

which in turn would break down thus

H
H:N:H:C1:

H:C1:

H:0:
H

{15)

H : O : ±5 NH4CI + H2O
H

(id)

Because the water molecule has both the plus charges of the hydrogen
nuclei and the negative charges of the lone oxygen electron pairs it will
hydrate any substance having a force field,

Bernal and Fowler (1933) conclude that all strongly polarized ions,
all divalent and trivalent positive ions, and the negative ions OH" and F~
are hydrated {see, however, page 7). The effect of hydration of ions is
to lower the refractive index of water by the coordination of water mole-
cules around these ions. This would tend toward an increase in the regu-
larity of arrangement {i.e., a shift in the direction Water III -^ Water II
-^ Water I) . (See page 12) .

In the past there has been a tendency to draw a distinction between
hydration and imbibition, the former term being applied to the association
of water with ions and molecules, the latter to colloidal materials. Modern
views would favor the definition of hydration as the resultant of any inter-
action between solute and water molecules tending to reduce the activity
of the latter. Such a definition erases any line that has been arbitrarily
drawn between imbibition and hydration. Though the extreme examples
of crystalloidal and colloidal hydration may be readily distinguished the

Grafts et al. — 28 — Water in Plants

fundamental nature of the process is the same in both cases, namely, a
satisfaction of electrical forces of attraction between molecules.

Some have pointed out the close connection between imbibition and
osmotic pressure. Heuser states (1944, p. 62), that thermodynamically
swelling (or imbibition) pressure appears to be identical with osmotic pres-
sure, and quotes Steinberger to the effect that "osmotic pressure is noth-
ing but swelling pressure, made evident by the external device of a semi-
permeable membrane."

Brooks and Brooks (1941) similarly point out the lack of a clear
distinction between the two phenomena: "Imbibition is simply absorption
of water by a system which is in effect an exceedingly concentrated solu-
tion, and can often be shown to obey the laws of ideal concentrated solu-
tions. ..." And "Imbibition, in any fundamentally sound sense, is not
a property of colloidal systems alone, but may be exhibited by homogeneous
solutions of crystalloids, among which are such diverse substances as
gases, molecules of solid or liquid non-electrolytes, and ions of electrolytes."
This constitutes a redefinition of imbibition. We favor the use of hydra-
tion in the sense that Brooks and Brooks define imbibition.

Bull (1943) uses an equation to calculate swelling pressures of a gel
that is identical with the vapor pressure equation for osmotic pressure
(equation 2, Chapter IV). This equation is:

Ph V = RT In Ho. <^-^'')

P

Where Ph is the swelling pressure and V is the partial molal volume
of the liquid. Since he found that the swelling of a gel can be evaluated in
terms of vapor pressure lowering of the solvent, regardless of the fac-
tors responsible, its close similarity to osmotic pressure is evident. Be-
cause such vapor-pressure lowering creates an energy gradient, it accounts
for water absorption by vacuolated cells and colloidal imbibants whenever
water at higher diffusion pressures is available.

At their extremes, osmotic and imbibition pressures are clearly differ-
ent. For instance, the retention of imbibitional water by cellulose fibers
involves little or no osmotic effect, and the reduction of the diffusion pres-
sure of water by the presence of ions or small molecules (i.e., in a solution
of NaCl) entails no imbibitional effect. However, when one considers
larger and larger molecules or molecular aggregates in solution, he en-
counters a marginal region where the two effects are not distinct. Just
as it is impossible to differentiate sharply between the crystalloidal and
colloidal states, so it is difificult to draw a line between osmotic and im-
bibitional forces. In the crystalloidal solution, all of the molecules are
relatively mobile and if a differentially permeable membrane is used, the
solute and solvent may be separated. In the colloidal solution, on the other
hand, some of the water of imbibition may be held so firmly that it is re-
movable from the colloid only by vaporization. Because of the localiza-
tion of imbibed water on the surface of the colloid, its solvent powers for
crystalloidal molecules may not be uniform throughout a given system.
Hence, where both crystalloids and hydrophyllic colloids are involved in
cells, although an equilibrium of forces exists, measurable in terms of the
osmotic concentration of the cell sap, some water may be held by imbibi-
tional forces, and such water must be accounted for in any attempt to
evaluate the water status of the cell. In this connection, it should be em-
phasized that the effects of both osmotic and imbibitional forces are simi-

Chapter III — 29 — Solutions

lar in that they bring about a reduction in the total volume of the system
(solute, or imbibant, plus solvent). This indicates an ordering of mole-
cules and an evolution of energy and invariably results in a reduction in
diffusion pressure. Thus water of imbibition includes "bound water" as
described by Gortner in the quotation on page 24. Though hydration
has been defined to include both osmotic and imbibitional phenomena, it
may be very convenient to make a qualitative distinction between them.

According to modern views on the structure of water, as explained in
Chapter II, forces of coordination resulting from hydrogen bonds tend to
hold water in a quasi-crystalline lattice and solutes capable of satisfying
these forces may enter into this structure. Numerical relations of coordi-
nation are of statistical significance only as the molecules are in a con-
tinuous state of flux, coordination becoming less and less definite with in-
creasing temperature. The firmness of binding is a matter of the nature
and intensity of the bonds involved. Hydrogen bonds are relatively weak,
covalent bonds stronger, and ionic bonds of great strength. When water
of hydration or imbibition near the saturation point is removed, little energy
is required; as more and more water is removed greater force is necessary.
For most colloids, however, water has little or no stoichiometric relation
to the hydrated or imbibing compounds. Probably both hydrogen and
covalent bonds account for its binding. With water of crystallization,
definite numerical relations exist and much energy is required to remove
such water from the crystal ; it is largely held by covalent bonds. Undoubt-
edly all possible types of bonds are involved in the hydration of cell walls,
protoplasm, and vacuole.

The eflfects of force fields upon the packing of water molecules have
been discussed by Bernal and Fowler (1933),, Morgan and Warren
(1938), and others. Though the arrangement of molecules may differ
with the resulting variation shown by the properties of liquid water, water
of crystallization, and ice, the state of aggregation does not change exces-
sively as shown by the limited variation in coordination throughout the
range from water at 100° C. to ice. Eley (1944) points out that the molal
heat capacity of hydrate water approximates the value -9 cal. deg'^ mol"^,
the value for ice. For further consideration of the effect of binding forces
upon the density of water as shown by adsorption on cellulose see Babbitt
(1942). Heuser (1944) devotes a complete chapter to the reactions of
cellulose with water.

In contrast to the solvent, the state of aggregation of the solute mole-
cules may vary widely and the definition of hydration involves solutes
that form ions, that dissolve as undissociated molecules, or that form
molecular aggregates of great magnitude. Though the forces causing
hydration obey a hyperbolic law and hence at certain proportions of solute
to solvent may exhibit a high rate of change, this region of high curvature
of the free energy : water content curve does not represent a break. Figure
9 shows two such curves for soils (Veihmeyer and Edlefsen, 1936).
Others are presented by Briggs (1932, Figures 1 and 2) and Edlefsen
and Anderson (1943, Figure 1). Greenberg (in Schmidt, 1938, page
472) presents a similar curve for swelling pressure.

There is considerable literature to indicate that a water content of 0.3
to 0.5 grams per gram of colloid may be a critical region for gelatin, casein,
acacia, fibrin, agar, and many other biocolloids (Gortner, 1938). Pos-
sibly this water content may fall within the region of maximum curvature

Crafts et al. — 30 — Water in Plants

of the free energy : water content curve and hence separate a region where
energy change with change of moisture is low from one where energy
change is high. The changes through these regions however are smooth
and continuous ; they are quantitative rather than quaHtative. Evidences
for breaks or plateaus on such curves usually involve changes of state
(sol ^ gel etc.) or very low water contents.

Early attempts to explain deviation of observed osmotic pressure meas-
urements from ideality involved association of the solvent and hydration
of the solute. The graph for observed osmotic pressure curved rapidly
upward at high sugar concentrations whereas the law of ideal solutions
followed a straight line. Differences between the curves represent losses
of water activity that could as well have been attributed to water binding.
These losses result from intermolecular forces characteristic of the solute
and solvent. Of the various methods for measuring hydration the vapor
pressure method should excel for it integrates these forces and gives a
true picture of water activity in the system ; it also avoids pitfalls such as
super-cooling in the freezing methods and molecular interaction where
a reference solute is used. Chandler (1941) has obtained accurate re-
sults by this method.

Greenberg (in Schmidt, 1938) states " — hydration does not appear
to be an important factor in determining the properties of protein solu-
tions." Bull (1943), on the other hand says "bound water may or may
not contribute greatly to the understanding of physiology and pathology,
but its importance for the understanding of protein reactions is extreme."
Greenberg along with many others has pointed out the shortcomings of
the various methods for determining bound water. If these methods are
inadequate for measuring the amount of bound water in a protein how
much less adequate are they for indicating its importance.

In conclusion it should be emphasized that the differences in degree of
hydration that have been measured and that surely exist between different
biological systems reflect differences in the intensity of binding forces and
in the packing and configuration of the molecules of solvent and solute that
these forces control. The nature of the forces and of the water molecules
doesn't vary ; the differences in state between the substrates however are
real and important. They typify the differences that exist between water
in the beaker, solutions as they occur in cells, and protoplasm, seat of the
complex and baffling activities of the living organism.

Mobility of Ions: — The migration of ions and molecules in aqueous solution is of
great importance to biology. Much research has been directed toward problems of
permeability and solute uptake by living organisms.

A perplexing problem in ion mobility is the high migration velocity of hydrogen
and hydroxyl ions. Velocities of K'^ and NH4"^ are quoted as 6.7 X 10'^ F cm. per sec.
where F =: the potential gradient in volts per cm. Those of H* and OH" are 32.5 X
10-1 p cm. per sec. and 17.8 X 10"* F cm. per sec. respectively. Bernal and Fowler
explain these high velocities by a mechanism involving a proton jump from one water
molecule to another when favorable configurations are presented. Such a mechanism
presupposed an appreciable degree of organization of the structure of water. Much
of modern physical chemistry involves the types and strengths of chemical bonds. The
structure of water as related to such bonds, and the forces between ions and water are
all concerned in ion mobility. Also involved are the membranes encountered and the
interfaces between them. It is evident from this discussion that the problems of the
mobility and uptake of solutes by plants are exceedingly complex when viewed from
the standpoint of molecular kinetics as exemplified by the process of diflfusion. When
one adds the metabolic activities of the growing cell to this picture it is easy to under-
stand the difficulties encountered in the study of plant nutrition. The material presented
in this chapter indicates the progress that is being made in the study of solutions. It

Chapter III — 31 — Solutions

is not the purpose of the present publication to go further into this subject. Interested
readers are referred to the books of Remick (1943) and Hober (1945) which have
been drawn on freely for the contents of this chapter. Further source material on the
dielectric properties of matter appears in Chemical Reviews of 1936. Articles by Debye,
SiDGwiCK, Wyman, Cohn, Kirkwood, and Scatchard in this review should all prove
of interest to biologists. A symposium on structure and molecular forces in pure liquids
and solutions held in 1936 in Edinburgh is reported in volume 33 of the Transactions
of the Faraday Society. Further detailed work on solutions is reviewed by Kincaid,
Eyring, and Stearn (1941), and by Scatchard and Epstein (1942). The thermo-
dynamics of electrolytic solutions have received excellent treatment in a recent mono-
graph by Harneo and Owen (1943).

Summary: — Two viewpoints are expressed in explaining the properties of aqueous
solutions. One emphasizes the importance of the molecular constitution of water re-
lating the discrepancies from ideal behavior to polymerization, hydration, and complex
formation involving water molecules. The other stresses the effects of intermolecular
forces between solute and solvent and their efifect upon colligative properties.

Molecular interactions involve electrical forces resulting from the charges on elec-
trons and atomic nuclei. They are comprised of ion to ion attractions, ion : dipole
forces, and interaction between various types of polar molecules. They result in the
numerous types of valence forces known to exist between molecules; ionic bonds,
covalent bonds, and hydrogen bonds. The latter are of great interest in biology as
they account for the coordination of water, the hydration of crystalloids and colloids,
and the formation of bridges responsible for the three dimensional archiecture of bio-
colloids.

Studies on the hydration of biological materials has given rise to the concept of
"bound water." Well deserved criticism has been directed toward certain of the meth-
ods used to determine this water. While some would deny its existence entirely, a more
plausible view is that a small amount of water can be bonded to colloidal surfaces by
intense forces. Such bound water must differ from free vrater in some of its physical
and chemical properties ; its molecular constitution, however, remains unchanged. No
sharp line can be drawn between bound and free water, there being a smooth deviation
in the average intensity of the binding forces as water is added to or withdrawn from
the system.

Hydration is defined as the resultant of any interaction between solute and water
tending to reduce the activity of the latter. Water does not vary greatly in closeness
of packing but the state of aggregation of solutes may vary from ions to colloidal mi-
celles. The forces causing hydration obey a hyperbolic law and the curve relating
free energy to water content is smooth and continuous. Breaks in such curves involve
changes of state or very low water contents.

The vapor pressure method should be best for measuring hydration because it inte-
grates all factors causing deviations from ideality. It avoids supercooling and the use
of reference solutes.

High ion mobilities of H+ and OH- are explained in the basis of a proton jump
along coordinated chains of water molecules when favorable configurations occur.

Chapter IV
OSMOSIS AND OSMOTIC PRESSURE

Introduction: — The Abbe Nollet (1748) is credited with performing
the first recorded experiments on the phenomenon of osmosis. Using an
animal bladder, he found that water would pass through but that alcohol
would not. Traube (1867) discovered the copper ferrocyanide membrane,
and Pfeffer (1877), by precipitating such a membrane in a porous pot,
was able to measure osmotic pressures up to several atmospheres. Exam-
ples of Pfeffer's results are given in Tables 5 and 6.

Table 5. — Osmotic pressures of sucrose solutions at constant temperature : —

Concentration (c)
g. per 100 g. H2O

Osmotic pressure
mm. of mercury

(Po) Po
C

1
2

2.74

4

6

535
1016
1518
2082
3075

535
508
554
521
513

Table 6

. —

Influence of temperature on osmotic pressure: —

Temperature
°C.

Temperature
absolute (T)

Osmotic
mm. (

pressure(Po) Pq
Df mercury T

6.8
13.7
22.0
32.0
36.0

279.8
286.7
295.0
305.0
309.0

505 1.80
525 1.83
548 1.85
544 1.79
567 1.83

Because the concentration of a solution in mols per liter equals the
reciprocal of the volume V in which one mol of solute is dissolved, it fol-
lows from Table 5 that PqV = a constant. This is analogous to Boyle's
law for gases. The proportionality shown in Table 6 between osmotic

pressure and absolute temperature shows that a relation analogous to Gay

p
Lussac's law for gases also applies to solutions ; that is -^ = a constant.

By combining these equations one obtains the relation PqV = RT
where R is a proportionality constant. For dilute solutions van't Hoff
gave the equation

P„V = nRT (i)

where n = the mols of solute in V liters of solution.

By a comparison of the osmotic pressure of a cane sugar solution with
gas pressure exerted by hydrogen gas at the same temperature and con-
centration van't Hoff showed that the constant R in the equation above
had the same value as the gas constant.

Table 7 from van't Hoff (1888) shows the results of such a com-
parison. The data are osmotic pressures of a sucrose solution containing
1 gram in 100.6 cc. of solution compared with pressures of an ideal gas
at the same concentration.

Chapter IV

33

Osmosis

Table 7.

— Comparison of osmotic and gas p

ressures: —

Temperature

Osmotic pressure

Ideal gas pressure

°C.

atmospheres

atmospheres

6.8

0.664

0.665

13.7

0.691

0.681

14.2

0.671

0.682

15.5

0.684

0.686

22.0

0.721

0.701

32.0

0.716

0.725

36.0

0.746

0.735

Though the paralleHsm is not perfect, these results show that, within
the Hmits of the methods available at the time, the osmotic pressure of a
cane sugar solution is equal to the pressure of a gas at the same tempera-
ture and containing the same number of molecules as there are solute mole-
cules in unit volume.

Starting with cane sugar, van't Hoff showed that approximately the
same relation could be calculated for other dissolved substances such as
invert sugar, malic acid, tartaric acid, citric acid, magnesium malate and
citrate, all of which de Vries had shown to be isotonic in equimolecular
concentrations. This offered confirmation for Avagadro's law as applied
to dilute solutions.

From the above work van't Hoff made the important deduction that
the osmotic pressure of a solution is equal to the pressure which the dis-
solved molecules would produce if they existed as a gas in the volume oc-
cupied by the solution. This kinetic view of osmotic pressure has been
termed the bombardment theory (Glasstone, 1940) or the solute pres-
sure theory (Meyer and Anderson, 1939) of osmotic pressure. Ob-
viously, as van't Hoff pointed out, his deduction applies only to very dilute
solutions ; theoretically, it would apply only to solutes that obey Henry's law
and to solvents whose vapors are perfect gases ; practically, as expressed
in van't Hoff's equation, it holds fairly well for aqueous solutions of non-
electrolytes below 0.1 M in concentration.

Refinement of the Osmotic Pressure Law: — In addition to the
solute pressure theory some advocate a solvent pressure theory (Meyer
and Anderson, 1939), whereas others advance the vapor pressure theory
(Callander, 1908). To one familiar with modern views of molecular
kinetics, it should be clear that the diffusion pressure of solute and solvent
molecules in a solution as well as their vapor pressures should all be inter-
related. And, theoretically at least, changes in concentration should alter
pressures not in proportion to the ratio of solute molecules to a fixed vol-
ume of solvent as is implied by van't Hoff's law, but in relation to the
mol fraction of solute in the solution (Lewis, 1908).

Assuming that the vapor of the solvent obeys the gas laws, the relation
between the osmotic pressure of a solution and the vapor pressure is as
follows :

P„V = RTlnA ^^^

P
where

Pq = osmotic pressure of the solution,

V = the partial molecular volume of the solvent under standard pressure,
p = vapor pressure of the solvent above the solution,
Po = vapor pressure of the pure solvent.

Grafts et al.

34

Water in Plants

Bancroft and Davis (1928) and Lewis (1908) have derived similar
equations.

By Raoult's law if — = xi = 1 — xo, where xi = mol fraction of

Po

the solvent and X2 the mol fraction of the solute, the osmotic pressure
equation becomes

P„V = — RTlnd— xo) (3)

for an osmotic system in which the vapor of the solvent obeys the gas law.
This gives the osmotic pressure law in terms of the mol fraction of solvent
in the solution. If one expands the term [ — ln(l — X2)] he obtains

P„V = RT (X2 + y2 X.J + Vi xo^ . . .) (4)

For dilute solutions the higher powers can be neglected and the equation
becomes

P„V = RTx2 (5)

Likewise, for very dilute solutions if n^ = number of mols of solvent in
the solution and n2 = number of mols of solute, then

no —

xo = — - and P„ V

RT

no

(6)

Again for dilute solutions m V may be replaced by Vj, the volume of
solvent associated with n2 mols of solute in the solution, and

P„ Vi = na RT (7)

This is the equation used by Morse (1914). It gives the relation of
osmotic pressure to concentration of the soluti'on in weight molality and is
accurate to a concentration of about 1 molal.

By one more assumption, namely that volume and weight molar solu-
tions of a solute do not differ (valid only for very dilute solutions) one
can substitute V, the volume of the solution, for Vj, the volume of sol-
vent, and obtain PV := nRT where n := the number of mols of solute in
the solution. This is the van't Hoff law ; as he emphasized, it applies only
to very dilute solutions. Its principal value is to show the analogy between
the gas law and the osmotic pressure law.

To indicate the type of results to be obtained by use of these equations,
Table 8 reports values obtained by Frazer and Myrick (1916) and pres-
sures calculated by several formulae.

Table 8. — Observed and calculated osmotic pressure values for sucrose solutions at
30° C. (R = .08206, V = 18.052. Vi = 1001.9, T = 303°) : —

Concentration,

Observed

Calculated

Calculated

Calculated

mols per

(Frazer

van't Hoff

Raoult law

Morse

1000 g. of water

and Myrick)

equation 1

equation 3*

equation 7

atm.

atm.

atm.

atm.

1.0

27.22

20.4

25.0

24.82

2.0

58.37

34.8

49.0

49.64

3.0

95.16

45.3

72.1

74.46

4.0

138.96

53.4

97.0

98.28

5.0

187.30

59.8

119.3

124.10

6.0

232.30

64.0

142.0

148.92

* These values were not corrected for the change in V with concentration.

From these results it is shown that van't Hoff's equation falls far short
of providing agreement with the observed values; Raoult's law is some-

Chapter IV — 35 — Osmosis

what closer ; Morse's equation is even nearer. This is to be expected be-
cause van't Hoff's law was proposed to apply only to very dilute solutions ;
equation (3) is calculated from the mol fraction of solvent but does not
take into account the departure from ideal behavior of the solvent water;
Morse's equation is based on weight normal solutions but does not include
a correction for hydration. Lewis shows that Morse's equation fails at
high concentrations of solute because upon expansion it gives, in contrast
to equation (4), the relation

P„ V = RT (X2 + X22 + X23 + . . . .) (8)

Whereas the higher powers of X2 may be neglected at low concentrations,
at higher ones the differences between equations (4) and (8) become great
and Morse's equation gives excessive values. For examples, see Lewis,
Tables V and VL

Further refinements of the osmotic pressure laws involved corrections
for hydration, association of the solvent, the volume occupied by the
solute molecules, and for the mutual attraction between solute and solvent
molecules as in the van der Waals equation for gases. An equation in-
volving a correction of the latter type is that of Porter (1917)

P„ (V-b) = RT (9)

where b represents the space occupied by solute molecules with a consequent
reduction in free space. Values for the osmotic pressure of cane sugar
solutions calculated according to van't Hoff (equation 1), Morse (equa-
tion 7), FiNDLAY (1919) (equation 3), and Porter (equation 9) are pre-
sented in Table 9.

Table 9. — Osmotic pressure of cane sugar solutions at 20° C; observed and calculated

values: —

Weight

NORMAL

Osmotic pres-
sure OBSERVED

Morse

Osmotic

pressure

calculated according to /'TnCTiI'^

7

N

CONCENTRATION

van't Hoff

Morse

FiNDLAY

Porter .' -X^'"'

/

/ S .) r; 1 •">

'/o.

/ ^* . ,u -

atm.

atm.

atm.

atm.

atm. / \

0.1

2.59

2.34

2.39

2.38

2.43(

0.2

5.06

4.59

4.78

4.76

4.9i

0.3

7.61

6.74

7.17

7.14

7.4i.

0.4

10.14

8.82

9.56

9.51

10.02 \- .

/

0.5

12.75

10.81

11.95

11.87

12.64 \

0.6

15.39

12.72

14.34

14.24

15.35 ^ *

0.7

18.13

14.58

16.73

16.59

18.09 X. ,/

0.8

20.91

16.36

19.12

18.94

20.87 N^ #

>

0.9

23.72

18.08

21.51

21.29

23.74 ^*"»*— ...

-^

^

1.0

26.64

19.73

23.90

23.64

26.64

The values presented by Porter show almost perfect agreement but this
does not represent a fundamental improvement in the osmotic pressure
formula for Porter selected by trial and error a value for b to bring about
such agreement. The value proved to be 0.310 liters per mol. The actual
volume of cane sugar is 0.214 liters per mol at 20° C. The difference
between these, 0.096 liters per mol, Porter assumed to represent water of
hydration corresponding to -^ or 5.3 molecules of water per molecule of
sucrose.

Assuming that sugar solutions are hydrated throughout the range of
temperatures from 0° C. to 60° C. and the range of concentration from
0.1 to 1.0 weight normal. Porter calculated a series of hydration numbers

Crafts et al. — 36 — Water in Plants

from Morse's data that give an idea of the corrections required to fit
formula (7) to observed values. Table 10 presents these values.

Table 10. — Calculated hydration numbers for sucrose solutions at various tempera-
tures and concentrations: —

Weight

CONCENTRATION

0° c.

10° C.

20° C.

30° C.

40° C.

50° C.

60° C.

0.1

53.0

43.0

45.0

3.0

6.9

8.8

11.5

0.2

15.8

16.0

16.0

7.0

5.5

4.5

5.8

0.3

10.3

10.3

11.2

6.7

6.0

4.1

3.5

0.4

7.7

8.0

8.3

6.3

6.4

4.3

2.8

0.5

6.9

6.8

7.2

6.0

6.0

4.3

3.0

0.6

6.4

6.3

6.5

5.7

5.7

4.3

3.2

0.7

5.9

6.0

6.3

5.6

5.2

4.3

3.2

0.8

5.7

5.7

6.5

5.6

5.0

4.2

3.3

0.9

5.5

5.5

5.8

5.4

5.0

4.4

3.3

1.0

5.5

5.5

5.8

5.4

4.9

4.5

3.6

Assuming again that hydration is a factor in determining the value of
b, hydration numbers have been calculated by Findlay (1919, Table 26)
for a set of determinations carried out by Berkeley and Hartley at 0° C.

Since all of these hydration numbers were calculated in order to make
equation (P) fit the observed values, they simply indicate the correction
in volume required to account for the departure from ideal behavior of
these sucrose solutions. Since the attractive forces between adjacent
molecules have not been considered in these calculations, the whole devia-
tion has been thrown into the hydration number.

The degree of hydration has not been established experimentally; the
above calculations are highly empirical.

Equation (4) can be expressed in the form

P„ = x(l + ^^x + ^x2 . . .) (Findlay, p. 61) (10)

o

If the concentration of the solution is expressed in terms of c gram-
molecules of solute in 1000 grams of water, x can be put equal to ^^ ^"^ ^
where 55.5 represents the number of gram molecules in 1000 grams of

1001.8
55.5

water. Since the molecular volume of water at 20° C. is ., , , equation

(10) may be written

(81.6) (293) (55.5)
P

1001.8 55.5 + c

1 +

\ 55.5 + c /

(11)

It has been assumed for years that water is associated. In order to
correct for association the term . in equation (11) has been altered to

^°°^'^ where a is the association factor. Using van Laar's value 1.65

55.5 -f-a

at 20° C. for a, Vo = ^°°^'^ and if the osmotic pressure of weight normal

sucrose is calculated by equation (11) the value of 23.50 is found for Po
in place of 23.64 (see Table 9). Since the observed value was 26.64, this
correction is of doubtful use. Evidently association is not important at
this concentration and at higher concentrations it should be lower accord-
ing to common opinion as to the effect of solutes on association at 20° C.
Correcting further by including a factor /3 for degree of hydration, the
term for the mol fraction takes the form .,,,"', ^ , > where c' is the

55.5 -|- c — p c

Chapter IV

37

Osmosis

concentration of the hydrated molecules. Assuming a hydration number
of 5, the value of Po for weight normal sucrose at 20° C. becomes 25.96
and including the association factor of 1.65 the value becomes 27.50.

Frazer and Myrick (1916) have used the same formula to calculate
osmotic pressure values for cane sugar solutions at 30° C. Table 11 gives
their data.

Table 11. — Osmotic pressure of solutions of cane sugar at 30° C: —

Weight
molar con-

Osmotic
pressure

Osmotic

pressure
calculated
according

OSM'

OTIC PRESSURE CALCULATED ACCORDING
TO EQUATION (10)

CENTRATION

a = 1

a = 1

a = 2

a = 2

a = 2

(H=l)

OBSERVED

TO Morse

^ = 0

0 = 6

^ = 6

^ = 5

/3 = 4

atm.

atm.

atm.

atm.

atm.

atm.

atm.

1

27.22

24.72

24.4

27.28

26.99

26.45

25.98

2

58.37

49.43

48.32

61.19

59.89

57.41

55.1

3

95.16

74.15

71.85

104.65

100.95

94.03

88.0

4

138.96

98.86

94.80

162.25

153.7

138.2

125.5

5

187.3

123.58

117.7

242.6

224.2

192.5

168.8

6

232.3

148.3

140.1

361.7

323.9

261.4

219.3

6.5

252.8

173.0

151.2

446.9

391.75

303.4

247.9

From these values it is apparent that no one hydration number or asso-
ciation factor will account for the deviations at these high concentrations.
Up to two molar, the osmotic pressures agree well with values calculated
on the assumption of the existence of hexahydrate, but at higher con-
centrations the best correspondence occurs where association is assumed
and hydration decreases as concentration increases. All of these assump-
tions are empirical however and when considered along with the hydration
numbers calculated by Porter (Table 10) they suggest a smooth and
continuous change of hydration and association with concentration such
as would occur if the increase in solute shifted the intermolecular force
fields and hence the coordination of the molecules of both solvent and solute.

Haldane (1918) has recalculated data of Berkeley and Hartley
according to the formula

0.082 NT . (12)

"' = Nr

n

where n, N, and Ni = the gram molecules of solute, pure solvent, and
solution per liter. This is identical with the formula of Morse. Haldane,
in assuming sucrose to exist as a pentahydrate, corrected for the space
occupied by the water of hydration. Table 12 gives Haldane's values.

Table 12. — Osmotic pressure of sucrose solutions at 0° C. (data of Berkeley and

Hartley, calculations by Haldane) : —

Concentration of sucrosh

-

Grams

sucrose

penta-

Osmotic pressure

in ATMOSPHERES

Observed by

Grams

hydrate

Berkeley

Calculated

Calculated

Calculated

Volume

Weight

sucrose per

per 100 g.

and

as per

as per

as per

molar

molar

100 g. water

free water

Hartley

van't Hoff

Morse

Haldane

atm.

atm.

atm.

atm.

0.097

0.0992

3.393

4.33

2.23

2.17

2.22

2.24

0.281

0.2978

10.180

13.21

6.85

6.29

6.66

6.85

0.533

0.6001

20.525

27.39

14.21

11.95

13.44

14.77

0.754

0.894

30.610

42.04

21.87

16.90

20.04

21.80

0.822

0.992

33.945

47.07

24.55

18.41

22.22

24.40

1.585

2.37

81.26

130.54

67.74

35.48

53.19

67.66

1.933

3.26

111.73

199.70

100.13

42.20

73.14

103.50

2.201

4.12

141.11

282.97

134.84

49.31

92.37

146.66

Crafts et al.

38

Water in Plants

These values of Haldane follow the observed values to a concentra-
tion about 2 molal and then depart, becoming higher than the observed
values. This probably results from use of the ratio of solute to solvent
molecules in place of the logarithm of the mol fraction of solvent as pointed
out by Lewis (1908) and explained on page 33.

All of the calculations so far presented have been based upon the con-
centration of solute in the solution. By shifting the basis for calculating
concentration and by assuming association of the solvent and hydration of
the solute fair approximations of the observed values have been obtained.

Another method of arriving at the osmotic pressures of solutions is to
use a different basis for starting calculations ; one that is more reliable than
concentration in reflecting the properties of the solution. One such basis is
vapor pressure. The vapor pressure of the solvent vapor above a solution
is an index of the escaping tendency of its molecules ; such forces as asso-
ciation and hydration, in other words, the intermolecular forces determin-
ing coordination of solvent and solute molecules are integrated into the
vapor pressure value. Though it tells nothing concerning mechanism, it
does reflect the true activity of the solvent in the solution and it is the
difference between this activity and the activity of pure solvent molecules
that determines the true osmotic pressure of the solution (Lewis, 1908).

Table 13 presents values for the osmotic pressure of sucrose at 30° C.
observed by Frazer and Myrick and values calculated from the vapor
pressure of solvent molecules above the solutions.

Table 13 — Observed and calculated osmotic pressure values for sucrose solutions at

30° C. : —

Concentration,

Observed

Calculated from vapor

mols per 1000 g.

of water

(Frazer and Myrick)

pressure (equation 2)

1.0

27.22

27.0

2.0

58.37

58.0

3.0

95.16

96.2

4.0

138.96

138.5

5.0

187.30

183.0

6.0

232.30

230.9

Tables 14, 15, and 16 present osmotic pressure values of cane sugar
at 30° C. and 0° C, methyl glucoside at 30° C. and 0° C, and calcium
ferrocyanide at 0° C. calculated from vapor pressure data compared with
directly observed values. These are from Berkeley, Hartley, and Bur-
ton (1919) and the formula used for calculating the o.smotic pressures

Table 14. — Observed and calculated values of the osmotic pressure of sucrose

solutions: —

At 30° C.

AtO°

C.

Weight con-

centration

Calculated

Calculated

Direct

g. per

osmotic

osmotic

osmotic

100 g. H2O

loge Po/P

S

pressure

loge Po/P

S

pressure

pressure

aim.

a tin.

atm.

34.00

0.0195

1.0029

26.82

56.50

0.0343

1.0010

47.25

0.0352

0.9951

43.91

43.84

8L20

0.0526

0.9987

72.59

0.0538

0.9916

67.43

67.68

112.00

0.0776

0.9955

107.55

0.0798

0.9869

100.53

100.43

141.00

0.1030

0.9918

143.33

0.1067

0.9832

134.86

134.71

183.00

0.1423

0.9865

198.98

0.1471

0.9784

186.86

217.50

0.1759

0.9736

249.16

0.1808

0.9740

230.70

243.00

0.2067

0.9712

264.46

Chapter IV

— 39 —

Osmosis

contained values of the partial specific volume of water S derived from its
compressibility instead of the usual mol volume V. The vapor pressures
were very carefully measured and the values were corrected for known
sources of error.

Table 15. — Observed and calculated vahies of the osmotic pressure of a-methyl

glucoside solutions: —

Weight con-

At 30° C.

Ate

)° C.

centration

Calculated

Calculated Direct

g. per

osmotic

osmotic osmotic

100 g. H2O

loge Po/P

S pressure logg Po/p

S

pressure pressure

atm.

atm. atm.

35.00

0.0359

1.0026 49.42

0.0383

0.9981

48.29 48.11

45.00

0.0473

1.0019 65.14

0.0515

0.9971

64.22 63.96

55.00

0.0593

1.0009 81.73

0.0645

0.9960

80.50 81.00

64.00

0.0701

0.9996 96.75

0.0770

0.9949

96.17 96.24

75.00

0.0835

0.9988 115.34

0.0925

0.9935

115.74 115.92

90.00

0.1024

0.9972 141.66

0.1137

0.9917

142.46

105.00

0.1214

0.9951 168.34

0.1355

0.9897

170.18

Table 16. -

— Observed and calculated values of the

' osmotic

pressure of calcium

ferrocyanide solutions at 0°

C.: —

Weight concentration

Calculated

Direct

g. per 100

g. H2O

OSMOTIC pressure

OSMOTIC PRESSURE

atm.

atm.

31.389

41.10

41.22

39.504

70.59

70.84

42.889

86.62

87.09

47.219

112.97

112.84

49.857

131.33

131.00

Calculations by the vapor-pressure formula (equation 2) follow the
observed values to a high concentration as shown in Tables 13-16. Where
vapor pressure values are available, this basis for calculation is more re-
liable because it does not throw the correction upon a postulated mechanism
(hydration, association, etc.) but deals with values that integrate these
physical properties of the solution in a reliable manner. Freezing point
values may be used in a similar way, as pointed out by Lewis.

The examples given are a sample of the calculations that bear a certain
resemblance to the gas laws. Many more such values have been calculated.
Over fifty modifications of the van der Waals equation alone have been
used to calculate osmotic pressure values. These different laws of solu-
tions deduced thermodynamically on various bases are in reality inde-
pendent of the phenomenon of osmosis ; they all entail a central concept,
namely, that there is a difference between the free energy of the solvent in
the solution and in the free state, and that a pressure imposed upon the
solution that is sufficient to equalize these free energies will vary with con-
centration of the solute in the same general way as does the partial pressure
of a gas under analogous conditions.

There is an obvious and fundamental weakness in this whole concept ;
whereas the gas laws apply to a single component, in solutions there are two
components to deal with. Because of the philosophy behind van't Hoff's
law of osmotic pressure there has been a tendency to treat the solution as
a one component system in which the solvent simply provides space for
dispersion of the solute molecules. The corrections for space occupied by
the molecules of the system and the forces between them as provided in

ft

Crafts et al. — 40 — Water in Plants

the van der Waals equation were applied only to the solute molecules and
neglected the solvent. And where corrections did consider the solvent
they were formulated in terms of stereotyped and arbitrary behavior as
in the corrections for association and hydration in the case of water.
HiLDEBRAND (1936) statcs, "Moreover, the concept of osmotic pressure
as an effect primarily of the solute, with the solvent simply furnishing
space, has obscured the effects of intermolecular forces and the inter-
changeability of solute and solvent. . . ."

Using the modified van der Waals formula

(P + -W^ • (V - b) = RT (13)

where P is the osmotic pressure and V is the volume of solution containing
one gram molecular weight of the solute, Bancroft and Davis (1928)
have calculated osmotic pressures for mixtures of benzene and toluene in
which the two materials are considered as interchangeable.

Although their V values at the high concentrations are smaller than the
possible volume into which the liquid solute could be compressed and con-
sequently improper for calculating R values, the latter values between mol
fraction values of 0.9 and 0.1 are fairly close to the true ones. By adding
two more constants to the equation Bancroft and Davis were able to cal-
culate R values from mol fractions of 0.99 to 0.01 that agreed very closely
with theory. For similar calculations involving ethylene chloride and bro-
mide in benzene, see Lewis (1908), Tables V and VI.

Fundamentally there is no difference between solute and solvent in a
solution. In an osmotic system the permeability of the membrane to one
constituent designates that constituent as the solvent. However, substitu-
tion of a different membrane may reverse the designation.

Practically, the physical state of the pure constituents at the operating
temperature and pressure may determine the more convenient designation ;
if one constituent is a solid or a gas it is most conveniently considered as
the solute. If both are liquids solubility relations may determine the desig-
nation, the least soluble being designated the solute. However, since in
liquid solution all constituents assume the liquid state, there is no unique
basis for naming the solute and solvent ; in osmotic systems the permeability
of the membrane constitutes the only true basis for designation. In bio-
logical systems water is usually the solvent; in some situations, however,
permeability of membranes to gases in solution may complicate analysis of
the osmotic system involved.

In the example illustrated by Bancroft and Davis, benzene and
toluene were selected because they form an ideal solution at the tempera-
ture used. In most osmotic systems the solutions are not ideal ; because
of the anomalous properties of water, all aqueous solutions are non-ideal.
Gases themselves, with a few exceptions, are not ideal in their behavior and
the corrections developed to take care of departures from ideality of gases
do not apply to two-component systems. While the use of the van der
Waals corrections for space occupied by solute molecules and intermolecu-
lar forces between them may give fair agreement between calculated and
observed values of osmotic pressure for aqueous sugar solutions (Table
9) , the agreement is largely coincidental. True correction for non-ideality
of such solutions should establish a proper balance between factors of the
van der Waals type and adjustments for the polarity of water molecules
and their high cohesional interaction forces.

Chapter IV — 41 — Osmosis

Causes for Non-Ideal Behavior of Solutions : — Two general ex-
planations may be given for departure of a solution from ideal behavior :
1 ) the two components may be so dissimilar in properties that they are in-
capable of forming an ideal system and so the escaping tendency of each
component is affected by the other; 2) the two components may have such
great attractive forces for each other that they form compounds {i.e., hy-
drates) or one component may associate or polymerize reducing the total
number of molecules present.

The internal pressure (cohesion) of a liquid is a measure of the attrac-
tive forces between the molecules of that liquid, and differences in internal
pressure between solute and solvent determine, at least roughly, deviation
from ideal behavior of the solution. Deviations caused by differences in
internal pressure, and hence escaping tendencies greater than ideal, result
in positive departures from Raoult's law. If one component is highly polar
and the other less so, a positive deviation from ideality will result.

On the other hand, attractive forces great enough to cause association
or compound formation alter the properties of solutions by reducing the
number of active molecules, and bring about negative deviations from
Raoult's law. Association of either solute or solvent leads to increase in
vapor pressure of the solution and positive deviations from Raoult's law
(Glasstone, 1942). Generally speaking, positive deviations from Raoult's
law result in osmotic pressures less than those calculated from concentra-
tion and negative deviations result in higher values. All of the deviations
mentioned are appreciable only as the concentration of the solute becomes
appreciable.

Thermodynamically, it is possible to derive equations for non-ideal
solutions that are independent of the factors causing departures. Though
such treatment gives no hint as to the mechanism of the behavior of the
solution, it does give a convenient method for calculating values for vari-
ous functions of the solution. Because vapor pressure seems most ac-
curately to mirror the true escaping tendency of solvent molecules, be their
behavior ideal or non-ideal, vapor pressure measurements of the solvent
above a solution are made ; their departures from ideality are determined
by calculation from concentrations on the basis of Raoult's law ; the de-
parture times the mol fraction is termed the activity and the corrected vapor
pressure is named the fugacity. Because activity is measured in terms of
concentration, it may be expressed in units of mol fraction, concentration,
or molality. It is more accurate as an expression of the physical properties
of a solution than is osmotic pressure, in the same degree as vapor pressure
exceeds concentration in accuracy as an index of escaping tendency.

Due to the untiring efforts of Berkeley and Hartley (1906), Morse
(1914), Frazer and Myrick (1916), Berkeley, Hartley, and Burton
(1919), and others, accurate measurements have been made on the osmotic
pressure, not alone of dilute solutions but, in the case of sucrose, of solu-
tions almost saturated. Values from these studies have been cited in tables
in this chapter.

Physical chemists, realizing the difficulties in osmotic pressure measure-
ment, have been interested in the properties of solutions from a broader
viewpoint and have devised many methods for studying them. Because
the surface layer of a solution, in contact either with the vapor or the solid
solvent, acts like a semi-permeable membrane with respect to the escaping
tendency of the solvent molecules, measurements of vapor pressure, freez-

Crafts et al. — 42 — Water in Plants

ing point, and boiling point of solutions may be used to calculate the activi-
ties of solutions. Such measurements may be used directly to indicate
concentrations, conductivities, or activities, or they may be converted to
osmotic pressures by appropriate formulae. Because such methods may
be more accurate or convenient than direct osmotic pressure measurements,
they have been widely used.

There remains one field of physico-chemical study in which osmotic
pressure measurements are of value, that is, in determining the molecular
weight of large molecules. Substances such as dextrin, albumin, and many
others that form colloidal solutions cannot be studied by the indirect meth-
ods mentioned above because the low molecular concentrations render the
readings so small, and minute traces of impurities may overshadow the
true determinations. Membranes impermeable to such large molecules yet
unaffected by the impurities may be readily prepared ; they are sufficiently
rigid to withstand the small pressures involved. Many descriptions of such
osmotic pressure measurements may be found in the current chemical litera-
ture. Although they constitute an appreciable portion of current research,
they in no way reflect the immense importance of osmosis and osmotic
pressure in biology. In the latter field the study of the mechanism of
osmosis as it relates to turgor of cells, translocation of solutes, absorption
of water, and the more complex functions of growth and movement seems
highly important.

Summary: — The Abbe Nollet performed the first recorded experiments on
osmotic pressure. Traube discovered the copper ferrocyanide membrane, and Pfeffer
using such a membrane precipitated in a porous pot made the first reliable quantitative
measurements, van't Hoff, realizing the significance of Pfeffer's measurements, de-
rived the simple relation PV = n RT relating the osmotic pressure of a solution con-
taining n mols of solute in V liters of solution at the absolute temperature T to the
constant R. He showed this R to be identical to the gas constant, van't Hoff's law,
as given above, applied only to very dilute solutions.

Many refinements of the osmotic pressure law have been made. Corrections have
been made to put the concentration on a weight molar basis, to correct for the forces
of attraction between solute molecules and for the space they occupy, for hydration of
the solute and association of the solvent and for other factors. The most satisfactory
measurements for determining the osmotic pressure of an unknown solution, in addi-
tion to direct measurement, which is difficult, are vapor pressure lowering, boiling point
raising, and freezing point lowering. The latter three types of determination may be
used in some form to determine the physical properties of almost any type of solution
and by proper formulae the osmotic pressure may be calculated.

A fundamental weakness in most considerations of osmotic pressure has been a
neglect of the role of the solvent. Molecules of solute and solvent are equally im-
portant in determining the osmotic properties of a solution.

Many gases are non-ideal in their behavior ; most solutions are non-ideal ; all
aqueous solutions are non-ideal because water is highly irregular in its role of solvent.

Solutions may be non-ideal 1) because the two components are so dissimilar that
they are incapable of forming an ideal system ; the escaping tendency of each com-
ponent is aflfected by the other; 2) because the two components have such attraction
that they tend to form compounds (i.e., hydrates). Dissimilarity of components re-
sults in positive deviations from Raoult's law and osmotic pressures are found that
are less than those calculated. Excessive attraction of components results in negative
deviations from Raoult's law and observed values of osmotic pressure are higher than
calculated.

Most osmotic pressure determinations are made by indirect methods ; the freezing
point method is probably most useful. Direct determinations have been made on su-
crose solutions and a few other solutes at ordinary concentrations. Such determina-
tions are laborious. Direct determination is used now only to determine molecular
weights of large molecules. Continued study of the mechanism of osmosis seems
justified in the field of biology, particularly in the field of cell water relations.

Chapter V
THE MECHANISM OF OSMOSIS

Introduction: — In an accurate description of a physical mechanism,
critical definitions of the processes involved are necessary.

A large number of plant physiologists are agreed on the terminology
of the Physical Methods Committee of the American Society of Plant
Physiologists as presented by Meyer (1945). This terminology is used
here and the following definitions are given for use in the discussion that
follows :

Osmosis "may be conveniently, but not rigidly, defined as the diffusion
of a solvent across a differentially permeable membrane" (Meyer, 1945).
It is a result of the kinetic energy of the molecules.

Osmotic pressure. — "Historically .... osmotic pressure has been
used to designate the maximum pressure which develops within a solution
under ideal conditions .... the solution must be confined within a mem-
brane permeable only to the solvent, .... the membrane [must] be im-
mersed in the pure solvent, .... and the construction of the osmometer and
attached pressure measuring apparatus [must] be such as to permit the
development of a pressure equilibrium without any appreciable dilution of
the solution. This concept of osmotic pressure is not a very useful
one "

"The term 'osmotic pressure' can be more usefully employed as an
index of certain physical properties of a solution. Osmotic pressure is an
evaluation of the potential maximum turgor pressure which will develop in
a solution if it is permitted to come to equilibrium with pure water in an
ideal osmotic system. . , ."

"Osmotic pressure is an index indicating quantitatively the amount by
which the diffusion pressure of the water in the solution is less than that of
pure water at the same temperature and under atmospheric pressure"
(Meyer, 1945).

Diffusion is the net movement of the molecules in one direction result-
ing from their inherent thermal or kinetic energy and a difference in activi-
ties. The direction of diffusion is from a region of greater partial pressure
to a region of lesser partial pressure.

Diffusion pressure. — Meyer defines diffusion pressure as "that physi-
cal property of a substance which is responsible for its diffusion whenever
other prevailing conditions permit the occurrence of this process." He
gives an extended discussion of the relation of diffusion pressure to diffu-
sion, osmosis, and the development of osmotic pressure.

We offer the following definition. Diffusion pressure is a function of
the free energy of a substance. The term may be applied to both solute
and solvent of a solution. Ideally, the diffusion pressure of the solute is an
expression of the driving force with which its molecules will diffuse into
the pure solvent. Diffusion pressure of the solvent in pure solvent or in a
solution is a measure of the driving force with which its molecules will
diffuse into pure solute.

For example, if pure water were separated from pure glycerine by a
membrane permeable only to water, the diffusion pressure of the water

Crafts et al. — 44 — Water in Plants

would be a measure of the tendency of water to cross the membrane into
the glycerine. Thus the diffusion pressure of pure solute or pure solvent
may be pictured as the driving force with which each would diffuse into
the other in pure phase and would be equal and opposite to the force that
would have to be applied on either pure phase to prevent entry of the other.
This latter force on a unit area basis is equal to the osmotic pressure of the
solution as its composition approaches pure solute.
From the formula

PV = RT In — (1)

it is obvious that the osmotic pressure of a solution approaches infinity
as the concentration approaches 100 per cent solute. The same is true for
the substance designated as the solvent if the differential permeability of
the membrane be reversed. It seems therefore that the diffusion pressure
of pure solute or pure solvent approximates infinity.

Ordinarily, diffusion pressures are not measured ; only differences are
of value in consideration of osmotic systems. Thus diffusion pressure
requires mathematical treatment in a manner similar to that of free energy
where only differences are measured. Furthermore, like free energy,
diffusion pressure may be influenced by any force tending to restrict the
activity of the molecules. Among such influences are osmotic pressure,
hydrostatic pressure, and the action of adsorptive and electrostatic force
fields. In a simple osmometer, osmotic pressure is the predominating force
in determining the diffusion pressure differences of solvent and solute in
the system. Cells in plants may also be under the influence of hydrostatic
pressure other than atmospheric (usually subatmospheric) and adsorptive
forces of colloidal hydration may also be involved.

Eyster (1940) would identify osmotic pressure with the diffusion pres-
sure of the solvent in an osmotic system. The distinction which must be
made between these terms for a clear analysis of the subject should be ap-
parent from the discussion that follows. We are in agreement with the
definitions of Hall (1940) but would prefer the term "diffusion pressure"
to "escaping tendency," which he favors.

The term diffusion pressure has been used for many years in publica-
tions on osmosis and diffusion. Van Laar used it in his contribution to
the Faraday Society symposium (1917); Haldane used it in his paper
(1918) ; Meyer and Anderson use it in their Textbook of Plant Physiology
(1939), to mention only a few.

"The diffusion pressure deficit of water [in a solution] .... is the
amount by which its diffusion pressure is less than that of pure water at the
same temperature and under atmospheric pressure" (Meyer, 1945).

This definition is adequate for the simple osmometer in contact with
pure water at atmospheric pressure. For cells in plants where the diffusion
pressure of water external to the cells may be above or below that of water
at atmospheric pressure the DPD of water in a cell due to solute should
be defined as the amount by which its diffusion pressure is less than that
of pure water under the same external pressure (stage A of Figure 15).
This DPD may not correspond to the DPD of water in the cell referred to
pure water at atmospheric pressure. For example, the DP of water in the
xylem conductors of plants may be lowered much more by transpiration
pull than by the solutes dissolved in it.

Turgor may be defined as the state of an osmotic system such as a cell,

Chapter V

45

Mechanism of Osmosis

/r\

f\

a tissue, an organ, or a whole plant, that relates to the hydrostatic pressure
of the free solution or liquid contained in it. Because of the tensile strength
and the elastic nature of plant cell walls, the plant cell usually exhibits a
certain degree of turgor.

Turgor pressure at full turgor is the excess pressure exhibited by solu-
tion inside a turgid cell or osmometer above the external pressure. The
equal and opposite pressure exerted by the walls upon the cell contents is
termed wall pressure. For cells in contact with free water the external
pressure is approximately one atmosphere. Cells within a plant must ob-
tain their water from the xylem in which the diffusion pressure of water
may vary between wide limits. Such cells are seldom at full turgor; at
times the DPD of water in the xylem may equal or exceed the osmotic
pressure of cells in surrounding tissues : such a condition brings on wilting.

The Kinetic Basis of Osmosis: — Although the kinetic view of osmotic pressure
has been criticised from many angles, it seems inevitable that any true explanation of
pressure must rest upon a kinetic foundation. Ultimately the manifestation of pres-
sure must be a result of bombardment by the molecules of the substance exerting the
pressure, be it gas, liquid, or solid.

The mechanics of the process of osmosis may be clarified by use of a model to
illustrate the difference in the rates of diffusion of different gases and the effects upon
pressure.

In Figure 11, A is a porous clay thimble closed
at its lower end with a rubber stopper through which
extends a glass tube B. The lower end of B is sub-
mersed in water in the beaker C. As the apparatus
stands the thimble and tube are filled with air — mostly
nitrogen and oxygen. If an inverted beaker filled with
hydrogen is lowered very slowly over A nothing hap-
pens, but if it is lowered quickly bubbles of gas pro-
ceed from the lower end of B. If the beaker of hydro-
gen is kept over A bubbling proceeds for a time, slows
down, stops, and then water enters B and is pulled up
to a considerable height above the level in the beaker.
If the hydrogen is removed from A quickly, bubbling
ceases at once and the water rises in B. In each ex-
periment water rises to a certain height in B and then
slowly lowers again until it comes to the same level as
it is in C.

The explanation of these phenomena is as follows.
As the apparatus stands at rest, the thimble A and
tube B are full of air and the molecules are diffusing in
and out through the pores of the thimble at equal rates.
When hydrogen surrounds A its molecules, having
higher velocities and being smaller in size, diffuse in-
ward more rapidly than air diffuses out ; an increased
pressure is built up in A and B and air bubbles from
the tube. Rapidly, if the hydrogen is removed, or
slowly, if it is allowed to dissipate, hydrogen concen-
tration decreases around A and inward diffusion of
molecules is lowered to the same rate as outward.
Bubbling ceases. Then, if no more hydrogen is in-
troduced, outward diffusion exceeds inward because,
again, hydrogen moves through the porous thimble
more rapidly than air. The pressure inside the apparatus becomes less than atmos-
pheric and water rises in the tube. Finally, as hydrogen moves out and is slowly re-
placed by air again the apparatus attains its initial state. If hydrogen were main-
tained around A for a long time bubbling would continue as long as any air was left
in the apparatus ; when all air had left, either as bubbles from B or by diffusion out-
ward through A, the water in B would return to the initial level in C and the apparatus
would come to rest, being completely filled with hydrogen. Then if the hydrogen
around A were removed the pressure would be reversed, as described above, the water

D

Oo

^5

'^C

Fig. 11. — Apparatus for
demonstrating differential diffu-
sion of gases. A is a porous
clay thimble, B a glass tube, and
C a beaker of water. For ex-
planation, see text. (Redrawn
from Meyer and Anderson,
1939).

Grafts et al. — 46 — Water in Plants

in B rising, then lowering until it finally reached the initial state as all hydrogen was
replaced by air in the thimble.

If, instead of a beaker of water, a water manometer is placed at the lower end of
C, both positive pressure (corresponding to the bubbling) and negative pressure (as
noted by the rise of water in B) could be registered. With a manometer in place, if
a coarse-pored thimble is used, response to change of gas around A is more rapid but
the maximum and minimum pressures measured in B would be less than with a fine-
pored thimble. Finally, if a differentially permeable membrane (popularly termed
semi-permeable) such as heated palladium or silica, which is permeable to hydrogen
but not to oxygen or nitrogen, is used in place of a porous thimble at A, the pressure
in B will rise to a high value, depending upon the partial pressure of hydrogen inside
the apparatus and this pressure will remain at its maximum height so long as hydrogen
is kept in contact with the differentially permeable membrane and no leaks develop.

Although the case ©f the porous membrane would be difficult to analyse because
of the different velocities of the gases involved, that of the differentially permeable
membrane seems clear. When the pressure within this gas osmometer reaches a maxi-
mum the partial pressure of hydrogen inside equals the pressure outside and movement
of the molecules in the two directions is equal. The excess gas pressure within the
apparatus must therefore be equal to the partial pressure of the molecules of air that
are unable to penetrate the membrane when the system is placed under the total external
applied pressure of the manometer.

Osmosis in Liquid Systems: — The problems of osmosis w^here a
liquid solvent and liquid or solid solutes are involved differ from the above
principally in the differences that exist between the gaseous state and the
liquid state. With liquids the vapor pressure is a manifestation of the
ability of the molecules to escape from the body of the liquid against at-
tractive forces within the liquid and surface tension forces at the surface.
It is the actual pressure (or partial pressure) of the vapor phase in equilib-
rium with the liquid and it reflects the kinetic energy of the molecules as
influenced by temperature and, to a slight degree, total pressure. Diffusion
pressure, on the other hand, is a measure of the average intrinsic energy
of all the molecules and is a function of internal pressure, the force that
renders all liquids relatively incompressible.

A gas under increasing pressure is readily compressed and eventually
changes state to become a liquid under proper temperature conditions. A
gas under reduced pressure will expand indefinitely. The repulsive forces
between molecules are much greater than the attractive ; both however are
relatively small, and the latter almost disappear as a gas is rarified. A
liquid represents a state of equilibrium between attractive forces and re-
pulsive forces. As the molecules are forced closer together the repulsive
forces increase very rapidly so that the pressure rises tremendously with
small decrease in volume. Under reduced pressure a liquid expands very
little and tensions of 72 atmospheres for ether and greater than 100 atmos-
pheres for water have been attained before the forces of cohesion are over-
come and a vapor phase appears. Hildebrand (1924, p. 102) has pic-
tured the relation of internal pressure and cohesion in the following man-
ner (Figure 12).

While the volume changes involved in the gas osmometer are different
in magnitude than those of the liquid, the underlying kinetic principles are
the same. As evidenced in (Chapter IV, the improved formulae for cal-
culating osmotic pressure from concentrations involve an expression of
the relationship between the numbers of solute and solvent molecules pres-
ent and corrections to account for the vokmies occupied by the molecules
and the attractive force between them. Though the formulae may not meet
the requirements for solutions of all concentrations, it is not because of the
failure of the basic principles upon which they are founded but because we

Chapter V

— 47

Mechanism of Osmosis

do not yet know enough about the properties of Hquids under all condi-
tions of pressure and concentration to formulate a perfect equation.

When a solute is dissolved in a
Hquid solvent its molecules disperse
throughout the volume of solvent.
The molecules of the solvent are
now dispersed through somewhat
more space than they were origi-
nally. And the solute molecules,
depending upon the concentration,
are dispersed through from some-
what more volume (for concen-
trated solutions) to very much more
volume ( for dilute solutions). The
total volume of solute plus solvent,
however, is usually somewhat re-
duced. Table 17 shows the mag-
nitude of this reduction for sucrose
solutions (cf. also Bousfield and
LowRY, 1910). Because of the dispersion of the molecules and of the
intermolecular forces of attraction between the molecules, the vapor pres-
sure of both solute and solvent is reduced. The relation of these for an
ideal solution is given bv the diagram from Raoult's law shown in Figure
13.

Table 17. — Volume of zveight-normal solutions of cane sugar at 0° C. {Sp. gr. cane
sugar at 0" C. = 1.59231 ; from Morse, 1914) :—

Fig. 12. — Diagram showing the relations
between attractive and repulsive forces between
molecules. (From Hildebrand, 1924).

Volume of

Volume of

Volume of

Differ-

Con-

Concentration

SOLVENT

SOLUTE

Sum

solution

ence

traction

cc.

cc.

cc.

cc.

cc.

p.ct.

0.1

1000.13

21.328

1021.458

1020.73

0.728

0.07

0.2

1000.13

42.656

1042.786

1041.25

1.536

O.IS

0.3

1000.13

63.984

1064.114

1061.80

2.314

0.22

0.4

1000.13

85.312

1085.442

1082.38

3.062

0.28

0.5

1000.13

106.640

1106.770

1103.01

3.760

0.34

0.6

1000.13

127.968

1128.098

1123.70

4.398

0.39

0.7

1000.13

149.296

1149.426

1144.39

5.036

0.44

0.8

1000.13

170.624

1170.754

1165.13

5.624

0.48

0.9

1000.13

191.952

1192.082

1185.91

6.172

0.52

1.0

1000.13

213.280

1213.410

1206.69

6.720

0.55

This diagram emphasizes that although the vapor pressure above the
solution may be higher (in the case of a more volatile solute) or lower
(in case of a less volatile solute) than that above the original solvent, the
individual vapor pressures of the two components are both lowered.

In liquids, diffusion pressure is an expression of the balance between
repulsive forces and attractive forces characteristic of the liquid state and
is responsible for its low compressibiHty. In solids the forces of mutual
attractions are so great and of repulsion so little that diffusion in the sense
of molecular migration as it occurs in liquids is slow and almost escapes
detection. The fact that the term diffusion pressure may be applied to the
molecules of a solid solute depends upon the fact expressed by Professor
Kendall (1937) that sohds in solution are in the liquid state. That is,
when solution takes place, the forces that serve to maintain the molecules
of a solid in that state are overcome and the molecules in solution act under
forces like those in the liquid.

Grafts et al.

48

Water in Plants

Practically all liquids become solids below the critical temperature when
pressure becomes sufficiently high, and so pass out of the liquid state where
the laws of diffusion pressure apply. In the liquid state, however, the
diiTusion pressures of both solute and solvent are lowered as the solute
dissolves, and it is this diffusion pressure lowering that becomes of para-
mount importance in the mechanism of osmosis.

Pi

^^^^

/?<■♦

ps

^^

^

/

r^t

r'K

^

/

/

^

^

/

/

<

py

/

— ^

\

^

/

/

X

V

/

/

/

\

nA

/

/

\-

\

/

\

N^

/

X

\

N, 0

N, ho

0.1
0.9

02
0.8

0.7

0.4
O.d

OJ
0.S

0.6
0.^

07 0.8

0.3 O.i

0,9
OJ

1.0
0

Fig. 13.— Diagram showing the relations of partial and total vapor
pressures of solutions according to Raoult's Law. pi° is the vapor pres-
sure of component 1 in the pure state; pi is its (partial) vapor pressure
in the solution, and Ni its mol fraction. p2°, P2, and N2 are similar
quantities for component 2.

The Mechanics of Osmosis : — The process of osmosis is independent
of the pressure state under which the total osmotic system operates; the
osmotic pressure and the turgor which the system is capable of developing
are the same whether the system is atop a high mountain, at sea level, or
within an evacuated chamber. This is true because turgor pressure is not
an absolute pressure but an excess or differential pressure, and osmotic
pressure is a physical property of the solution nearly independent of pres-
sure. For convenience, it is usual to consider the system at atmospheric
pressure and measure pressure differences from that base line setting the
diffusion pressure of water under one atmosphere at zero. The principal
difficulty that the plant physiologist encounters is in the treatment of prob-
lems in plants where the water is subject to tension and is not at atmospheric
pressure.

Chapter V

49 —

Mechanism of Osmosis

In the following treatment the osmotic system will be considered as
operating at atmospheric pressure. If a solution is placed in an osmometer
surrounded by pure solvent an unbalance in forces is apparent. The dif-
fusion pressure of the solvent on the outside is greater than that on the
inside and solvent molecules diffuse inward. This is osmosis.

As osmosis continues and the mass of liquid inside the osmometer
becomes greater, if the osmometer is closed and rigid, pressure is built up
on the inside. This pressure, forcing all molecules of the solution closer
together, increases the diffusion pressure of both solute and solvent in the
osmometer. When the solvent molecules inside the osmometer have in-
creased in diffusion pressure until they are in pressure equilibrium with
those on the outside, the system is in osmotic equilibrium and the difference
in pressure betzveen the liquid inside and the pure solvent outside is com-
monly termed the osmotic pressure of the solution. Other terms that have
been applied to this equilibrium pressure are hydrostatic pressure and turgor
pressure. It should be noted that this is equal to the osmotic pressure of
the solution as it exists at the equilibrium concentration, and not that of
the original solution. To measure the osmotic pressure of the original
solution it would be necessary to impose pressure upon it so that no solvent
enters the osmometer.

Returning to the initial effect of adding a solute to a solvent, many writ-
ers have emphasized the resulting reduction in diffusion pressure of the
solvent molecules. Haldane (1918) by a rigorous mathematical analysis
has shown that the reduction in diffusion pressure of solvent molecules
brought about by the addition of the solute is mathematically equal to the
osmotic pressure of the solution as defined above. Obviously osmotic pres-
sure has been defined in two distinct ways. Can they be reconciled ?

R

w

\ ■•'■

M ,

Pz

Fig. 14. — Apparatus useful in analysing problems of osmosis. M repre-
sents a differentially permeable membrane separating a cylinder into two
chambers W and S containing pure water and sugar solution, respectively.
Pi and P2 are frictionless pistons.

In Figure 14, M represents a differentially permeable membrane sepa-
rating a cylinder into two cells W and S containing pure water and sugar
solution respectively. If these two cells are closed by pistons Pi and P2,
an osmotic system is provided that may be used in analysing the problem
stated above. Starting with the fluids W and S both at atmospheric pres-
sure and the same temperature, one can see that a difference in the diffusion
pressure across the membrane M exists, water in the solution S having
the lower diffusion pressure.

Obviously, too, the diffusion pressure of water across the membrane
can be equalized in two ways :

1.) If pressure is applied to S by means of P2, W remaining at atmos-
pheric pressure, the diffusion pressure of the water molecules in S may be
raised to equal that of W — namely, 1 atmosphere. And the hydrostatic
pressure (above 1 atmosphere) in S at this state (water equilibrium)

Grafts et al.

— 50

Water in Plants

equals numerically the osmotic pressure of S. This is the pressure that the
physicist has termed osmotic pressure and the physiologist has termed
turgor pressure at full turgor.

2.) If pressure is lowered in W by moving Pi to the left, S remaining
at atmospheric pressure, the diffusion pressure of the water molecules in
W may be lowered to equal that of the water in S. The hydrostatic pres-
sure (subatmospheric) in W now equals the difference originally existing
between the diffusion pressure of water molecules in W and those in the
solution S — namely, the diffusion pressure deficit of the water in the system.
As Haldane has shown, this diffusion pressure deficit of the water in the
system numerically equals the osmotic pressure of the solution (turgor
pressure at water equilibrium).

PPP = OP

Fig. is. — Relations among osmotic pressure, diffusion pressure deficit, and
turgor pressure for an ideal osmometer.

To illustrate the numerical relationships between these values, plant
physiologists have long used a simple diagram (see Figure 19, page 73).
As presented by Meyer and Anderson ( 1939, p. 149) , this diagram has a
curved sloping line to designate osmotic pressure, a feature necessary to
account for the changes in concentration resulting from increase in volume
as the cell becomes more turgid. For years this diagram in varied forms
has aided the student in understanding the osmotic relations of the vacuo-
lated plant cell. It is introduced here in idealized form to aid in an under-
standing of the fundamental mechanics of osmosis and osmotic pressure
as they occur in physical systems.

If we visualize an ideal osmometer having a perfect differentially per-
meable membrane and rigid enough to resist volume changes, Figure 15
illustrates the relations ainong osmotic pressure (OP), diffusion pressure
deficit of water (DPD), and turgor pressure (TP) for the system. The
expression diffusion pressure deficit has been given a number of names
by physiologists including suction force, suction tension, suction pressure,
turgor deficit, water-absorptive power, net osmotic pressure, osmotic dif-
fusion pressure, and functional pressure. It is of great importance in plant
physiology, as it is a measure of the force with which a cell may absorb
water. For convenience it has been contracted to DPD.

Chapter V — 51 — Mechanism of Osmosis

Figure 15 reveals the following relationships existing among diffusion

pressure deficit, turgor pressure, and osmotic pressure of an osmotic system :

OP = DPD + TP (2)

When TP = O, OP = DPD (state A) (i)

And when DPD = O, OP = TP (state B) (4)

At any point intermediate between the two limits indicated by equa-
tions (3) and (4) (state A and state B), the osmotic pressure is equalled
only by a summation of two lesser pressures — namely, the DPD and the
TP of the system at that particular state, as is indicated by equation (2).

That any of the states of Figure 15 may be set up as equilibrium states
is illustrated by the following example. Referring to Figure 14 and as-
suming that solution S has an osmotic pressure of 10 atmospheres, state B
may be accomplished by applying a positive pressure of 10 atmospheres,
by means of piston P2 leaving P^ at atmospheric pressure as indicated
under 1 ) on page 49. State A may be accomplished by lowering the pres-
sure on W by means of Pi to the extent of 10 atmospheres leaving S at
atmospheric pressure (see 2, page 50). To visualize the intermediate
states, if Figure 14 is turned so that M is horizontal with S on top and
W below, (neglecting pressure due to gravitational force) by using a
total weight of 10 atmospheres, if it be divided into two of 5 atmospheres
each and one applied to P2 and the other hung on P^ then as equilibrium
is attained the situation illustrated by the state DPD ■=. TP or the midpoint
of Figure 15 is depicted. By dividing the total 10 atmosphere weight in
other ways, any of the innumerable possible states of Figure 15 may be
depicted.

Other methods more comparable to those used in plant physiology may
be employed to bring about such equilibria. For instance, instead of hang-
ing weights on Pi it is possible to substitute solutions of equivalent DPD
values in place of W; gases containing water vapor of equivalent DPD
values could be used ; colloids of like water deficiencies would serve.

In this analysis it should be emphasized that the concentration or osmotic
pressure of the solution is the constant characteristic that is unique ; it de-
termines the distance between the parallel lines A DP of solvent and A DP
of solute and so fixes the value of OP (or DPD -f TP). The total pres-
sure upon either or both phases at equilibrium as determined by the exter-
nal pressure may vary either above or below the values of one atmosphere
as indicated by the dotted extensions shown in Figure 15. In consider-
ing plant cell water relations, the concentration or the pressure of the ex-
ternal phase (comparable to W) may be of great importance as will become
evident in subsequent chapters.

That the osmotic system of the plant may be described in terms of
standard physical units is evident from the following analysis. If A f is
defined as the partial specific free energy of the solvent in a solution, A fn
as the partial specific free energy of the solvent due to turgor pressure, and
A fo the partial specific free energy of the solvent due to osmotic pressure
(presence of solute), then

A f = A fn + A fo (5)

Transposing in (2) we have

—DPD = —OP -f TP (<5)

Af = V (—DPD) (7)

Afo = V (-0P) (5)

••■ A fjj = A f — A f^ = V (—DPD -I- OP) = V (TP) (9)

Where V = specific volume.

Crafts et al. — 52 — Water in Plants

Evidently the osmotic pressure of a solution can be equated to the sum
of two definite measurable quantities, the diffusion pressure deficit and
the turgor pressure of an osmotic system (equation 2). Osmotic pressure
is equal to the DPD when turgor pressure equals zero (equation 3) or to
the turgor pressure when DPD equals zero (equation 4). The conditions
designated by equations (3) and (4), however, represent two different
states of the osmotic system. And definitions of osmotic pressure based
upon these two distinct states should recognize their mechanical differences.

The three terms osmotic pressure, diffusion pressure deficit (of water),
and turgor pressure seem necessary in a consideration of an aqueous osmot-
ic system. Each is a distinct property of the system. The identification of
osmotic pressure with diffusion pressure deficit (cf. S. C. Brooks, 1940;
C. J. Lyon, 1941) or with turgor pressure (cf. Dutrochet as quoted by
Palladin, 1923; and Eyster, 1943) has undoubtedly caused the greatest
confusion in discussions of osmotic systems. Wann's (1943) attempt to
do away with the concept of osmotic pressure does not eliminate the diffi-
culty.

Because of its relation to turgor in cells, the condition of osmotic equilib-
rium termed "state B" in Figure 15 is of much interest to the physiologist.
In this state the turgor pressure in the solution is such that movement of
water molecules through the membrane is equal in each direction. This
is the pressure that is commonly termed the "osmotic pressure" of the cell.
Since only at water equihbrium is this turgor pressure equal to the osmotic
pressure of the cell, it seems best to term any hydrostatic pressure above
the diffusion pressure of the pure solvent, as arbitrarily designated by the
base line in Figure 15, the turgor pressure, for at all states but full turgor
this is only one component of the value that equals osmotic pressure. The
term could serve equally well in describing the properties of a purely physi-
cal osmotic system (cf. Meyer, 1945, page 154). The turgor pressure of
an osmotic system varies through a range of pressure values which lies
above the one atmosphere reference level numerically paralleled by those
traversed by the diffusion pressure of the solvent in the region below one
atmosphere. And as diffusion pressure deficit of a solution is a measure
of the excess diffusion pressure of the pure solvent over that of the solvent
in the solution in "state A," turgor pressure measures the excess diffusion
pressure of the solute in the osmometer in "state B" over the diffusion
pressure of the solute in the solution at atmospheric pressure. In this way
the activity of both solute and solvent are expressed in their logical rela-
tionship. To limit the term diffusion pressure to the solvent is arbitrary ;
the custom has resulted from the unbalanced view of osmosis that grew
out of the van't Hoff relation, and from the fact that most membranes used
in osmotic pressure studies have been permeable to water, so that osmotic
adjustments occurred mainly through water diffusion.

The "Solvent" and "Solute" Pressure Theories : — Many have re-
jected the kinetic view of turgor pressure development in an osmotic sys-
tem, chiefly on the basis that the pressure results from the entrance of the
solvent into the solution through the semipermeable membrane. Findlay
(1919) has pointed out that this criticism rests on a misunderstanding,
(See also footnote f, p. 110, of Palladin, 1923).

When turgor pressure is at its maximum ("state B") and is therefore
equal to the osmotic pressure, water is in equilibrium ; hence turgor pressure
at this stage cannot be explained on the basis of excess of entry over loss

Chapter V — 53 — Mechanism of Osmosis

of water. And when the tendency for water to enter the osmometer
(DPD) is at a maximum ("state A") and therefore numericaUy equal to
osmotic pressure, there is no turgor pressure, and hence no excess pressure
(above the diffusion pressure of pure solvent) resulting from the diffusion
of solute molecules.

Some confusion results from the search for the source of energy re-
sponsible for the expression of turgor. When an osmotic system in the
initial stage ("state A," Figure 15) is allowed to come to water equilib-
rium ("state B") by the absorption of water, the energy required to ac-
count for the osmotic work that the system can perform comes from the
water molecules that enter. This does not prove that the turgor pressure
against the membrane results solely from diffusion of water molecules. In
the first place, the membrane is permeable to water and will not sustain a
static pressure from water molecules. And, secondly, the diffusion pressure
of water in the solution has arisen from an initially low value. With the
absorption of water to attain water equilibrium, all molecules in the osmom-
eter are raised in energy value. Water molecues in the solution attain
a new energy level equal to those outside the membrane. The solute mole-
cules pass through an equivalent increase in energy. Being the molecules
that cannot pass through the membrane, these are responsible for a static
pressure equal to the turgor pressure in the solution. Whether their in-
crease in energy comes from the water molecules entering the osmometer,
or from work done in compressing the solution by means of a piston, the
end result is the same, namely, an increase in the diffusion pressure of the
solute. This increase in the diffusion pressure of the solute equals the
turgor pressure which in turn equals in value the diffusion pressure deficit
of the water in the solution at the same concentration and under only at-
mospheric pressure.

Controversy still exists over the "solute pressure" and "solvent pres-
sure" theories of osmotic pressure (Beck, 1928; Meyer and Anderson,
1939). Early physicists, following van't Hoff defined osmotic pressure
as the hydrostatic pressure in an ideal osmometer at water equilibrium.
They attributed the pressure to bombardment by solute molecules. Op-
ponents of this view define it in terms of the lowered vapor pressure,
diffusion pressure, or activity of the solvent. This viewpoint was carried to
the extreme by Bousfield (1917) who stated, "It has also been shown that
the osmotic phenomena may be interpreted as resulting from the activity of
the steam (monohydrol) molecules in the molecular interspaces of the solu-
tion, which when subjected to external pressure approximately obey the
gas law. This leaves us free to conclude that osmotic pressure has no real
existence as an expansive force in the interior of a solution, attributable
to the molecules of a solute behaving like an enclosed gas." In view of the
permeability of the membrane to water this interpretation is difficult to
understand.

Study of the literature indicates that the controversy has been based on
confusion of the two definitions that have been discussed. Advocates of
the "solute pressure" mechanism have pointed out that a static pressure
such as those measured by Morse, et al., Berkeley and Hartley, and
others, could not be caused by bombardment of the membrane by water
molecules because the membranes are permeable to water. Those support-
ing the "solvent pressure" hypothesis insist that it is the water entering
the osmometer that brings about the pressure. Both groups have failed to

Grafts et al.

54 —

Water in Plants

appreciate the significance of the range of states through which the osmom-
eter passes from "state A" to "state B." Both are right insofar as their
definitions go but both definitions are deficient. The only complete defini-
tion involves the concept expressed by the relation

OP = DPD + TP (2)

In words, osmotic pressure is a physical property of a solution. For
an osmotic system it measures the limiting value that the turgor pressure,
or the diffusion pressure deficit, or their sum, may attain.

zoo

5

Ho

(3

160

\

<nlOO

'so

/

V

/"

y

s

y

^

^

^

c

^^

"

Ho
O

o'-«- — I 1

100 zoo 300 ■400 JOO 600 700 300

Crams sucrose per 1000 ml. of <2olution

Fig. 16. — Calculated and observed values of osmotic pressures of cane
sugar solutions. A, observed values of Frazer and Myrick. B, values
calculated according to Morse. C, ideal values from van 't Hofif's law.

The "solute pressure" theory attempts to explain turgor pressure at
water equilibrium ("state B") whereas the "solvent pressure" hypothesis
tries to rationalize osmotic pressure in terms of the diffusion pressure
deficit of the water in the solution ("state A"). The force causing osmosis
is obviously this excess diffusion pressure of the pure solvent. And at the
instant the differentially permeable membrane comes in contact with pure
solvent this force is numerically equal to the osmotic pressure of the solu-
tion. But it is directed inward and it is not the osmotic pressure of the
system as commonly defined by physicists. Only after the solution is com-
pressed until it is in water equilibrium across the membrane is osmotic
pressure in the classical sense manifested and that pressure — the hydro-
static or turgor pressure of the system — is an outwardly directed force of
the proper dimensions.

Calculation by Indirect Methods : — Many methods have been used to avoid the
controversial aspects of osmosis. One has been to work in terms of vapor pressure.
Because the reversible work done in a change of state does not depend upon the process,
vapor pressure measurements may provide accurate data from which to calculate
osmotic pressure values, as shown by Table 13. They are, however, of little aid in
clarifying the mechanics of osmosis. Callendar (1908) has gone so far as to pro-
pose that osmosis takes place as passage of water vapor through minute capillaries in
the membrane. A vapor phase seems highly improbable as an essential feature of the
artificial membranes used to measure osmotic pressure (copper ferrocyanide in col-
loidal form), and of plant membranes in view of their hydrophilic nature.

Another method is to calculate osmotic pressure values from indirect measurements
upon thermodynamic principles avoiding the question of mechanism. Although such
values may be very accurate, they do not give the answer that the biologist is seeking,

Chapter V — 55 — Mechanism of Osmosis

namely, a picture of the underlying principles of osmosis upon which the mechanism
is based.

Evaluation of Diffusion Pressure: — If, in considering an osmotic
system, one attempts to assign values to the diffusion pressures of both
solute and solvent, the latter presents difficulties for in dilute solutions the
value approaches infinity. However, in practical problems only differences
in diffusion pressure are of significance. Therefore, for convenience in
handling such problems, it may be desirable to assign the pure solvent in
some standard state an arbitrary value which may be used as a base line
for further calculations. We follow Meyer, who states (1945, footnote,
page 151) "The diffusion pressure of pure water at atmospheric pressure
and at the same temperature as the water, the diffusion pressure of which
is to be designated, is conveniently taken as the zero point on the diffusion
pressure scale." Using this convention, with a solution having an activity
of 1 mol per liter, when TP = 0, OP = DPD = 22.4 atmospheres (neg-
lecting dissociation, hydration, association, etc.).

If solutes in the liquid state followed the gas law one could readily
calculate an ideal diffusion pressure for any substance by the formula

-j^ • 22.4, in which d =: density and M = molecular weight. This would
give water a diffusion pressure of 1243.2 atm. at 0° C, the value for ethyl
alcohol would be 383.7 atm., glycerine 306.5 atm., sucrose 103.9 atm. The
actual deviation from such a law is shown in Figure 16, where calculated
and observed values for osmotic pressure are plotted against concentra-
tions. Curve C represents the ideal values obtained by equating OP to

-j^j^ • 22.4 for sucrose. This follows the van't Iloff law. Curve B shows

the values calculated according to Morse, and curve A the observed values
of Frazer and Myrick given in Table 8.

If water at atmospheric pressure is assigned a diffusion pressure value
of zero and water in all aqueous solutions at that pressure negative values
(DPD's), solutes may be assigned zero values at zero or any other specified
concentration and positive values (TP's) at all higher concentrations. By
this convention, solutions may be assigned osmotic pressure values in the
common units of pressure. It should be realized, however, that both of
these conventions are arbitrary and somewhat illogical for they fail to take
into consideration the numerical relations between solute and solvent
molecules that have been shown to bear some relationship to their relative
diffusion pressures.

The Diffusion Pressure of the Solvent: — The concept of the diffu-
sion pressure of the solvent, i.e., water, is often difficult for the student to
grasp. While a solution in a beaker shows no evidence of its potential
capacity for work, if placed in an osmometer in contact across the semi-
permeable membrane with pure solvent it will Hft a piston. The pure sol-
vent in the beaker exhibits no such capacity.

Referring back to Figure 14, if the solution S is removed and the mem-
brane is placed in contact with a very large reservoir of dry air, water will
evaporate from the surface of the membrane, the pressure in W will lower
and Pi will move to the right if left free. If water is allowed to continue
evaporating and the piston Pi is held in a fixed position the pressure in W
will lower until a tension of many atmospheres is developed providing the
water and apparatus contain no bubbles or unwet surface upon which a
vapor phase may be initiated. In fact the cohesion of water, calculated to

Grafts et al.

— 56

Water in Plants

be in the neighborhood of 17,000 atmospheres, is the limiting factor in
determining the pull which the molecules will develop as a result of evapora-
tion from the surface. In practice tensions of from 100 to 300 atmospheres
have actually been demonstrated. This is the mechanism which the plant
utilizes to supply water to its uppermost parts. It is a practical demon-
stration of the diffusion pressure of the water molecules.

The equilibrium vapor pressure of the water molecules above the mem-
brane constitutes a measure of the diffusion pressure of the water inside.
The relation between the diffusion pressure deficit of the water inside the
membrane and the vapor pressure lowering is given by the equation

Po

RT In p

{10)

DPD =

V

If the base line for establishing DPD values is obtained by setting the
diffusion pressure of pure water at atmospheric pressure equal to zero,
DPD values for water may be calculated. Homan, Young, and Shull
(1934) give a series of such values terming them "stromogenic tensions."
Meyer and Anderson (1939) give a more detailed series (p. 207, Table
24). Table 18 presents values at 20° C. calculated by the above equation.
Each atmosphere of diffusion pressure deficit shown in the fourth column is
equivalent to a column of water over 30 feet in height. This indicates that
in the case of the experiment mentioned above, if air having a relative
humidity of 99 per cent were used in contact with the membrane of Figure
14 evaporation would support a column of water approximately 400 feet
in height ; an atmosphere at 98 per cent would maintain a column 800 feet
high and lower humidities would support columns of proportionate height.
This concept of the diffusion pressure or driving force of water is extremely
useful in problems of plant physiology as it provides a basis for the en-
trance of water into plants, its movement to the leaves, and its evaporation
into the air. It should also prove valuable to the animal physiologist and
soil scientist.

Table 18. — Relation between relative humidity, vapor pressure, and diffusion pressure
deficit of atmospheric moisture at 20° C. : —

Relative

Po

Diffusion

HUMIDITY

Vapor pressure

LOGe

PRESSURE DEFICIT

per cent

mm. Hg.

P

in atmospheres

100

17.54

.00000

00.00

99

17.36

.01004

13.43

98

17.19

.02019

26.89

97

17.01

.03043

40.53

96

16.84

.04075

54.28

94

16.49

.06184

82.38

92

16.14

.08332

110.99

90

15.79

.10535

140.33

85

14.91

.16245

214.00

80

14.03

.22314

297.24

75

13.16

.28765

383.17

70

12.28

.35662

475.04

60

10.52

.51078

680.39

50

8.77

.69314

923.31

40

7.02

.91629

1220.56

30

5.26

1.20396

1603.76

20

3.51

1.60943

2143.87

10

1.75

2.30258

3067.19

In spite of the arbitrary assignment of zero for the base line of water,
it is evident that pure water has a very high diffusion pressure. It is this

Chapter V — 57 — Mechanism of Osmosis

high energy that causes water molecules to break away from the surface
in the process of evaporation; it is manifested in Brownian movement;
it is the same high energy that causes water to enter the solution in the
osmometer through the differentially permeable membrane. The arbitrary
assignment of zero diffusion pressure to the solvent in many considerations
of osmotic pressure has probably been largely responsible for the erroneous
concept that the solvent does not enter into the energy relations of the
process of osmosis but simply supplies space for the dispersion of the solute
molecules. It should be evident from the above discussion that the excess
energy of the pure solvent molecules over that of the solvent molecules in
solution is the cause of osmosis. And after osmosis has occurred and the
solution has become more dilute, the only way to return to the original state
is to put energy back into the system either as heat to evaporate water
from the solution until it is at its original concentration, or as mechanical
energy to press pure water from the solution through the differentially
permeable membrane.

Although this discussion of the mechanism of osmosis may be long and
involved, it seems to be necessary to clarify certain aspects. Osmosis, as it
relates to cell turgor, is of paramount importance for an understanding
of water relations. The ability of the cell to absorb and retain water is
critical in its function in the plant. Upon this ability depends its turgidity,
its form and size, its relation to the mineral nutrients supplied by the en-
vironment, its capacity to expand and grow, and, in the final analysis, its
power to compete for water and so to survive. From this standpoint the
biologist needs to understand the mechanics of osmosis, the function of
membranes, and the energy relations of aqueous osmotic systems as re-
lated to living cells.

Summary: — A simple gas osmometer is a convenient mechanism to illustrate the
principles of osmosis. When a gas osmometer is in equilibrium across the membrane
with the pure gas to which the membrane is permeable, it seems evident that the partial
pressure of that gas on the two sides of the membrane is equal and that the excess
pressure of the confined gas is due to its partial pressure.

In the liquid state when a solute is added to a solvent, the diffusion pressure of
both is reduced. If the solution is now placed in an osmometer and the latter sur-
rounded by the liquid to which the membrane is permeable (the solvent), this liquid
moves in until the diffusion pressure of solvent on the inside attains its initial value,
that of the pure liquid outside.

The difference in pressure between the solution inside and the pure solvent outside
is commonly termed the osmotic pressure of the solution. Turgor pressure at water
equilibrium is probably a better designation. Haldane has shown that the reduction
in diffusion pressure of the solvent resulting from the addition of a solute also equals
the osmotic pressure of the solution. This difference is more correctly termed the
diffusion pressure deficit of the solvent in the solution. A more valuable definition of
osmotic pressure is contained in the following equation:

OP = DPD + TP

Only when TP = O does DPD = OP, and when DPD = O does TP = OP. At
all intermediate stages the three terms are needed to describe an osmotic system.

The "solvent" and "solute" pressure theories concern the two extreme states of
the osmometer, namely when TP = O and when DPD = O. A complete consideration
of osmosis must include all intermediate states as well.

The energy responsible for entry of water into an osmometer is resident in the
molecules of the pure solvent. The force expressed by the turgor pressure is due to
energy of the solute molecules, energy transmitted to them from the entering solvent
molecules.

Arbitrary reference levels for measuring changes in diffusion pressure of both
solute and solvent must be established. A customary practice is to set the level of
diffusion pressure of the solvent under atmospheric pressure at zero; that of the solute

Grafts et al.

58 Water in Plants

at zero concentration at zero. Though illogical, this is convenient; it should be under-
stood that it is merely an expedient. . ^ . u n :^„ ;f +«
The diffusion pressure of the solvent may be demonstrated by allowmg it to
evaporate through a membrane permeable only to liquid when wet; the energy of the
molecules is exhibited by the great lifting force shown. Addition of a solute reduces
this pressure and the only way to recover it is to put energy back as by evaporating and
condensing it again in pure form, or by forcing pure solvent from an osmometer by
pressure upon the solution.

Chapter VI

WATER AS A PLANT COMPONENT — INTRACELLULAR

DISTRIBUTION OF WATER

Introduction : — Because water occurs so commonly, it is often taken
for granted and little attention is paid to its properties. Especially is this
true in biology where water may make up the bulk of the living organism.
The content and character of water are too often considered subsidiary to
the properties of other structural components and its role in the structure
and function of the organism neglected.

The foregoing chapters have stressed the unusual properties of water,
not only as an individual chemical compound but as a solvent of other com-
pounds. In the living organism water must play a vital role in determining
the nature of the physiological environment wherein metabolism takes place
and various functions are carried on. The intermolecular forces between
individual water molecules and between water molecules and molecules of
other structural materials such as cellulose, pectin, lignin, etc., must have
an important influence in the design of the physical mechanism of such
complex organisms as plants. In fact, water, as the liquid medium for
growth, provides an essential continuity for the transport of solutes, the
transmission of impulses, and the coordination of correlative influences that
distinguish the organized plant from a mass of individual cells.

The continuity of the protoplasm may rightfully be considered the
critical mechanism in the above coordination. However, certain structural
features such as the moist cell walls upon which CO2 molecules are ab-
sorbed in leaves and the thoroughly wet pectic surfaces of root hairs that
provide intimate contact with colloids in the soil for ion exchange, con-
stitute connections between the protoplasm and these colloids that cannot be
neglected.

And, finally, the very character of life itself may depend upon the unique
properties of water and the interrelations between water molecules and
those of proteins, lipoids, carbohydrates, and the various mineral elements
that go to make up the living organism.

As a beginning to the presentation of the water relations of plants it
seems proper to consider the water of cells, its distribution, the forces
determining its absorption, movement and loss, and the dynamics of balance
between these forces.

Anatomy of Cells : — It would be impossible to give detailed consideration to the
water relations of the great number of cell types found in plants, a few of which are
shown in Figure 17. The complexity of form and function as related to specialization,
adaptation, and division of labor among cells and tissues makes generalization difficult.
As a compromise an attempt will be made to treat in detail a common cell type with
occasional reference to important exceptions. The type selected is the mature, active,
vacuolated parenchyma cell common to the pith, phloem, and cortex, and also occurring
in the xylem of succulent organs. Though more highly specialized, the mesophyll
cell of the leaf may be considered to fall within this same category. Such a cell is
conveniently described under three phases : cell zvall — the enclosing membrane, some-
what elastic but having considerable tensile strength; protoplasm — the semi-fluid living
substance including cytoplasm, nucleus, plastids, and other inclusions ; and vacuole — the
central region, filled with cell sap.

In certain water storage cells, such as those making up most of the leaf and stem
tissue of succulents, the vacuoles may be relatively large, the protoplasm and cell wall
consisting of thin layers. In others, for example the meristematic cells of stem and
root tips, the vacuoles are small and scattered. In these the protoplasmic phase pre-

1

0.1

<»l

m

c

Fig. 17.— Several types of parenchyma cells found in plants. These cells are A,
Elodea leaf parenchyma, B, Iris leaf parenchyma, C, tobacco phloem parenchyma D,
a root hair, E, a meristematic cell from the root tip of onion, and F, a stamen hair
from Tradescantia.

Chapter VI — 61 — Intracellular Distribution

dominates and most of the cell's water is held as part of the living substance. In cells
that have lost their living function and possess little more than a cell wall skeleton,
such as vessel segments, tracheids, and sclereids, water may be present only in the im-
bibed state in the wall ; it may fill the lumen or not, depending upon the condition of
the tissue. These are some of the extreme variations ; others will be mentioned later.
While the cell is usually described as the unit of plant structure and function, the
so-called building block, it is becoming increasingly evident that caution is demanded
in such usage. The classical cell theory is being replaced by the concept that the
organism as a whole is the important unit, and that the interconnected protoplasts con-
stitute an integrated mechanism representing a higher order of organization than any
summation of the individual parts. This concept has been used by Munch (1930) in
consideration of the mechanism of solute movement in plants, his term for the inter-
connected protoplasm being the symplast as contrasted with the apoplast — the sum
total of non-living substances of the plant. This change has resulted from the realiza-
tion that a plant cell cannot be considered an independent entity but rather that it is a
highly coordinated part of the whole (symplast), connected to adjacent cells by proto-
plasmic .strands and also by continuous water. At the same time, in spite of its inter-
connections, each cell maintains a certain degree of individuality. The use of cells as
functional units in experimental work (plasmolysis, etc.) is a matter of convenience.
The reliability of the results of such work often depends to a large extent upon the
degree of injury to the cells from isolation of the tissue.

Water Content: — Water is included among tlie components of all
plants but the variation in composition is so great that it is impossible to
give general figures for the content. One example that might be considered
typical of many field crops is corn, which Latshaw and Miller (1924)
found to contain 71.4 per cent water when harvested at the time the grain
matured. Many forage crops contain 75 to 80 per cent moisture when cut
for hay, and fruit and vegetable crops contain even more water. The water
content of plants usually shows a periodic diurnal variation and a progres-
sive decrease with maturity. Pisek and Cartellieri (1931, 1932) found
the water content in the leaves of a number of herbaceous mountain plants
growing in full sun to be 69 to 83 per cent of the fresh weight ; for cer-
tain shade plants it was greater, 80 to 86 per cent. Leaves of trees gen-
erally contain less water than those of herbaceous species. The authors
just cited reported (1939) a range from 50 to 70 per cent (of fresh weight)
for youngest and oldest leaves of Fagus sylvatica. Water in the trunks
of trees is lower in amount than in leaves. Gibbs (1935) gives figures
varying in the range 40 to 55 per cent of wet weight for trunks of quaking
aspen.

At the lower extreme of water content, some of the simpler plants can
survive almost complete desiccation during long dry periods. Scofield
and Yarman (1943) report values for the lichen Umhilicaria as low as 6.1
per cent of the fresh weight ; for the terrestrial alga Pleurococcus, Fritsch
(1922) found a lower limit of about 5 per cent.

A general idea as to the water content of certain plant organs used as
food materials may be had from Table 19.

Binding Forces : — The absorption and retention of water by plant
cells depend upon forces that have been, for convenience, termed osmotic,
imbibitional, and chemical. Whether or not there is any fundamental
difiference between them, the net effect of all these forces, including ionic,
covalent, dipole, and hydrogen bonds, microcapillary forces, and the col-
ligative effect of solutes, is to lower the free energy and hence the diffusion
pressure of water.

Osmotic pressure has been treated in detail in the two preceding chap-
ters. As previously stated, the addition of a solute to a solvent causes an

Grafts et al. — 62 — Water in Plants

Table 19. — Water content, in per cent of fresh weight, of some common food materials

(data of Chatfield and Adams, 1940) : —

Water content
as per cent of
Plant fresh weight

Lettuce (inner leaves) 94.8

Tomato (red, ripe fruit) 94.1

Radish (edible portion root) 93.6

Cabbage (edible portion) 92.4

Watermelon (fruit flesh) 92.1

Carrot (edible portion root) 88.2

Onion (edible portion bulb) 87.5

Apple (edible portion fruit) 84.1

Potato (whole tuber) 77.8

Prune (fresh, flesh and skin) 76.5

Field corn (dry whole grain) 110

Common bean (dry seeds) 10.5

Barley (hulless type grain) 10.2

Peanut (raw, with skin) 5.1

increase in the average distance between the solvent molecules and hence a
lowering of the diffusion pressure of the solvent. Forces of attraction be-
tween solute and solvent molecules cause further diffusion pressure lower-
ing. Any such lowering measurable by separating the solution from the
pure solvent by a membrane permeable only to the solvent will be consid-
ered as due to osmotic force.

Imbibitional forces result from the attraction between solvent molecules
and colloidal substances. Their magnitude may be such that the diffusion
pressure of the imbibed water is lowered to the extent of hundreds of atmos-
pheres. Most plant colloids are of the hydrophilic type, that is, they have
a strong attraction for water. Because of their structure, colloids are char-
acterized by a high surface : mass relation and surface energy undoubtedly
enters into the binding or immobilization of water molecules.

The absorption of water by a hydrophilic colloid results in swelling,
and pressures of great magnitude may be exerted by swelling seeds, wood,
etc. Imbibitional forces are of great importance in the retention of water
by cell wall substances. The initial pull developed as a result of evapora-
tion of water from the mesophyll of leaves is imbibitional in nature and only
after transmission of this pull or deficit through the continuous aqueous
phase of cell walls, protoplasm, and vacuole do the osmotic and hydrostatic
aspects of transpiration pull become manifest.

By chemically bound water is meant that water which enters into true
hydrate formation. Depending upon the nature of the substances involved,
this water has also been termed water of constitution and water of crystal-
lization. In dealing with cellulose and similar colloidal materials the con-
cept of chemically bound water may be difffcult to visualize. Such water
we understand to be that which differs from surface bound water only in
that it combines in definite proportion to the bonding material as if it were
localized in and limited by the lattice structure.

Water of Cell Walls : — The water retaining function of the plant cell
wall is closely tied up with its chemical and physical makeup. The wall is
pictured as consisting of three layers :

1 ) The middle lamella, or intercellular substance. This consists almost
entirely of pectins and lignins deposited by the cytoplasm.

Chapter VI — 63 — Intracellular Distribution

2) The primary wall, the first wall layer containing cellulose. This
wall is infiltrated by pectic materials, and is characterized by its ability to
undergo increase in surface area, and to exhibit elasticity, plasticity, and
reversible changes in thickness. It may also contain lignin.

3) The secondary wall, a thickening layer deposited on the primary
wall. It is composed of cellulose, lignin, and sometimes other materials.
It is not capable of reversible changes in thickness.

Both primary and secondary walls are believed to be composed of a
skeletal framework or matrix of cellulose forming a continuous system, the
interstices of which form another continuous system, of pores or micro-
capillaries (Bailey, 1938). Filling these may be found a variety of sub-
stances, namely water, pectic materials, lignin, hemicelluloses, cutin, suberin,
and sometimes small amounts of other organic or inorganic substances.
Resins, gums, tannins, callose, fats, oils, pigments, ethereal oils, proteins,
phospholipids, and salts of sodium, potassium, and silicon have been identi-
fied. Very young primary walls may contain protoplasm in the intermicel-
lar system (Frey-Wyssling, 1939).

Most of the important advances made in recent years toward an under-
standing of cell wall properties have dealt with fibers of various origins —
wood, cotton, ramie and flax, materials with thick secondary walls and of
high cellulose content. Hence much of the information does not directly
relate to the problems of primary cell walls.

Agreement has been reached that the ultimate units composing cellulose
are long chains of glucose residues, the so-called "molecules" of cellulose.
However, the number of residues in any chain has not been established with
certainty. Estimates vary from 50 to several thousand, or possibly the
number is indefinite. Due to unavoidable degradation of the cellulose in
preparing samples for particle size analysis, the more likely figure is doubt-
less in the thousands.

It is further agreed that these chains or molecules are aggregated
parallel to one another into groups called micelles, but the manner of group-
ing is in question. One theory holds that the glucose chains form elongated
submicroscopic micelles. Another theory states that the chains are aggre-
gated into definite microscopically visible cellulose particles, separated by
infiltrating substances (Farr, 1944). Frey-Wyssling has proposed (see
1939) that the micelles are made up of very long slender cellulose chains,
which are arranged in a manner such that there are alternately crystalline
and amorphous regions. In the latter, the chains are not sufficiently close
or parallel to form a crystal lattice. Aggregates of micelles, termed fibrils,
may be observed under the microscope in many fibers. These are the small-
est visible structures in the cell wall. Their orientation within the wall may
explain many of the physical properties of cellulose.

The forces binding water to cellulose have been considered as residing
in the amorphous regions of the micelle and in the intermicellar amorphous
material. The forces holding the glucose residues together in the chain
are strong primary valence (covalent) linkages between carbon and oxygen.
The forces binding the chains together in the micelles are hydrogen bonds,
the weaker attractions of OH dipoles, and the permanent electric moment
of the C-O-C groups (Mark, 1944). In the crystalline regions, the lattice
energy is such that water does not cause a separation of the chains. Swell-
ing consequently would be due entirely to the increase of the distance be-
tween the micelles resulting from the absorption of water by the amorphous

Grafts et al. — 64 — Water in Plants

material filling the interstices, and by the binding of water by such OH
groups as are exposed on the micellar surface. But in the amorphous
regions of the micelle, where the chains are somewhat curled and arranged
in a random manner, water can enter to cause swelling. This has recently
been demonstrated by Hermans and Weidinger (1946).

According to Stamm (1936), there are three principal ways in which
water is held by cell walls: (a) water of constitution, (b) surface ad-
sorbed water, and (c) capillary condensed water. No sharp line separates
(a) from (b), nor (b) from (c). The graphical relation between water
content of the cellulosic material and relative water vapor pressure of the
surrounding atmosphere is represented by a smooth sigmoid curve (Figure
10). According to Babbitt (1942), the initial portion (first water taken
up) of the adsorption isotherm of cotton fibers is best explained by a
monomolecular adsorption, the middle portion by a polymolecular surface
orientation. Finally, capillary condensation predominates to produce the
steep upper portion of the curve.

Often it is not possible to distinguish between adsorptive and chemical
binding of water. Bull (1943, p. 214) has clearly stated the viewpoint
that there are varying affinities between adsorbent and adsorbate particles
due to the degree to which the latter are exposed on the surface, and the
resulting degree to which their force fields have been satisfied. The forces
are the same for both types of binding.

From the above description, it is obvious that cell wall material is highly
colloidal and characterized by immense internal surface. Stamm (1936)
states that the internal surface of one gram of cellulose is between one and
ten million square centimeters. On much of this surface must be exposed
the polar OH groups of which there are three per glucose unit. These
hydroxyls are hydrophilic and experiment has shown that potentially each
may coordinate up to three water molecules.

Studies on sorption of water vapor by cellulose have indicated only one
half to two thirds of the theoretical water uptake, based on that expected
by the attraction of one water molecule by each hydroxyl (Stamm, 1944).
Since only an estimated half of the OH groups are on the micellar surface,
and some are used in bonding the cellulose together, Stamm estimates that
only about one out of four hydroxyls is effective in holding a water mole-
cule, an amount of surface bound water which he states is equal to a mois-
ture content of about 8 per cent. From this it is apparent that not a great
amount of water is held on the surface of the micelles.

The intimate relation between the cellulose fibrils and the infiltrating
substances would suggest a partial satisfaction of OH bonds by attraction
between the two materials. The subsidiary substances of the nature of
pectic compounds, hemicelluloses, and gums are more hydrophilic than
cellulose, and like cellulose, are polymeric substances composed of chains
of sugar or uronic residues. Hence in the primary wall particularly they
would account for considerable uptake of water.

Water condensed in the smallest capillary spaces is held relatively firmly,
but as the water content increases the successive increments of water fill-
ing the increasingly larger capillaries may be considered as approaching
the nature of water in bulk, mechanically held.

In studies on hydration of cellulose a strong hysteresis is noted, more
water being held by cellulose that has not been subject to drying. Urqu-
HART (1929) attributes this to the condition of the secondary valence bonds

Chapter VI — 65 — Intracellular Distribution

of the hydroxyl groups of the cellulose molecules. In the original water
soaked condition these bonds are saturated ; when the cellulose is dried these
groups are freed of water and, as a result of shrinkage, pairs are drawn
together so that the individual groups of adjacent cellulose molecules mu-
tually satisfy each other. Upon remoistening, some of the bonds originally
binding water are not readily freed for water absorption. This results
in reduced uptake. Other factors affecting hydration of cell walls are the
presence of the infiltrating materials listed above (page 63) the osmotic
and imbibitional properties of the protoplast, the presence of ions and
molecules passing through or along the walls, and the hydrostatic status
of water in the tissue. The latter is considered important for cells with
thick walls, as in certain marine algae (de Zeeuw, 1939).

The ability of cell walls to hold water in competition with the evaporat-
ing power of the air in the stomatal chambers of leaves and the colloids of
the soil is of great importance in the economy of the plant. Apparently
the imbibition process in living plants is completely reversible for even
after severe wilting, if the plant is supplied with water the wall shows no
appreciable damage except where collapse and death of cells has destroyed
the organized structure. Though considerable water may be lost from the
walls, they apparently can maintain enough to retain their normal properties.
No hysteresis effects comparable with those noted for dried cotton fibers
have been observed in connection with wilting of plants.

The amount of water held by cell walls may be considerable. Crafts
(1931) found that phloem walls of potato stolon were relatively thick in
their natural condition, and that their loss of volume on drying indicated
a high water content. While considerable shrinkage resulted when they
were dried in alcohol, drying in air at 80° C. produced a 50 per cent re-
duction in volume (Table 20). This indicates a minimum water content
of 50 per cent in these walls.

Table 20. — Walls of the phloem of potato stolon expressed

cross section of the phloem: —

as

percentage of total

Fresh Dehydrated in alcohol

Dehydrated
IN AIR AT 80° C.

36.8 ± 0.44 25.8 ± 0.55

18.5 db 0.32

The above is cited to illustrate the condition in one type of tissue.

Water held by hydrogen linkages along the cellulose chains of cell walls
is probably relatively immobile. On the other hand, considerable water
must exist in microcapillaries between the fibrils and this water should be
fairly free. Such water plays an important role in water movement along
cell walls. This movement occurs during absorption by roots, from root
hairs across the cortex and into the xylem. It also takes place laterally
through the wood, across the cambium, and in the bark. It undoubtedly
accounts for flow from the last tracheids at the bundle ends to the meso-
phyll of leaves. And it probably occurs in a large measure during develop-
ment of fruits, tubers, and other storage organs. The microstructure of
many cell walls seems highly adapted to such water movement.

Protoplasmic Water: — Considering the properties of protoplasm, it
is remarkable that its water content often attains values above 90 per cent ;
and that an average value for many plant cells may be as high as 85 per cent.

Grafts et al. — 66 — Water in Plants

Furthermore, account should be taken of the fact that the water content
may vary between wide limits. Some evidence suggests that changes of
physiological significance occur only at certain critical contents. For in-
stance Sponsler, Bath and Ellis (1940) state that the distance between
polypeptide chains of gelatin increases markedly at 33 per cent but remains
constant from that water content up to one of about 90 per cent. Prob-
ably most remarkable is the fact that the thin layer of protoplasm of a
highly vacuolated cell may control the movement of salts and hence of
water against gradients of a hundred fold or more where the protoplasm
itself is so highly hydrated.

Chemically, protoplasm is a heterogeneous mixture of water, proteins,
phosphatides, lipids, sugar, salts, and other compounds. Protein makes up
about two-thirds of the non-aqueous substances.

Proteins are composed of amino acids united through peptide linkages.
A peptide chain may contain several hundred amino acid residues arranged
somewhat as follows :

R' H O R"' H O

I I II I I ii
C NHC C NHC

/|\/\|/\/|\/\l/\/

HC C NHC C N

O R" H O R'^ H

In the protoplasm these chains are probably coiled or folded into vari-
ous shapes and sizes. When spread in a film they are stretched out more
nearly straight. The R residues consist of, or bear, both hydrophihc and
hydrophobic groups. Oxygen and nitrogen atoms coordinate water by
virtue of their ability to form hydrogen bonds. Each oxygen may hold 2
water molecules, and the NH groups a like number. OH and NH2 groups
potentially hold three molecules each and COOH groups four. Further-
more, layers of water molecules may form bridges between the backbones
of adjacent polypeptide chains to produce a vein of strongly bonded water
separating the two chains by the width of a water molecule. Hydration
centers on the side chains are fewer and attractive forces somewhat weaker
than on the backbone.

From x-ray data on gelatin (Sponsler, Bath and Ellis, 1940), the
spacing between backbone layers is constant at 4.4 A up until a hydration
of about 33 per cent is reached, at which time the spacing increases to 7.0 A,
which holds up to a water content of 90 per cent. Other properties of
proteins that change as water content passes through the critical range of
30 to 35 per cent are heat of imbibition, imbibition pressure, freezing
point, expansion upon freezing, etc. When less than 30 per cent water is
present in proteins the freedom of movement of water molecules is appar-
ently restricted.

There is speculation as to the grouping of the chains of a protein, the
shape of the particles or fibrils formed, the degree of continuity, and the
forces holding them together. Sponsler (1940) and Sponsler and Bath
(1942) picture an enormous number of molecular and submicroscopic par-
ticles to be present in protoplasm, grading in size up to visibility. These
are composed mostly of protein displaying various shapes, monolayers,
packets, etc. The chain is the fundamental unit of structure just as in
cellulose, the principal difference being that the protein molecule is looped
or folded. These units are aggregated into (a) super molecules, small

Chapter VI — 67 — Intracellular Distribution

packets consisting of monolayer polypeptide chains folded back and forth,
or groups of monolayers of shorter length, in size about 50 A on a side ;
(&) compact aggregates, due to grouping of the supermolecules, particles
that may possibly be visible with the ultramicroscope ; and (c) associations
of super molecules and compact aggregates with water channels between.
Such groups may be microscopic in size.

Forces accounting for the grouping of the chains into particles, and of
the particles into the various aggregates, are attributed by Sponsler to
several types of bonds: primary valence {e.g., cystine) bonds; electrostatic
forces due to ions, etc. ; hydrogen bonds ; and van der Waals forces of
cohesion. The complex aggregates are probably porous structures, inter-
penetrated by water channels, which widen in certain regions to form sub-
microscopic vacuoles of varying size. Composing the walls of these vac-
uoles, which, according to Sponsler, Warburg has termed "reaction cham-
bers," are the active groups which are intimately tied up with respiratory
and other vital reactions, including hydration phenomena.

Frey - Wyssling (1940) has pro-
posed a structure for proteins com-
bining corpuscular and reticulate proper-
ties. His structure is more rigid and con-
tinuous than that visualized by Sponsler,
consisting of loosely interwoven molecu-
lar strands of protein chains (Figure
18). The high water content is related
to reticulate structure ; fluidity and viscos-
ity changes are explained by shifting
bonds.

Fig. 18.-Protein structure pic- BeRNAL (1940) propOSeS that long

tured by Frey-Wyssling (1940). \ / f f t>

particles, aggregates of polypeptide chains,
are oriented into spindle-shaped bodies termed tactoids which can have
either positive or negative charges.

Any postulated structure must conform with the many extraordinary
properties exhibited by protoplasm. Among these are elasticity, plasticity,
viscosity (and the ability to vary in this respect with no change in concen-
tration), anisotropy, a widely variable imbibitional capacity, rigidity, tensile
strength, adhesiveness, thixotropy, and differential permeability. All of
these properties are dependent on or related to the amount of water present,
and to the types of forces responsible for the retention of water. Because
protoplasm may exist in the liquid state, the gel state, and in intermediate
states, and may flow or stream, its structure cannot be described in general
terms. It neither conforms to a reticulate structure nor a corpuscular one
but at times appears to shift from one to the other. Rigidity must be due
to the presence of a submicroscopic framework wherein the molecules are
coordinated to a lattice structure. Hydrogen bonds may account for such
coordination.

Fluidity, according to modern theory of liquid structure, should result
from a breaking down of the lattice to a closer packed structure in which
points of abnormal coordination constituting cavities occur. In simple
liquids these cavities have been pictured as providing space for the rota-
tion of paired molecules in a mechanism postulated to account for viscous
flow (Hirschfelder, Stevenson, and Eyring, 1937). If one attempts
to apply this theory to fluid protoplasm, he might visualize pairing and

Grafts et al. — 68 — Water in Plants

rotation of water molecules, inorganic ions, and simple organic molecules,
as occurs in simple liquids. Long-chain molecules may be pictured as bend-
ing in such regions, globular molecules as turning or shifting. Involved
also must be a loosening of the framework to allow structural units to
glide by each other, separated by layers of water molecules in which true
viscous flow occurs.

In streaming protoplasm viscous flow can be observed not only along
the moving strands but also at the surfaces of plastids, nuclei, vacuoles,
and other cellular structures. Such flow must result in the breaking and
shifting of many bonds and the activation energy of streaming required to
overcome cohesional and lattice forces is presumably derived from cellular
oxidations. Frey-Wyssling (1938) proposes that the high content of
water in protoplasm may be due not alone to the retention of water by the
hydrophilic groups, but to the fact that the meshes produced by the poly-
peptide framework are of considerable size, making possible the holding
of water and its dissolved salts in these meshes unattached to framework
bonds. Such water would be comparatively free and might readily move
by replacement. He (1940) attributes decrease in water content, such as
occurs during dormancy, to gradual narrowing of the meshes and masking
of the hydrophilic groups by polar radicles. Such dehydration would occur
gradually without disturbing molecular configuration.

The views of Lepeschkin (1936, 1938) on the structure of proto-
plasm are somewhat different. He proposes that water is not present as
a dispersion medium, but that it enters into a loose combination with
lipoids and proteins, complexes which comprise the dispersion medium and
which he has termed "Vitaids." His theory rests on several lines of evi-
dence (a) the relatively rapid movement of lipoid soluble materials through
the protoplasm compared to that of water suggesting that water is only one
constituent of the dispersion medium and that fats are also a part (b) the
typical behavior toward protoplasm of weak acids and other substances,
which affect lipoids relatively little. Furthermore, water is believed by
Lepeschkin to be absorbed by protoplasm up to a certain point, above which
it separates as vacuoles. By placing certain marine foraminifera and algae
in dilute sea water he was able to observe vacuolization after limited swell-
ing of the organisms. In addition to the water-protein-lipoid dispersion
medium, there is postulated to be a variety of dispersed phases, including
substances of a hydrophilic, as well as those of a hydrophobic nature.

M. H. Fischer (1923) advanced similar views of protoplasmic struc-
ture on the basis of studies of phenol-water systems: "It is essentially not
a solution of protoplasmic material dissolved in water (like phenol-in-
water) but one of reverse type, namely, water dissolved in protoplasmic
material (like water-in-phenol)." Mason and Phillis (1939) believed
that data which they obtained in sap pressing experiments (see later) on
cotton leaves seemed to be more in accord with the "Vitaid" hypothesis
of Lepeschkin, than with the assumption that water forms the dispersion
medium.

These ideas of Lepeschkin and Fischer have not had widespread
acceptance. Brooks and Brooks (1941) reject the viewpoint in question
on the basis that "(a) It is improbable that the phenol-water systems are
analagous to the other systems mentioned . . . . (b) the abundance and
small size of water molecules make it, a priori, improbable that they would
be completely separated into separate phases by the large and complex pro-

Chapter VI — 69 — Intracellular Distribution

tein molecules; (c) water soluble dyes and salts diffuse freely and rapidly
within the cell; and (d) the evidence .... shows that protoplasm without
a special surface membrane is freely miscible with water." Chambers
(1944) also regards water as the continuous phase in protoplasm, as evi-
denced by microinjection experiments. The ready diffusion of aqueous
solutions thi'ough cytoplasm, the formation of discrete droplets by water
immiscible liquids, the rapid freezing of water in cytoplasm when inocu-
lated, all are strong evidence.

We must therefore at present accept the working hypothesis that water
forms a continuous phase in protoplasm.

It should be obvious from the foregoing discussion that forces of many
types are involved in the hydration of protoplasm, and at present it is im-
possible to analyse completely the various interrelations of these forces.
Keeping in mind the unique nature of the living protoplasm, the intricate
and involved processes going on, and the almost insurmountable difficulties
presented by the complexity of the energy relations, it seems that a clear
picture of protoplasmic function must await years of patient research.

Water in Vacuoles : — Vacuolar sap may consist of as much as 98 per
cent water ; it has the highest water content of the three cellular phases. In
it are found a miscellaneous and variable assortment of sugars, salts, acids,
pigments, etc. Its composition among different plants is extremely variable.

The colloid content of vacuolar sap may be appreciable in some species.
In conifers, tannins, gums, resins, and other substances have been demon-
strated, and some (e.g., Roberts and Styles, 1939) have, correctly we
believe, stressed their importance in the water relations of the cell.

The substances which most commonly comprise the hydrophilic col-
loidal fraction of vacuoles, at least in the higher plants, are proteins, tannins,
and mucilages. Tannins may be found combined to some degree with pro-
teins or mucilages. Other colloidal substances of a fatty nature such as
lipids, phosphatides, and phytosterols may be present (cf. Guilliermond,
1941, p. 164).

An especially high vacuolar colloid content in petal cells of certain
Boraginaceae can be demonstrated (Gicklhorn and Weber, 1926). Ap-
parently the sap may even solidify. The solidification follows a spontaneous
contraction of the vacuole, plasmolysis, or vital staining. On treatment
with neutral red solutions the sap separates into two phases, an inner gel
and, between the gel and the cytoplasm, an optically clear watery solution.
Hofmeister (1940a, b), who has recently investigated the problem by
micrurgical methods, concludes that the neutral red effect is a precipitation
reaction (coacervate formation), while the plasmolytic and vacuolar con-
traction phenomena are of the nature of syneresis.

Microchemical tests by Hofmeister (1940&) on cells of Symphytum
officinale showed no tannin, protein, or cellulose in the vacuolar sap. Large
quantities of pectin, however, were indicated. Other examples of vacuoles
containing sap of a jelly-like consistency are known (Guilliermond, 1941,
p. 154).

The vacuolar sap of most cells has been considered to be essentially a
true solution. The researches of Hoagland and Davis (1923, 1929),
Collander (1939), Lundegardh (1940), and others indicate the high
content of nutrient salts that occur in solution in plant cells. In addition
to salts, sugars are of great importance in determining the water retention
of cells. On the other hand, one must recognize the complex nature of the

Grafts et al. — 70 — Water in Plants

vacuolar contents and the existence of phenomena associated with the col-
loidal state.

In addition to osmotic water, certain authors suggest that water may
be held in vacuoles by entirely different mechanisms in which metabolic
energy plays a dominant part {cf. Bennet-Clark, Greenwood, and
Barker, 1936; van Overbeek, 1942). These theories, involving so-called
"active" water relations, will be discussed in Qiapter VIII.

Methods for Determining Water Partition:— No method has been devised by which
the relative amounts of water in cell wall, protoplasm, and vacuole may be determined
accurately. A common method has been the expression of sap from whole tissues,
either in the living condition or after killing by freezing, boiling, or treatment with
ether, etc. The fluid thus obtained must be termed whole cell sap since it is a com-
posite liquid having its origin in all three cellular phases. That such sap is a true or
representative sample of vacuolar contents is erroneous even though, for tissues com-
posed of mature vacuolated cells it may consist predominately of vacuolar liquid. Sap
expression techniques will be considered in Chapter VII.

Perhaps the most reliable information on the composition of vacuolar sap has been
obtained by the use of large cells of certain algae. Nitella. with cells up to 1^/^ inches
long and % inch in diameter, has been used; Valonia, a large spherical coenocyte, is
even more convenient. These large cells may be broken open and slightly compressed
whereupon from a drop to a cubic centimeter or more of sap is obtained. This repre-
sents a fairly reliable sample of the vacuolar sap of such plants, and many experiments
dealing with permeability and solute absorption have been made using these cells. Re-
cent studies have involved absorption of radioactive isotopes and accurate measures
of the partition of these elements have been obtained.

Micrurgical techniques have been devised {see for example Livingston and Dug-
gar, 1934) whereby rather pure vacuolar sap may be secured from even small cells,
e.g., hair cells of tobacco, by inserting a small hollow needle into the vacuole and draw-
ing out the liquid. The method is laborious and provides very small samples that re-
quire analysis by micro methods.

Chibnall (1923) treated spinach leaves with ether as a means of separating
vacuolar from protoplasmic sap. The cells rapidly plasmolyzed, the vacuoles mark-
edly decreased in volume, and a clear liquid could be obtained on applying pressure.
Examination showed no rupture of cells, and the protoplasm remained essentially in-
tact. He admitted that the "vacuolar" liquid contained some protoplasmic constituents,
but believed that the expressed sap was almost entirely vacuolar in origin. The ground,
pressed residue gave a measure of the protoplasmic and wall sap.

Mason and Phillis (1939) estimated the partition of water between the vacuole
and protoplasm of the cotton leaf in two ways. First, by determining the concentra-
tion of chlorine and potassium in press sap assumed to be vacuolar in origin, in sap
assumed to be protoplasmic in origin, and in whole cell sap expressed from tissue that
had been frozen. They calculated the amount of water associated with the quantities
of CI" and K* found. Vacuolar sap they designate as that expressed from living leaves
where shearing forces are avoided. Protoplasmic sap they take as that originating
from the frozen and thawed press cake remaining after expression of vacuolar sap.
Second, by plotting the weight of sap (believed to be vacuolar) expressed from living
leaves against pressure applied in uniform increments. The total weight of vacuolar
sap was calculated by extrapolation.

The authors concluded that only about 30 per cent of the total cell water is present
in the vacuoles of cotton leaves. This value seems low and both procedures for its
calculation are subject to criticism. The possibility of water in amount disproportional
to solute expressed from cell walls and protoplasm, due to the behavior of cell colloids
toward water and dissolved substances at the higher pressures seems likely. It also
appears certain that some filtration of solutes by the cytoplasm would occur when liv-
ing cells are submitted to pressure. The fact that solute concentration of the vacuolar
sap was only one-fifth that of protoplasmic sap could be explained in this way. Be-
cause residues from expression of living leaves absorbed water, even from protoplasmic
sap, it seems that the pressures used must have brought about a separation of water
from the solutes in the cells, giving a false measure of the concentration of the so-
called "vacuolar" sap.

Determination of the volume of each cellular phase will give a rough indication of
the partition of water in the cell ; there would be little error in assuming that the vol-

Chapter VI — 71 — Intracellular Distribution

ume of the vacuole is a measure of its water content ; the method is more questionable
in the case of the cell wall and cytoplasm.

In many tissues the protoplasm of mature vacuolated cells forms a thin parietal
layer, comprising only a small portion of the total volume. Values between 10 and 30
per cent are common in storage parenchyma ; palisade cells of leaves have a greater
proportion of protoplasm.

Equilibrium Between Phases : — Finally, attention should be drawn
to the fact that, at least in active cells, there is a constant trend toward an
equilibrium between the forces attracting water to the three cellular phases.
We use the word trend because an actual equiUbrium probably never ob-
tains, due to varied activities of the hving organism of which the cell is a
part. With Krogh (1939), we consider "steady state" a better term, but
even this is not completely valid.

The most useful concept in a consideration of the mechanics of inter-
phasal water balance, and for the balance between various tissues and or-
gans of the plant is that of Meyer ( 1938), namely diffusion pressure deficit.
As explained in Chapter V, water, regardless of its state, location, or tem-
perature, has a definite diffusion pressure. When this diffusion pressure
is decreased, as by addition of solutes or imbibants, by decrease in turgor
or temperature, or by any other means, it develops, with respect to its own
previous state, or with respect to water in any phase which has not under-
gone change, a difference in diffusion pressure. The difference may be
termed a deficit if the previous state is designated as standard. For con-
venience, this term is contracted to DPD.

With this concept in mind it is not difficult to explain the maintenance of
a dynamic equilibrium in plant cells. If water is evaporated from the cell
wall, the DPD of water in the wall will increase, and water will move in
from the protoplasm to make up at least a part of the deficit. In turn, water
will leave the vacuole to satisfy the DPD of water in the protoplasm. And
if water is moving through an adjacent tracheid there will be a movement
to satisfy the developing deficits in all three cellular phases.

Other activities which may change the DPD of water in cells and shift
the equilibrium are: 1) water exchange with other cells; 2) solute meta-
bolism, including condensation, hydrolysis, accumulation, or loss; 3) water
metabolism, anabolic {e.g., photosynthesis) or catabolic {e.g., respiration) ;
4) changes in turgor; and 5) changes in hydrophily of the protoplasm.
Under certain conditions the volume of the protoplasm may increase at the
expense of water in the vacuole, a phenomenon known as vacuolar contrac-
tion. As a consequence the solute concentration in the vacuole increases ;
presumably water moves into the cell to satisfy the DPD gradient. Con-
traction of the protoplasm may occur under other conditions. Chloro-
plasts of Spirogyra readily contract to give up water to the rest of the cell
(OsTERHOUT, 1945). This may occur normally, or it may be induced ex-
perimentally through the action of certain salts.

The DPD concept emphasizes the fact that water in the free state under
atmospheric pressure has a greater diffusion pressure than does solvent
water in vacuoles, imbibed water in cell walls and protoplasm, or water
under tension in the xylem. Thus water tends to move along gradients of
diffusion pressure from regions where it is relatively free to those where
it is highly bonded or relatively deficient. Considering the individual cell
and its aqueous medium, such movement is opposed by turgor within the
cell and hydrostatic tension outside, and between these forces the dynamic
equilibrium known as water balance is in constant flux. Scarcely an ac-

Grafts et al. — 72 — Water in Plants

tivity takes place in the plant which does not in some way influence this
balance.

Summary: — Water plays a vital role in determining the nature of the environ-
ment wherein various cell functions are carried on. It forms a continuum in which
transport of solutes and stimuli occur. The protoplasm also constitutes an inter-
connected unit termed the symplast.

Water makes up as much as 75 to 90 per cent of the total mass of many common
plants. Water is retained in cells by osmotic, imbibitional, and chemical forces, all
of which lower its free energy. At their extremes and in simple systems these three
types of forces are distinct and are easily distinguished ; in complex systems, however,
they overlap. Various types of bonds are involved in the hydration of cells among
which the hydrogen bond is probably predominant.

Plant cell walls may consist of pectins, lignins, cellulose, and a number of other
infiltrating substances. Cellulose and pectin have strong affinities for water. Water
also exists in the microcapillaries of the cell walls. Much of this water is relatively
free, and it may translocate during absorption and movement of water in the plant.

Protoplasm may consist largely of water. Yet it may regulate the movement
and accumulation of solutes and hence of water. Certain evidence indicates that a proto-
plasmic water content of around 30 to 35 per cent is critical ; water held at lower con-
tents is less mobile. Proteins are pictured as forming interconnected chain structures
similar to, but more complex than, those of cellulose. Many types of radicles, both
hydrophilic and hydrophobic, may be included in protein structure. Hydrated protein
may have sol or gel structure with the molecules in various states of aggregation.
Protoplasm may have similar but more complex structure. It may shift from reticulate
to corpuscular structure, or from a lattice having a high degree of coordination to a
fluid containing holes resulting from abnormal coordination. Capillary forces may
also come into play, holding water in the structural meshwork of protoplasm.

Vacuolar sap is high in Vi^ater but it also contains sugars, salts, acids, pigments,
tannin, protein, gums, resins, fats, etc. Water is held osmotically by the solutes in the
vacuole. The osmotic pressure also reflects an equilibrium with inbibitional forces
in those cells in which vacuolar colloids occur. Active forces have been postulated by
some to account for the high water content of vacuoles in certain plants.

Vacuolar sap from large algal cells has been used in studies on sap composition
and solute uptake. Sap from smaller cells has been withdrawn micrurgically for
analysis. Sap obtained by pressure may contain materials from cell walls, protoplasm,
and vacuole, and it does not give an accurate indication of concentration in any of the
three phases.

A tendency toward water equilibrium in the three cell phases is maintained by a
tendency for diffusion pressure equilibrium. Factors that shift this equilibrium are
loss or gain of water, solute metabolism, water metabolism, changes in turgor, and
changes in the hydrophily of the protoplasm.

Chapter VII
THE OSMOTIC QUANTITIES OF PLANT CELLS

A Description of the Quantities and of their Interrelations : — The

osmotic quantities of plant cells are osmotic pressure (OP), diffusion pres-
sure deficit (DPD), and turgor pressure (TP). That the dynamics of
water movement and retention can be explained by these three quantities
has been shown by the work of Thoday (1918) and Hofler (1920), and
later by Beck (1928), Ursprung (1935), Meyer (1938), and others. A
diagram that has proved extremely valuable in presenting these concepts
and their interrelations is shown in Figure 19. One fundamental equa-
tion necessary to the interpretation of this figure is

OP = DPD + TP (i)

This diagram resembles Figure 15 of Chapter V with the exception
that the diffusion pressure deficits are shown as positive quantities and are

/./ /.z /.a

Relative cell volume

/■s

Fig. 19. — A diagrammatic presentation of the relations among osmotic
pressure, diffusion pressure deficit, and turgor pressure as applied to plant
cells. Redrawn with modification from Meyer and Anderson (1939).

placed above the base line. The curved upper boundary labeled osmotic
pressure designates the concept used by Meyer (1945), that is, the index
of the value which the sum DPD + TP may attain in the cell. It is sloping
to account for the increase in volume and decrease in concentration that
occurs as water passes into the cell between the states of limiting plasmol-

Grafts et al.

— 74 —

Water in Plants

ysis and full turgor. It is curved because the relation betwe^en osmotic
pressure and change in volume is hyperbolic (OP2 = OPi~), where
subscript 1 represents the initial state and 2 the subsequent state.

JOQ

1-04

/.OS

l/Z

/■24

""/To

roz 104 ^06 /od flo
Relative Cell volume

Fir 20 -Three forms of TP-volume curves for plant cells. A, curve for cells
havS waUs of htgh tensile strength. B and C, curves showing complex relations
between elasticity and tensile strength.

Starting at the condition of limiting plasmolysis (relative cell volume
1.0) the DPD is high and equals the osmotic pressure of the cell contents
There is no turgor at this point. As water is taken up by the cell turgor
increases due to' the opposing force which the cell wall exerts on the cell
contents.

Chapter VII — 75 — Osmotic Quantities of Cells

The TP-volume relation in Figure 19 is represented by a straight Hne,
a relation which holds only if the cell wall is perfectly elastic. Such is not
usually the case and Figure 20 shows three such curves derived from ex-
perimental results. The first (Figure 20A) shows a curve that bends up-
ward with increasing volume. This represents a condition exemplified by
a rubber film where, as the elastic limit is approached, the high tensile
strength limits expansion. Another example would be a group of spherical
cells surrounded by a tissue of great tensile strength such as the epidermis
of a leaf or the bark around a stem. As such cells expand and fully occupy
the space within the limiting tissue the force of wall pressure that opposes
turgor is shifted to the confining influence of the stronger tissue and ex-
pansion is limited.

The inverse curve (bending downward) would represent a situation
such as exists with a bubble where the film becomes thinner with stretch-
ing and, losing in tensile strength, finally bursts. The second curve (Fig-
ure 20B) starts like the first but shifts to the inverse form at the condition
of full turgor. The third (Figure 20C) appears to start by bending down-
ward and then changes as if the tensile strength were increasing. Other
physiological processes may be involved and the time element also may in-
fluence the form of this curve. If the DPD of the external environment is
rapidly reduced as occurs experimentally when flaccid cells are immersed
in water, the shape of the TP curve may be concave upward because time
is not allowed for plastic stretching. As full turgor is approached the slow
extensibility of the wall causes an increasing slope. Hofler (1920),
Ernest (1934c), and Broyer (1946) present such curves.

A particularly good example of this behavior was observed in Nitella
cells by Stowe (from Tamiya, 1938).

With increasing volume of the cell the DPD decreases rapidly because
of dilution of the cell sap and increase in turgor pressure, both of v/hich
increase the diffusion pressure of water in the cell. Any curvature in the
TP line will be reflected in the DPD since the two are related.

Hasman (1943) used the weight method in plotting DPD curves ex-
hibited by storage tissues of potato tuber and roots of beet and carrot.
Using molarities of KNO3 and sucrose solutions as abscissae, she found
that positive parts of the curves may be straight, concave upwards or con-
cave downwards. Various causes are discussed.

The osmotic pressure also decreases as the cell expands and its contents
are diluted. At full turgor cell volume and TP are at a maximum ; osmotic
pressure reaches a minimum and DPD = 0. In this state the diffusion
pressure of water in the cell equals that of pure water at the same tempera-
ture and at the reference pressure.

From this discussion it is evident that there is danger of oversim.plifying
the relations between the osmotic quantities of the cell. The diagrams
shown are useful only when the time period required for the volume change
involved is short. Under such conditions plastic stretching and the actual
expansion due to growth are minimized so that they can be neglected.

Under certain conditions the turgor pressure may pass the zero refer-
ence level and become subatmospheric ; in such an event the DPD is in-
creased over its value at incipient plasmolysis. It seems doubtful if this
reduction in pressure can go very low in cells such as root hairs, mesophyll,
and the like because of the nonrigid condition of the walls ; as the pressure
drops below atmospheric such cells will fold, wrinkle, or collapse and the
pressure remain approximately constant. In rigid, thick-walled xylem con-

Grafts et al. — 76 — Water in Plants

ductors, on the other hand, tensions of many atmospheres have been pic-
tured in tall trees and plants suffering permanent wilting. The contents of
such cells are virtually pure water and a dynamic equilibrium exists between
the water in such cells and that in the more concentrated vacuolar sap of
surrounding parenchyma. Tension in the xylem depends upon the ability
of water to exist in a metastable state when no unwet surface is present on
which the vapor phase may initiate. The ability of living parenchyma to
retain water in competition with forces of such magnitude depends upon
the osmotic and imbibitional character of their constituents.

It follows from the discussion in Chapter VI that if a complete picture
of the water relations of the cell is to be embraced by the three terms OP,
DPD, and TP, it is necessary to include under OP all forces leading to
water absorption by the cell, i.e., leading to a lowering of the DP of water ;
where tension exists, its value must be added to the OP. Under TP are
included all forces acting in the opposite direction. DPD then represents
any difference occurring between these forces, and finds expression as a
net force causing water to enter the cell.

Some prefer to treat problems of plant water relations on a thermodynamic basis,
following the general scheme presented on page 51. One such treatment (Edlefsex,
1941 ; Edlefsen and Anderson, 1943) deals particularly with the use of soil moisture
by plants, and interprets the movement of water on a free energy basis. Water tends
to move in a direction such that its free energy is lowered. The partial specific free
energy of water in a solution is a function of several components : various force fields
(gravitational, electrical, adsorptive) ; dissolved material giving the solution an osmotic
value ; and hydrostatic pressure.

Broyer (1946) has applied these principles directly to the problem of cell water
relations in a comprehensive treatment. He aptly describes the free energy concept as
applied to movement of water through the plant as follows : "The fundamental principle
underlying the movement of materials is that each molecule possesses a total internal
energy equal to the sum of its internal kinetic and potential energies .... and the
molal (or partial molal) free energy is equal to the product of the mean free energy
of the particles and the number of particles in one mole. A system is subject to spon-
taneous change if there is any conceivable process whereby the internal energy of the
constituent molecules can be effectively reduced. The action, here especially that con-
cerned with translation of the particle in space — its escaping tendency — which could
be produced by such a conceivable process is determined by the internal free energy
of the individual molecules. The free energy of the particles may be modified by any
change in condition of the external environment."

Distinction is made between those partial specific free energies which efifect move-
ment of water into the cell, and those producing outward movement. The difference
and sign represent the magnitude of the escaping tendency gradient, and the direction
of water movement. Thus

NIF = 2 IF — SEF (2)

Net influx specific Sum of influx specific Sum of efflux specific

free energy free energies free energies

The principal partial specific free energies identified are

osmotic solute specific free energy,

hydrostatic specific free energy,

metabolic specific free energy,

non-metabolic specific free energy (effect of colloids).

Each of these may act inwardly or outwardly depending upon the state of the cell and
external conditions.

The relationship between the osmotic specific free energies (F) and osmotic pres-
sure (P) is given by Broyer as

f — P

P = p - po = = F, (i)

V° (1.013 X 109)

Chapter VII — 17 — Osmotic Quantities of Cells

where

f = partial molal free energy of water in the given state, in ergs;

i° r= molal free energy in its standard state, in ergs;
V° =: the molal volume of water in its standard state, in liters;
po = the pressure on the water in its standard or reference state, in atmospheres;
p = the pressure on the medium in the given state, necessary to make f equal to f°, in atmos-
pheres.

The osmotic "solute" specific free energy is therefore equal to the change in free
energy brought about by the formation of the solution (addition of solute to solvent).
In the state as defined, the free energy change due to solute is equal in magnitude to
the DPD of water in the solution at the reference pressure, or to the TP of the system
at water equilibrium, both of which are numerically equal to the osmotic pressure as
indicated by Figure 15 of Chapter V. As with diffusion pressures, free energies are
not determined; only the differences between the given and some arbitrarily chosen
standard state are considered.

Another system is that employed by Brooks and Brooks (1941). The terms and
concepts selected to explain movement of water and solutes are fugacity and activity.
Fugacity (f) is defined as a corrected vapor pressure, corrected for deviation from
ideal gas behavior. Activity (a) is defined as a corrected concentration, and propor-
tional to a fugacity ratio, a =: -t^, where f° is the fugacity in an arbitrarily chosen

standard state. The close connection between these two quantities is apparent, since
both are functions of partial molal free energy of a constituent in a solution, and from
the fact that both are used as measures of what is qualitatively termed the "escaping
tendency." By definition f° may be set equal to one, whereupon the activity numerically
equals the fugacity.

The use of "fugacity gradients," "activity gradients," "escaping tendency gradi-
ents," in explanations of water and solute movement in osmotic phenomena, is sound,
and the concepts which these terms represent are essential in a clear understanding of
the problems.

In addition to the above terms. Brooks and Brooks use osmotic pressure, but
they have given it a meaning different than that used in this volume (cf. Chapter V),
tending to identify osmotic pressure gradients with activity gradients (1941, p. 6, 32).
In animal cells, where there is little or no effect of turgor on the activity of water in
the cell, this may be justified. But the significant turgor in plant cells requires a clear
distinction between these two quantities. Osmotic pressure, in our usage, is not a
function of pressure, except to the extent that concentrations may be altered. Activity
is a definite function of pressure.

There is much to be said for the adoption by physiologists of the terminology of
physical chemistry. It would have a unifying eifect and would provide a series of
accurately defined units for expressing quantitative results. On the other hand, since
the methods of measurement remain the same and the same numerical values must be
relied upon, no new or more accurate information is acquired. Measurement of the
forces involved in movement and retention of water by plants does not submit to highly
accurate methods and certain qualitative aspects such as the health and vigor of tissues
do not come within the scope of physico-chemical determination. There are many
plant functions that cannot yet be measured by physical or chemical methods.

Furthermore, compared with the OP = TP -f- DPD system, the physico-chemical
terminology is not more simple. The concept of DPD which is now accepted by many
plant physiologists (Meyer, 1945) is represented, at least partially, in the terms net
influx specific free energy, activity gradient, fugacity gradient, and escaping tendency
gradient. And, finally, in view of the inadequate preparation of undergraduate stu-
dents in biology, it may be premature to attempt a thermodynamic treatment of biologi-
cal systems, except at the graduate level.

Membranes and Permeability: — In order that the osmotic pressure
of a solution may find expression as turgor, there must be a membrane
having differential properties in the osmotic system. Ideally a differentially
permeable membrane is one that allows the passage of solvent but not of
solute. In the living cell this role is largely played by the protoplasm, which
is relatively permeable to water, but much less permeable to solutes. The
selective capacity of the cell is believed due, more specifically, to two

Grafts et al. — 78 — Water in Plants

"plasma" membranes. One, termed the ectoplast (or plasmalemma) , com-
prises the outer limiting layer of the cytoplasm immediately within the cell
wall ; the other is the tonoplast, the inner limiting layer bathed by vacuolar
sap.

While these membranes are too thin to be detected microscopically,
evidence for their existence has been obtained in several ways. More con-
vincing is the micro-injection of aqueous solutions of certain dyes, which
distribute themselves throughout the protoplasm, but do not pass out of the
cell (Chambers, 1944). Micrurgical manipulations have demonstrated
the tough elasticity of these layers. Seifriz (1928), using onion scale
tissue, was able to strip off the protoplast from the vacuole by means of a
micro-needle, leaving a free floating sac, limited by what appeared to be
the tonoplast. The appearance of such structures is evidently rather com-
mon (ZiRKLE, 1937). They are often produced on thin sectioning of tis-
sue; those from red beetroot retain pigmented vacuolar sap, and several
may be produced by cutting one cell. Kuster (1928) has further described
these structures as vacuolar envelopes (Vacuolenhiillen), and gives meth-
ods for isolating them from ripe berries of several species of Solanuiu.
Whether the limiting membranes of these structures actually represent only
the tonoplast is questionable. When such structures break under the
microscope, a very thin membranous remnant is left. If the tonoplast
consists of a membrane but a few molecules in thickness, as some have
postulated, it seems doubtful if any such remnant would be detectable.

The phenomenon termed "Intrabilitat," leading to vacuolar contraction,
suggests a differential permeability between the inner and outer membranes.
Various substances such as dyes are absorbed freely from the bathing
solution by the protoplasm but do not diffuse into the vacuole. This has
been presented as evidence that the ectoplast is permeable to such sub-
stances but the tonoplast is not (c/. Osterhout, 1943). As pointed out
by Stiles (1937), other factors — adsorption, solubility, and partition co-
efficients— also could conceivably contribute to this effect. Electrical
measurements have demonstrated a difference in behavior of the two proto-
plasmic surfaces (Osterhout and Harris, 1927).

There has been hesitancy on the part of some to accept the existence of
these membranes (Fischer, 1921; Lepeschkin, 1938). To some extent
the confusion has been a result of failure to accurately define what is meant
by the term "membrane." If reference is made to definite visibly distinct
layers, then there is little supporting evidence from plant cells. But it
is not necessary to postulate such structures. At the limiting surface or
interface of any complex solution there is a tendency for orientation and
accumulation of substances which act to reduce the interfacial tension.
Consequently, the chemical and physical properties of the interface will
differ from those in the body of the solution. The composition of this sur-
face will, therefore, depend on the substances present in the solution. In
this sense it is difficult to visualize the absence of membranes.

The composition of the plasma membrane has not been established with
certainty. The best evidence points to an association of protein and lipoid
(Davson and Danielli, 1943). The lipoid molecules are believed to form
a film in such a way that they have a certain freedom of orientation. Ad-
sorbed to this film is the protein layer, composed of long polypeptide chains,
cross-linked so that a heterogeneous pore or sieve effect is obtained. The
hydrocarbon groups of the chains are oriented so as to be in the oil layer,

Chapter VII — 79 — Osmotic Quantities of Cells

thus acting as anchors ; the polar water soluble groups remain in the aqueous
phase. The forces holding the complex (protein-fat-water) together must
be of an exceedingly intricate nature. Other substances may be present at
the surface in addition to the three indicated. The complexity is increased
when one considers that the composition may differ from plant to plant,
varying with internal and external conditions.

A similar view is the lipoid-sieve theory of Collander and Barlund
(1933). A lipoid-protein mosaic is postulated as comprising the surface
of the protoplasm, a view which tends to reconcile the essential features
of the "sieve" and "lipoid" theories. On such a basis many of the results
of permeability experiments may be explained, especially when the molecu-
lar volume of the permeating substances is considered. Details may be
found in books by Davson and Danielli, and by Hober (1945).

Artificial membranes of many sorts have been tested in attempts to
simulate such properties of the protoplasm. And membranes have rendered
valuable service in the physical measurement of osmotic pressure (Morse,
1914; Tinker, 1917). Among artificial membranes that have been used,
some certainly partake of the nature of sieves for the pore size can be
regulated in their preparation (Elford, 1937; Sollner, Abrams, and
Carr, 1941) ; others act as selective solvents, for instance water between
layers of chloroform and ether (Meyer and Anderson, 1939), or oil be-
tween aqueous phases (Overton, 1902) ; air or other gases in a closed
container will act as selective diffusion media for passage of solvents be-
tween solutions of different concentrations.

From the complex nature of protoplasm, as described in Chapter VI,
it seems possible that all mechanisms that are compatible with a continuous
liquid system may be involved in the protoplasm. Furthermore, from
studies on the nature of chemical bonds (Pauling, 1939; Remick, 1943)
it seems that electrostatic and other bonding forces, by repelling some com-
pounds, holding others in close association with the constituents of a mem-
brane, and allowing free passage of others, may play a role in differential
permeability. Ultimately the properties of all membranes must be explain-
able in terms of the chemical and physical properties of their constituent
molecules.

Permeability to Solutes : — The limiting surfaces of protoplasts were long con-
sidered to be readily permeable to solutes, and absorption was pictured as taking place
by diffusion. Since the memorable work of Hoagland and Davis (1923), Brooks
(1929), Steward (1932), Collander (1939), Lundegardh (1940), and many others,
it is now recognized that most solutes enter the plant as a result of an active absorption
process utilizing metabolic energy and often acting against strong gradients in con-
centration. Demonstration of active solute absorption can be accomplished using vital
stains that accumulate in cells to concentrations far above that of the bathing solution.

Thus, whereas the cell walls are pictured as readily permeable, the cytoplasmic
layer has distinctive properties which make it different from any artificial membrane
that has been prepared. And careful researches have established a long list of en-
vironmental and internal factors that determine the permeability and absorptive capacity
of plant cells for solutes (Hoagland, 1944). Principal among these are oxygen
supply, temperature, organic nutrients, the status of the cells with respect to previously
absorbed solutes, and the nature and concentration of solutes in the external medium.

In the absorption of nutrient ions from soils, not only the solutes present in the soil
solution but the composition of clay colloids in intimate contact with root surfaces
is involved. Studies by Jenny and Ox'erstreet (1939) show that contact exchange
phenomena may enable the roots to take up ions from the solid phase, their release
into solution being accomplished only after they have reached the vacuoles of the root
cells. Though the protoplasm itself may not extend through the walls of root hairs
to come into actual contact with the soil, the presence in the wall of polyuronides

Grafts et al. — 80 — Water In Plants

provides a highly hydrated matrix along which ions may migrate much as they do in
the protoplasm itself.

Thus, it seems that the protoplasm is highly impervious to the outward migration
of solutes and that the inward movement is usually efifected by an absorptive process
that is active and hence not subject to the ordinary laws of diffusion. When the proto-
plast is subject to adverse conditions such as lack of oxygen, excessive temperature,
excessive concentration of hydrogen or hydroxyl ions or toxic substances, or to lack
of organic nutrients, its metabolic activity may be so reduced that loss of nutrients
occurs. Response to such conditions may be reversible up to a certain point; beyond
this, permanent injury occurs and complete loss of control over solute movement in-
dicates death of the cell.

In view of these properties of the living protoplasm, it seems that the term per-
meability, in the classical sense of a passive control or selective action upon passage of
ions or molecules, has little to do with the functioning of the living cell. Most cell
walls may be considered permeable to water and salts in the classical sense. And loss
of solutes from injured or senescent cells takes place by diffusion. But uptake by the
active living cell, and possibly migration via the symplast, are active processes in-
volving the use of metabolic energy.

Permeability to Water: — Water passes through the plasma mem-
branes with relative ease, although its rate may change due to various in-
fluences. For example, the protoplasm of young cells appears to be more
permeable to water and salts than that of older cells ; protoplasm of sene-
scent tissue is again more permeable (Maximov and Mozhaeva, 1944).
Cells in a frost-resistant condition seem to possess increased permeability,
and this is proposed as one explanation of hardiness (Levitt and Scarth,
1936).

The permeability of cells to water may be determined in several ways:
1) rate of plasmolysis or of deplasmolysis, 2) by change in volume of
protoplast (plasmometric method), ^) by change in volume or weight of
bulky tissues, 4) by means of conductivity and osmotic pressure measure-
ments of expressed sap. 5) Special methods have been devised for studies
on large algal filaments by de Zeeuw (1939). He determined the water
exosmosis from Chaetomorpha linum by sensitive refractometric and dilato-
metric procedures. The first is based on extent of dilution of the external
medium, the second on change of cell volume.

The rate of water movement through isolated protoplasts of onion
bulb scale was found by Levitt, Scarth, and Gibbs (1936) to be 0.3
cubic microns per minute, passing through one square micron of cell surface
under a DPD difference of one atmosphere. Such values are commonly
termed diffusion constants. Other reported values of diffusion constants
are for Fiicus eggs 0.16 (Resuhr, 1935) ; leaf cells of Salvinia auriculata
0.55 (Huber and Hofler, 1930) ; internodal cells of Tolypellopsis stelli-
gera 1.08 (Palva, 1939). The above value of 0.3 represents a linear rate
of 20 microns per hour for water movement through the cell membrane.

Brauner and Brauner (1940) found that exposure to light increased
the water permeability of plant tissue and postulated an electro-osmotic
mechanism as an explanation. Others {e.g., Weber, 1929a) have found
opposite results, and still others report no apparent effect of light on per-
meability.

Permeability of cells toward water may vary from cell to cell and from
tissue to tissue. Huber (1933) found for Vallisneria leaf a rate 30 to 40
times less in the mesophyll than in the epidermis. L. and M. Brauner
(1943&) have made a significant contribution toward an understanding of
permeability of plant cells to water. They state that passage of water
through membranes is determined by two factors, each operating in an op-

Chapter VII — 81 — Osmotic Quantities of Cells

posite direction: (a) an "electrostatic valve" effect, a function of the in-
tensity of the electrokinetic potential at the pore walls, and (b) a mechani-
cal filter effect. It is shown that, as is true for certain cellophane mem-
branes, the permeability to water exhibited by plant cells is determined
largely by factor (o) above. Permeability is thus greatest at the iso-electric
point of the protoplasm ; hydration and viscosity are minimal at this pH.
In dense protein membranes, such as calf's bladder, the degree of hydra-
tion (swelling) and water permeability run hand in hand, i.e., factor (b)
predominates. Contrary to the finding of the above authors, de Haan
(1933) found that the water permeability of onion bulb scale epidermis
increased as the hydration of the protoplasm increased. There are many
factors to consider in interpreting permeability phenomena.

Permeability of Cell Walls: — It has been conveniently assumed, in
discussing the passage of substances into and out of cells that the cellulose
walls are truly permeable to water and solutes alike. Actually all cell walls
would be expected to offer some resistance to the passage of water and dis-
solved materials, but in most instances this resistance is not important when
compared to that of the outer surface of the protoplasm. For most par-
enchyma cells, with normally hydrated walls, this is certainly true. On the
other hand, certain cell walls, those containing cutin or suberin (i.e., cork),
and those in seed and fruit coats may be almost completely impermeable.
Some are clearly differentially permeable. The work of Brown (1909)
showed that barley grains readily absorbed water from a number of salt
solutions, but the salt remained excluded. Shull (1913) lists a number
of substances to which the seed coat of cocklebur is impermeable, as well as
several which can penetrate. Denny (1917) found great variation in the
passage of water through seed coats and the outer bulb scale of onion. A
difficulty resulting from differential permeability of cell walls has been met
in plasmolytic studies (see page 82).

Osmotic Pressure. Methods of Measuring Osmotic Pressure in
Plants. The Plasmolytic Method: — Though discovered previously by
Pringsheim and Nageli, the plasmolytic method for determining osmotic
pressure in plants was not employed until about 1877, when de Vries and
Pfeffer used it in their studies. Because little equipment is required, and
because observation of living cells is fascinating, studies on plasmolysis
have been popular with cell physiologists ; there is a wealth of literature on
the subject. Mention should be made of Ursprung's (1938) monograph
on the measurement of osmotic properties of cells, in which practically all
important findings are reviewed. Other general works which may be con-
sulted are Brauner (1932), Strugger (1935), Kuster (1935), Hober
(1945), and the periodical Protoplasma which contains one or more papers
on plasmolysis in almost every issue.

Plant cells immersed in hypertonic solutions exhibit plasmolysis. This
phenomenon is characterized by separation of the protoplasm from the cell
wall due to loss of water from the cell contents and contraction of the
vacuole and surrounding cytoplasm. Movement of the water is caused by
the gradient of diffusion pressure established when the tissue is immersed
in hypertonic solution. Onset of plasmolysis, due to excess shrinkage on
the part of the protoplast over shrinkage of the cell wall, depends upon the
relative elasticity and rigidity of these two phases. Cell walls are usually
the more rigid.

Grafts et al. — 82 — Water in Plants

An external solution which reduces the turgor of the cell to zero, but
does not cause plasmolysis, is said to be isotonic with respect to the cell sap ;
the cell assumes the condition of limiting plasmolysis. The osmotic pres-
sure of the bathing solution at this state is designated by the symbol Og.
In practice the condition of limiting plasmolysis is detected with difficulty ;
a slightly more advanced stage known as incipient plasmolysis wherein the
protoplasm first begins to draw away from the walls is used as a standard
reference state. Since this is taken for the condition of isotonicity, there is
by definition a slight difference between the true osmotic pressure of the
flaccid cell, and that measured at incipient plasmolysis.

Limitations of the Plasmolytic Method: — Although the plasmolytic
method has been criticised as involving too many errors, many of the im-
portant cell-water concepts have been developed by its use. It has several
advantages: 1) measurements on single cells are possible, 2) intact living
cells may be investigated, 3) permeability and other types of studies may be
made in conjunction with the method. The following criticisms have been
ofifered to its use : 1 ) the actual measurement is restricted to the state of
incipient plasmolysis. Only by correcting for the change in volume may
values be computed for cells in any state of turgor. — 2) Permeability
changes may be involved, which, while of interest from the standpoint of
permeability, may bring about errors due either to absorption of the plas-
molyzing solute or to loss of cell solutes or both. — 3) It may be inapplic-
able to cells possessing large protoplasm : vacuole ratios (Oppenheimer,
1932a). — 4) Adhesion of the cytoplasm to the cell wall, at least for cer-
tain types of cells, allegedly may result in excessive osmotic pressure values
(BuHMANN, 1935). — 5) The cell wall may be so impermeable to the
solute of the plasmolyzing solution that it crinkles (Huber and Hofler,
1930; Pringsheim, 1931). — 6) The exact determination of incipient
plasmolysis is often difficult because of inability to observe the very first
separation of the cytoplasm from the wall. — 7) Mechanical shock from
cutting or isolation of sections may produce abnormal conditions in the
protoplasm (Lucke and McCutcheon, 1932). — 8) Sap released from
cut cells may exert injurious efifects on intact cells. — 9) There is no critical
point in the plasmolysis time curve (Ernest, 1935). — 10) Plastic stretch-
ing or shrinking of cell walls may invalidate the results. (Oppenheimer,
1930&).

Because of the long list of possible errors, one may question the accuracy
of measurements made by this method. However, some of the criticisms
are unjustified ; errors involved in others are so small as to be insignificant.
Material may be selected that does not sufTer from all of the disadvantages
noted. Tissues with pigmented vacuoles have been popular because in-
cipient plasmolysis is easily recognized. The leaf epidermis of Rhoco dis-
color is a classical material.

As a plasmolyzing substance, sucrose has been almost universally em-
ployed. It is nontoxic, does not appreciably penetrate the protoplasm, and
its physical and chemical properties are well known. Beck (1927), after
critically comparing sucrose with potassium nitrate solutions, concluded
that results obtained with the latter were unreliable because of an excessive
penetration into the cells. Many of the early plasmolytic data were ob-
tained through the use of KNOo. Peters (1942) states that "sucrose
appears to be more completely excluded from cells than any of the other
substances." To mannitol has been ascribed similar properties (Collander

Chapter VII — 83 — Osmotic Quantities of Cells

and Barlund, 1933; van Overbeek, 1942). Sodium chloride, calcium
chloride (and their mixture), glycerin, and urea have also been employed.
The time necessary for equilibrium between the cell contents and external
solution has been variously reported to be from 20 minutes to 2 hours.
Ernest (1935) objects to the plasmolytic method on the ground that there
is no critical point for incipient plasmolysis in the plasmolysis-time curve,
and that the time necessary for equilibrium may be so great that changes
occur, introducing errors. This is opposed by Bennet-Clark, Green-
wood, and Barker (1936), who point out that after 30 minutes the amount
of water still to be transferred is insignificant. See also Oppenheimer,
1936.) Generally, for suitable tissues and under proper conditions, water
transfer in plasmolysis tests should attain equilibrium in 30 minutes. Red
beetroot cells have been shown to remain in a condition of incipient plas-
molysis for 16 hours with no detectable change (Currier, 1944c).

Penetration of sucrose or loss of solutes are more likely to occur where
cells are bathed in hypertonic solutions or pure water than in isotonic
solutions (Meyer and Wallace, 1941). Steward (1928) found that
potato tissue discs placed even in distilled water failed to lose significant
quantities of solutes during reasonably long periods. As an indication of
the low permeability of cells to sucrose, Huber and Hofler (1930) cal-
culated the permeability of stem cells of Majanthemum for water to be
10,000 times greater than for sucrose.

That adhesion of the cytoplasm to the walls occurs in certain cells upon
plasmolysis has been frequently reported (literature by Buhmann, 1935).
ScARTH (1923) observed that the protoplast of Spirogyra separated from
the wall smoothly when plasmolyzed with salts of monovalent alkaline
earths, but di- and trivalent cations increasingly produced adhesion. Ex-
posure to very dilute solutions of di- and trivalent salts, followed by plas-
molysis in a solution of the monovalent salt, similarly resulted in adhesion.
The efifects were reversible and could be removed by washing in water.
Methylene blue was found to completely prevent the trivalent salt effect,
and to remove the effect once established. Scarth suggested that in some
cases the adhesion may be due to the imparting of a positive charge to the
cell wall, which would attract the protoplasm, and that this may be accom-
panied by changes in viscosity or adhesive properties of the protoplasm.

Quantitative estimations of the magnitude of adhesion pressure were
sought by Buhmann {loc. cit.). Values found for leaves of Rlioeo dis-
color were of the order of 1 atm., for Bergenia cordifolia 1-3 atm., Hook-
eria lucens 3-6 atm., and for Pinits laricio 5-10 atm. Her method involved
1) determination of the limiting plasmolysis value, POg, 2) plasmolysis in
hypertonic solution, 3) redetermination of the limiting plasmolysis value,
DOg, this time approaching it from the plasmolyzed state, 4) complete
deplasmolysis in hypotonic solution, and 5) again determining the limiting
plasmolysis, ROg. It was assumed that the POg, DOg, and ROg values
should be identical in the absence of adhesion. Actually, POg values ap-
peared to be generally higher. ROg values slightly exceeded DOg values ;
this was attributed to a new, but weaker, adhesion with the wall.

Buhmann's adhesion values for beetroot tissue amounted to one to
three atmospheres (Table 21). Using the same tissue and a similar tech-
nique, one of us obtained somewhat lower values as shown in Table 33,
Chapter VIII (Currier, 1944a). A complicating factor in such tests in-
volving de- and replasmolysis relates to the extensible nature of the cell

Grafts etal. —84— Water in Plants

wall. Walls exhibit, in varying degree, both elasticity and plasticity, the
latter defined as "the ability to undergo permanent irreversible changes in
size and shape" (Heyn, 1940). Cells whose walls exhibit excessive plastic-
ity during severe plasmolysis or deplasmolysis are not suitable material for
this type of experiment.

Table 21. — Measurement of adhesion pressure in Beta vulgaris root
(data of BuHMANN, 1935) : —

Plasmolysis (POg) Deplasmolysis (DOg) Adhesion pressure

atm. atm. atm.

19.61 16.35 3.26

18.13 15.64 2.49

18.87 17.42 1.45

18.13 17.06 1.07

Such plastic stretching has been observed in beetroot tissue by Pring-
SHEiM (1931). Some representative data are presented in Table 22.
Stretching would be expected to aflfect measurements of Og. Sections of
beet tissue soaked in tap water for an hour gave an Og value as much as

Table 22. — Longitudinal strips of beetroot (Futterriibe) 14 cm. long, soaked for

various times in water, then overnight in 0.5 M KNOs- Change in length is expressed

in per cent of initial length (data of Pringsheim, 1931) : —

Hours

IN WATER

0

1 2 2.5 3 4

5

6

7

cm.
%

14.00
0

14.50 14.77 14.75 14.75 14.75
+3.6 +5.3 +5.3 +5.3 +5.3

14.80

+5.7

14.85
+6.1

14.95

+6.8

13.62
—2.7

Now overnight in 0.5 M KNO3 : -

—

cm.

%

13.82 13.82 13.90 13.90 13.87
1.3 1.3 —0.7 —0.7 —0.9

13.95
0.04

13.95
—0.04

14.05
+0.04

5.7 atmospheres less than sections not soaked in this way. Table 23 shows
these results. Unless otherwise stated, the symbol Og refers to the osmotic
pressure at limiting plasmolysis approached from the normal state. This
is interpreted as due to plastic stretching of the cell wall, causing plasmoly-
sis to set in at a greater than normal cell volume. Such an interpretation
neglects any error caused by loss of vacuolar solutes during soaking. Com-
plete absence of anthocyanin in the tap water is taken for evidence that no
injury was present. Presumably loss of solutes was slight, if present at all
during the one-hour period.

Table 23. — Effect of zvater saturation on Og values (Currier, 1944a) : —

Og (usual method) Og (water saturated) Difference

ATM. ATM. ATM.

14.5

12.3

2.2 ■

22.6

16.9

5.7

17.5

12.3

5.2

Volume change studies on isolated mesophyll cells of Sedum nicaense
have shown that considerable plastic stretching of these cells occurs upon
soaking. Incipient plasmolysis of such cells often occurs at cell volumes

Chapter VII — 85 — Osmotic Quantities of Cells

greater than normal (Currier, 1943). Martens (1931) demonstrated a
marked extensibility in staminal hairs of Tradescantia virginica, amounting
to increases in length of 100 to 150 per cent for cells freed of their cuticle.
The review by Heyn (1940) and Ursprung's monograph (1938) may be
consulted for a discussion of plastic and elastic stretching of cell walls.

Similarly it seems certain from the work of Delf (1916) and Pring-
SHEiM (1931) that plasmolysis in strongly hypertonic solutions causes
plastic shrinking of cell walls, tending to increase somewhat the DOg value
in comparison with Og values.

Gasser (1942) was unable to demonstrate adhesion pressure in several
plants which he investigated. Bennet-Clark, Greenwood, and Barker
(1936) definitely oppose the idea that it may invalidate plasmolytic meas-
urements, at least for the plant material used {see Table 30). They give
the following reasons: "the magnitude of the difference [between cryo-
scopic and plasmolytic measurements of OP] is too great; deformation of
the cell wall such as is demanded by the adhesion view is not observed ; it
should be impossible to observe cells in equilibrium in the condition of limit-
ing plasmolysis, which in actual fact are seen in large numbers."

Sufficient evidence has been reported to indicate that adhesion does
occur but it probably has not been a serious factor in most of the measure-
ments made by the plasmolytic method. Actually the nature of the asso-
ciation between the cell wall and cytoplasm is not well understood. If, as
has been suggested, the wall is a living part of the cell, there is good reason
to postulate an intimate structural bond between the two. Balazs (1943),
investigating epidermal cells, believes that the fine threads connecting the
separated protoplast with the wall ( Chodat-Hecht filaments) are derived
from the wall and not from the protoplast. Careful observation of plas-
molysed onion bulb cells on the other hand indicates that these threads may
be continuous with the plasmodesmata of pits and hence have an intimate
relation with the protoplasmic connections normally present.

L. and M. Brauner (1943&) believe that there is no general explana-
tion for the adhesion of protoplasm to walls, since various conditions may
act to produce different behavior in the same material.

Of the various kinds of plants to which plasmolytic methods have been
applied, the marine algae {Phaeophyceae, Rhodophyceae) have perhaps
yielded the most unreliable data (Kotte, 1915; Hofler, 1930, 1931;
Hoffman, 1932; Bunning, 1935). The trouble appears to lie in a rela-
tively high permeability towards the plasmolyzing solute, in marked swell-
ing of the wall and protoplasm, depending on the solution employed, in
volume changes that are difficult to measure due to irregular shapes of cells,
and in a strong adhesion of the protoplasm to the wall. According to de
Zeeuw (1939) the wall swells as plasmolysis is approached due both to
release of turgor pressure, and to the imbibing of the relatively pure water
passing through from the vacuole. The relationship may be more than
adhesion; Bunning (1935) states that the protoplasm may permeate from
one third to one half of the wall in Callithamnion roseum. It is therefore
not surprising that plasmolysis is injurious to these plants.

Plasmolytic Method — Experimental Procedure: — A recommended procedure for
determining the limiting plasmolysis value Og of a tissue is as follows : sections of
tissue are cut to such a thickness that one to four layers of intact cells remain. They
are quickly distributed among graded, weight molar sucrose solutions, the concentra-
tions of which vary by steps of 0.02 to 0.05 M. Infiltration by means of a vacuum
pump will shorten the time necessary to attain equilibrium and will reduce optical

Grafts et al.

86 —

Water in Plants

difficulties. After 30 minutes (or longer, if necessary) the sections are examined
microscopically for evidence of plasmolysis. The concentration range of the solutions
should be chosen so that no plasmolysis occurs in the more dilute while the highest
causes plasmolysis of all cells. Between the two extremes, sections will show plas-
molysis of some cells, but not of others. By carefully scrutinizing 100 cells of each
section under high power, and plotting the number of plasmolyzed cells against
the sugar concentration, a curve may be drawn from which the concentration effecting
plasmolysis of 50 per cent of the cells may be found. Figure 21 shows such curves.
This concentration value is taken as the Og value for the section. It should be clear
that it is an average value since each cell behaves somewhat differently. The degree
of precision obtainable by this method can be of the order of ± 0.1 atmosphere.

The assignment of OP values to
sucrose solutions may be based on:
1) osmometric measurements (Morse,
1914; Berkeley and Hartley, 1916;
Frazer and Myrick, 1916) ; 2) vapor
pressure measurements (Berkeley,
Hartley, and Burton, 1919) ; 3)
freezing point data (Int. Crit. Tables,
1933) ; or 4) formulas corrected for
the anomalous behavior of aqueous su-
crose solutions (see discussion in
Chapter IV). Study of data from
these four sources shows that the va-
por pressure data most nearly agree
with the directly measured values.
Freezing point data are a close sec-
ond ; values calculated from concen-
trations require correcting, particu-
larly above 1 molal. At concentrations
below this, no great error is intro-
duced by any of these methods. Phys-
iologists owe a great debt of gratitude
to the workers listed above who pains-
takingly determined by direct meth-
ods highly accurate values for the os-
motic pressures of sucrose and other
solutions over so wide a range of con-
centrations. Tables of osmotic pres-
sure values of volume molar sucrose
solutions at 20° C. may be found in
MoLZ (1926) and Ursprung (1938, page 1275). It may be concluded from Morse's
work that while sucrose solutions do not obey the ideal gas law equation with respect
to concentration, they do for temperature, at least from 0° C. to 25° C. The correction

293.1 _ „

factor for plasmolytic experiments carried out at 20 C. amounts to ^yj^ — l.U/J.

De Vries in 1888 proposed the name isotonic coefficient to describe the relative
molar amounts of substances forming isotonic solutions. Thus a molal solution of
KNO3 exhibits a greater osmotic pressure than a molal solution of sucrose. This

Table 24. — Isotonic coefficients of various salts calculated on a zvcight molar basis.
Sucrose = 1.00 {data of Fitting, 1917) : —

O.Z

o.d 0.4 o.s o.c

M0I3 6ucrosQ

0-7

Fig. 21. — The relation between con-
centration of plasmolyzing solution and
degree of plasmolysis. Curves showing
the 50 per cent value.

Salts to which Rhoeo ct:lls are
relatively permeable

Salts to which Rhoeo c^ells are
relatively impermeable

Salt

By plasmolysis By cryoscopy

Salt

By plasmolysis By cryoscopy

KNO3 1-69

KCl 1.74

KBr 1.14-5

KCIO2 1.73

K2SO4 2.27

NaNOs 1.7

NaCl 1.71

LiNOs 1.83 (?)

LiCl 1.8

1.78 MgS04 105

1.84 Mg(N03)2 2.54

1.84 Ca(N03)2 2.43

1.75 Ba(N03)2 2.23

2.36 BaClj 2.42

1.80-2 MgCl> 2.49

1.84 Sr(N03)2 2.35

1.77-8 SrCl2 2.49

1.85-7 CaClo 2.46

1.1

2.SS

2.4

2.22

2.46

2.64

2.43 (?)

2.55

2.59

Chapter VII

87 — Osmotic Quantities of Cells

results from the dissociation of the former into ions resulting in a greater number of
solute particles per unit volume. With sucrose taken as one, Fitting (1917) calcu-
lated isotonic coefficients of many solutes by comparing their ability to produce limit-
ing plasmolysis in leaf cells of Rhoeo discolor. Table 24 lists some of his values, as
well as those calculated from freezing point measurement. There is good agreement
in most instances. De Vries used this method to determine the molecular weight of
raffinose.

Further Uses of Plasmolysis: — In addition to osmotic pressure
measurements, the plasmolytic method may be used in permeability studies
to determine the effect of solutes on the behavior of protoplasm, and as a
criterion of the physiological state of cells.

Fig. 22. — Plasmolysis types as illustrated by Strugger
(1935). Convex plasmolysis, a; concave plasmolysis, b;
"krampf" plasmolysis, c and d.

The form which the cytoplasm assumes upon contraction and separa-
tion from the wall is usually indicative of its viscosity (Weber, 1924),
which in turn may reflect other conditions in the cell. Its form is also re-
lated to the nature and concentration of solutes in the plasmolyzing solu-
tion, and often is characteristic for any one type of cell. Figure 22 illus-
trates some of the forms which have been described. Convex plasmolysis
(Figure 22a) is attended by, or is the result of, low viscosity, and the
separation from the wall appears to progress smoothly and without per-
ceptible adhesion. It is generally observed in normal uninjured cells, and
particularly in mature cells. Concave plasmolysis (Figure 22b) indicates
a relatively high viscosity, w^here the separation occurs with more difficulty.
The form identified as spasmodic ("Krampf") by the German workers
(Figure 22c and d) is an advanced concave type which indicates an even
higher viscosity. Weber (1925) made the interesting observation that
guard cells of Vicia faba in the closed condition exhibit a convex form when
plasmolyzed, while those in the open condition showed the concave or
spasmodic form.

Several forms of plasmolysis are allegedly due to differential permeabil-
ity of ectoplast and tonoplast. Solutes may diffuse into the protoplasm but
not into the vacuole ("Intrabilitat").

Grafts et al.

88

Water in Plants

In cap plasmolysis (Figure 23) the cytoplasm swells markedly at the
ends of the cell, where there has been separation from the wall. This effect
can be induced by using a plasmolyzing solution containing salts of K, Na

or Li (HoFLER, 1939). Calcium salts antagonize
the action. In tonoplast plasmolysis (Figure 24)
two or more distinct vacuolar membranes are formed.
Still other types, limited to certain kinds of cells,
have been described. Strugger (1935) is a source
of literature on plasmolysis types.

Several types of response are included under the
term "stimulative" plasmolysis. Separation of the
protoplast from the wall may result from a number
of kinds of stimuli : mechanical, chemical, photo, and
thermal. Plasmolysis is usually observed at a time
when the cells are immersed in water and hypotonic
solutions. The reaction is often reversible. Several
explanations of this phenomenon have been oflfered :
release of imbibitional water by the protoplasm, an
active contraction of the protoplast, and an increase
in protoplasmic permeability, permitting solutes to
leak out.

Under certain conditions, some cells exhibit vacu-
olar contraction, a marked reduction in size of the
vacuole due to swelling of the cytoplasm. There is
no separation from the wall as in plasmolysis. Ex-
amples of vacuolar contraction are illustrated in Figures 25, 26, and 27.
Of this, and of stimulative plasmolysis, more will be said in Chapter VIII.

Fig. 23.- — Onion epi-
dermal cell showing cap
plasmolysis. Redrawn
from Strugger (1935).

Fig. 24. — Tonoplast plasmolysis of onion epidermis.
K, the hyaline, strongly swollen nucleus ; P, the cyto-
plasm, and T, the tonoplast. (Strugger, 1935).

It seems obvious from this and the previous discussion of plasmolysis
that the protoplasm cannot be regarded as merely a thin membrane per-
mitting passive diffusion of water to and from the vacuole. In all experi-
mental work with plasmolytic methods or vital staining, utmost care must
be taken against unconscious injury of the cells by use of unanalysed "tap"
and "distilled" water. Where cells are to endure long exposure, salt solu-

Chapter VII

89

Osmotic Quantities of Cells

Fig. 25. — Vacuole con-
traction in a cell of the fruit
flesh of Ligustrum vulqaire.
Redrawn from Strugger
(1935).

tions should be buffered to a favorable pH value and physiologically bal-
anced with respect to uni- and bivalent cations. Toxic solutes should be
avoided as far as possible and temperature and
light should be regulated to avoid injury of the
cells.

While plasmolytic methods are of interest in
the study of cell processes, plasmolysis is not of
normal occurrence in plant tissues. There may be
exceptions, e.g., spore formation in certain algae
(Gross, 1940). Generally, it is considered to be
injurious to cells, either by rupture of the plasmo-
desmata connecting them and by rupture of what-
ever union exists between the unpitted wall and
the protoplasm, or by effecting certain colloidal
changes in the protoplasm. Scarth (1941) pre-
sents evidence that the degree to which a cell may
be plasmolyzed without undue injury "is deter-
mined by the point at which an irreversible stiffen-
ing, presumably coagulation, of the protoplasm
occurs," and that "the immediate cause of death
is usually the rupture of the rigid ectoplasm on
deplasmolysis." Bennet- Clark and Bexon
(1943) have shown that there is a rapid increase
in the respiratory rate on plasmolysis of beetroot

cells. The question as to whether osmotic pressure
determined at limiting plasmolysis could be converted
to tlie value at normal volume merely by considering
volume change has always been troublesome to the
investigator. An increased respiration rate is one
of the first definite indications that marked changes
do occur during plasmolysis. Such a response may
effect, or may be the result of, other reactions which
conceivably are involved in the water balance of the
cell.

Returning to the plasmolytic measurement of os-
motic pressure, it can be safely stated that the anom-
alies discussed above are not often observed when
proper precautions are taken and when suitable ma-
terial is employed. As a rule, best results are ob-
tained where the plasmolysis form is convex.

The Plasmometric Method: — Introduced by
HoFLER (1917) this procedure is adaptable to cer-
tain types of cells. The technique involves rather
strong plasmolysis, followed by measurement of the
respective volumes of cell and protoplast. The value
of Og is obtained from the following relationship :

Vol. of protoplast

Fig. 26. — Vacuole
contraction in an onion
epidermal cell having the
vacuole strongly stained
with neutral red. Drawn
from a photograph by
Strugger (1935).

Og = OP of plasmolyzing sol. X

Vol. of turgorless cell

The method is claimed to be highly accurate, and
may be more suitable for single cells than the limiting plasmolysis method.
However, there are certain disadvantages. Only cells of regular shape

Grafts et al.

— 90 —

Water in Plants

a

b

Fig. 27. — Vacuole contraction as shown
by leaf cells of Elodea canadensis after
staining with neutral red. Unplasmolyzed
cell, a; plasmolyzed, b. Redrawn from
Strugger (1935).

whose volume may be determined with fair accuracy are suitable. Large
cells with a low protoplasm/vacuole ratio are recommended. The possi-
bility of injury and subsequent changes due to strong plasmolysis must

be considered. Levitt and Scarth
(1936) used the plasmometric method
to investigate the bound water content
of hardened and unhardened cells. It
is a preferred procedure in permeabil-
ity studies, but may not necessarily in-
dicate permeability in the normal state.

Gryoscopy : — The cryoscopic meth-
od has been widely used for indirect
measurement of osmotic pressure in
plants. It involves the determination
of the freezing point of expressed cell
sap, the osmotic pressure being directly
proportional to the freezing point low-
ering. The important investigations
of Dixon and Atkins (1913) in Eng-
land, Walter (1931^) in Germany,
and Harris (1934) in this country utilized this method and have con-
tributed to its improvement.

The following advantages over the plasmolytic method may be noted: 1) measure-
ments may be made at normal cell volume; 2) no errors due to change in permeability
are encountered; 3) changes resulting from injury due to shock are avoided; 4) deter-
mination of average OP values of whole tissues or organs is possible; 5) freezing
point lowerings may be determined with a high degree of accuracy.

Possible errors and limitations of the method include : 1 ) inherent difficulties con-
nected with expression of plant saps. Small samples from a single tissue type are more
significant than large mixed samples. With any sample, dilution of the vacuolar sap
by water liberated by alteration in physical organization of colloidal matter may con-
stitute a real limitation (Newton, Brown, and Martin, 1926; Meyer, 1928; Jaccard
and Frey-Wyssling, 1934 ; Roberts and Styles, 1939 ; Kerr and Anderson, 1944 ;
Currier, 1944a). Walter and Weismann (1935) consider this not to be a source of
error. — 2) If killing is accomplished by heat, water loss from the sample may result in
too great a depression. — 3) Chemical changes such as hydrolysis, mostly enzymatic in
nature, may increase the amount of osmotically active solutes. Condensation reactions
or production of volatile substances might lower the osmotic concentration. — i) Con-
tamination of sap by contents of vascular elements is a possible source of error. — 5)
Use of different pressures for expression of sap may give discordant results because
varying amounts of water of imbibition from protoplasm and walls are included in the
samples. — 6) Filtration, and adsorption of solutes by dead cellular membranes during
pressing may result in lower values. There is evidence to the contrary {e.g. Thren,
1934) but more information is needed. — 7) The press metal may have a catalytic action
on the sap.

When these errors are all reduced to a minimum, valuable data can be obtained by
the cryoscopic method. In spite of its drawbacks it is the most practical method for
measuring the osmotic pressure of bulk tissue.

Expression of Sap: — Dixon and Atkins (1913) recommended that sap be ex-
pressed from tissues that have been frozen ; if the tissue is not killed in some manner
filtration of solutes by the living cell membranes may lead to low values. The succes-
sive fractions of expressed sap from untreated tissue show a progressive increase in
OP. GoRTNER, Lawrence, and Harris (1916) showed that while this is true for
most plants, for some the concentration remains constant.

Other means of rendering the cell membranes permeable prior to sap expression
are: grinding (Hibbard and Harrington, 1913; Newton, Brown, and Martin, 1926;
Sayre and Morris, 1932) ; and exposure to toxic vapors (Chibnall, 1923; Goldsmith
and Smith, 1926). Heating the samples (Thren, 1934; Doneen, 1934; Mallory,

Chapter VII

— 91

Osmotic Quantities of Cells

1934) gives satisfactory results, and in some cases may be preferred. Freezing is the
method commonly employed, and the use of liquid air or dry ice is recommended.
While for some plants an ice-salf bath at — 15° to — 20° C. is sufficient (Gortner and
Harris, 1914), for others such as the needles of Pinus rigida in the winter condition
the lower temperature of dry ice is required (Meyer, 1929). Broyer (1939) has re-
viewed methods of sap extraction. 9

Hydraulic presses are commonly employed in connection with press cylinders of
various design. Figure 28 shows a semi-micro press cylinder useful in expressing sap
in volumes of 0.5 to 5.0 ml. In order to gain the desired precision, the magnitude of

Top v/ev)/ of di\3C C

Top view of baje D

0 1 z d cm

Fig. 28. — Press cylinder outfit used to express small amounts of sap from plant
tissue. From Currier (1943), modified after Broyer and Furnstal (1941).

the pressure used should be known within narrow limits. This has special significance
in dealing with cells where the proportion of cell wall and protoplasm to vacuole is
high. The possibility of imbibed water diluting the expressed sap was considered by
MosEBACH (1936) to be a major error in cryoscopic investigations of certain brown
and red marine algae. Pressures should be expressed in common units, as pounds per
square inch, based on the area of the press cylinder. Pressures of 10,000 to 16,000
pounds per square inch are recommended.

The Cryoscope: — Various cryoscope designs are available. The German workers
have preferred the Burian-Drucker (1909) apparatus, which accommodates 1.5 ml.
of sap, and the Drucker-Schreiner (1913) adapted for small volumes (about 0.005
ml.). The latter is less accurate and not as generally useful as the first. Figure 29
shows a semi-micro cryoscope accommodating 0.5 to 1.0 ml. of liquid. Mosebach
(1940) designed an apparatus which employs a fraction of a drop of sap obtained
from succulent tissue without pressing. The sap is frozen in a capillary tube, and the
melting point is accurately observed by means of a horizontal microscope. Where
larger amounts of sap are available (15-25 ml.) the standard method described in
manuals of physical chemistry is suitable. With this a differential thermometer of
the Beckman type, or a Haidenhain thermometer with a fixed zero point may be em-
ployed.

All of the above apparatus depends upon a mercury thermometer for determination
of the freezing point. Another method employs the thermoelectric principle, and sev-
eral designs are available (Dixon, 1911; Herrick, 1934). In this procedure a fine
wire thermocouple consisting of two kinds of metal is inserted into the solution, an-
other is held in a reference liquid at constant temperature. The difference in tem-
perature causes current to flow through the circuit and this is measured by a sensitive
galvanometer.

Where freezing points of "living" tissues are to be determined the thermocouple
has been preferred. However, because the thermocouple point requires rupture of
cells, whereby the needle is flooded with sap from a limited number of cells which
freezes ahead of the uninjured cells, Walter and Weismann (1935) claim that a
mercury thermometer inserted into a relatively dry cavity in the tissue gives a truer
freezing point value. Regardless of which method is employed, the data obtained in
the freezing of "living" tissues is difficult to interpret. This problem is further con-
sidered in Chapter VIII.

Crafts et al.

92 —

Water In Plants

Mair, Glasgow, and Rossini (1941) successfully employed a platinum resistance
thermometer for measuring freezing points of hydrocarbons. Their paper should be
consulted for discussion of the theoretical aspects of freezing point measurements.

Physical Principles : — The principle involved in the cryoscopic method
is that the presence of a solute lowers the vapor pressure (and diffusion
pressure) of a solvent. Since the vapor pressure of water is equal to that
of ice when both phases are in equilibrium, the addition of a solute to the
liquid lowers its vapor pressure. This causes the ice to melt, transforming
it to liquid. The melting process absorbs heat and the temperature drops
to a point at which the vapor pressures of the solid and liquid phases are
again equal. This is termed the freezing point of the solution.

Fig. 29. — Construction of cryoscopic apparatus. The insulated vessel H is fitted
with a top D with holes drilled to receive a heavy wire stirrer A, an ordinary ther-
mometer B to record bath temperature, a glass air jacket K, and a small glass tube
I closed at one end, in which a piece of capillary tubing G containing water may be
frozen. Within the air jacket the sample tube I is held in place by a cork fitted with
holes to receive the freezing point thermometer C and a fine wire stirrer E, as well as
a small hole F for inserting the frozen capillary to initiate crystallization.

Raoult's law states that the vapor pressure of water is lowered in pro-
portion to the mol fraction of solute added.

Pq — p ^_ "2 ("f)

p ~ ni + no

where po and p are the vapor pressure of pure solvent and solution and
ni is the number of mols of solvent containing n2 mols of solute.

Chapter VII — 93 — Osmotic Quantities of Cells

Vapor pressure: temperature curves of dilute solutions are approxi-
mately parallel with those of pure water. Therefore, for dilute solutions

rp ^^^ rp

-^-—f = a constant (K) (5)

where To and T are the freezing temperatures of pure solvent and solu-
tion. If — ^ — be substituted for po — p in Raoult's law, then

To-T no (d)

K Pq m + n2

T„-T = Kp„

o

or
n2 (7)

ni + no

For dilute aqueous solutions K has been determined to be 1.86° C. per
mol of undissociated solute. Where the solute is an electrolyte, dissocia-
tion into ions increases the total number of particles, each of which has
an equal effect in lowering the vapor pressure of the solvent. Since the
ratio of the number of solute particles to the number of solvent particles is
the important factor in determining the freezing point of the solution,
electrolytes are more effective than nonelectrolytes. It has been noted
earlier that a molal solution of KNO3 has a much lower freezing point
than has a molal sucrose solution ; if ionization were complete, and other
factors did not interfere, the lowering would be twice as great. Before
the introduction of the activity concept, freezing point determinations were
used to measure the so-called "degree of ionization." More recently they
are used to measure activities, the discrepancy between theoretical and
actual measured activity being ascribed to the influence of intermolecular
force fields upon the escaping tendency of the molecules.

Conversion of the freezing-point lowering of a solution to osmotic
pressure values at 0° C. is easily accomplished by the following relation :

0F=^- 22.4 (S)

or
OP = 12.04 A (9)

where A = freezing-point depression.

Lewis (1908) offers the more accurate form of this equation

OP = 12.06 A — 0.021 A- (10)

which has been used by Harris and Gortner (1914) and Harris (1915)
to compile a table of values convenient for converting freezing point data
to osmotic pressures. In correcting for change in osmotic pressure from
0° to, for example, 20° C, the assumption is made that expressed tissue
saps, like sucrose solutions, obey the gas law for change in temperature.
The validity of this assumption is of special importance where the sap is
high in colloids.

Freezing-Point Determination: — The procedure generally used for determining
the freezing point of expressed sap is as follows. The sap is placed in a small tube,
and a thermometer is inserted with the bulb immersed, together with a small wire
stirrer. This tube is placed within another larger tube, providing a dead air space
to preclude too rapid cooling. The jacket tube is now immersed in a freezing mixture,
usually ice and salt, and the temperature of the sample slowly falls; stirring is con-
tinuous. Upon crystallization, the mercury column rises abruptly, steadies, remains
constant for a short time, then falls slowly. The plateau, or highest temperature
reached is recorded as the observed freezing point. Crystallization at temperatures
below the freezing point can be readily induced by introducing the tip of a fine glass
capillary containing ice into the solution.

Crafts et al.

94

Water in Plants

The value so obtained is not the true freezing point, because of (a) supercooling
of the solution, and (b) absorption of heat by the apparatus. Harris and Gortner
(1914) proposed a correction for the supercooling. Since crystallization usually be-
gins at a temperature lower than the true freezing point, the liquid phase at the ob-
served freezing point, in equilibrium with the ice crystals, is more concentrated than
it would be at the true freezing point. Since one eightieth of the weight of water
solidifies per degree of undercooling,

(^-^)

or

V

Al — 0.0125 u Al

ai)

(12)

where A^ and A = observed and true freezing point depressions, V = volume of
solution, and u = number of degrees of undercooling. This correction has been
widely used in cryoscopy. If undercooling can be avoided by salting with ice crystals,
jarring, etc., the correction is unnecessary.

O-Z

0.^ o.c o.a

Mots 6ucro6e per IQOOg water

1.0

Fig. 30. — Calibration curve for the apparatus shown in figure 29. The
lower curve was constructed from data from the International Critical Tables
(1933) ; the upper curve presents the observed values.

An approximate correction for heat capacity eflfects may also be calculated if the
following values are known : 1 ) the weight and heat capacity of glass sample tube,
thermometer, and stirrer in contact with the solution; 2) volume of solution; J) tem-
perature at which freezing begins; 4) temperature of bath; and 5) observed freezing
point. The heat capacity effect is small and the correction unnecessary where the
volume of solution is large in comparison with the volume of the thermometer and
glass. For small amounts the error may be serious.

By use of a modified procedure (Currier, 19446), it has been found possible to
eliminate these two corrections. The semimicro apparatus (Figure 29) was standard-
ized by recording the observed freezing points of carefully prepared molal sucrose
solutions, ranging in concentration from 0.1 M to 1.0 M in steps of 0.1 M, and com-
paring them with the accurately known theoretical values (Int. Crit. Tables, 1933).
The results of this comparison are shown in Figure 30. By referring an observed
value to this graph, the corrected value may be obtained at once. The increasing dis-
crepancy between A obs. and A corr., as the solutions become more concentrated, is not
due to supercooling effects as the correction for supercooling, when calculated accord-
ing to Harris and Gortner's formula, remains at about 0.01° C. for all concentrations.
The probable explanation is that with increasingly concentrated solutions a greater
amount of heat is lost to the system because crystallization is slower, and the time re-
quired to reach the apparent freezing point is longer. Walter (1931o) recommended
that in the cryoscopic determination the amount of supercooling be constant and ap-

Chapter VII

95

Osmotic Quantities of Cells

proximately 1° C. This procedure requires a preliminary trial to determine where
freezing must begin to be 1° C. below the freezing point.

As proof of the validity of correcting observed freezing point values as described,
freezing curves, such as that shown in Figure 31, were constructed according to the
recommendations of Mair, Glasgow, and Rossini (1941). Extrapolation of the part
of the curve where thermodynamic equilibrium exists between the solid and liquid
phases of the system, back to the initial downward part of the curve, gives a close
approximation of the true freezing point. The above authors point out that in a strict
sense the true freezing point is slightly higher than this, but they have proved that
the difference is insignificant when the amount of supercooling is small in comparison
with the head of temperature (difference between the temperature of freezing bath and
that of the test solution). With a head of 10° to 15° C, this error was found to be
insignificant in the determinations made as described. True freezing points of both
sucrose solutions and beet juice, obtained by plotting freezing curves, agree with those
corrected by means of the calibration curve of Figure 30.

^/.u

0.0

^

o

1

1

L

\-l 0

r

"^

--^

.^

\

^

"■^

^^

"■^

a.

,

•--

^

■^

L

>

s.

[

-PA

\

£.,<J

\

c

)

T/

0

me

— n

linu

'fej

2

0

Fig. 31. — Freezing curve constructed according to recommendations
of AIair, Glasgow, and Rossini (1941). Extrapolation of the latter
portion back to the initial downward line gives a close approximation of
the true freezing point of the solution.

In dealing with plant saps of high viscosity, separation of ice may occur more
slowly, resulting in a slower rise of the mercury column and a greater proportion of
heat lost to the system. Under these circumstances a calibration curve prepared by use
of sucrose may not be valid. The error would likewise not be corrected by use of the
formula for supercooling. Where doubt exists, comparison of freezing curves like that
of Figure 31 with similar curves for sucrose solution will indicate the error involved.

Vapor Pressure Methods : — A unique method for determining
osmotic pressure is the vapor pressure method devised by Barger (1904,
1924) for the purpose of calculating molecular weights, and adapted by
Halket (1913) to plant saps. Small glass capillaries of uniform bore

Crafts et al.

96

Water in Plants

are prepared, and into them are drawn, first, a droplet of unknown (sap),
followed by an air space, then by a droplet of sucrose solution of known
concentration, and so on. Each tube then contains alternating layers of
sap, air, sugar solution, air, sap, etc. (Figure 32). The tubes are sealed in

Fig. 32. — The Barger Halket method for determining
the osmotic pressure of plant sap. Capillary tubes contain
alternate columns of standard sucrose solutions and sap.
In this figure the black columns represent stock solution,
the clear columns sap. Each tube contains stock solution
of a different concentration.

a flame and mounted on a slide. Several tubes are prepared for each de-
termination, each varying with respect to the concentration of the sugar
solution. By means of a. binocular microscope and micrometer, the length
of the sap columns is measured before and after equilibrium has been at-

FiG. 33. — Osmotic pressure determination by the method
of Ursprung and Blum (1930). Standard reference solutions
are contained in capillary tubes c, attached to a cover glass d
by plastic cement p. The cover glass with attached capillaries
is sealed on top of a shallow dish by grease f. The depression
in the dish contains the expressed sap I.

tained. The results are placed on a graph, and that concentration that pro-
duces no change in the length of the sap columns is considered to be iso-
tonic with the sap, i.e., having equal vapor pressure and osmotic pressure.
The method depends upon the diffusion of water vapor through air, which
acts like a semi-permeable membrane. Though the method is tedious and
subject to error from mixing of the droplets and movement of films along

Chapter VII — 97 — Osmotic Quantities of Cells

the walls of the capillaries, it is useful where only small volumes of sap are
available. Wildervanck (1932) preferred it over other methods for de-
termining the osmotic pressure of Nitella translucens sap.

Modifications of this method are described by Signer (1930) and by
Berl, et al. (1930). The capillary method of Ursprung and Blum
(1930) dififers in that the expressed sap or other solution to be investigated
is placed in the shallow depression of a small moisture chamber, and that
the standard reference solutions are contained in a series of capillaries at-
tached to an upper glass cover (Figure 33). Technical details may be
found in the original paper.

Procedures have been perfected for measuring osmotic pressure thermo-
electrically, based upon differences in vapor pressure between the solution
to be investigated and some standard reference solution (Hill, 1930;
Baldes and Johnson, 1939). Either a sensitive thermopile or thermo-
couple is used, and very small amounts of liquid can be investigated. The
principle involved {cf. Baldes, 1939) is that differences in temperature
between the two droplets under standard conditions for evaporation can be
measured by a delicate galvanometer. This reading is converted to vapor
pressure or osmotic pressure by standardizing the apparatus with known
solutions. RoEPKE (1942) has provided refinements in apparatus and
method.

Osmometric Methods : — Direct determination of osmotic pressure by
means of osmometers is not particularly adaptable to plant saps. Bour-
dillon (1939) describes a micro apparatus utilizing a flat horizontal mem-
brane, which will accommodate as little as 0.2 cc. of protein solution in
concentrations down to 0.025 mm. Blegen and Brandt Rehberg (1938)
used specially prepared collodion tubes. Results compared well with Hill's
vapor pressure method. Ordinarily osmometric procedures are successful
only where the sample is a fairly pure colloidal solution, where significant
amounts of dissolved electrolytes are absent. Levitt (1946) has investi-
gated the colloid osmotic pressure of proteins extracted from potato tuber
tissue in this manner.

Other Methods for Osmotic Pressure: — The "simplified" or
"minimum cell volume" method, designed by Ursprung (1923), consists
of measuring the change in length of strips of tissue immersed in solutions
(usually sucrose) of varying concentration. The lowest concentration of
sugar that produces minimum length is considered to be isotonic with the
cell sap at limiting plasmolysis. There are two serious objections to the
method. First, it is not valid to assume that a tissue strip in equilibrium
with the most dilute solution effecting "minimum cell volume" is in a con-
dition of limiting plasmolysis. There is assumed to be a point, as the con-
centration is increased, where shrinkage ceases, and volume remains con-
stant. Table 25 shows, however, that within the concentration range
used, potato tissue may continue to shrink with increasing concentration
of the bathing solution. Although a constant condition is approached, the
fact that higher and higher concentrations are capable of causing reduc-
tion in imbibition of the walls indicates that the method cannot produce
highly accurate results. On the other hand the simplified method is a
valuable means of determining DPD values at normal volume (see later).

The second objection is that change in length of a tissue is not always
a true indication of change in volume. Many strips exhibit distortion

Grafts

et al.

98

Water in

Plants

Table 25. — Change in volume of potato tuber tissue cylinders immersed 8 hours in
sucrose solutions of varying concentration at 20-22° C. Values are based on normal

volume (Currier, 1943) : —

CONC

. OF SOLUTION

Per cent change

Tap water +12.4

0.1 M + 6.5

0.2 + 3.4

0.3 + 0.8

0.4 — 9.6

0.5 —19.5*

0.6 —21.9

0.7 —27.6

0.8 —33.0

1.0 —33.6

* AH cells plasmolyzed at this point.

when permitted to swell or to shrink. Especially is this true of epidermis
with its cuticle on one side. That of Sedum nicaense has been shown to
decrease in the two surface dimensions upon swelling (increase in total
volume) in water. Table 26 shows the measurements.

Table 26. — Changes in dimensions of epidermal strips from Sedum nicaense leaves.
Values are arbitrary units (Currier, 1943) : —

Normal

s.

ATURATED

Incif

'. plasmol.

L

W

D

L

w

D

L

W D

7.75

4.00

1.01

7.30

3.90

1.35

7.70

4.00 0.84

9.21

4.21

0.90

8.86

4.00

1.24

9.00

4.10 0.73

10.85

2.14

1.00

10.78

2.08

1.37

10.70

2.14 0.81

5.71

2.36

5.85

2.43

• • •

5.85

2.50

6.94

2.00

6.85

2.07

6.78

2.14

L = Length; W = Width; D = Depth or thickness.

The simplified method may theoretically be extended to measurement
of all three osmotic quantities in the following manner. Letting O =
osmotic pressure of the tissue, DPD =: diffusion pressure deficit of water
in the tissue, T = turgor pressure of the tissue, V = volume of the tissue,
and the subscripts g = limiting plasmolysis, n := normal state, and s ^
water saturation, the following relations should be valid.

Og = OP of plasmolyticum causing minimum volume {13)

On = Og • -^ {14)

Vn

Os = Og • 4^ {15)

Vs

DPDg = Og {16)

DPDn ^ On — Tn = OP of plasmolyticum causing no change in Vn {17)

DPDs = zero {18)

Tg = zero {19)

Tn = Ts • ^^'" — Vg^ = On - DPDn {20)

(Vs — Vg)

Ts = Os {21)

This method proves convenient for rapid estimation of approximate
values for these states of a given tissue.

Chapter VII — 99 — Osmotic Quantities of Cells

The Magnitude and Variation of Osmotic Pressure in Plants: —

To secure quantitative data relative to the role of osmotic pressure in plant
functions, and in plant distribution, an enormous number of measurements
have been made. Many were obtained by plasmolytic methods, especially
where it was necessary to work with single cells. The cryoscopic pro-
cedure has been preferred by ecologists, since it is more adaptable to field
conditions and because average values of plant organs and whole plants are
more easily obtained. In such studies, osmotic pressure data are used as a
criterion of the plant's ability to withdraw water from the soil, and to re-
tain it against the evaporative capacity of the atmosphere. For reasons
already presented, neither the plasmolytic nor cryoscopic method is without
objection. The former indicates the absorptive capacity at limiting plas-
molysis only ; the latter must rely on total expressed sap with the obvious
associated errors. In both, the normal turgor pressure is unknown. It
would appear that the simplified method theoretically would be expected
to give the most valuable ecological data, but from a practical viewpoint the
cryoscopic procedure is to be preferred.

Osmotic pressures of plant tissues have been found to vary from values
of around one atmosphere to an extreme of 202.5 atm. reported for the
expressed sap of Atriplex confertifolia, growing in an alkali region of
Utah (Harris, 1934, p. 70). The sap contained 67.33 grams of chloride
per liter. However, most mesophytes exhibit concentrations within the
range of 5 to 30 atmospheres. Desert species yield higher, hydrophytes
usually lower values than mesophytes. It should be stated that these gen-
eralizations represent averages or trends. Exceptions must be expected be-
cause of the multiplicity of factors exerting an effect on sap concentration.

Contiguous cells, even in a homogeneous tissue, may not have identical
cell sap concentrations. Regardless of protoplasmic connections, each cell
maintains a structural and functional individuality, reflected in one way by
the osmotic pressure of its contents. Plasmolytic studies clearly demon-
strate this fact ; all cells do not reach incipient plasmolysis simultaneously,
but Og values distribute themselves in a typical S-shaped curve (Figure
21 ). It is usually found that Og values are greater for small than for large
cells within a single tissue.

Gradients of osmotic pressure have been reported within individual
tissues. While such gradients may be real, they are constantly changing,
and may even reverse in direction due to seasonal and diurnal changes and
to environmental and internal factors.

Ursprung and Blum (1916a) reported a radial gradient in the corti-
cal region of Helleborus foetidus root, and in xylem parenchyma of Fagiis
sylvatica stem, the Og values increasing toward the center. Vertical gradi-
ents were found in the stem of Urtica dioica, Og increasing from tip to base
for all tissues. A gradient decreasing downwardly was reported for root
tissues of Helleborus, Urtica, and Fagus.

There may be considerable variation in osmotic pressure among the tis-
sues composing an organ. The distribution of Og values among leaf tissues
assumes the following pattern : palisade parenchyma > spongy parenchyma
> upper epidermis > lower epidermis (Ursprung and Blum, 1916a;
BUHMANN, 1935).

Ursprung and Blum found for root tissues of Helleborus, Urtica and
Fagiis highest Og values in xylem and phloem parenchyma, and in com-
panion cells ; lowest in the outer cortical region and epidermis.

Grafts et al. — 100 — Water in Plants

In nonwoody stems of Helleborus and Urtica, minimum values appeared
in epidermis, cortex, and pith, with highest values in xylem and phloem
parenchyma, companion cells, and cambium. Woody twigs (Fagus)
showed highest concentrations in xylem parenchyma cells, lowest in phloem
parenchyma.

Averages of values exhibited by leaf, stem, and root tissues of four
plants are shown in Table 27. Figures represent "osmotic values," i.e.,
number of mols of KNO3 (per liter of solution) isotonic with the cell sap
at incipient plasmolysis. (Note, however, remarks concerning KNO3
as a plasmolyzing agent, page 82).

Table 27. — Osmotic pressure of various tissjies determined plasmolytically, expressed
as mols of KNOs per liter of solution. Data of Ursprung and Blum (1916a). For
conversion to atmospheres, osmotic pressure values of KNO3 solutions (Ursprung,

1938, p. 1290) are included: —

Helleborus Urtica Fagus Sedumacre

Epidermis -484

Spongy parenchyma -575

Palisade parenchyma 871

Outer cortex 522

Inner cortex 532

Phloem parenchyma

Companion cells -577

Cambium 558

Xylem parenchyma 567

Pith 521

Rays, cortex

Rays, wood

.473

.371

.278

.635
1.015

.571
1.017

1 ■

.330

.472

.671

.348

.495

.679

.377

.518

.573

• • •

.556

.722

.492

.548

.634

.411

.59

.983

.495

.471

• • •

> • •

.869

■ • •

• ■ •

.938

20° C. : —

Concentration

OP

volume molar

atm.

0.7

27.0

0.8

30.5

0.9

33.9

1.0

37.2

1.1

40.4

1.2

43.5

KNO3 solutions at 20

Concentration OP

volume molar atm.

0.1 4.3

0.2 8.3

0.3 12.3

0.4 16.1

0.5 19.8

0.6 23.4

Sap contained in the conducting elements of the xylem is very dilute.
Maximov (1929a, p. 54) reports osmotic pressures in root sap (exudate)
of Xanthium strumarium, Impatiens halsamina, and Zea mays of 0.67, 0.36,
and 1.46 atm. respectively. Van Overbeek (1942) recorded 0.42 atm.
for the initial exudate of decapitated "low salt" tomato plants, 1.32 atm. for
"high salt" plants. The dry matter content of xylem exudate of squash was
found to vary between 0.102 and 0.23 per cent (Crafts, 1936). Stock-
ing (1945) determined a value of 1.9 atm. for this plant growing in culture
solution. Crafts (1932) found an average of 7.0 atm. in the phloem exu-
date of pumpkin and cucumber.

Variation of osmotic pressure among dififerent organs of plants has
been studied. Values exhibited by leaves generally exceed those of roots
(Hannig, 1912; Dixon and Atkins, 1916) ; older leaves exceed young
leaves (Dixon and Atkins, 1912; Korstian, 1924; Gail and Cone,
1929). Illuminated leaves show a higher OP than those growing in the
shade (Dixon and Atkins, 1912; Lutman, 1919; Halm a and Haas,

Chapter VII — 101 — Osmotic Quantities of Cells

1928; Marsh, 1941). Fruit tree leaves exhibit a higher OP than the im-
mature fruits (Chandler, 1914).

With respect to average osmotic pressures of entire plants, individuals
of unlike species growing under the same environmental conditions may
display significant differences in sap concentration (Harris, 1934).

In general, a decrease in osmotic pressure is expected in the sequence
trees, shrubs, herbs (Harris and Lawrence, 1916). Winter annuals
show a decrease over perennial herbs. Succulents and hydrophytes are
characterized by rather low, xerophytes by high sap concentrations ; meso-
phytes He between (Maximov, 1929a, p. 274).

Many studies have dealt with the effect of environmental factors on
osmotic pressure. Any change or condition leading to the development of
a water deficit in the plant is generally attended by an increase in sap con-
centration. The OP tends to vary directly with the concentration of the
soil solution, and is increased by those factors which cause the plant to
transpire at a greater rate.

The cell sap concentration of leaves generally varies from a maximum
in the early afternoon to a minimum in the early morning hours ( Stoddart,
1935; Marsh, 1940). The rise during the daytime is due both to photo-
synthetic activity and to increasing water deficit within the leaves. Such
fluctuations are less pronounced in roots than in aerial organs.

In addition to diurnal variation, plants show annual or seasonal fluctua-
tions in OP. In some — certain conifers for example — the sap concentra-
tion may be higher during the winter months (Gail, 1926; Meyer, 1928).
In others, a maximum is reached in the summer (Mallery, 1934). Ac-
cording to Marsh (1940), the osmotic pressure of lowland prairie species
which he examined increased during the growing season about one half over
that at early summer; for upland species the concentration doubled or
tripled.

The variation in osmotic pressure as a result of extended water deficit
may be very great. Gasser (1942) used the limiting plasmolytic method
to determine the direction and extent of changes in Og of detached plant
organs (mainly leaves) placed under conditions which permitted slow
water loss. In an extreme instance, cells of the terrestrial alga Trentepohlia
aurea increased in Og from 123.4 atm. to 291.4 atm. in ZZ days. Out of
95 plant species, 69 indicated a rise of 15 to 223 per cent of the initial
value. The rest either showed no change or a slight decrease. Where a
condition of water excess was induced by placing the leaves in moist air,
72 out of 87 plants exhibited a decrease in Og, within a range of 5.3 to
66 per cent of the initial value.

The changes in Og (the author uses Szg) are ascribed to processes em-
braced by the term osmotic regulation, wherein the solute concentration
varies due to hydrolysis or synthesis of polysaccharides, and by exosmosis
or endosmosis of solutes. The responses shown can possibly be attributed
in some degree to plastic stretching or shrinking of the walls which were
not conclusively shown to be inoperative. However, it is not unlikely that
other factors, as suggested by the author, are primarily responsible for the
changes observed.

Diffusion Pressure Deficit. Methods of Measuring DPD : — The

diffusion pressure deficit of the water in plant cells is a measure of the abil-
ity of those cells to absorb water. Its value may be measured directly, or it
may be obtained by difference if the osmotic and turgor pressures of the
cells are known. When TP = zero, as at limiting plasmolysis, OP = DPD,

Grafts et al.

102

Water in Plants

but at all other states the hydrostatic pressure or turgor of the cells lowers
the D'PD of the water in the cells. At full turgor, or what is termed water
saturation, the diffusion pressure of water in the cell sap is equal to that of
pure water at the same temperature, and the DPD = zero. Cells in the
normal state are neither at full turgor nor at limiting plasmolysis ; proce-
dures employed in estimating DPD must be applicable to cells in a state of
partial turgor. The following methods have been used in DPD measure-
ments. In all of them, the water absorbing power of cells, tissues, or organs
is balanced by various means, such that there is no change in size, weight,
turgor, or vapor pressure.

5^ ^o.osY

*0.06

*0.04-

■^O-OZ -

\

\

I

I

U e 4 6 S /O /2 /4- /6 /8 £0 22 24 26 28 30 J2

Os/7?o//c Pressure faf/n. of £0° C.J

Fig. 34A. — Diffusion pressure deficit values determined by the vol-
ume method. (Meyer and Wallace, 1941).

The Cell Method: — Although the idea was suggested by de Vries (1884), this
method was first employed in actual measurements by Ursprung and Blum (1916fc).
A thin section of tissue, a bit of thin leaf, or any cells that may be observed under the
microscope may be used. The tissue is placed in paraffin oil on a slide to prevent
evaporation of water. One or more cells are selected, and their surface outlines are
drawn on paper by means of a camera lucida. The tissue is then placed in sucrose
solutions of varying concentration until one is found in which the cells do not change
in volume. The OP of the solution causing no change in volume is recorded as equi-
valent to the DPD of the cells.

This method has certain weaknesses. Ernest (1931) has pointed out that upon
removing a tissue section from a plant organ, a change in equilibrium conditions occurs
and volume measurements may become invalid. In fact, gradients in DPD may be
reversed due to changing intercellular pressures and to the osmotic effect of sap liberated
by sectioning on the cells. She further demonstrated that water-saturated tissue, on
sectioning, can absorb yet more water due to partial removal of intercellular pressure.
Thus DPD values measured may be too high. Ernest (1931, 1934a, 6) used an epi-
dermal strip method, whereby intact mesophyll cells adhere to the epidermis, and found
leayes of Iris and Crocus, among others, to be suitable material. It has been pointed
out by Oppenheimer (1936), however, that even here there is a release of intercellular
pressure. This author (1930a, \9Z2a,h) further criticises the cell method. Volume
changes are difficult to detect and are often so small as to be insignificant within the
range of experimental error. Changes in the three dimensions are often disproportional,
so that volumes determined by surface measurements are erroneous.

Chapter VII

— 103 — Osmotic Quantities of Cells

It therefore appears that values obtained by this method and reported in the litera-
ture are open to some question.

Strip or Simplified Method: — The advantage of the strip method (Ursprung,
1923) is that the measurement embraces hundreds of cells and is more successful in
estimating the average DPD of organs. Tissue strips in size approximately 20 mm.
long and 2 mm. wide are removed from leaves, stems, or fleshy storage organs. Fila-
ments and slender roots are cut into suitable lengths. Vascular elements should not
run parallel or at right angles. The length of the sections in paraffin oil is imme-
diately determined by means of a microscope and a stage micrometer ; the strips are
then transferred to a series of sugar solutions. The DPD of the water in the tissue is

I

\^OJ

K

V

\

x\-

%

x\

1-

1

-

\

5.

1-

-

\

1

1

1 1 1 1 1

till)

1

1 1 1 1 1

2 ^ 6 S /O /£ /4 /6 /S £0 22 24 26 28 JO J2

Osmo/'/c Pressure (oJ^m. a/^ ^O*^ C)

Fig. 34B. — Diffusion pressure deficit values determined by the
weight method. (Meyer and Wallace, 1941).

equivalent to the OP of that solution which effects no change in length. Usually 30 to
45 minutes is sufficient time; often 5 to 10 minutes is enough.

A simple modification of the above method is useful where massive tissues are
studied (Lyon, 1936). CyHnders of tissue 5 to 10 mm. in diameter and several centi-
meters long are removed from organs by means of a cork borer, and are immediately
measured for length by means of a binocular dissecting microscope fitted with an
ocular micrometer. They are then distributed among sucrose solutions of varying con-
centration. After several hours, the solution in which the cylinders show no change in
length is assumed to be equal in DPD to the tissue. In place of tissue cylinders, square
sticks may be used, prepared by means of a double-bladed knife (Lyon, 1940). Check
strips are kept in paraffin oil.

Instead of measuring length changes, volumes may be determined by an immersion
method. Cylinders of tissue upon removal from organs are immediately immersed in
calibrated glass tubes partially filled with sugar solutions. A diameter of 10 mm. is
a convenient size for the tubes. The sugar solutions are in a graded series of known

Crafts et al. — 104— Water in Plants

concentrations and temperature is held constant. The difference in liquid level before
and after immersion of the cylinder is recorded as the initial volume of each cylinder.
The cylinders are next transferred to larger volumes of the same solutions and are
permitted to come to equilibrium, the time being related to the diameter. For beetroot
cylinders 9 mm. in diameter and 28 mm. long, 8 hours were found to be sufficient (Cur-
rier, 1943). The cylinders are then freed of excess moisture, and their volumes again
determined. Absence of any change in volume is assumed to mean that the cylinder is
immersed in a sucrose solution equivalent to the DPD of water in the tissue. If such
a solution is not included, a graph may be prepared from which a fairly accurate esti-
mate of the DPD may be made. Errors due to non-uniform swelling of the tissue are
eliminated by this immersion method.

Change in volume of a tissue piece may be affected by intercellular air. Increase
in turgor reduces the size of the intercellular spaces, as evidenced by the escape of gas
bubbles. Thus, the actual change in cell volume would be greater than that indicated
by tissue measurements. Similarly, the spaces might increase in size on shrinkage of
the tissue, giving low values. There should be no error in DPD measurements where
no, or only slight, volume change occurs.

While the simplified or strip method involves fewer errors than the cell method,
there remain the difficulties due to removal of intercellular pressure at the cut surfaces,
and the low order of volume change of some tissues due to inelastic walls. The suit-
ability of each tissue must be determined in advance.

The Weight Method: — This method involves the same principle, that is a sugar
concentration is sought which produces neither gain nor loss in weight of water by the
tissue. A major source of error is in reproducing comparable conditions of surface
moisture at the time of weighing the tissue pieces. Also, any surface active material
released from cut or injured cells that reduces the surface tension of the sugar solu-
tions to a point where wetting of the intercellular capillary spaces takes place will in-
troduce an error. This infiltration can be quite significant under some conditions
(AsHBY and Wolf, 1947).

Meyer and Wallace (1941) found that the volume method (based on change in
length) and the weight method gave practically the same results when applied to
potato tuber tissue (Figure 34). Also that permeation of sugar into or loss of solutes
from the tissues at concentrations near the DPD were insignificant, and even at other
concentrations were not sufficient to change the trend of results.

Other Methods for DPD: — Stocking (1945) devised a procedure
for studying the DPD of water in tissues of intact plants growing in
the field. Using squash plants, he injected sugar solutions of known con-
centrations into the hollow petioles of leaves of comparable size and age.
At periodic intervals thereafter he withdrew small samples of the solutions
and determined their concentrations using an Abbe hand refractometer.
Not only could he measure the DPD of water in the tissues at a given time,
but he could follow changes throughout a considerable period, dependent
mainly upon the volume of solution that could be held by the hollow petiole.
From his data he determined the diurnal trend in water deficit in the leaves
and calculated tensions in the xylem that corresponded to these. Values
varying between 0.2 and 9.1 atm. were calculated.

A method for examining the DPD or water absorbing power of rela-
tively intact root tissue is credited to Sabinin (1925). When plants grow-
ing in water culture are excised above the root they usually exhibit exuda-
tion as a result of the phenomenon termed "root pressure." If sucrose or
mannitol is dissolved in the solution surrounding the roots, exudation
ceases and if a potometer is affixed to the bleeding stem it will be found
that the movement of water through the root may be reversed. By care-
fully adjusting the concentration of the bathing solution until movement
ceases entirely, a value will be obtained that might be considered in equilib-
rium with the DPD of the root system. However, van Overbeek (1942)
has shown by cryoscopic determination of the osmotic concentration of the
exudate and of the bathing solution that an active mechanism may be in-

Chapter VII — 105 — Osmotic Quantities of Cells

volved, amounting to as much as 71 per cent of the average root pressure
(see also Chapter IX, page 150). White (1938) also postulates an active
component in absorption of water by roots. Probably the method is more
valuable in studies on the physiological activity of roots than in osmotic
pressure or DPD determinations.

Several other techniques have been employed in DPD measurements:

a) Hard leaf method (Ursprung and Blum, 1927). This method is adapted to
such structures as sclerophyllous leaves, conifer needles, grass leaves, and horsetails.
Changes in thickness instead of length are determined.

b) Lever method (Ursprung and Blum, 1930). A modification of the above;
changes in thickness are indicated by a delicate pointer.

c) Tissue tension (de Vries, 1884). Release of tension on splitting a growing
shoot tip produces a curvature v^-hich can be sketched on paper (Livingston, 1906) or
projected (Bouillenne, 1932).

d) "Schlieren" method (Arcichovskij and Ossipov, 1931a), whereby the con-
centration of the reference solution is balanced against that of the plant sap so that
their refractive indices are the same. This method is designed for use on growing
stems.

e) Potometer method (Nordhausen, 1921; Arcichovskij and others, 1931). In
this a reference solution is brought into contact with the tissue being studied and change
in volume is registered on a potometer tube. This method is adapted to measuring the
DPD of water in tissues of attached stems.

/) Vapor pressure methods. Here the vapor pressure of water in the air in a
small enclosed chamber is regulated such that there is no change in volume of a tissue
piece (usually area or length is measured) (Ursprung and Blum, 1930; Renner,
1932) ; or weight (Arcichovskij and Arcichovskaja, 1931) of cells. The difficulties
inherent in the method are related to temperature control of the apparatus and the
rather long duration of time required for equilibrium. If the tissue is weighed, con-
densation of water in the intercellular spaces may give trouble.

Methods for DPD determination in intact organs and whole plants will
be discussed in more detail in Chapter IX.

The Magnitude and Fluctuation of DPD Values : — Diffusion pres-
sure deficit values lie in the same general range as that found for osmotic
pressures. The latter are commonly employed as an index of maximum or
potential DPD's which can be developed in cells. Although in most cells
the DPD should not ordinarily exceed the OP, instances have been re-
ported where this seemed to be true. Pisek and Cartellieri (1931)
noted such a result in leaves of several plants, and for the most part in the
afternoon. In their opinion, the simplified method, used for DPD, yielded
values which were too high, especially when leaves in a wilted or nearly
wilted condition were measured. Kerr and Anderson (1944), finding
that DPD values exceeded OP values in developing cotton seeds, concluded
that imbibitional phenomena were the cause, and that cryoscopic values
were too low.

That a considerable proportion of the DPD may at times be due to the
development of high tensions has been emphasized by some. Such negative
wall pressures, in intact leaves of trees, especially during periods of ab-
normally high water loss, may exceed the influence of osmotic effects on
the DPD (seeCuv, 1936).

The variation, in particular the diurnal variation, is much greater for
DPD than for OP. This is made clear by visualizing a cell which at limit-
ing plasmolysis exhibits an OP of a given value, say 20 atm. As water is
absorbed, the OP decreases from 20 to perhaps 16 atm., while the DPD
decreases from 20 to 0 atm. In cells where tensions can develop, a greater
range is possible. Thus it becomes evident why OP values obtained by use

Crafts et al. —106— Water in Plants

of the cryoscopic or plasmolytic methods fail to reveal the actual capacity
of the cells to absorb water.

The effect of environment on DPD is therefore more pronounced than
on OP. The important external factors are soil moisture, atmospheric
humidity, and light. Li (1929), using leaves of Syringa ohlata, concluded
from statistical studies that from 6: 30 A.M. to 10 A.M. light and relative
humidity exert an important effect, while from 10 A.M. to 2 P.M. light
becomes insignificant. Temperature was considered relatively unimportant
in its effect on DPD.

Although this statement is true for purely liquid systems where DPD
varies as absolute temperature in systems involving the atmosphere, tem-
perature, through its relation to the degree of saturation, may have a pro-
found effect upon transpiration and hence upon the DPD of the tissues.
For further discussion of this point see page 190 and Figure 52, Chapter
X. Table 28 indicates the effect on DPD of varying amounts of moist
soil.

Table 28. — Effect of varying soil moisture supply upon the DPD of plant tissues

(MoLZ, 1926): —

Variation in suction force of the leaves

Amount of water given

daily

of each Bcllis perennis plant after

to individual plants

for

receiving the given amounts

period of 2 weeks

of water daily for 2 weeks

150 g.

4-1.37 atm.

75 g.

-t-2.40 atm.

50 g.

-f3.81 atm.

5g.

-f 4.20 atm.

Wide diurnal fluctuations in DPD values of plant cells have been re-
ported as might be expected. The maximum is reached about noon or in
the early afternoon (Molz, 1926; Li, 1929; Herrick, 1933) under most
conditions.

Table 29. — Osmotic quantities in the hypocotyl of Helianthus amiuus seedlings,
determined at normal volume (data of Beck and Andrus, 1943) : —

Millimeters

from base of

cotyledons

Epid.

On

Cortex

DPDo

TPn

Zone

Epid.

Cortex

Epid.

Cortex

1

0-5

7.Z

11.35

5.3

11.15

2.0

0.23

2

5-10

6.64

11.14

5.2

10.93

1.44

0.21

3

10-15

6.49

10.72

5.1

10.60

1.39

0.12

4

15-20

6.14

9.52

4.9

9.40

1.14

0.12

5

20-25

6.05

9.37

4.0

8.95

2.05

0.42

6

25-30

6.08

8.47

2.7

8.20

2.28

. . .

7

30-35

6.08

8.02

3.4

7.60

2.58

0.42

Meyer and Wallace (1941) found DPD values of approximately 8
atm. in potato tuber tissue. This closely approached the OP of the ex-
pressed sap measured cryoscopically, indicating a low order of turgor.
Lyon (1942) obtained values of roughly the same magnitude but found
considerably higher OP values, using the minimum cell volume (simplified)
method.

Gradients of DPD are often of interest to the physiologist in studies
of translocation and other functions. Beck and Andrus (1943) measured

Chapter VII — 107 — Osmotic Quantities of Cells

values for Helianthus annuus seedlings. DPD's (suction tensions) were
determined, by use of Ursprung's simplified method, in seven zones of the
hypocotyl, as indicated in Table 29. It will be noted that the values de-
crease consistently from the region at the base of the cotyledons to a region
35 mm. below. This is true for both the epidermis and cortex. Values of
the other two osmotic quantities are also included in the table for com-
parison.

Turgor of Plant Cells : — Turgor plays several important roles in the
structure and function of the plant. Because no rigid skeleton is possessed
by herbaceous plants nor by the tender parts of woody species, the swollen
condition of the cells acting against the restraining influence of the cell
walls provides support. Growth of plants is related to turgor expansion
of their cells. Plant movements of various sorts may be due to changes in
turgor. Opening and closing of stomata are the result of turgor variation
of the guard cells. Seed dispersal may be facilitated in certain species by
"exploding" of fruits {e.g., the squirting cucumber). Quality of many
succulent vegetables such as celery, lettuce, cucumbers, and the like may be
related to their state of turgor so that some extremely important problems
confront the handlers of fresh vegetables, problems related to water loss
at varying humidity and temperature. Severe losses are sometimes caused
by splitting of certain fruits and vegetables, for example cherries, water-
melons, and tomatoes. Complete explanations for this are not known, but
excess water and high humidity are causative factors in that they result
in high turgor states. Anatomical factors are also involved (Kenney and
Porter, 1941). The keeping qualities of cut flowers depend on the mainte-
nance of turgor, and this presents serious problems to the commercial florist.

Finally, it has been observed that plants do not thrive and their cells do
not function properly when their turgor is low or lacking. Although this
may relate more directly to the low water content of such plants and the
effects of water deficit upon hydration of the protoplasm, imbibition of the
walls, and concentrations of solutes in the protoplasm and vacuoles, study
of water deficient plants emphasizes the intimate and complex relations
existing among these several factors. Therefore, though water deficiency
and the attendant wilting may injure plants in more important ways than
through loss of turgor, the state of turgor of the plant is a valuable index
of its water content and supply, and hence of its physiological well-being.

Intercellular pressure (A) may be regarded as an additional osmotic
quantity, related in the following manner :

OP — DPD -f TP ± A. (22)

It seems best, however, to consider this factor simply a component of turgor
pressure whenever cells are associated in a tissue.

Measurement of Turgor: — Few direct measurements of cell turgor
have been made. Values of 3 to 10 atm. were obtained by a manometric
procedure in the massive-celled M^e/Za (Arens, 1939). Overbeck (1930)
measured the turgor of the fruit of the squirting cucumber by means of a
manometer. He found a value of 2.4 atm., which was sufficient to throw
the seeds 12.7 meters. Most measurements of turgor are made by differ-
ence, using the simplified method, to determine OP and DPD values. In
the normal state, OP measured cryoscopically and DPD determined by the
simplified method enables one to calculate turgor pressure from the rela-

Grafts et al. — 108— Water in Plants

tion TP = OP — DPD ; and at water saturation, the cryoscopic value is
a measure of the degree of turgor. In both of these instances, the accuracy
of the measurement depends on the extent to which expressed sap is rep-
resentative of vacuolar contents.

Bennet-Clark and Bexon (1940) investigated turgor pressures of
leaves by determining the amount of added pressure necessary to express
juice from the cells. In this method a round piece of leaf is placed hori-
zontally between two solid brass cylinders. Weights are added to the upper
cylinder until liquid is first exuded by the cells into the intercellular spaces.
A horizontally placed microscope is employed to detect this point. The
pressure imposed was believed to be equal to the turgor pressure (and
hence equal also to OP) of those cells possessing the lowest osmotic pres-
sure. For this reason it was termed the "minimum hydrostatic pressure"
of the fully turgid leaf.

The method operates on the assumption that cell sap exudes only when
the externally imposed pressure equals the osmotic pressure of the vacuole.
However, this can be true only when the cells are initially in a turgorless
condition; otherwise the observed values would be low. It must also be
assumed that the elastic stretching of the wall is equal throughout the tissue.
The authors state that "... application of a pressure lower than the hydro-
static pressure of the contents of the fully turgid tissue expresses no juice."
It would seem that any amount of pressure, when applied to fully turgid
cells, would express some liquid, although admittedly more from cells hav-
ing low osmotic pressures. Therefore, the method of Bennet-Clark and
Bexon can hardly be considered critical for determining turgor pressure.
Furthermore, the technical difficulties involved would seem almost insur-
mountable.

The term plasmoptysis designates the bursting of cells due to the in-
ability of the cell wall to resist turgor pressure. The phenomenon has
been observed on immersing cells in water (pollen grains, ripe pulp cells of
watermelon fruit, certain marine algae). Some solvents such as alcohol
may cause bursting due to rapid entry into the cells. In other instances,
swelling of the protoplasmic and cell wall colloids may be the cause. This
has been demonstrated in barley root hairs by immersion in a weak acetic
acid solution (Strugger, 1935).

Intercellular Movement of Water: — It has been clearly pointed out
(Meyer, 1938) that the DPD gradient existing between two cells is the
cause of water movement from one to the other; the osmotic pressure
difference may not enter the problem. Under certain conditions it is pos-
sible for water to move from a cell with a relatively high to one with a
relatively low osmotic pressure due to the existence in the first cell of a
higher turgor than in the second. The diagram of Figure 35 illustrates
an example.

Ursprung and Blum (19165') pointed out that the concentration of
osmotically active solutes in a cell is not necessarily the determiner of water
movement, for if the cell is saturated with water it has no further ability
to absorb ; water, on the other hand, may pass through such cells hindered
only by the resistance to movement through the membranes involved. Ab-
sorption of water by roots undoubtedly involves movement across tissues
that vary in osmotic concentration, and finally, the water is delivered into
xylem vessels where the concentration of solutes, though higher than in the
soil, may be much lower than in the cortical cells across which the water has

Chapter VII

109 — Osmotic Quantities of Cells

moved. Priestley (1920) and Crafts and Broyer (1938) have attempted
to picture mechanisms to explain the uptake of vi^ater by roots (cf. Chapter
IX, pages 149 and 150).

"V

O P=/2fltmos.

WP«6

CPD'6

OP^IO/itmoi.

-A^

Direction of water movement

Fig. 35. — Diagrammatic illustration of water
movement along a gradient of DPD. (Meyer, 1938).

Summary: — The osmotic quantities of plant cells are osmotic pressure, diffusion
pressure deficit, and turgor pressure. The dynamics of water movement and retention
can be explained in terms of these quantities.

As with an osmometer also with cells the relation OP = DPD + TP is funda-
mental to an understanding of osmotic relations. Under certain conditions of water
stress TP may pass the zero level and pressure may become subatmospheric.

Under OP all forces leading to water absorption by the cell are included. When
tension exists, its value must be added to the OP. Increase in hydrophily of the proto-
plasm results in water uptake. The OP of the vacuolar sap reflects the integrated re-
sultant of the interaction of all forces causing water to enter the cell. Under TP are
included forces acting in the opposite direction.

In order that osmotic pressure of a solution may find expression in turgor a differ-
entially permeable membrane must separate the solution from a more dilute solution
or from pure solvent. In the cell the protoplasm serves as this membrane. Its selec-
tive capacity is believed due to two cytoplasmic membranes, the ectoplast, and the
tonoplast. These are pictured as a complex of lipoid and protein. Some physical
membranes act as sieves, others as selective solvents. The lipoid-sieve theory com-
bines the essential features of both to explain the permeability of protoplasm.

Active absorption is responsible for most solute uptake by the cells of living
plants. Essential to this process are oxygen supply, temperature, organic nutrients,
the salt status of the cell, and the nature and concentration of solutes in the external
medium.

Some ions may be taken up from the solid phase of the soil by contact exchange.
Polyuronides in the root hair walls make a connecting medium between the soil and
the cell protoplasm capable of ion transfer. The passive permeability of cells to ions
does not account for active absorption by living cells.

Cells are generally readily permeable to water. Cells may vary among themselves
with respect to water permeability.

Tugor is contingent upon osmotic water uptake; absorption and movement are
dependent upon osmosis. An external solution which reduces turgor to zero is said
to be isotonic with respect to the cell sap. Such a solution is used in determining the
osmotic pressure of the cell by the plasmolytic method.

Many objections have been raised to the plasmolytic method, nevertheless it has
proved valuable in studies of cell physiology. Some of the more pertinent criticisms
of the method involve adhesion of the protoplast to the wall, plastic stretching of the
wall, and the technical difficulties in correctly observing the critical state of incipient
plasmolysis. By plotting the number of plasmolyzed cells against sugar concentration
an average OP value for many cells may be obtained.

Several forms of plasmolysis have been observed, the more important being con-
vex and concave. Plasmolysis may follow chemical or physical stimulation. Vacuole
contraction is another type of response.

The plasmometric method involves strong plasmolysis followed by measurement
of cell and protoplast. Osmotic pressure is calculated from these.

The cryoscopic method is widely used. It is relatively free of errors but involves
the expression of sap and this has definite limitations. A differential thermometer is
used to determine freezing point lowering of sap; a thermocouple is usually used to

Grafts et al. — 1 10 — Water in Plants

find the freezing point of living cells. From the freezing point lowering the osmotic
pressure can be calculated by a simple formula.

Errors in cryoscopy involve supercooling and absorption of heat by the apparatus.
Methods are available to correct both of these; by a calibration technic they may be
avoided.

A vapor pressure method has been devised to determine the osmotic pressure of
small samples of sap. Another sensitive method involves the temperature low^ering
due to evaporation.

A simplified tissue method involves measuring tissue strips in series of solutions
of graded concentrations and calculating to the original volume. Another method
involves balancing root pressure against an external solution of known concentration.
This method has led to detection of a discrepancy postulated to result from active
water uptake by root cells.

Most plants have osmotic concentrations in the range of 5 to 30 atmospheres. An
extreme value of 202.5 atm. has been recorded. The distribution of osmotic pressure
values in the leaf assumes the following pattern: palisade parenchyma > spongy
parenchyma > upper epidermis > lower epidermis. Xylem sap is very dilute. Phloem
exudate has a higher concentration.

Diffusion pressure deficits of cells and tissues may be determined by surrounding
them with solutions which cause no change in volume or in weight. Another method
is to inject a solution into the hollov^f petiole or stem and follow changes in concentra-
tion. Variations in DPD resulting from diurnal fluctuation in transpiration may be
followed by this method. Several other methods have been described.

Changes in DPD values are greater than in OP values. They result from varia-
tions in soil moisture, humidity, and light. Cell walls of some xerophytes show little
elasticity, hence DPD values fluctuate sharply and water is conserved.

Turgor plays an important role in growth and development of plants. Quality of
vegetables depends on turgor. Growth hormones may act through turgor expansion.

Direct measurement of turgor is difficult. Values from 3 to 10 atm. have been
recorded. Intercellular water movement follows DPD gradients; it may be inde-
pendent of OP.

Chapter VIII
ACTIVE CELL WATER RELATIONS

Introduction : — Recent developments, most within the last decade,
necessitate re-examination of certain aspects of cell water relations. Spe-
cifically considerable doubt has been cast on the classical concept of the
mechanism of uptake and retention of water by plant cells. This classical
view has been fully discussed in the two preceding chapters. Simply stated,
it holds that water is retained in the vacuole osmotically by reason of the
presence of solutes in the sap ; that at limiting plasmolysis the osmotic
pressure of the sap is equal to that of the bathing solution ; and that, at full
turgor, the hydrostatic pressure developed in the vacuole is equal to the
osmotic pressure of the vacuolar sap. In simpler terms, for the range from
full turgor to incipient plasmolysis. DPD = OP — TP. The protoplasm
is considered to be passive in its behavior to water ; it constitutes a thin
layer, differentially permeable, retaining solutes within the vacuole but per-
mitting ready passage of water.

Most interpretations of cell water studies have rested upon these assump-
tions. However, sufificient evidence has now accumulated to cause serious
doubt concerning the passivity of protoplasm with respect to water move-
ment and retention. Plant physiologists are asking themselves i) do some
living cells retain in their vacuoles more water than can be accounted for
by their solute content; 2) what role does metabolism play in water ex-
changes; 3) exactly how does protoplasm function in the water balance of
cells ; 4) how do certain alleged vital water functions relate to other physio-
logical processes — namely absorption, movement, and loss of water? Con-
cisely, we would like to know if protoplasm can act differentially toward
both water and solutes. Secretion of water is particularly difficult to visual-
ize in view of the high content of water in the protoplasm itself.

Some of the early workers in plant physiology attempted to explain cer-
tain aspects of water absorption and translocation on the basis of a vital
control of water (Hales, 1738; Knight, 1801 ; Godlewski, 1884; Janse,
1913; BoSE, 1923), but the evidence has never been considered valid. In
the present chapter current research leading to evidence for active control
of water will be presented and discussed.

The water specifically referred to in the phrase "active water relations"
we consider to be that water which may be transported or retained against
an apparent diffusion pressure gradient, by processes requiring the expendi-
ture of metabolic energy. It includes water held in the protoplasm by forces
involved in living processes which may bring about a separation of water
and solutes. Practically, it is reflected in an apparent inequality between
the DPD and the quantity OP — TP. A distinction is thus attempted be-
tween active forces and passive (osmotic) forces, a distinction which is
difficult in the present state of knowledge, and which may of necessity be
somewhat arbitrary.

Evidence from Plasmolytic and Gryoscopic Measurements: — To

determine the nature of the forces accounting for turgor, promising results
have been obtained by comparing the osmotic pressure of the living cells

Grafts et al.

— 112

Water in Plants

measured plasmolytically (Pg) with that of the expressed cell sap measured
cryoscopically (On). From such experiments Bennet-Clark, Green-
wood, and Barker ( 1936) were led to the conclusion that the protoplasm
of certain cells, using metabolic energy, was able to "secrete" water into
their vacuoles. It follows from this conclusion that such cells could retain,
within their vacuoles, more w^ater than would be expected from purely
osmotic forces. Using root tissues of Beta vulgaris and Brassica napobras-
sica, and petioles of Begonia rex, Rheum rhaponticum and Caladium hicolor
they measured cryoscopically the osmotic pressure of the expressed sap.
After correcting these values for change in volume (Vn to Vg) they com-
pared them with values obtained plasmolytically. As shown in Table 30,
marked discrepancies occurred, particularly in Beta, Brassica, and Begonia.

Thus Og was greater than On

Vn

v^

by significant amounts. This dis-

crepancy Bennet-Clark, Greenwood, and Barker attributed to a force
termed "water secretion." In place of the classical equation DPD = OP —
TP they proposed DPD :=r OP — TP -f X, and X they described as "an
unknown additional pressure sending water into the cell."

Table 30. — Cryoscopic and plasmolytic measurement of osmotic pressure in various
plants (data of Bennet-Clark, Greenwood and Barker, 1936). Values are atmos-
pheres at 20° C, and in most instances represent averages of several tests: —

OP FROM

freezing point op from

depression plasmolysis x

Plant (On) (Og) (Og — On • Vn/Vg*)

Beta vulgaris A 15.5 23.4 7.1

Beta vulgaris B 9.5 12.6 2.6

Beta vulgaris C 12.0 18.4 5.8

Brassica napobrassiea A 11.4 17.8 5.9

Brassica napobrassiea B 11.85 17.0 4.6

Begonia rex A 5.3 8.0 2.4

Begonia rex B 5.5 8.0 2.2

Rheum rhaponticum A 7.5 8.1 0.2

Rheum rhaponticum B 8.6 9.5 0.5

Caladium hicolor A 5.8 5.5 — 0.5

Caladium bicolor B 5.8 6.4 0.4

* A constant factor of 1.05 was employed in calculations to correct for shrinkage from Vn to Vg.

The discrepancies for Caladium and Rheum range from — 0.5 atm. to
-f 0.5 atm., values that probably are not significant. For Begonia, Brassica
and Beta on the other hand the discrepancies are all positive and range from
2.2 to 7.1 atm., values that are highly significant and constitute from 25 to
30 per cent of the DPD at limiting plasmolysis. These results are of im-
portance in pointing out the magnitude of errors that may occur in the usual
interpretation of such experiments ; they are of greater importance in that
they challenge the accepted views of the mechanics of water absorption and
retention by cells and call for re-examination of the data upon which these
views were built. In a later paper (Bennet-Clark and Bexon, 1940)
the nature of the secretion process is treated in more detail, the proposition
being made that it does not constitute an exception to the physico-chemical
laws of diffusion but rather that water is transported into the vacuole by
the concurrent diffusion of another component which acts as a "carrier."
Discussion of the theoretical considerations of the secretion theory is pre-
sented later.

Chapter VIII — 113 — Active Relations

Studies dealing with growth processes in tomato have led Went (1944)
to admit the possibility of a non-osmotic force contributing to the DPD of
cells. Sap from killed leaves had an OP (determined cryoscopically ) of
slightly over 9 atm., while a sucrose solution of about 25 atm. was required
for plasmolysis of mesophyll cells.

The presence of hydrophilic colloids in the vacuole has been advanced
as an explanation of discrepancies between the plasmolytic and cryoscopic
methods for determining osmotic pressures. For ten species of conifers
whose expressed cell sap was low in mucilaginous colloids Roberts and
Styles (1939) found fairly good agreement between the two methods;
the average discrepancy amounted to 2.6 atm. in favor of the plasmolytic
determination. On the other hand, plasmolytic values w^ere around 14 atm.
higher, or over 40 per cent of the DPD for seven species possessing much
colloid. The colloid content was measured by the somewhat questionable
method of comparing outflow times for sap through a capillary tube. Other
substances, for example sugars, also have marked effects upon viscosity and
further analyses of the saps would have aided in interpreting the data.
Although this method possibly involves an error, the fact that cryoscopic
values of the two groups of conifers were of the same order of magnitude
is evidence that colloids are probably responsible for the viscosity differ-
ences. While recognizing water secretion as a possibility, Roberts and
Styles favor the presence of colloids in the sap as an explanation for the
differences obtained. They believed that the plasmolytic method embraces,
in addition to osmotic pressure of the cell sap, swelling pressures developed
by vacuolar colloids which the cryoscopic procedure fails to measure. The
difficulty of distinguishing between osmotic and imbibitional forces in com-
plex solutions was emphasized in Chapter III. Although Roberts and
Styles did not make it clear, it should be emphasized that in order for the
above explanation to be valid, water must have been removed from the
colloid during preparation of sap for the cryoscopic determination.

Using leaves of several tree species, Maximov and Lominadze ( 1916)
compared osmotic pressure values of pressed sap determined by the Barger-
Halket capillarimetric method with plasmolytic values. The latter values
averaged 1.7 atm. higher than those of the pressed sap and this discrepancy
accounts for a force representing about 8 per cent of the water absorbing
power (DPD) of the cells at limiting plasmolysis. Though one may ques-
tion the use of whole leaves for expression of sap while only epidermis is
used for plasmolytic measurements, the fact shown by Ursprung and Blum
(1916a), BuHMANN (1935), and others that the osmotic pressure of the
mesophyll exceeds that of the epidermis, indicates that the discrepancy
found was too low.

Comparisons of the plasmolytic and Barger methods in studies on
Nitella, where volume changes were considered, gave almost identical values
(WiLDERVANCK, 1932). Similar results were obtained by comparing
plasmolytic and cryoscopic methods (Currier, 1943). It is perhaps sig-
nificant that in this case sap may be obtained without pressing, so that it
is almost identical with vacuolar sap in the intact cells.

Oppenheimer (1932a) thought that good agreement between plas-
molytic and cryoscopic osmotic pressure values could be obtained if suitable
plant materials were employed, and when the various sources of error were
accounted for. He investigated twelve plants, employing mostly leaf tis-
sues. With a few exceptions his values agreed within 1.5 atm. In most
instances the plasmolytic value was the higher of the two.

Grafts et al.

114

Water in Plants

This discrepancy was attributed to various causes: 1) adhesion of the
protoplast to the wall ; 2) the slight difference in osmotic pressure between
a solution effecting limiting plasmolysis and that causing incipient plas-
molysis; 3) time of treatment, insufficient or excessive; 4) failure to ac-
count for the volume change between the normal and incipiently plasmolyzed
states; 5) excessive proportion of protoplasm in the cells; 6) inelastic
stretching of cell walls.

/atmospheres
IS 6 10

Fig. 36. — Osmotic pressure values of leaf cells of Bergenia cordifolia. Solid lines
represent initial plasmolysis, dashed lines d'eplasmolysis, and dotted lines cryoscopic
values on the expressed sap. (Buhmann, 1935).

The sampling error involved in making plasmolytic tests usually one
day after the cryoscopic tests, and using different leaves for each, may
have influenced these results. Epidermis was chosen for many of the
plasmolytic determinations; these were compared with cryoscopic values
obtained on sap expressed from many whole leaves. Oppenheimer's

Chapter VIII — 1 15 — Active Relations

discrepancies would probably have been greater had he used a mean plas-
molytic value for mesophyll and epidermis.

A somewhat different interpretation was offered by Buhmann (1935).
Investigating various types of tissues, discrepancies varying from a frac-
tion of an atmosphere to as much as 60 atmospheres were found. Plas-
molytic values were, on the average, in the order of 5 to 7 atmospheres
higher than the cryoscopic. The discrepancies were ascribed to adhesion
of the cytoplasm to the cell wall, producing a delay, and hence an over-
estimation of the OP at incipient plasmolysis. For a discussion of the ad-
hesion problem, see Chapter VII. Figure 36 shows a series of measure-
ments on four tissues of Bergenia cordifolia leaves showing values of POg,
DOg, and cryoscopic On. In Finns laricio where, in one instance, the dis-
crepancy attained a value of 60 atm., Buhmann attributed 13.5 to 27 atm.
to adhesion.

Using Beta vulgaris root, and neglecting change in volume, Buhmann
found discrepancies in the plasmolytic and cryoscopic values ranging from
5.25 to 7.78 atm., the plasmolytic values being higher. Adhesion pressure
amounted to 1.07 to 3.26 atm. An unusually high shrinkage (Vn/Vg =
1.22 to 1.36) was reported for this tissue which, when applied as a further
correction, produced good agreement between plasmolytic and cryoscopic
values.

In a study of the water relations of red beet root tissue one of the pres-
ent authors (Currier, 1944a) verified the occurrence of plasmolytic-
cryoscopic discrepancies (PCD's) in measurements of osmotic pressure.
However, there were unexpected variations among different lots of beets
tested. After volume corrections had been applied, the value at limiting
plasmolysis exceeded the cryoscopic value by as much as 5.9 atm. for two
of the lots tested ; for other beets the average discrepancy did not exceed
one atmsphere. In some instances the cryoscopic value was the higher one,
but here the difference rarely exceeded a fraction of an atmosphere
(Tables 31 and 32).

Table 31. — Description of beets employed in PCD determinations, and periods during

zvhich tests zvere made: —

Age Stage

Lot Variety in weeks of growth Time of tests

a

Detroit

35-36

Pre-bolting

Mar. 15-22

B

Detroit

59-63

Dormant

Apr. 23-May 21

C

Detroit

59-63

Bolting

Apr. 23-May 21

D

Crosby

36

Bolting

May 23-26

E

Detroit

17-18

Vegetative

July 7-15

F

Crosby

7-2S

Vegetative

May 21-Sept. 13

G

Detroit

20-22

Vegetative

July 13-29

H

Detroit

15-31

Vegetative

Sept. 15-Jan. 3

The excessive plasmolytic values could not be attributed to any of the
explanations previously offered. Using a technique similar to that of
Buhmann (1935), no significant adhesion pressure could be detected
(Table 33). The method involved the use of plasmolyzing and deplas-
molyzing solutions having osmotic pressures about 4 atm. greater and less
respectively than the predetermined limiting plasmolysis value, which in-
sured complete plasmolysis and deplasmolysis without stretching or shrink-
ing the wall to any great extent. Adhesion pressure values lay within 0.5
atm., and were no greater for beets exhibiting a -f-PCD (plasmolytic value
> cryoscopic value corrected for volume) than for those having a — PCD

Grafts et al. — 116 — Water in Plants

(cryoscopic > plasmolytic). Nor was there visible evidence that the cyto-
plasm adhered to the wall.

With one exception, the highest -|-PCD values were found in beets
yielding the most concentrated expressed sap (Lots D, E, G), the lowest
PCD's in beets with least concentrated saps (Lots B, C, F). Lot A, with
the highest average discrepancy, had a relatively dilute sap.

Table 32. — Summarised results of plasmolytic and cryoscopic tests on various lots of

beets: —

Number

Average

Average

OF EXPERI-
MENTS

OSMOTIC

PRESSURE

Change

IN VOLUME

Average
PCD"

deviation

Lot

Plasmolytic

Cryoscopic

Average

Range

PCD"

atm.

atm.

%

%

atm.

atm.

A

4

16.2

10.4

3.0

1.0-5.5

+ 5.3

0.7

B

9

10.2

9.1

5.2

3.6-11.3

-1-0.6

0.4

C

6

10.0

8.9

3.2

1.4-4.8

-1-0.8

0.7

D

8

22.8

17.2

7.3

3.0-10.6

+ 4A

0.9

E

4

1S.6

13.9

5.4

1.9-7.5

-1-0.9

1.1

F

21

12.6

12.0

2.7«

0-7.7

+ 0.3

0.8

G

12

17.9

16.1

2.2«

0-5.9

+ 1.4

1.0

H

4

14.5

13.9

4.3

3.S-S.0

0

1.1

" Cylinders of four roots of Lot F and of six roots of Lot G were soaked in isotonic sucrose
solution prior to sectioning, hence no volume change is involved. When these are not considered,
average changes in volume are somewhat higher, 3.3 and 4.4 per cent respectively.

^ PCD is introduced as a convenient contraction of the term plasmolytic-cryoscopic discrepancy.

The suggestion was made by Bennet-Clark, Greenwood and Barker
(1936) that water secretion may be a function only of potential growing
tissues. This does not appear to be true for beet root tissue. Lot F was
tested beginning when the plants were 3 weeks old, and continuing until
they were 25 weeks old. The youngest plant examined showed a PCD of
— 0.6 atm. Out of twenty determinations, nine showed negative PCD's
and an average discrepancy of near zero. During this growth period, both
Og and On increased at about an equal rate.

Table 33. — Results of deplasmolysis and replasmolysis tests on beet root tissue.

Atmospheres at 22.5° C: —

Beet No.

PCD

PO,

DOg

ROg

POg-DOg

POg-ROg

39

—0.1

13.0

12.5

12.8

0.5

0.2

40

+ 0.7

13.8

13.6

13.6

0.2

0.2

41

—0.1

12.5

12.0

12.2

0.5

0.3

42

—0.3

12.5

12.0

12.2

0.5

0.3

47

—1.0

13.0

12.5

12.5

0.5

0.5

54

—0.8

13.8

13.8

0

■ •

55

+ 0.2

17.2

16.9

17.4

0.3

—0.2

56

+ 3.6

19.4

19.3

19.2

0.1

0.2

SSa

+ 1.6

17.7

18.0

17.7

—0.3

0

61

+ 1.4

16.4

16.3

16.0

0.1

0.4

62

—0.8

14.1

13.6

13.6

0.5

0.5

Plants yielding the highest -j-PCD values (Lots A and D) were in a
prebolting or bolting stage of growth. These lots were fall planted, the
others spring planted. That temperature was not directly associated with
the discrepancy is shown by the fact that Lot A was subjected to winter
temperatures, while D grew under warm greenhouse conditions, yet both
yielded high PCD's.

On the basis of this and other experimental evidence presented else-
where in this chapter, it was postulated that the discrepancies (PCD's) are
related to contamination of expressed sap by liquid held by and expressed

Chapter VIII — 1 17 — Active Relations

from the protoplasm ; that the discrepancy may be positive or negative de-
pending on whether the contamination was less or more concentrated than
the vacuolar sap ; that the nature of the contaminating protoplasmic sap
depends on the state of hydration of the cytoplasm ; and that where the dis-
crepancy is positive (protoplasmic sap less concentrated than vacuolar) the
cells may be in a cold hardy condition. The possibility of dilution by wall
imbibed liquid is not precluded; however, morphological differences (cell
size, wall thickness) sufficient to account for significant variation in PCD's
were not observed.

While this monograph was in press a paper by Levitt (1947) appeared,
in which he throws doubt on the possibility of plant cells maintaining water
secretion pressures in excess of about one atmosphere. This conclusion is
based on calculations which indicate that there is insufficient energy re-
lease, judging from maximum observed respiratory rates, to make such
work thermodynamically possible.

In summary, it is evident that with few exceptions, osmotic pressures
of plant cells measured plasmolytically exceed those determined cryoscopic-
ally, even when volume changes are taken into consideration. Explanation
of the discrepancy based on active secretion of water by the protoplasm
into the vacuole, adhesion of the cytoplasm to the wall, the presence of
colloids in the vacuole, various errors in method, and a high variable im-
bibitional capacity of the protoplasm, have been suggested.

While admitting that both of these methods of measuring the OP of
plant tissues are beset with numerous errors, it is possible that where com-
parative values are secured from similar plants in different states of growth
many errors will cancel out. Additional data are needed.

Interpretation of Sap Expression Data : — The meaning of results of
osmotic pressure measurements, where plasmolytic and cryoscopic methods
are compared, is confused by the questionable origin of expressed sap. If
plasmolytic values could be compared with osmotic pressure determinations
on sap known to have originated in the vacuole, the discrepancies, if any,
would have greater significance.

Several methods have been devised in an attempt to obtain expressed
sap of known origin from plant tissue (cf. Chapter VI). Knowledge as to
the distribution of solutes and water between the protoplasm and vacuole
is greatly needed, for it would be of assistance in many kinds of physiologi-
cal investigations.

The problem has been attacked by refining the pressing technique. The
usual method has been one in which frozen and thawed plant organs, or
parts of organs, are wrapped in cheesecloth and pressed, with no particular
attention to the arrangement of the individual pieces. Mason and Phillis
(1939) found that if cotton leaves were carefully stacked, and pressure
applied slowly so as to prevent shearing forces, a very dilute sap could be
obtained. They postulated that such sap was vacuolar in origin, and that
it was expressed through "fissures" produced in the protoplasm. On this
basis, in one experiment a difference between plasmolytic and cryoscopic
osmotic pressure determinations of 20.4 atm. was found (OP of plas-
molyzing solution = 22.8 atm., OP of press sap == 2.4 atm.). This led
them to suspect that the water secretion force was "much greater than any-
thing contemplated by Bennet-Clark, Greenwood and Barker." At
the same time they agreed that no actual evidence has been produced to

Grafts etal. —118— Water In Plants

prove that such a process, dependent on metabolic energy, actually exists,
and suggested that high imbibitional forces might also be involved.

In other sap-expression experiments designed to shed more light on the
proposed water-secretory phenomenon, Bennet-Clark and Bexon (1940)
distinguished betvi^een pressure slowly applied, and pressure quickly ap-
plied. Leaves containing anthocyanin in their vacuoles were found to be
suitable material. Slow increments of pressure applied to carefully stacked
leaves produced a colorless sap approaching pure water in composition, up
to a certain point which was termed the "break." Here the yield of sap
per pressure increment increased, the osmotic pressure was higher, and
anthocyanin appeared in the juice. The fraction expressed prior to the
break was interpreted to consist primarily of vacuolar water, from which
many of the solutes had been filtered by the cellular membranes, and pos-
sibly some protoplasmic water. Sap appearing after the "break" was at-
tributed to breakdown of the protoplast.

Sudden increments of pressure, on the other hand, released a sap con-
taining anthocyanin. with a higher osmotic pressure than occurred with
slow increments. This was interpreted to be primarily vacuolar in origin.
Bennet-Clark and Bexon feel, with Mason and Phillis, that the vacuo-
lar contents are probably liberated through fissures formed in the proto-
plasm without serious injury thereto. Sap from the killed residue of leaves
was considerably more concentrated than the "vacuolar" sap.

Cryoscopic determinations on these various sap fractions gave some
interesting results, especially when compared to plasmolytic values. A
sample of these is shown in Table 34.

Table 34. — Comparison of osmotic pressure values determined plasmolytic ally and

those determined cryoscopically on various fractions of expressed leaf sap

{data of Bennet-Clark and Bexon, 1940) : —

Fagus Beta Parthcnocissiis Gossypium

sylvatica vulgaris quinquifolius barbadense

atm.

atm.

atm.

atm.

14

15-16

22

25

17-18

7

5-7

9-10

9

8-10

12-14

Mean plasmolytic value . . . 30-32

OP cytoplasmic sap 18-24

OP vacuolar sap 5-8

OP entire cell sap 15

It is evident from these results that even where entire cell sap (from
frozen leaves) is used for comparison, the cryoscopic value amounts to only
40-50% of the plasmolytic value. The discrepancies are even greater with
respect to "vacuolar" sap. If the inference drawn here is true, that the
water absorbing power of the cell markedly exceeds the osmotic pressure of
expressed sap, then the plasmolytic method and other procedures involving
equilibrium between living cells and bathing solutions are to be preferred
in measurements of diffusion pressure deficits but are not suitable for
osmotic pressure determinations.

Phillis and Mason ( 1941 ) postulate three kinds of sap which may be
expressed from cotton leaves :

a) vacuolar sap, obtained by rapid application of relatively high pressure to care-
fully stacked living leaves,

b) injury sap, the result of rather low pressures for long periods of time, origi-
nating partly in the vacuole and to some extent in the protoplasm; they agree with
Lepeschkin (1937) that pressure first results in vacuolar contraction, but after a time

Chapter VIII

— 119

Active Relations

water separates in the form of small vacuoles, and it is this liquid which is believed to
compose a portion of injury sap,

c) death sap, sap expressed from the killed residue after the vacuolar sap has been
expressed, and originating almost entirely in the protoplasm.

Phillis and Mason, while in accord with Bennet-Clark and Bexon
that vacuolar sap may be expressed by rapid increments of pressure, differ
with reference to the nature of "injury sap." On the basis of volume of
liquid obtained, and of the chlorine content of successive fractions, they
assume the absence of filtration effects, and prefer to consider the sap a
mixture of protoplasmic and vacuolar solutions.

It is not at all clear, if solutes are filtered out at low pressures, why
some filtration would not occur at more rapidly exerted high pressures.
Similarly, even if fissures are produced in the protoplasm of some cells, it
does not appear likely that all cells would be aft'ected in the same way. And
if the protoplasm yields liquid when low pressure is applied for a rela-
tively long period, it seems possible that some protoplasmic sap would also
be expressed by quick pressure.

More accurate methods for separating the liquid present in the various
cellular phases are needed. Until these are found, sap from frozen and
thawed tissue may be preferred over that from fresh tissue for use in com-
parisons with plasmolytic values. Since it generally contains more solutes
than that from living tissue, there can be Httle claim that vacuolar sap from
frozen cells is diluted by filtration. Furthermore, there is less variation
in concentration with different pressing techniques. The fact remains
however that it is whole cell sap from many cells — a mixture, that is worth-
less in interphasal distribution studies.

While fluid expressed from living tissue usually exhibits a significantlv
lower osmotic pressure than that from killed tissue, there are some plants
for which this is evidently not true. Examples are given by Walter
(1931&, p. 36), and his data are presented in Table 35. The plants rep-
resented are of a type which one might expect to behave in this manner.
Rich in colloids, they hold relatively large amounts of water by imbibitional
forces. The equality in osmotic pressure between saps from living and
dead tissue could in part be due to the liberation of colloidally bound
water on heating or freezing. It could also reflect the anatomy of succu-
lent plants, made up of large thin-walled cells rich in water. Such cells,

Table 35. — Effect of killing tissue on the osmotic pressure of expressed sap

(data of Walter, \93lb) : —

Treatment

Optmtia
phaeacantha

Opuntia
versicolor

Living

30 min. at 100° C. . . .
24 hrs. at —20° C. . . .

atm.

9.56
9.63
9.92

atm.

10.89
11.54
11.10

Aloe

Rhoeo
discolor

Taxus
baccata

Living

atm.

5 71

atm.

6.48
6.87

6.58

atm.

18.46
18.36
18.40

100° C. heat

Liquid nitrogen

5.33
5 06

Grafts et al. — 120— Water in Plants

upon pressing, might easily rupture and yield a sap more nearly like that
existing in the vacuoles. It is difficult to see, however, how this would be
true for plants such as Taxus. Finally, there could be, as Walter suggests,
sufficient filtration of solutes in expressing viscous sap from killed tissue to
explain the similarily of osmotic pressures obtained.

For purposes of comparison, some representative data to be found in
Dixon (1914, p. 177, 182) and in Thren (1934) are presented in Tables
36 and 37. It is clear that in most instances there is a considerable dis-
crepancy between the concentrations of sap from living and killed tissues.
Furthermore, the magnitude of the discrepancy varies markedly among the
different plants.

Table 36. — Comparison of saps expressed from living and killed tissue

(Dixon, 1914) : —

Freezing point depression °C.
Plant Organ Living Killed

Ilex aqiiifolium leaves 0.667 1.225*

Hedera helix leaves 0.728 1.031**

Iris germanica rhizome 0.450 0.829

Pyrns mains fruit 1.507 1.919

Citrus limonum fruit 1.033 0.588

Solamim tuberosum tuber 0.523 0.588

Vitis vinifera fruit 2.567 3.185

Chamaerops humilis leaf 0.365 1.529

Beta vulgaris root 1.473 1.761

* Heated.
** Desiccated, all others frozen.

According to Thren the more compactly the leaf tissues are arranged,
the greater the difference expected between the two values. That is, Biixus,
with compact leaves, showed the greatest deviation. But this would not
seem to satisfy all of the results.

In this connection, the work of Newton (1924) is recalled; he found
an inverse correlation between cold hardiness and volume of sap expressed
from living wheat leaves. The difference was attributed to a greater amount
of bound water and dry matter in the more hardy tissue. Unfortunately
no osmotic pressure values were determined.

Table 27. — Osmotic pressure of sap expressed from living vs. killed {heat) leaves

(data of Thren, 1934) : —

Living Killed

atm. atm.

Buxus sempervirens 3.1 24.2

Vinca minor 5.1 14.4

Linaria cymbalaria 5.4 11.2

Sedum reflexum 7.9 11.3

Sedum album 6.2 6.2

Also pertinent to the problem are data reported by Meyer (1928), the
result of sap expression studies on the leaves of Finns rigida, with special
reference to cold hardiness. The total water content of the leaves varied
but little throughout the year, so that the development of cold resistance
during the winter was not due to this factor. The proportion of sap ex-

Chapter VIII

121

Active Relations

pressible from living leaves was much less during the winter months than
during the summer. This suggested, as did Newton's data, that the pres-
sure method may be useful in estimating cold hardiness in plants. The
explanation that the proportion of coUoidally (gel) bound water was greater
in winter was substantiated by the increased difficulty experienced in freez-
ing the leaves. Meyer's results are shown in Figure 37.

loo

90

80

70 ■

60

<0

SO

bUtL

III

c

D

£

^0

30

20-

10

^f\ug, ^ept Oct. Nov. Dec. Jan. Feb. Han flpKMcfy June July

Fig. 37. — Seasonal variations in the dry matter content, inexpressible water, and
water expressed as sap after different treatment of leaves of Pinus rigida. A, volume
of sap expressed from unfrozen leaves; B, additional sap expressed from leaf samples
frozen in an ice-salt bath; C, additional sap expressed from samples frozen in solid
carbon dioxide; D, inexpressible water; E, dry matter content. Pressure, 5000 pounds
per square inch. Data of Meyer (1928).

Later (1932) Meyer attempted to demonstrate a correlation between
cold resistance in pine and the amount of bound water detectable by the
calorimetric method. By this method there was a slightly greater amount

Crafts et al. — 122 — Water in Plants

of bound water in the summer leaves, attributed to a somewhat greater
total water content. Nor could a clearly significant difference be shown by
pressure-dehydration tests on living (ground) leaves in the summer as
compared to the winter condition. Amounts of sap expressible were greater
from summer leaves, but a part of this may be accounted for by the greater
total hydration of those leaves. The disrupting effects of grinding should
not be overlooked. Difficulties met in interpretation of bound water data
obtained by such methods as the calorimetric are indicated in Chapter III
(see also Weismann, 1938). Levitt (1941) interprets such results as
obtained by the pressure method, employed also by Newton (loc. cit.) and
by Martin (1927), as a reflection of the degree of injury suffered by the
protoplastic membranes from the application of pressure. Yet the behavior
of living leaves in freezing and pressing studies must reflect properties
other than a variable resistance of membranes. If in the hardened condi-
tion the permeability of the plasma membranes is increased, as indicated by
studies in frost resistance, one should expect that expression of sap from
living tissue should proceed more easily than from unhardened leaves.
This is not true, as Newton and others have shown.

The difference in osmotic pressure between sap expressed from living
and dead (frozen and thawed) beet root was found to be 0.1 atm. for tissue
exhibiting a higher plasmolytic than cryoscopic value. For another beet
root where the cryoscopic value was the higher, the difference was 3.1 atm.
(Table 38). Sap in the first instance was believed to have been diluted by
protoplasmic water to a greater extent than was true for the latter.

Table 38. — Osmotic pressure of sap pressed from liz'ing as against dead (frosen)
tissue, comparing two types of beets. Values in atmospheres at 22.5° C.

(data of Currier, 1943) : —

Expressed

SAP

PCD

Fresh

Killed

Difference

5.8
0.7

8.4
13.2

8.5
16.3

0.1
3.1

Consideration of the data and discussion presented in this section brings
out the following necessary conclusions :

i) No method that is above criticism has been devised for obtaining pure vacuolar
sap.

2) The unknown quantities are filtration of solutes, especially by the semi-
permeable membranes of living cells ; the diluting effect of coUoidally held water,
especially as may occur on freezing or otherwise killing the tissue followed by pressing ;
and other changes which might decrease or enhance the osmotic pressure, e.g., enzymatic
reactions.

3) It would appear that plants vary markedly with respect to differences in con-
centration of saps expressed from living and dead tissue.

4) The potential errors involved appear to be serious enough to throw doubt on
much of the mass of osmotic pressure data obtained by the cryoscopic method.

Plasmolytic Behavior of Cells : — In conformity with the classical
view of plant water relations, a cell in the condition of limiting plasmolysis
immersed in a sap identical with that contained in its own vacuole would be
expected to maintain a condition of limiting plasmolysis. In the case of a
turgid cell such a result would not be expected for while the osmotic pres-
sures may be equivalent within and without the cell, the diffusion pressure
deficit is less within. Hence water would tend to move outward into the

Chapter VIII

123 —

Active Relations

external solution, increasing the osmotic pressure of the vacuolar sap. At
equilibrium there would be a balance with respect to DPD, but not to OP,
and the cell will exhibit some turgor.

Experimentally, the vacuolar concentration of contiguous cells may be
assumed equal to that in the cells under observation. Providing sap were
expressed from the tissue in the condition of limiting plasmolysis, and pro-
viding the section to be immersed in the sap is also in this condition, fifty
per cent of the cells should plasmolyze. It is necessary that the volume of
external juice be large enough to preclude error due to dilution by water
leaving the cells.

There are few reports in the literature of such a procedure. To show
that sap expressed from living tissue is not isotonic with respect to living
cells, Dixon (1914, p. 184) mounted sections of beet root and leaves of
Chamaerops humilis in whole sap expressed from frozen tissue. Absence
of plasmolysis under these conditions was taken to substantiate the claim
that freezing had no concentrating effect on the cell sap. Sap from the
living tissue gave a freezing point depression of 0.599° C, while that from
frozen tissue was 1.517°.

Bennet-Clark, Greenwood and Barker (1936) made use of this
procedure as an argument for water secretion. Cells of plants which dis-
played a marked discrepancy between plasmolytic and cryoscopic OP
values, failed to plasmolyze in sap expressed from frozen contiguous tis-
sue, while those in which the two measurements agreed well were approxi-
mately 50 per cent plasmolyzed.

That there may be different responses to this type of treatment in rela-
tion to the magnitude of the plasmolytic-cryoscopic discrepancy (PCD)
was suggested by tests on red beet root (Currier, 1944a). It was shown
that where the plasmolytic OP was the higher, fewer cells mounted in ex-
pressed sap became plasmolyzed than where the reverse was true (Table
39).

Table 39. — Plasmolysis of beet cells in their ozvn sap: —

Beets showing negative

Beets showing

POSITIVE

PCD

PCD

Beet

Per cent

Beet

Per cent

number

plasmolysis

number

plasmolysis

41

41

51

18

, ,

37

52

15

42

54

56

16

49

9

54

45

63

4

, ,

30

^ ,

9

68

100

64

6

Although the above investigators make no mention of any injurious
effect, this type of experiment may not be valid because of the alleged toxic
action of expressed sap on living cells (Prat, 1927; Ernest, 1935). Ex-
pressed beet root sap is reported to markedly increase the respiratory ac-
tivity of discs of tissue immersed in it (Bennet-Clark and Bexon, 1943).
Diluting, boiling and filtering, and sterilizing the juice failed to significantly
retard the effect. The active constituents present were found to be organic
acids, malic especially. Just how an increased respiration would affect the
plasmolytic response is not known. The malate effect resembles the ability

Grafts etal. —124— Water in Plants

of auxin to increase respiration (Commoner and Thimann, 1941), and
water intake may be tied up with respiratory activity (van Overbeek,
1944). Further evidence relative to the effect of certain solutes on water
balance in cells (Bennet-Clark and Bexon, 1946) is discussed later under
the subject of mechanisms.

One of the errors claimed to be inherent in any measurement of osmotic
pressure is the response described as "stimulative" plasmolysis (cf. Chap-
ter VII). Under certain conditions, and in certain tissues, cells plasmolyze
in apparently hypotonic solutions, and even in distilled water. Osterhout
(1913) observed such phenomena in root tips of eel grass (Zostera marina).
The effect was ascribed to an unbalanced ionic environment, which in-
creased the permeability of the protoplasm, permitting osmotically active
solutes and water to escape and resulting in the shrinking of the protoplast.
Weber (1929&) observed a similar behavior in fruit flesh cells of Poly-
gonatum officinale that appeared to be perfectly normal. Plasmolysis oc-
cured when intact cells were mounted in a drop of tap or distilled water
containing a small amount of macerated tissue. That the effect was not
limited to these cells was shown by placing an Elodea leaf in the drop of
water, whereupon plasmolysis also appeared. Weber thought that an ac-
tive substance might have been released by the addition of water or by
slight mechanical pressure applied to the pulp, further suggesting that cer-
tain specialized cells contained the active material. No single explanation,
however, could adequately account for the phenomenon.

Gross (1940) concluded that the plankton diatom Ditylum hrightwellii
does not behave as an osmotic system, due to an anomalous plasmolytic be-
havior which he observed. Rapid shrinking and rounding up of the proto-
plast was found to occur in hypotonic sea water, in a fresh water medium,
and even in distilled water. The response could be initiated by several
treatments and conditions: (a) pressure applied to the organisms by means
of a micro-needle, (&) change in the ionic environment — NaCl alone was
effective, (c) the pH of the medium — at pYi 8 the effect usually disappeared,
and (rf) light, prolonged absence of which caused plasmolysis. The process
was described as similar to resting spore formation in every respect. The
anomaly was such that less shrinking and plasmolysis occurred in molar su-
crose solutions than in more dilute solutions. The fact that rounding up
occurred upon transfer to dark conditions only after 16 to 24 hours, sug-
gested to Gross that the turgor mechanism involved osmotic work, and that
reserve substances present in the cells were exhausted after this length of
time.

Relatively pure water is generally believed to be withdrawn on plas-
molysis of cells. Gross states, however, that from this particular organism
whole vacuolar sap is lost. No change in protoplasmic volume could be de-
tected; evidently imbibitional forces are not responsible. Gross favors a
mechanism similar to the "water secretion" of Bennet-Clark, et al.
(1936).

Other examples of anomalous plasmolysis are known. Kuster (1929)
and Prat (1934) may be consulted for literature. It is at the present state
of our knowledge unprofitable to speculate as to the meaning of these so-
called anomalies. Nor are generalizations permissible. Just as each plant
has its peculiar physical and chemical architecture, so will it necessarily
have its own particular variation of function. Prat states that "the only
thing about which all the authors agree is the irregularity of the reaction."

Chapter VIII — 125 — Active Relations

He suggests a mechanism operative by a sudden increase in permeability,
whereby water, and solutes, are permitted to leak out. This would not ex-
plain plasmolysis in distilled water. An active contraction of the protoplast
seems to be a justified assumption in many instances.

The phenomenon to which the name "vacuolar contraction" has been
given (KiJSTER, 1926), although differing fundamentally from plasmolysis,
deserves discussion at this point. This is a marked swelling of the proto-
plasm with reduction in vacuolar size but with no essential change in cell
volume nor separation of cytoplasm from the wall. It is not a simple
osmotic process, since it can occur in a hypotonic environment as a result of
some sort of stimulation. While onion scale epidermis and Elodea leaves
have been preferred materials, the reaction may be observed in many types
of cells (KiJSTER, 1940). Arens and de Lauro (1946) report that "hy-
dropot" cells in the epidermis of Sagittaria leaves offer favorable material.
Contraction may be experimentally induced by immersing sections or whole
leaves in 1 : 1000 neutral red solutions {pH 7 to 10) for a short time. The
vacuoles shrink often to a small fraction of their original size.

Apparently the increase in protoplasmic volume occurs at the expense of
the vacuolar contents. Akerman (1917) was able to demonstrate an
osmotic pressure increase of 5 atmospheres in vacuoles of Drosera rotundi-
folia tentacles, due to vacuolar contraction. Arens and de Lauro {loc. cit.)
support the theory that water alone is absorbed by the cytoplasm, the tono-
plast restraining the passage of solutes. This is based only on the retention,
during the process, of neutral red or of anthocyanin in the vacuole. Hart-
MAiR (1937) found that in Elodea such a transfer of water from vacuole
to cytoplasm occurred, but that in onion epidermis there was a decrease in
plasmolytically determined osmotic pressure, hence the cause of contraction
could have been a sudden increase in permeability of the tonoplast.

The underlying mechanism could well be an increase in the imbibition
pressure of the protoplasm. Arens and de Lauro prefer to regard it as
the result of oxidation-reduction processes, since many of the conditions
leading to vacuolar contraction are associated with respiration — /^H, wound-
ing, age and activity of the cells, and the specific effect of neutral red.

The use of "cap" plasmolysis (page 88) as a device to study the effect
of ions on protoplasmic hydration has been made by Hofler (1939) and
his associates Kaiserlehner (1939) and Houska (1940). Swelling of
the cytoplasm of onion epidermis to 5-10 times the original volume may be
experimentally induced by the use of hypertonic solutions of K, Na, or Li
salts. Ca, Sr, and Ba salts do not act in this way. Accompanying effects
are an increased fluidity of the protoplasm and a cessation of streaming.
It is of interest that the reaction is entirely reversible. According to Hofler
the K salt might raise cytoplasmic hydration by becoming chemically bound
to some protoplasmic building block, resulting in an enhanced attraction
between this component and water. The cap plasmolysis method should
prove valuable in other studies of this kind.

There is a great deal of physiological interest in both stimulative plas-
molysis and vacuolar contraction, since they clearly demonstrate the con-
tractile nature of the protoplasm and its wide variation in capacity to im-
bibe water. In certain instances the action might be interpreted as a defense
mechanism where the cell is exposed to adverse conditions. The possibility
should not be excluded that such variation may be associated with normal
functioning of certain cells. The observations do suggest, however, that

Crafts et al.

— 126 —

Water in Plants

there are as yet too many unknown factors related to protoplasmic struc-
ture, permeability, and imbibition to allow a simple osmotic explanation of
all cell water relations.

Gryoscopy of Tissues and their Equilibrium Bathing Solutions: —

If cells at limiting plasmolysis possess in their vacuoles a sap equal in
osmotic pressure to the external solution, the same should hold true where
the external solution is hypertonic. Results of such tests utilizing beet root
tissue (Currier, 1944a) indicated the expressed sap (after freezing) to
be somewhat more dilute than an external "isotonic" solution for tissue
exhibiting a +PCD (Og > On • Vn/Vg). For —PCD tissue (Og < On ■
Vn/Vg), the expressed sap was slightly more concentrated. Where the
tissue cylinders were treated with weakly hyper- and hypotonic sucrose

expressed sap O P

ratio was

solutions, there was also evidence that the .,., . , ,■ r. x.

' equuibriu'.Ti solution O F

higher in the case of the — PCD beets. The data are shown in Table 40.
Each value represents an average of five determinations.

Table 40. — Osmotic pressure detei
bathing solutio,

'rninafions of tissue cylinders ana
ns. Atmospheres at 22.5° C: —

' oj tlicir e

'quihbrium

Livi

NG tissue

PCD negative

Iso- Hypo-
tonic TONIC

OR ZERO

Hyper-
tonic

PCD positive

Iso-
tonic

Hypo-
tonic

Hyper-
tonic

Limiting plasmolysis .

Expressed sap

External sol'n (initial)
External sd'H (final) .

12.7
13.1
12.7
12.8

12.1
8.3
8.8

15.5
16.9
16.8

17.5
16.4
17.5
17.5

14.8
13.6
13.7

19.4
22.4
21.5

Dead tissue

PCD

negative

PCD

positive

Expressed
External s(
Difference

sap

Dlution . . .

16.4

16.7

0.3

20.2

21.2

1.0

Eaton (1943) has suggested that the water secretion values of Bennet-
Clark are invalidated by the questionable assumption that plasmolytic
values measure the water retaining force of cells in the normal condition.
He believes that solute accumulation during the immersion of sections in
plasmolyzing solutions is a real possibility, and cryoscopy of tissues removed
from such solutions, rather than of tissues of normal volume, would have
led to different conclusions. The data in Table 40 do not bear this out
with respect to one type of tissue. It is doubtful that sucrose would be ab-
sorbed from near-isotonic solutions sufficiently to be troublesome.

When equilibrium values were determined for dead tissues, there was no
great difference between the two kinds of beets mentioned above. How-
ever, in seven out of a total of eight tests, the expressed sap was from 0.4
to 1.7 atm. more dilute than the external solution.

Some equilibrium studies on certain marine algae have been reported.
For some materials, the expressed liquid may be considered almost pure
vacuolar sap. Mosebach (1936) so views the sap obtained from the swim-
bladder of Sargassum Unifolium, where the expressed sap amounted to

Chapter VIII — 127 — Active Relations

25.2

S

25.1

6.7

25.1

6.6

25

4.6

25.2

3.8

about 96 per cent of the fresh weight. For other materials, he believed
that imbibed water in cell walls and protoplasm, released on pressing,
could produce deviations much greater than purely methodical errors in
technique. Hence, values obtained for several species (Table 41) are, if

Table 41. — Cryoscopy of sap expressed from several marine algae and of the sur-
rounding sea water. Atmospheres at 20° C. {data of Mosebach, 1936) : —

Expressed
Brown alga£ sap Sea water Difference

Cystosira harbata (shoots) 30.2

Sargassum linifolium ("leaves") 31.8

Sargassmn linifolium (swimbladders) . . . 31.7
Red algae

Rytiphlaea tinctora 29.6

Spyridia filamentosa 29

anything, too low. This table indicates that the osmotic pressure of these
plants varies from 3.8 to 6.7 atm. above that of sea water. This infers that
turgor pressures are also in this range of magnitude, but there is no proof
that this is so. Valonia macrophysa and V, utricularis show a difference
of only about an atmosphere between the surrounding sea water and sap,
which can be obtained in an almost pure state.

Osmotic regulation in these forms must be largely one of accumulation
of solutes, mostly salts. Osmotic adaptation by Nitella translucens to
changing external concentration has been demonstrated (Wildervanck,
1932) ; a much greater regulative capacity is exhibited by many land plants
(Gasser, \9A2—see Chapter VII).

These results were interpreted to mean that imbibitional phenomena
must be taken into account in measurements of osmotic pressure of ex-
pressed sap, although they do not preclude the possibility that water secre-
tion forces may also be present. The differences demonstrated may actu-
ally be small compared to those which might obtain in other plants.

Auxin and Water Uptake : — The storage parenchyma of the tuber of
Solanum tuberosum is a favorite experimental object for certain types of
water relation studies, since it provides a large amount of homogeneous
tissue. Stiles and Jorgensen (1917) observed that the swelling of potato
tissue in tap and distilled water did not conform to current ideas of an
osmotic system. A temperature coefficient (Qio) of about 3 was indicated.
This is not characteristic of purely physical systems, and has been inter-
preted by Commoner, Fogel, and Muller (1943) as pointing to an en-
zymatic process associated with water absorption.

Several investigations have been undertaken to demonstrate the effect
of auxin on water and salt uptake in potato. While the effect of auxin on
growth processes has been a fertile field of research for many years, its
role in the water relations of the plant cell has been recognized only re-
cently. Reinders (1938) found that potato tuber tissue showed an in-
creased water uptake under the influence of auxin. In preliminary experi-
ments, tissue discs 1 mm. thick and 17 mm. in diameter were permitted to
remain in "stagnant" tap water for 24 hours, then were transferred to
aerated distilled water, whereupon a considerable increase in wet weight
occurred in the amount of from 18.6 to 30.5 per cent of initial weight after
8 days. Slight increases were noted even when the transfer was to non-

Grafts et al.

128 —

Water in Plants

aerated distilled water and to oxygen-free distilled water. A definite tem-
perature effect was observed. As an example, the increase in fresh weight
of discs kept in aerated water for two days at 21° C. amounted to 10.3 per
cent, compared to 4.7 per cent for a temperature of 10-11° C. In another
experiment, carried out at 21° C. and 1-2° C, the respective amounts were
11.3 and 1.6 per cent for the same period of time. Addition of heteroauxin
produced a much greater increase in wet weight, and resulted in a greater
loss of dry weight. Aeration of the bathing solution induced cell divisions
and disappearance of starch from the surface region of the tissue disc. No
cell divisions were observed in unaerated cultures. The conclusion of the
author is that the auxin effect is on respiration itself, and that the addi-
tional energy released is somehow utilized in uptake of water.

•f-ZO

I

I °

■<: -/O

I -20

-30

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^

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■ — o* *■"*

1

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-^

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\

\.

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so

/£0

/60 200

Hoars

Fig 38.— The effect of 0.2 M sucrose (S), 0.2 M sucrose plus 10 mgm. of indole-
3-acetic acid per liter (SA), these concentrations of sucrose and auxin plus 10 3 M.
KCl (SAK), these concentrations of sucrose and auxin plus 10-4 M. K fumarate
(SAF) on changes in the wet weight of aerated potato slices. Data of Commoner,
FOGEL, and Muller (1943).

Steward, Stout and Preston (1940) were unable to concur that
Reinders' data proved water uptake to be an active process in potato tissue,
because respiration was measured only by loss of dry weight. In their
opinion the fact that auxin induces an influx of water into the tissue is no
proof that the mechanism is not one of simple osmosis.

Steward and his collaborators however report data which do support
the possibility of active water absorption by potato in the absence of added
auxin. Discs placed in aerated 0.005 N solutions of KBr and KNO3
showed increases in fresh weight surpassing those of discs in distilled
water. A specific effect of the ions involved was suggested as a possible
explanation. Whereas potassium increased water absorption, calcium acted
as an inhibitor. The effect of potassium ion was increased when accom-
panied by nitrate ion.

Chapter VIII _129— Active Relations

These authors could not escape the conclusion that water absorption
under the conditions of their experiments was in some manner linked to
vital processes. Among these processes, respiration and protein synthesis
appeared to proceed proportionately to water intake, even though the con-
centration of respirable reserves (sugars, amino acids) were depleted to a
greater extent than in other cultures where protein synthesis and water
absorption were less. The suggestion was offered "that actively metaboliz-
ing cells which can grow may absorb water in a manner which has but little
relation to any conventional osmotic or suction pressure theory but may
be more directly linked with metabolic processes (respiration and protein
synthesis) ; processes which are determined by oxygen and affected by the
nature of the salts present in the external solution."

In the view of Reinders (1942), increased hydration of the protoplasm
may possibly result from the formation of osmotically active solutes in the
"so-called free water" of the protoplasm.

The problem was further investigated by Commoner, Fogel and Mul-
LER (1943), who reported an "active absorption of water against an osmotic
gradient." A decrease in wet weight by tissue immersed in aerated 0.2 M
(hypertonic) sucrose solution could be prevented by the addition of auxin
(indole-3-acetic acid). Where KCl or fumarate were also present, the
tissue increased in wet weight. In the absence of auxin, water was lost.
The authors postulated an active process, one closely associated with solutes,
especially the four-carbon dicarboxylic acids, to be responsible. The sug-
gestion that auxin controls absorption or accumulation of salt, thus affect-
ing the intake of water, does not concur with the statement that water enters
actively against an osmotic gradient. Figure 38 graphically represents
some of the data of this investigation.

Van Overbeek (1944) criticized the work of Commoner, et al., for
two reasons. The lack of aseptic conditions during the tests prolonged for
a week or more might introduce complicating factors. And since Reinders
observed marked absorption in distilled water, the suggestion that the auxin
effect is on salt metabolism must be erroneous. The experiments were re-
peated under both aseptic and non-aseptic conditions, with the result that
the effect of auxin on water absorption of potato was confirmed. Greater
regularity, however, obtained under aseptic conditions (Figure 39). Van
Overbeek was unable to demonstrate an accelerating effect due to the addi-
tion of KCl or fumarate to distilled water, 0.2 M sucrose or 0.2 M mannitol
solutions containing auxin. Naphthalene acetic acid acted similarly to in-
doleacetic acid. By means of cryoscopic determinations, sap expressed from
the auxin treated discs was found to have a lower osmotic pressure than that
from the control discs, so that on this basis the effect of auxin is not one of
increasing the amount of osmotically active solutes. The decrease in OP
was in line with the increased volume of the tissue so that probably there
was no change in total solute.

We must conclude with van Overbeek that the effect of auxin must be
either (a) one of decreasing the wall pressure or (^) an effect of "active"
pressure. While the first is a definite possibility, there is also reason to be-
lieve that water absorption may be in some way associated with the increase
in respiratory activity produced by auxin.

Active forces leading to retention of water against an apparent osmotic
gradient would, on first consideration, be sought in rapidly growing tissue.
The potato tuber is a dormant structure, yet potentially it is capable of

Grafts et al.

130

Water in Plants

growth. In this respect it differs from mature tissues, for example that of
apple fruit parenchyma, where further growth is not normal.

60

40

^ ZO
0, 0

S/er//e

Co//l/res

I/7occ//o/'ec/

D/sr/7/ea^ ^a/-er

1

A.

20

//^O /VAA ^/iA

/ /Omy. per L.

0.2M Ma/7r7/fo/

fel

B.

I

H^O N/IA A//IA

/ /O/TJ^. per L.

40

£0

0.2 A/ Sc/crose

I

I

c.

/y^O //lA MA //^A

/ /O /OO/ny. per L.

Fig. 39. — Effect of auxiss on water uptake of potato tissue
under sterile and non-sterile conditions. The height of the bars
extending above the dotted line indicates the auxin-induced water
uptake of the sterile cultures. A. Experiment in water and 1 and
10 mg per liter naphthaleneacetic acid. Duration of test, 4 days.
B. Test carried out at same time and with same material as A;
0.2 M mannitol used as basic medium. C. Test similar to A and
B, but made at a different time; 0.2 M sucrose used as basic
medium, and 1, 10, and 100 mg per liter indoleacetic acid as auxin.
Data of VAN Overbeek (1944).

It is evident that at least in some instances the phenomena are not due
to solute accumulation. To say that auxin accelerates the respiratory rate
carries little meaning without knowing how the extra energy output is
related to water retention. If the auxin effect is one of increasing wall
plasticity only, it is clear that no swelling could take place in hypertonic
solution. The fact that expressed sap is slightly more dilute for the auxin-
treated discs (van Overbeek) is still not conclusive evidence that the
vacuolar OP did not increase, due to the possibility of active uptake of
water by the protoplasm. Data on this point would be helpful.

Chapter VIII _ 131 _ Active Relations

Measurements of Diffusion Pressure Deficits : — Another manifesta-
tion of an "active" force has been observed in potato tuber tissue by Lyon
(1942), who used the simplified method (cf. Chapter VII). Comparison
was made between the observed DPD value (OP of sucrose solution effect-
ing no change in the volume of the tissue), and the calculated DPD value,
derived as follows :

DPNn = On — TPn (i)

where On = Og • — ^ (2)

Vn

^ Vs (Vs — Vg) ^^

The resuhs indicated that calculated DPDn values exceeded observed values
by as much as 14 atm. for tubers in warm (70° F.) storage, or over half
again as much as the observed DPDn. The average discrepancy was about
3 atm. for tubers which had been in storage at 42° F. and about 6 atm. for
those at 70° F.

Certain trends are apparent in the data — the significantly higher dis-
crepancy at the warmer storage temperatures ; its absence at harvest time ;
apparent increase in turgor simultaneous with water loss. These observa-
tions are difficult to explain by means of the classical theory of osmotic pres-
sure. The author suggests that while the water secretion hypothesis is a
possibility, its proof is lacking, and that "the force could be one of pure
chemistry, such as an effect of colloids on the diffusion pressure of water
within the cells or a difference in electrical potentials, either of which could
vary during storage through physiological changes."

Lyon points out that the discrepancy could reasonably be due to: (a)
underestimating the calculated normal wall pressure or (b) overestimation
of the osmotic pressure at normal cell volume. As one limitation of the
simplified method, there is the possibility of excessive Og values, for rea-
sons stated on page 97 (Chapter VII). Such an error, if sufficiently great,
would account for the discrepancy. It may be a matter of plasticity of
cell walls, the degree varying under differing conditions. One might also
question the validity of assuming a direct proportionality between volume
and osmotic pressure at the three measurement states. The non-solvent
fraction in potato tuber cells would seem to be significant. Regardless of
these suggestions with respect to the method, there are indications here of
some kind of active regulation. The wide variation in the calculated DPD
value and the consistent trends are otherwise difficult to explain.

The water secretion theory of Bennet-Clark finds support in some
investigations of Brauner and Hasman (1946), who have proposed that
an electrokinetic component is partly responsible for the DPD of cells.
Using potato and carrot tissue in isotonic solutions of K2SO4, sucrose and
CaCl2, the DPD (gravimetric method) varied in a manner suggesting that
the charge on cell membranes may exert a control on the movement of
water. The differences were not attributable to permeability factors. Small
amounts of CaCl2 had a marked eft'ect in lowering the DPD. In potato,
about 10 per cent of the total DPD was ascribed to the electroosmotic fac-
tor. Somewhat higher values were subsequently (1947) demonstrated in
beetroot and in carrot.

Crafts et al.

132 —

Water in Plants

Kerr and Anderson (1944) studied the osmotic quantities of cotton
seeds in various stages of growth. In seeds less than 24 days old OP values
exceeded the DPD, a relationship to be expected. But in older seeds the
reverse was true to a marked degree (Table 42). Osmotic pressures were
determined cryoscopically, using sap expressed from frozen tissue ; DPD's
were calculated by use of the change-in-weight method (Qiapter VII).
The authors considered two possible explanations: (a) some sort of active
water absorption, and (b) imbibition. Use of respiratory inhibitors, as
KCN and ethyl ether, indicated that respiratory activity was probably not
directly related to the phenomenon. It was pointed out that the embryo
remains very small during the first 24 days of its growth. The excessive
DPD values appeared after this period, at a time when the embryo was
rapidly developing and coincident with the breakdown of endosperm. The
high colloid content of the embryo cells suggested the possibiHty that water
liberated on freezing and pressing produced OP values which were too
low. Imbibitional forces thus were believed to account for the difference
between the two measurements, which is "more apparent than real."

Table 42. — Osmotic pressures of cell sap from cotton seeds of three ages before and
after immersion in sucrose solutions {data of Kerr and Anderson, 1944) : —

OP OF SAP

EXPRESSED FROM

OP OF SAP

CONC.

OP

SEEDS AFTER

Age OF

EXPRESSED

DPD

OF SUCROSE

OF SUCROSE

IMMERSION

SEEDS

FROM SEEDS

OF SEEDS

SOLUTIONS

SOLUTIONS

IN SUCROSE

days

atm.

atm.

M

atm.

atm.

18

9.63

7.0

0.25

6.7

9.63

23

8.07

10.2

0.40

11.1

8.35

30

7.77

13.7

0.50

14.3

9.12

This work emphasizes the importance of colloidal hydration in the water
relations of some plant tissues, a force which may be generally under-
estimated.

Evidence from Studies of Frost and Drought Resistance: — The

freezing point of living tissue has usually been found to be lower than that
of dead tissue or its expressed sap. Muller-Thurgau (1886) found
— 0.98 and — 0.55° C. for living and dead potato tuber tissue respectively,
and — 0.8 compared to — 0.4 for Phaseolus leaves. Maximov (1914) re-
ported —2.15° and —1.25° respectively for red beetroot, and —1.21° for
the extracted sap. Similar data are reported by Lewis and Tuttle ( 1920) ,
Carrick (1930), Curtis and Scofield (1933), Jaccard and Frey-
Wyssling (1934), Luyet and Gehenio (1937).

An adequate explanation of this behavior has not been ofifered, but there
are several hypotheses which variously attribute the difference to 1 ) capil-
lary attraction of water in the intercellular spaces, 2) the resistance offered
by protoplasmic membranes to the movement of water out of the cells, 3)
non-solvent (bound) water in living cells liberated upon death, and 4)
errors in method. The reader is referred, for an adequate discussion of
this subject, to reviews by Walter and Weismann (1935), Luyet and
Gehenio (1939), and Levitt (1941).

The display of a double freezing point by living tissue, confirmed by
several workers, is a complicating feature in the interpretation of results.

Chapter VIII — 133 — Active Relations

After study and review of existing data, Luyet and Gehenio (1937) favor
the theory that the first freezing point represents the congelation of inter-
cellular liquid ; the second is due to freezing of the intracellular water.
Also confusing the attempt to interpret freezing behavior of living tissue
is the fact that variation of experimental conditions will produce different
results. Luyet and Galos (1940) studied the effect of rate of cooling on
the freezing point of living and dead potato tuber tissue, concluding that
the difference between the two values became less with decrease in cooling
rate, and suggesting that at a velocity of about 0.1° C. per minute, the two
should be approximately equal. Dead tissue did not behave in this manner.
This confirmed earlier results of Walter and Weismann (1935), who
state that while the freezing point of living tissue invariably is lower than of
dead tissue, no conclusions should be drawn from this as to the osmotic
concentration of the cell sap in living and dead tissue. They attribute the
discrepancy to the impossibility of stirring in such determinations, and to
the varying speed at which ice is formed. The latter in turn depends on
the permeability of the protoplasm to water, and on the DPD of the cells,
since freezing first occurs in the intercellular spaces. They present data
to show that under the same freezing conditions the same amount of ice
formed in living as in dead tissues, and that the osmotic pressure of the
cell sap is the same in both. This conclusion was necessitated by the ob-
servation that when cooling curves indicated an equilibrium between the
tissue and cooling bath, the areas under the two curves were the same.
Walter and Weismann's data are criticized by Levitt (1941) as not
conclusive, since it was not determined whether the tissue was alive at the
end of the experiment, a doubtful assumption in his opinion.

Contrary to results obtained with tissues as experimental material,
Gehenio (1941) reported a higher freezing point for living myxomycete
Plasmodium than for dead. No adequate explanation seemed possible. One
suggestion was that freezing caused the disintegration of protoplasm with
the reduction in the size of particles and an increase in their number, thus
augmenting the effective concentration. The phenomenon might also be
explained by the preferential adsorption of water by the dead protoplasmic
colloids.

There is better agreement between the freezing points of dead tissue and
expressed sap, although the former yields lower values. This has been
variously interpreted ; according to some (cf. Jaccard and Frey-Wyssling,
1934), it is due to the liberation of colloidally bound water, or to some other
type of disintegration or disturbance such as ruptured xylem vessels. With
this "dilution theory" Walter and Weismann disagree, pointing out that
cryoscopy of dead tissue, as of living tissue, is less accurate than of ex-
pressed sap because of the impossibility of stirring, etc. ; but when special
precaution is taken the two values agree well. Their view, then, is that the
concentration of cell sap remains constant throughout the living, dead, and
expressed sap states.

In this connection tests carried out on beet root tissue may be of interest
(Currier, 1944a). A mercury thermometer with a small (5 mm. diameter)
bulb, and calibrated in fiftieths of a degree, was used. A small hole in a
tissue cylinder accommodated the bulb. Freezing was initiated by inserting
a small glass capillary filled with ice into the tissue. After the freezing
point of the living tissue had been determined, the cylinder was frozen, and
the freezing point of the dead tissue was ascertained in a similar manner.

Grafts et al. — 134— Water in Plants

Subsequently the sap was expressed and a third value obtained. From
Table 43 it is clear that the living tissue froze in every instance at a lower
temperature than did the dead tissue. The beet roots employed had pre-
viously been investigated with respect to plasmolytic and cryoscopic deter-
mination of OP. There was found to be considerable resistance to freez-
ing in the case of -j-PCD beets, with particular difficulty in inoculation.
The reverse was true of — PCD plants, where in some instances freezing
was spontaneous, i.e., required no inoculation. In line with other evidence,
the differences were believed due to dilution and concentration effects as
a result of freezing.

Table 43. — Freesing points of living fisstie, dead tissue, and expressed sap.

Atmospheres at 22.5° C: —

PCD Living Dead Sap

Positive 26.4 22.8^ 18.0

Positive 23.6 15.3 13.3

Zero 21.2 14.6 14.3

Negative 12.9 11.7 12.9

Negative 18.6 11.5 13.0

• Freezing not complete.

Recent reviews (Levitt, 1941 ; Scartii, 1944) on the subject of cold
hardiness of plants have emphasized the fact that resistance to freezing tem-
peratures by certain plants is due primarily to protoplasmic factors. While
there has been shown to be some correlation with other characteristics ac-
companying the hardened condition, e.g., small cells, lower moisture content,
higher sap concentration, they are considered to be of only secondary im-
portance. Considerable significance is attached to the physical state of the
protoplasm in hardened as compared to unhardened cells. Associated with
the former are (a) an increased permeability and (5) decreased consistency,
both of which are attributed to (c) a greater hydrophily of the protoplasm.
By means of this theory many of the observations on cold hardiness may be
explained.

The experimental basis for this hypothesis is derived for the most part
from application of micrurgical and plasmolytic methods applicable to liv-
ing cells. Certain advantages are thus gained over the considerable number
of chemical and physical procedures that have been employed with dead
tissues and extracts. The following additional characteristics were found
to be associated with the hardened condition of living plant cells : increased
thickness of the cytoplasmic layer, high resistance to deplasmolysis injury,
ability of the protoplast to round up on plasmolysis, greater density of
protoplasmic strands, less rigidity of ectoplasm on dehydration, lower re-
fractive index of ectoplasm, less clumping of plastids and granules on plas-
molysis, greater non-solvent space (in some), less tendency of colloids to
coagulate.

The literature of frost resistance is so extensive that only one phase of
the problem will be considered here — the matter of hydrophily of the proto-
plasm. Frost resistance implies ability to withstand freezing temperatures.
It means that the tissues are able to prevent formation of ice, or have an
ability to withstand it. The term hydrophily, used in connection with proto-
plasm, refers to the total water content or hydration ; it carries no implica-
tion as to the nature of the forces holding this water. The relationship

Chapter VIII — 135 — Active Relations

between the degree of hydrophily developed by hardening cells and the
amount of "bound" water in the protoplasm is in question. Levitt con-
cludes from study of the literature that the reduced rate of water loss
sometimes observed in hardy plants can usually be explained on the basis
of some morphological character, rather than on bound water. It is well to
emphasize again (Qiapter VI) that knowledge concerning the water hold-
ing mechanisms in protoplasm is limited. While the evidence for water
binding in dead systems such as expressed saps may be questioned, in proto-
plasm we are dealing with a system possessing active forces, a system that
is able to perform osmotic work with regard to solutes, and probably also to
water.

Several observations indicate that protoplasmic volume increases dur-
ing hardening. Levitt and Scarth (1936) found this to be true for
cortical cells of Catalpa, where the protoplasm occupied about 50 per cent
of cell volume. In this amount it cannot help but exert a marked influence
on water control. The work of Kessler and Ruhland (1938) indicated
a greater volume of protoplasm in hardened tissues. The fact that smaller
cells, with greater relative volume of protoplasm, are the more hardy, is of
significance. On the other hand, Scarth (1941), on the basis of an ob-
served lower refractive index and a greater permeability of the ectoplasm,
has suggested that the increased hydration on hardening may be limited
to this outer protoplasmic layer.

Levitt (1941) finds it difficult to reconcile the decreased water con-
tent of hardy plants with increased hydration of the protoplasm ; he sug-
gests that the walls may dry. A better explanation might picture the
protoplasm as varying its hydrophily allowing reduction of water in
vacuole and walls at the same time that its own water content increases.
Such a process might involve unfolding of protein chains or opening of
ring structures to present more points for hydration, the more active struc-
ture being maintained by metabolic energy.

Another explanation for increased hydrophily is possible. It has been
reported that the respiration of hardened cells is of lower intensity than
in non-hardened cells {see review by Levitt, 1941, p. 124). This might
suggest that respiratory energy is utilized in masking hydration points,
and that a low rate would permit greater hydration. There seems to be
no agreement however on the relation between respiratory rate and degree
of hardiness. Regardless of the type of bonding involved the retention of
water, as a part of protoplasmic structure, must be influenced by metabolic
forces. The possibility must not be lost sight of that water may be held
in living protoplasm by forces so intimately related to the living state
that they cannot be studied in dead or injured cells, or in extracts.

In many respects drought resistance is similar to frost resistance. It
has seemed logical to many investigators that a considerable amount of the
water of true xerophytes must be bound to colloids. In spite of the nu-
merous criticisms {e.g., Weismann, 1938), methods for estimating bound
water are still being employed in studies of drought resistance (Whitman,
1941, MiGAHiD, 1945). The latter author has aptly stressed the point
that where sap is expressed, bound water can be determined only when
the press cake residue is also taken into account. He concludes that the
bound water content of xerophytes is significantly higher than that of
mesophytes. In several instances values in the order of 17 per cent of
total leaf water was reported as bound.

Crafts et al. —136— Water in Plants

The preceding discussion can lead to no definite conclusions as to the
existence of active control of water in frost or drought resistance. The
explanations listed above are highly speculative ; they must await new
methods for substantiation ; only as the molecular structure of protoplasm
in the living state is investigated and described may we hope to build a
clear picture of the forces maintaining its structure and accounting for its
remarkable properties.

Evidence from Animal Physiology: — Animal cells dififer from plant
cells in two distinct ways: they possess neither a semi-rigid wall, nor a
large central vacuole. In many ways, however, they are functionally
similar.

LucKE and McCutcheon (1932) consider plant and animal cells to
behave as osmotic systems if experimental conditions are carefully con-
trolled so that injury does not result in loss of semi-permeability, and if
correction is made for osmotically inactive contents. For example, they
found normal uninjured erythrocytes to swell and shrink in hypo- and
hypertonic solutions to a lesser degree than would be expected of an ideal
osmometer, but when a correction for osmotically inactive material was
made approximate agreement obtained. Brooks and Brooks (1941) make
the same general conclusion — that only osmotic forces are acting, but point
out that some anomalous results are still unexplained.

Ponder (1944) discusses three possible explanations: a) the anomalies
are due to varying amounts of bound water, b) the cells lose or gain solutes
during the experiments, c) elastic structural forces residing in the proto-
plasm oppose uptake of water from hypotonic solutions as well as loss of
water to hypertonic solutions. The last explanation was considered the
most plausible.

Correction for non-solvent volume in marine invertebrate eggs did not
produce the expected agreement in the pressure- volume relations of the
cells (Leitch, 1934). For some cells the calculated amount of osmotically
inactive material, based on the total protein and fat content, appeared to be
too low ; for others it was too high. He suggested that several factors might
affect water exchanges ; where volume changes were below expectation
surface forces might prevent penetration of water. Brooks and Brooks
(1941, p. 75) conclude that "most eggs do not behave as perfect osmometers
even after allowance is made for the known amount of water and solids in
them."

Differential behavior of animal cells and tissues toward water has been
reported in many instances. Krogh (1939), in a monograph on osmoregu-
latory processes in aquatic animals, points out that most animal cells are
in osmotic equilibrium with their bathing solutions ; when the concentra-
tion is modified swelling or shrinking must occur. Some cells, however,
are able to maintain hypo- or hypertonicity with their liquid environment.
Since rigid walls are absent, neither turgor nor tension is great enough to
be important in the attainment of a steady state with respect to water.
Such a state can exist only because osmotic regulatory devices requiring
energy are able to eliminate water or solutes as the case may be; these
processes involve the utilization of metabolic energy.

Though the over-all evidence for active water regulation by the lower
aquatic animals is inconclusive, several cases may be mentioned that are
strongly suggestive. Noctiluca miliaris, a marine protozoan has a lower
specific gravity than sea water, differing from most other members of the

Chapter VIII _137_ Active Relations

group. When mechanically or electrically stimulated its density increases,
and this is accompanied by an excretion of acid. Thus there seemed to be
some relation between the distribution of acid in the cell (around pH 3)
and its ability to float or sink (Gross, 1934). Harvey (1917) found that
cyanide treatment or absence of oxygen did not alter the density of the
organisms, indicating that respiratory energy is not a factor.

The contractile vacuole of many fresh water protozoa is believed to
function as an osmoregulatory device (review by Kitching, 1938). Ap-
parently it is able to control body volume by excreting water (or a very
dilute solution) as fast as it enters by osmosis through the surface mem-
brane much as water may be pumped from a leaky vessel. That cyanide
slows the vacuolar rhythm and results in swelling may be taken to mean
that osmotic work is involved. Transfer to hypertonic solution causes
slowing or cessation of vacuolar activity.

Beadle (1934) studied the water relations of Giinda ulvae, a small
worm living in estuaries and adapted to existence both in fresh and sea
water. He concluded that on change from sea water to fresh, water enters
through the skin and the animal swells. The additional water is absorbed
by cells of the gut and secreted into intercellular vacuoles by expenditure
of energy. Following this original restoration of balance, the ectoderm is
believed to inhibit further entry of water because of a reduced permeability.
The latter is visualized as an "osmotic resistance" of the ectoderm cells
similarly dependent on energy. The secretion of water into vacuoles seems
to resemble that of plant cells postulated by Bennet-Clark, et al. (1936).

Krogh states that in higher aquatic animals, most of the known osmo-
regulatory functions are confined to a more or less advanced type of kidney.
In some organisms extra-renal excretion apparently occurs. One form of
kidney characteristic of fresh water organisms produces a urine markedly
hypotonic to the blood, sometimes nearly as dilute as the surrounding water ;
another type, as in mammals, is able to produce a concentrated urine hyper-
tonic to the blood.

Other organisms adapted to changing environment are certain teleost
fishes. Study of the eel Anguilla vulgaris (Keys, 1932) revealed mechan-
isms by which the animal is able to maintain a fairly constant internal con-
centration when changed from fresh to sea water, and vice versa. Fresh
water is hypotonic, sea water hypertonic. Keys states that in the latter,
water is swallowed and the excess sodium chloride is subsequently excreted
by the gills. In fresh water the kidney is capable of filtration, and salt
conservation.

According to Adolph (1930), a skinless frog may act more or less as
a perfect osmometer. To the skin is assigned a role of active water trans-
port, water passing inward more readily than outward.

In man the kidney is believed to perform a highly specialized type of
osmotic work. According to common understanding the process involves
filtration by the glomeruli and subsequent absorption of water by the
tubules. The latter operate against an osmotic gradient. Other examples
of active water transfer may be recognized in the function of the salivary
glands and possibly the intestine.

The part played by hydrophilic colloids in the water balance of man
has been stressed by some investigators (Fischer, 1921 ; Gortner, 1938).
For example, in many instances transfer of water from one part of the
body to another is explained by imbibitional phenomena. Since the ability

Crafts et al. — 138— Water in Plants

of colloids to hold water varies greatly with changing environment such as
salt concentration, etc., it is proposed that water may be taken up at one
point and released at another. In a strict sense this cannot be considered
as active water transport.

PiCKEN (1936) stated that the mviscles of higher invertebrates appear
to be "considerably less hydrated than would be the case if they were in
simple equilibrium with the surrounding protein solution" and visualized
some active regulation of hydration. This suggests that energy is expended
in decreasing the imbibition pressure of the protoplasm. In a recent per-
sonal communication this author suggests that "regulation" may probably
be achieved in the first place by the synthesis of structurally different pro-
teins which differ in their affinity for water.

While ultimate explanations for the phenomena listed above are not at
hand, it seems that active processes involving water are numerous in the
animal organism. For a thorough discussion of active transport of solutes
and water in animals, see Hober (1945).

Relation to Solute Accumulation: — Though solute accumulation
cannot receive detailed treatment in this volume, it seems appropriate to
attempt a reconciliation of the processes of water and solute absorption
since both may apparently be active processes. Since solute accumulation
implies absorption against a concentration gradient, the dynamic "steady
state" maintained by the plant is usually characterized by a higher solute
concentration within the cell than without. The analogy between this and
water secretion should be clear.

The known facts concerning solute accumulation by plant cells may be
briefly stated :

1) Plants under the appropriate conditions retain within their cells higher con-
centrations of certain ions than exist externally (Osterhout, 1922; Hoagland and
Davis, 1923a). Furthermore, the total ion concentration in the cell sap may exceed
the external concentration by as much as 25 times (Hoagland and Davis, 1929).

2) The accumulation process has been demonstrated to depend on metabolic energy
(Steward, 1932; Hoagland and Broyer, 1936). This conclusion has resulted from
tests utilizing cyanide, oxygen deficiency, temperature variation, and limited respirable
food reserves. Light was found by Hoagland and Davis (19236) to be an important
factor, probably through its relation to food synthesis.

3) Solute accumulation takes place in both aquatic and terrestrial plants. Many
investigators have used massive-celled algae such as Nitclla, Valonia, Chara, and
Halocystis since vacuolar sap in a fairly pure state may be obtained in sufficient quanti-
ties for analysis. For a review of such studies, see Osterhout (1936, 1947o). Barley
roots have been used with success, sap expressed from frozen and thawed roots being
employed for analysis (Hoagland and Broyer, 1936). Potato tuber tissue has been
used in studies on salt uptake by Steward and his associates.

4) The preponderance of the ions determined in such studies are believed to exist
within the vacuoles of the cells almost entirely in the free state (Hoagland and Davis,
1923a; Osterhout, 1936).

5) The process appears to be limited to growing tissues or to those potentially able
to grow.

6) While most investigations have dealt with electrolyte absorption, other solutes
may be accumulated.

7) The underlying mechanism is not yet clear. Steward and Harrison (1939)
distinguish between "primary" and "induced" absorption. The first refers to an active
process dominant in accumulation phenomena of living cells. The second implies ab-
sorption due to the physical and chemical properties of the tissue with no dependence
on metabolic energy. Absorption of ions by dead tissues comes in this category. The
forces involved in induced absorption are those of dififusion, complex formation, col-
loidal adsorption, ionic exchange, and Donnan membrane potentials.

Chapter VIII _139— Active Relations

The mechanism underlying primary ion absorption is very complex and
involves the little understood properties of living protoplasm. While the
protoplasm must be the locus of the energy release, apparently a higher
level of accumulation exists in the vacuole (Hoagland and Broyer, 1942).
Though in the initial stages of accumulation by low salt plants, concentra-
tion in the protoplasm may greatly exceed that of the vacuole (Brooks,
1938) in the steady or equilibrium state the vacuolar concentration is
greater. Though this implies accumulation in the cytoplasm, followed
possibly by passive release into the vacuole, the end result is equivalent to
secretion. Intimately associated with the active process are problems of
permeability, interionic relations, m.etabolic level, active water control, etc.

Steward, et al. (series of papers to 1943) concluded that ion accumu-
lation is related to protein metabolism. The capacity to synthesize protein
is considered essential to cells that accumulate and retain solutes in their
cell sap.

Accumulation or active solute uptake by plants is an established fact.
Until recently workers have directed little attention to the water relations
of this process. From the similarity of ion uptake and water secretion one
is prompted to ask, if ions are actively absorbed, cannot water also be ac-
cumulated by living cells ? Hoagland has stressed the independence of ion
and water uptake. Are these processes as independent as might appear?
Or may they not be simply different aspects of a single process, the differ-
ences in rates being reflections of differences in availability or of transport
away from absorbing organs ?

Steward, Stout, and Preston (1940), as previously noted, observed
an anomalous water uptake in potato discs. The presence of potassium
accelerated absorption whereas calcium had a depressing effect. Nitrate
ions accentuated the acceleration due to potassium. The suggestion is made
that in addition to solute accumulation, water absorption may also be re-
lated to respiration and protein synthesis. The results, they state "suggest
that aerobic respiration, protein synthesis, water absorption, and salt ac-
cumulation are all mutually dependent processes which occur in all cells
which are not subject to equilibrium conditions but the behavior of which,
at constant temperature, is regulated by oxygen tension and the nature and
concentration of the salt solution in which they are immersed."

The relation between water absorption (by roots) and active solute
absorption becomes even more intimate in the theories of Lundegardh
(1946). Anions are pictured as actively absorbed by the protoplasm,
energized by a special "anion respiration," Cations then move more or
less passively from negative points on the surface to negative points in the
protoplasm, being "dragged along" due to potential differences maintained
by anion accumulation. The ions which move from the root hair across
the cortex, K% NO3", HCO3", etc., carry with them spheres of water, the
amount related inversely to the osmotic pressure of the cell sap. As the
ions are permitted to "leak" into the vessels they carry this water along.
They carry in addition what Lundegardh terms "extra water," which in-
cludes metabolic water, some carried to the roots by sugars, and some due
to changes in the swelling power of the protoplasm. In this manner, the
bleeding sap (exudate) may be more dilute than the medium bathing the
roots.

Relation to Other Plant Functions: — The possible role of active
water control in other plant functions may be briefly indicated. Interrela-

Crafts et al. — 140 — Water in Plants

tions have been implied by various investigators with respect to the general
processes of absorption by roots (van Overbeek, 1942 ; White, 1942) ;
to transpiration (Dixon and Barlee, 1940) ; and to translocation (God-
LEWSKi, 1884; Janse, 1913; and Bose, 1923). These will be considered
in the two chapters that follow.

Some who have studied mechanisms of cell elongation, or cell growth,
have been inclined to suggest processes involving active control of water.
The theory that auxin acts to increase wall plasticity and promote active
deposition of wall substance has many adherents, notably Heyn (1940).
Evidence that plastic extensibiHty in potato tuber tissue is greater under
aerobic conditions than where oxygen is excluded has been supplied by L.
and M. Brauner (1943a). Others believe that auxin enhances the im-
bibition pressure of the protoplasm. Strugger (1934), for example, found
by plasmolytic methods that an increase in protoplasmic viscosity accom-
panied cell enlargement, and concluded that an enhanced sweUing pressure
is directly involved. He suggested that acids resulting from respiratory
activity might promote swelling. Other explanations of cell enlargement
include an increase in solute concentration and the existence of electrical
potentials across the cell membranes produced by auxin, either of which
might bring about increased turgor, resulting in growth.

Growth is a complicated process. Perhaps several of the above mechan-
isms are operating simultaneously, all influenced by auxin directly or in-
directly. The problem is one of the most difficult in physiology ; neverthe-
less there has been much progress.

The striking movements exhibited by some plants have been of consid-
erable interest to physiologists. The sudden drooping of leaflets and
petioles of Mimosa pudica upon stimulation has caused much speculation
as to the cellular mechanics involved. It is generally agreed that the re-
sponse is the direct result of the loss of turgor by the cells comprising the
lower region of the pulvinus. This may be due to an active contraction of
the protoplast (Bose, 1928; Weidlich, 1930) whereupon liquid is secreted
into the intercellular spaces. Bose has been able to identify the contracting
cells by staining methods, and claims to have found by actual measurement
a decrease in cell diameter due to the contraction.

At the same time, the cells comprising the upper region of the pulvinus
expand somewhat. This is due in part to stretching resulting from the
force of gravity on the drooping leaf, and an attendant increased DPD,
which causes an additional uptake of water (Weidlich, 1930). During
the recovery process, which is of considerably longer duration than the
initial response, the cells in the lower part of the pulvinus apparently re-
absorb liquid and regain their normal turgor. That this liquid is relatively
pure water, and that the contraction is not a result of changes in permeability
which would permit whole cell sap to flow out, is the view of Weidlich
(1930).

Glandular secretion is another process in plants poorly understood at
the present time. According to Blackman (1921), Pfeffer in 1877 pro-
posed three hypotheses to account for the exudation of fluid from living
cells :

1) An unequal osmotic pressure developed by the membrane in different parts of
the cell,

2) Unequal distribution of osmotic material in different parts of the cell,

3) Presence of osmotic substances in cell wall outside of the membrane, causing
water to move out of the cell.

Chapter VIII

141

Active Relations

Blackman favored theory No. 2, where a steady concentration differ-
ence would be maintained by osmotic work requiring energy. Evidence
supporting this view has been supplied by Osterhout (1947&). By treat-
ing one end of a Nitella cell with an isotonic sucrose solution, the internal
osmotic pressure at that end was apparently increased. On removal of the
sugar solution, so that water completely surrounded the cell, water entered
at the treated end and was expelled at the other.

Mechanisms : — Few mechanisms have been proposed to account for
the active regulation of water in living organisms. There has been more
speculation on active solute uptake than on water absorption, first, because
of its relative importance in the nutrition of plants, and, second, due to
the paucity of evidence on active water relations. Especially is this true in
the plant field where there is little clear evidence for active water regula-
tion. Eaton (1943) thinks there are insufficient data to warrant postulat-
ing "uncertain physical and chemical forces" in such plant functions as
bleeding and root pressure.

Some have attacked the concept of "vital" functions in physiology as
unnecessary and even absurd. They imply a meaning different than the
one used in this chapter. Although our usage of the term indicates the lack
of a mechanical explanation at the present time, it presumes that an ex-
planation may be forthcoming. To us, the term "active" implies a process
involving the utilization of metabolic energy. In this sense salt accumula-
tion and water secretion are both active processes.

Adolph (1943, p. 255) comments that, in addi-
tion to osmotic pressure, "other sorts of forces, both
known and unknown to physicists, may be simul-
taneously present," and he is not optimistic that all
of them are ever identified.

Anomalous osmosis might prove a starting point
for an attack on active cell processes, for, in it, water
moves in a direction contrary to that expected on the
basis of concentration differences. Anomalous os-
mosis is a well-established phenomenon. It may be
due to processes of the nature of electroosmosis or
"anaphoresis" as described by Osterhout and Mur-
ray (1939). They used the apparatus diagrammed
in Figure 40 in which A represents an aqueous solu-
tion of trichloracetic acid separated from water, C,
by a layer of guaiacol, B. As expected, acid moved
to the right. Water, however, moved in the same
direction at a greater rate so that its mol fraction in
A decreased. The probable mechanism is that water
of hydration is carried along with the solute. Cal-
culations proved that over 25 mols of water would
have to be transported by 1 mol of acid to account
for some of the results obtained. Since hydration
did not remain constant, definite hydrates were probably not concerned.

Some have considered that electroosmosis may be a major factor in con-
trolling the water balance of cells. It may act to implement osmotic pres-
sure (positive anomalous osmosis), or to oppose it (negative anomalous
osmosis). Under proper conditions a transport of water through a mem-

f

/?

I

Fig. 40. — Apparatus
for demonstrating "ana-
phoresis." A, an aqueous
solution of trichloracetic
acid; B, a layer of guaia-
col; C, a layer of water.
From Osterhout and
Murray (1940).

Grafts et al.

142

Water in Plants

brane from a concentrated to a more dilute salt solution may be demon-
strated.

Electroosmosis may be defined as the movement of liquid along a
.barged solid surface (e.g., pore walls in a membrane) due to a potential
difference across a membrane exerted tangentially to the interface (that is,
the pore walls). The difference in potential, termed membrane potential
or liquid junction potential, is dependent on the presence of electrolytes
in the solution ; non-electrolytes are inefifective. Differential ion migration
velocities and a heterogeneous pore system contribute to the production of
the potential difference.

+ + + + + +

- H + - +

+ - H +

■^f:

OoHCL _ 4. _ ^. _ ^

+ -t- H^ + "-

C0//CI

av/CL

Co> C/

Co> C/

CiHCL

Fig. 41. — Diagrammatic representation of two membranes,
(A) permeable to cations only, the other, (B) anion permeable.
Each separates two HCl solutions of concentrations Co and Ci
where Co > Ci. A diagrams positive anomalous osmosis and B,
negative anomalous osmosis. The mechanisms are based on
electroosmosis. From Hober (1945).

The electroosmotic flow is dependent on the production of an electrical
double layer at the solid-liquid interface. On immersing the membrane in
a solution containing electrolyte the surface assumes a charge (usually
negative for natural membranes) which may result from ionization of the
membrane or adsorption of ions from the solution. The liquid at the inter-
face carries the opposite charge and becomes, at least in part, a mobile
layer. The electrokinetic potential across the interface (double layer) is
determined by the concentration and kind of electrolyte, the nature of the
membrane, and other factors. Figure 42C diagrams the nature of this
double layer within the pore of a membrane.

In order that electroosmosis may proceed, there must be a flow of cur-
rent across the membrane ; that is, there must be a closed circuit. Bartell
(1923) suggested that the return circuit occurs through the medium of the
double layer. However, Sollner (1930) and Sollner and Grollman
(1932) pointed out that a heterogeneous system of different sized pores
might account for return flow. Figure 41 helps to explain this mechanism.

Figures 41 A and B represent membranes having pores of unequal size.
Each separates two HCl solutions, those on the left being the more con-
centrated. In A the smaller pore is permeable to cations only ; the wall is

Chapter VIII

143

Active Relations

negatively charged; the movable liquid carries plus (H+) ions. The mem-
brane potential (E) across this pore is greater than that (e) across the
larger pore setting up a driving force that produces a movement of the
positively charged liquid toward the left. Since the normal flow is also in
this direction, the residual flow is termed positive anomalous osmosis.

The membrane in B is positively charged. The smaller pore prefer-
entially transports anions, therefore exhibits a smaller positive potential
(e) than the larger (E). Movement of the vein of liquid in this pore is
toward the more dilute solution, and is termed negative anomalous osmosis.

Vacuole \ Cytoplasm

JugariiA— Sugar

t

Malic Ik Malic
acid I ^ acid

£yternally
supplied
matate

B

+ +
+ +
+ +
+ +

4^

f\

+ +

+ +

Fig. 42. — Electroosmotic flow according to Bennet-Clark and Bexon (1943).

Bennet-Clark and Bexon (1943) have introduced a theory of water
movement by which they attempt to explain secretion across the tonoplast
into the vacuole due to electrokinetic activity. Instead of the classical model

solution I semipermeable membrane | water,
they prefer

solution I permeable membrane | water

as being more characteristic of the cell. They propose that the excess
turgor observed in their earlier work (Bennet-Clark, et al., 1936) is due
to continuous diffusion of solute. This might account for a potential differ-
ence across the tonoplast and provide the driving force for electroosmosis.
Or the water might move as water of hydration with the solute, a process
similar to Osterhout and Murray's anaphoresis. Turgor in cells is pic-
tured as resulting from such a continuous diffusion process rather than
from a "static osmotic equilibrium which could only be attained with an

Crafts et al. — 144 — Water in Plants

ideal semipermeable protoplast." To organic acids such as malic is assigned
an important role in this process.

Figure 42 from Bennet-Clark and Bexon (1943) will aid in follow-
ing the suggested series of events. In Figure 42A, malic acid diflfuses
from the vacuole into the protoplasm where it is enzymatically converted
into carbohydrate. The latter moves into the vacuole, whereupon it is
broken down to malic acid again. This completes a continuous diffusion
cycle of non-electrolyte inward and electrolyte outward. In this way a
potential difference might be maintained which in turn would account for
an electroosmotic flow into the vacuole. Figure 42B represents a simple
model used by Bennet-Clark for illustrative purposes. A container, the
bottom of which comprises a permeable ("oxidized collodion") membrane,
contains a saturated salicylic acid solution with excess of solid. This is
immersed in another vessel containing pure water. A hydrostatic pressure
is developed as indicated by the manometer, which is greater than the
osmotic pressure of the solution. This excess pressure is due to an electro-
osmotic flow through the pores of the membrane. One such pore is indi-
cated as Figure 42C.

In a fourth paper on the water relations of plant cells, Bennet-Clark
and Bexon (1946) give further evidence that bioelectric forces exert an
influence on water movement. Inner epidermis of onion bulb scale was
mounted in a perfusion apparatus designed for rapid replacement of the
bathing solution while the tissue was under microscopic observation. When
tissue, which had been treated with KCl solution (27.9 atm.) long enough
to cause complete plasmolysis (40 minutes), was exposed to a sucrose
solution of the same osmotic pressure, a temporary swelling of the vacuole
was observed. After 15 to 30 minutes the initial volume was reestablished.
Transfer back to KQ produced the reverse effect — a shrinkage of the
vacuole, followed by a return to the initial volume. That this result was
not simply a diffusion effect on the OP of the solution between the wall
and the protoplast was shown by the fact that isolated protoplasts behaved
in the same way.

Bennet-Clark and Bexon give the following explanation. Assuming
a negative charge on the membrane, the permeability to K"" is greater than
to CI". On transfer from sucrose to KCl, the greater penetration of K""
produces a positive charge on the inner membrane surface. This potential
difference effects an electroosmotic flow outward, and cell volume is reduced.
When KCl is replaced by sucrose, preferential diffusion of KCl outward
causes the inner surface to become negatively charged, and the electro-
osmotic flow is directed inwardly. Equilibrium obtains in both instances as
the membrane potential drops to zero.

One point made insufficiently clear is whether the volume of protoplasm
remains approximately constant during swelling and shrinkage of the
vacuole. Vacuolar contraction, and its reversal, frequently reported in
onion epidermis, could conceivably account for at least some of the volume
changes observed.

Brauner and Hasman, in a series of papers {see 1947) have defended
the view that an electroosmotic flow of water is a significant component of
DPD of plant cells. In their theory, bivalent cations should act to neu-
tralize the electric potential in the pores of the double membrane (cell
wall-cytoplasm). The difference between gravimetric determinations of
DPD, one in sucrose solution, another in CaCl2 orMgCl2, in their view

Chapter VIII — 145 — Active Relations

gives a measure of the electrokinetic component. As an example, for red
beetroot the DPD in sugar solution was 7.90 atm. ; in CaCl2 it was 6.75
atm. Possibly one should inquire if at least some of the effect of the salt
is not due to its penetration into the interior protoplasm, with a resulting
change in capacity to hold water.

The suggestion that electroosmosis and related phenomena are operative
in the control of water by plant cells is very plausible; their relative im-
portance, however, is difficult to evaluate. The requirements for electro-
osmotic flow include: a permeable membrane with a heterogeneous pore
system ; electrolytes of suitable concentration on each side ; and continuous
diffusion of electrolyte demanding some device for maintaining concentra-
tion differences. It is this last that designates the process in living systems as
an active one. If a mechanism suited to the maintenance of such concen-
tration differences can be demonstrated, the above requirements can be met
by living cells. Hober (1945), who has very adequately discussed mechan-
isms of electroosmosis, suggests, with reference to frog skin, that a con-
tinuous production and removal of hydrogen and bicarbonate ions would
establish a permanent concentration gradient across the protoplasm and so
produce the potential difference required for such flow.

Up to now our discussion of mechanisms has dealt with active transport
of water across membranes. At the beginning of the chapter, we included
in a definition of active water that water held within or outside the proto-
plasm due to the expenditure of energy.

The water retaining mechanisms in protoplasm are almost unknown.
The views, however, of Sponsler and Frey-Wyssling, outlined in Qiap-
ter VI with respect to binding of water by protoplasm, aided by modern
concepts of the structure of water and of proteins, are very suggestive.
The ability of the protoplasm to vary its volume is known from many
observations — stimulative plasmolysis, vacuolar contraction, frost harden-
ing, etc. It is true that some of these responses could be the result of
change in solute concentration but it is just as likely that water is actively
moved, as revealed for example, by vacuolar contraction studies (see page
125, Chapter VIII). Perhaps a distinction is unnecessary in the light of
recent suggestions of Steward and of Lundegardh that water and solute
uptake may be intimately related. Furthermore, salts can markedly affect
the imbibitional properties of colloids.

Northern (1942) found that stimulation decreased the structural
viscosity of protoplasm, and concluded that this result was conditioned by
dissociations of cellular proteins, at least in part. It was suggested that
such dissociations produced a greater imbibition pressure in the protoplasm.

In their studies on foliar hydration, Phillis and Mason (1945) enter
into this matter, with the thesis that the hydration of cotton leaves is largely
controlled by protoplasmic imbibition. The latter is in turn controlled by
the amount and kind of salts present. Sugars are ineffective in causing
absorption of water. An imbibitional mechanism is favored in which micro-
scopic vacuoles in the protoplasm are the proposed loci of accumulation of
both water and salts. Since water in some instances was absorbed without
an increase in the concentration of salt, an enhanced OP as the cause of
water intake was ruled out. Following a suggestion of Lloyd and Pleass
(1927) the effect of salt was considered as weakening the protoplasmic
framework.

Reinders subsequently (1942) verified her earlier results and by ex-

Crafts etal. —146— Water in Plants

tending the experiments to include six other storage tissues, concluded that
water intake as observed in potato is a general phenomenon. Especially
striking behavior was shown by tuber tissue of Helianthus tuberosus.
While confirming the correlation with respiration, it seemed " . . . . most
probable that water-intake is only a secondary phenomenon, a consequence
of other metabolic processes, by which osmotically acting substances arise."
These substances would more logically be formed in the protoplasm than
in the vacuole, according to Reinders.

Information relative to the hydration of protoplasmic proteins has been
obtained by Levitt (1946). Isolation of the proteins from potato tubers
was apparently accomplished without denaturing them. The protein solu-
tions were introduced into microcups where their osmotic pressures were
determined manometrically. There was some indication that proteins from
external tissues, allowed to form new periderm, exhibited greater hydration
than those from internal tissues that were dormant. This is a new method
of investigating the water relations of protoplasm ; it should lead to other
interesting results.

Movement of water from cell to cell could conceivably result from ac-
tive processes causing inequalities in osmotic pressure within the cell, in-
equalities of membrane permeability, or changes in imbibition pressure.
Reference is made to these possibilities in chapters which follow.

In conclusion, it seems evident that if active water movements, meta-
bolically controlled and energized, are demonstrated beyond doubt in plants,
it will be necessary to revise the basic concepts underlying many plant
processes. Though momentarily seeming to confirm vitalistic ideas on
water relations of plants, such a demonstration will require, in the long run,
a deeper search into the physical and chemical mechanisms involved. And
although certain classical views may seem already to be outmoded, it must
be admitted that the evidence for active water movement is still far from
complete as are many other aspects of protoplasmic activity.

Summary: — In the classical concept of cell water relations the protoplasm is
passive with respect to the passage of water, and consequently water movement and
retention are determined by forces of osmosis, imbibition, etc.

Recent work indicates that the protoplasm may play some vital role in water dis-
tribution.

There is a fairly consistent discrepancy between tissue osmotic pressures deter-
mined plasmolytically and cryoscopically. This has been interpreted by some as in-
dicating that water may accumulate in vacuoles due to a secretory action of the proto-
plasm. Other explanations proposed for the discrepancy are : the presence of colloids
in the vacuole ; dilution of expressed sap by protoplasmic water, producing cryo-
scopic values which are too low ; adhesion of the protoplasm to the wall, resulting in
excessive plasmolytic values ; and methodical errors in both procedures.

Refinement of sap expression technics has produced saps termed "protoplasmic"
and "vacuolar" by some investigators. The results have been interpreted as support-
ing the secretion hypothesis. It seems questionable if any clear distinction of this type
can be made on sap fractions obtained by pressure.

While plasmolytic methods by classical theory should provide an accurate picture
of vacuolar concentration, many anomalous results have been obtained which may re-
flect some kind of active control of water by the protoplasm.

When expressed saps are compared with equilibrium bathing solutions by cryo-
scopic methods, discrepancies appear that indicate possible imbibitional or secretory
activity of cells.

Auxin has been shown to increase water uptake by living cells. This has been
considered as evidence for active secretion of water into the vacuole, but other ex-
planations are possible.

The freezing point of living tissue is usually lower than that of dead tissue. The
most plausible explanations are : difficulties exhibited by living tissue toward freezing

Chapter VIII — 147 — Active Relations

point determinations, and protoplasmic factors. Forces regulating the state of water
in protoplasm may constitute one of the latter.

Frost resistance in plants may be related to protoplasmic factors including in-
creased permeability and decreased viscosity, both of which are attributed to a greater
hydrophily of the protoplasm. Such a condition might involve a reversible increase in
protoplasmic water at the expense of the vacuole brought about by unfolding of pro-
tein chains or opening of ring structures to expose more points for hydration, such
structure being maintained by metabolic energy.

The evidence for active control of water by animal cells and tissues is such that
water secretion, or active transport, appears likely in several instances.

A comparison of solute and water absorption has suggested that aerobic respiration,
protein synthesis, water absorption, and salt accumulation are mutually dependent
processes.

Active cell water relations may affect many other plant functions. Transpiration,
translocation of salts and water, plant movements, and glandular secretion are a few.

Several mechanisms have been suggested to account for active water control by
the protoplasm. Anomalous osmosis involving the carrying of water of hydration by
ions during accumulation is one. Electroosmosis, the movement of liquid along a
charged solid surface (pore wall) due to a potential difference across a membrane is
another. Differential ion migration and a heterogeneous pore system contribute to
such a potential difference. Continuous diffusion of solute may account for a potential
difference and provide the driving force for electroosmosis. A continuous cycle in-
volving enzymatic conversion of malic acid to carbohydrate in the protoplasm, diffu-
sion of the latter into the vacuole and breakdown to malic acid again is one suggestion.
Salts probably play an important role in any anomalous movement of water across
membranes. The continuous production and removal of H+ and HCO3" ions may be
one source of solute for continuous diffusion from the cell.

Though the argument for active water regulation in plants is supported by an in-
creasingly wide array of data, the overall evidence at present is inconclusive. One
does, however, gain the impression that the classical views of cell water relations can-
not account for many of the recent findings. There may be as yet too many unknown
factors related to protoplasmic structure, permeability, and imbibition to allow a simple
osmotic explanation of all cell water relations.

Chapter IX
UPTAKE AND MOVEMENT OF WATER IN PLANTS

Introduction : — The simple lower plants, having no specialized tissue
for absorption and movement of v^^ater, are either limited in their habitat
to a moist environment or they remain small in size and are adapted to
periodic desiccation. Our common higher plants have developed specialized
tissues and organs for the rapid uptake and transport of water ; by means
of these, and special protective layers that greatly restrict water loss, they
are able to survive with a large portion of their surface exposed to the rel-
atively dry environment whilst their living cells enjoy virtually an aqueous
medium. In such plants there is a continuous but varying competition for
water between the plant as a whole and the environment, and the struggle
for water throughout the life of the plant is not confined to living cells but
exists among dead cells and their aqueous contents, cell walls, vacuoles,
protoplasm, and air spaces. When the absorption rate is high and transpi-
ration low, competition is at a minimum. When uptake is reduced or water
loss increased, competition for water increases and reaches a maximum dur-
ing advanced stages of wilting if the water supply to the roots fails. Periods
of stress occur diurnally in most plants due to excess transpiration during
midday ; seasonal trends are noted as annual plants pass through stages of
maximum growth and maturation; annual trends take place in perennial
plants during similar growth stages. Seedlings growing in moist soils and
a moist atmosphere show low stress ; in fact, they often have excess pres-
sure in their xylem conductors as evidenced by the forcing of liquid drops
out of hydathodes (guttation) or by exuding liquid from the xylem when
excised. Plants in water culture may exhibit guttation even when fully
grown, particularly during the night and early morning hours; stress in
the xylem of such plants may be demonstrated during midday by the rapid
inrush of dye when the stems are cut under a dye solution. Plants growing
in soil will guttate only if the soil is maintained near its field capacity and
atmospheric humidity is high; as soil moisture is depleted stress mounts
in the xylem, and throughout the plant. When the moisture content of the
soil reaches the range known as the permanent wilting percentage, the plant
is nc longer able to absorb appreciable amounts of moisture ; if more is not
supplied it eventually dies.

Water Absorption: — Most plant physiology texts {cf. Miller, 1938;
Meyer and Anderson, 1939) describe the structure of roots and explain
the relation of gross structure to the function of water absorption. Kramer
has reviewed the literature on soil moisture in relation to plant growth

(1944) and on the absorption of water by plants (1945). M agist ad

(1945) has covered the relations of salinity and alkalinity to plant growth.
Water absorption will receive limited treatment here, emphasis being placed
on recent experimental work on active absorption and the forces opposing
absorption.

It has long been recognized that water uptake by roots ma> be active
or passive (Renner, 1915; Kramer, 1945). Active absorption results
from a metabolic functioning of the roots and is evidenced by root pres-
sure, xylem exudation from excised roots and under some conditions, gut-

Chapter IX — 149 — Uptake and Movement

tation. The term "active" as used here is not identical with that used in
Chapter VIII because, in this case, a complex cellular mechanism is in-
volved. The extent to which water secretion, etc., enters into the mechan-
ism of active water absorption by roots has not been determined.

Passive absorption goes on when transpiration exceeds absorption and
results from a DPD gradient developed in the leaves and transmitted to the
roots through continuous columns of water under reduced pressure. Be-
cause conditions under which active absorption predominates occur only
rarely in the life span of most plants, a large portion of the water used is
taken up by passive forces. Nevertheless, active absorption has been stressed
in physiological studies (White, 1942) and it may play an important role
in the growth of seedlings, lawn grasses, plants of the tropical rain forest,
and by most crop plants during rainy spring weather.

Active Absorption: — Priestley (1920) was one of the first to give
a comprehensive picture of a mechanism of active water absorption by the
root in which structure and function were adequately related. According
to this picture, water entering the root hairs passes inward across the cortex
and endodermis and enters cells of the stele surrounding the non-living
xylem vessels. This passage Priestley attributes to an osmotic gradient.
Because the concentration of solutes in the xylem vessels may be less than
in the surrounding parenchyma, and because, for continuous absorption,
solutes must be supplied to the xylem, the cells surrounding the vessels
are postulated to be more permeable to solutes on their inner surfaces and
hence they would allow a "leakage" of solution into the xylem. The dif-
ferential permeability might result from the fact that the cytoplasm of the
xylem parenchyma cells is in contact through the outer walls via protoplas-
mic connections with living protoplasm ; on the inner walls it faces a dead
lignified layer. Furthermore, metabolism of the root cells might lead to
oxidation of sugars to organic acids ; osmotic activity would thus be en-
hanced because several acid molecules would result from splitting of a
single sugar molecule ; protoplasm is relatively permeable to organic acids
and might release them in solution into the xylem elements.

If the above mechanism could provide solutes to the xylem in sufficient
quantity to maintain a higher osmotic concentration than occurs in the soil
solution, water should move in to satisfy the osmotic gradient, and higher
osmotic concentrations within intervening tissues would not interfere be-
cause water movement is determined by gradients of diffusion pressure and
not osmotic pressure.

Crafts and Broyer (1938) elaborated somewhat the above picture bas-
ing their theory on two essential assumptions (1) that the interconnected
protoplasts of the living cells of the root form a continuous protoplasmic
system (the symplast) extending from the root hairs to the xylem paren-
chyma; and (2) that the close relation of the cortex with its system of air-
filled intercellular spaces with the soil atmosphere makes possible a higher
metabolic status than occurs within the endodermis where intercellular
spaces are lacking and where water movement would tend to sweep in CO2
formed by respiration in the cortex. Since anatomical and physiological
considerations seem to substantiate these conditions, functioning of the root
mechanism is pictured as follows. When solutes are absorbed and accumu-
lated in the root hairs a diffusion gradient is established across the symplast
and movement inward occurs via the interconnected protoplasm, the tend-

Grafts et al. — 150— Water in Plants

ency being to establish a uniform concentration. The metabohc gradient
across the tissue on the other hand as determined by the O2 : CO2 rela-
tions would enhance accumulation of solutes to a concentration that could
not be retained within the stele ; leakage of solutes must take place and
osmotic movement of water would tend to flush these along the cell walls
and into the xylem vessels for these constitute the only avenue of escape at
a pressure near atmospheric. This mechanism seems quite compatible with
known root structure and it fits in fairly well with the gross picture of
solute absorption by roots, xylem exudation by excised roots, and guttation
from intact plants.

McDermott (1945j has shown that exudation from the roots of excised
sunflower plants growing in soil is greatest when the soil moisture is near
the moisture equivalent. At higher moisture contents exudation is lower,
probably because of poor aeration. At lower moisture contents exudation is
reduced until it stops. The exudation stream reverses as the wilting percent-
age is approached. The moisture equivalent mentioned by McDermott
is a water content near field capacity, the amount of water a soil can hold
against gravity. For an extensive report of experimental work on root
pressures and exudation see Sabinin (1925).

A detailed study of the dynamics of xylem exudation indicates a more
complex mechanism than the above discussion would indicate. Experi-
ments by Grossenbacher (1938, 1939) prove that xylem exudation fol-
lows a fluctuating diurnal pattern that seems to be impressed upon it by the
previous history of the plants. And auxin has been shown to greatly stimu-
late the flow of xylem exudate (Skoog, Broyer, and Grossenbacher,
1938). If it could be shown that the diurnal fluctuation in root pressure is
caused by an auxin induced fluctuation in metabolic rate of the root cells,
the above observations might still fit into the mechanism of Crafts and
Broyer.

An even more serious departure from a strictly osmotic mechanism
is indicated by results reported by van Overbeek (1942). Tomato plants
growing in culture solution were decapitated and the excised roots placed in
distilled water whereupon each plant was attached by rubber tubing to a
small bore U-tube and two or more milliliters of xylem exudate collected.
Then the U-tubes were replaced by potometer tubes and mannitol was mixed
in the distilled water around the roots until flow in the potometer stopped.
When flow had remained static for 15 minutes the mannitol solution was
sampled and its osmotic pressure determined cryoscopically as was that
of the exudate. Theoretically, if water movement into the roots were
osmotic, these two solutions should be isotonic. In the experiments the
concentration of mannitol required to stop flow was from twice to several
times that of the exudate. However, the rate of exudation and concen-
tration of the exudate were related to the concentration of mineral nutrients
in the solution bathing the roots, the rate being lower and the concentration
higher if culture solution were used in place of distilled water, van Over-
beek concluded that the pressure with which tomato plants absorb water is
composed of two fractions 1) an active pressure and 2) a pressure of
osmotic origin. The nature of active water movement has been considered
in a previous chapter ; the above case is a good example of the way in which
such active water movement may afifect the water economy of plants.

In contrast to the above explanation of the differences in the concen-
trations of exudation sap and external solution, Lundegardh (1946) sug-
gests that the bleeding sap is frequently more dilute than the external solu-

Chapter IX — 151 — Uptake and Movement

tion as a result of the presence of "extra water." This is brought about by
disappearance, through metabolic processes, of osmotically active sub-
stances in the stele.

Passive Absorption : — As emphasized in Chapter V, the diffusion pres-
sure of water may be lowered in several ways i) by adding solute, 2) by
lowering temperature, 5) by reducing hydrostatic pressure. When evapora-
tional loss from leaves exceeds uptake by the roots, the diffusion pressure is
lowered throughout the plant and particularly through the elongated tubes
of the xylem is this pressure reduction equaUzed between leaves and roots.
As tensions of a few atmospheres are developed in the xylem, the forces
responsible for active absorption are exceeded and passive flow to satisfy the
hydrostatic gradient becomes the dominant factor in water uptake.

Not only is the active mechanism no longer effective ; experiments where
living and dead root systems have been compared have led Kramer (1933)
to believe that the osmotic mechanism responsible for active water absorp-
tion actually constitutes a resistance for the more rapid passive uptake ; dead
roots allow more rapid flow than live ones.

These and other experiments of Kramer (1932 to 1939, see 1945) and
work by many others including the very practical process known as the acid-
arsenical method for kiUing weeds (Kennedy and Crafts, 1927, 1931;
Crafts and Kennedy, 1930; and Crafts, 1933a, b, 1937) all emphasize
the fact that under most conditions in the field the movement of water into
and through the plant as a result of transpiration is a reversible process, and
that the water in the plant is in a state of tension stretched between the
menisci in soil colloids and in hydrated cell walls of the mesophyll. The
mechanics of water absorption and movement represent an adaptation on
the part of the plant to the demands of transpiration and the limitations of
the soil reservoir. The extent to which various plants have been able to
meet these conditions determines to a considerable degree their distribution
with respect to characteristics of the environment and their productivity
within any circumscribed range.

Methods of Estimating the Availability of Water: — Moisture is
retained in the soil by adsorptive and capillary forces. Its rapid absorption
by plants is also hindered by the low permeability of living plant roots
(Kramer, 1933). Richards (1941) has described a pressure-membrane
apparatus for studying soil-moisture availability at tensions of one atmos-
phere or above. The method consists of enclosing a thin layer of air-dry
soil in a cylinder on a permeable cellulose membrane that is supported by a
brass screen. The soil is saturated with distilled water and then the excess
moisture is forced out by gas pressure. The moisture retaining force at any
given soil moisture content ("moisture tension" of Richards and Weaver,
1944) is measured by the gas pressure required to attain that content.

This method is weak in that it is useful only on laboratory prepared soil
samples in which the natural structure has been destroyed in preparation;
the pressure applied tends further to destroy structure ; the effect of solutes
on the diffusion pressure of water is not included in the measurement. The
latter effect may be measured cryoscopically and the freezing point depres-
sion plus the moisture tension measure the DPD or total moisture stress
of the soil.

Physiologically, soil moisture has been classified as -7 ) that available for
vegetative growth, 2) that which may be used by certain plants to maintain

Grafts et al. — 152 — Water in Plants

life under conditions of low turgor, and 3) that totally unavailable to plants
(FuRR and Reeve, 1945). The existence of a definite wilting coefficient
or initial permanent wilting point has been contested since Briggs and
Shantz (1912) first proposed the term. Furr and Reeve (1945) define
the first permanent wilting point of the sunflower plant as permanent wilting
of the basal leaves, a moisture status that practically marks the end of
vegetative growth. For all useful purposes, this designates the point at
which the soil must be irrigated if growth of a crop is to continue.

They designate ultimate wilting as the soil moisture content at which
apical leaves wilt permanently. This stage is usually accompanied by irre-
versible wilting and, finally, death of the lower leaves. Between initial and
ultimate permanent wilting lies a range of soil moisture contents for the
study of which Furr and Reeve describe a standardized procedure. For
light and medium textured soils this range is narrow and though it repre-
sents a significant shift in the DPD of soil moisture, the actual amount of
water involved is relatively small. For heavy soils, because of lack of uni-
formity of soil moisture around absorbing roots, slow moisture movement,
and strong adsorptive forces, this range may be significantly broad. Further-
more, cases are on record where crop yield has been reduced before any
appreciable evidences of wilting could be detected, as will be mentioned later.

At field capacity (maximum moisture content in equilibrium with the force
of gravity) the DPD of the soil moisture is between 0.1 and 1.0 atmospheres
in most soils (usually less than 0.5 atm.). As water is taken from the soil,
the total moisture stress mounts hyperbolically (see Figure 9, Chapter III).
Because of the rapid change of stress with decreasing water content, it has
proved difficult to reach an agreement as to the significance of plant re-
sponses in the field, especially in the range between 5 and 10 atmospheres.
Study of soil moisture in this range is difficult because of the rapid increase
in DPD with small decreases in moisture content ; also because it is im-
possible to maintain a constant water content within this range with plants
growing in the soil ; estimates of the absorbing power of plants at permanent
wilting range from 4 atmospheres to 20 and higher.

Evidence regarding the availability of soil moisture for plant growth is
conflicting. Apparently from the standpoint of crop production, in light and
medium textured soils, moisture is equally available to plants throughout the
range from field capacity to permanent wilting (Veihmeyer, 1927; Con-
rad and Veihmeyer, 1929; Hendrickson and Veihmeyer, 1942; and
Veihmeyer, Edlefsen, and Hendrickson, 1943). Much recent evidence
has been advanced, however, to indicate that deep-rooted plants on heavy
textured soils may show a marked response to water deficit before the soil
mass has reached the permanent wilting percentage (PWP) (Furr and
Degman, 1932 ; Furr and Taylor, 1929 ; Aldrich, Lewis, Work, Ryall,
and Reimer, 1940; Schneider and Childers, 1941; Davis, 1940; and
Wadleigh, Gauch, and Davies, 1943).

Certain apparent differences in plant response to water deficits are to be
expected ; they probably result from inaccuracies in the interpretation of
moisture determinations. The fact that, in light soils, water movement and
root growth are rapid results in a fairly uniform depletion of moisture
throughout the soil mass occupied by roots, and the bulk of the soil reaches
the permanent wilting percentage at about the same time. In heavy soils
where root growth and moisture movement are slow, the upper zone where
the bulk of the roots occurs may become depleted (to or below the PWP)
before the lower horizon reaches this point. As a result, most of the absorb-

Chapter IX — 153 — Uptake and Movement

ing roots may occupy soil at or near the wilting range, whereas the deeper
ones may penetrate to depths where there is ample available moisture; the
root system may be somewhat restricted ; and crop yield may be reduced
before the average moisture content reaches a critical point. There may be
no visible evidence of wilting on the part of the plant. Actually, one cannot
measure the true PWP of the soil reservoir of a plant unless the whole soil
mass is uniformly permeated with roots so that moisture is taken out evenly.
Physiological responses have been observed, however, even though wilting
was not visible. Wadleigh and Ayers (1945) have reviewed some of this
work.

RosENE (1941) and Hayward and Spurr (1944) have investigated the
influence of the DPD of the external medium upon the rate of water entry
into roots of water culture plants. Although Veihmeyer and Hendrick-
SON (1934) have shown that sunflowers grown in sand cultures did not wilt
when placed in sucrose solutions until DPD's of 16 to 20 atmospheres were
reached (approximately equivalent to the DPD of the soil at PWP), ab-
sorption may be reduced at much lower tensions. The fact that such reduc-
tion in water uptake does not affect crop yields until forces from 16 to 20
atmospheres are involved indicates that plants normally absorb and transpire
more water than they need for carrying on the various functions in
which water takes part. The limits of deficit indicated by the results of
Veihmeyer and Hendrickson show that plants may endure high stress
and still grow normally ; somewhat higher values have even been found
occasionally (Richards and Weaver, 1944).

Though growth of most plants is not markedly hindered at soil mois-
ture contents above the PWP, plants vary with respect to root distribu-
tion, root hair formation, and other factors that affect their utilization of
water. One plant that is commonly known to require frequent irrigation
is onion; this is partially caused by limited root growth; Rosene (1941),
however, found that DPD's of 4.2 to 5.7 atmospheres represent the limits
above which intact roots of onion were unable to absorb water. Isolated
roots, dependent upon active absorption could only acquire water from solu-
tions having DPD's below 1.8 to 3.3 atmospheres. An OP of 6.5 atmos-
pheres limited absorption by intact roots of plants whose leaves were in an
atmosphere of 50 per cent relative humidity and at a temperature of 25 ° C.
Hayward and Spurr (1944) found that increasing the OP of the culture
solution from 0.8 to 4.8 atm. caused an 80 per cent reduction in water ab-
sorption by corn roots. An OP of 6.8 atm. stopped absorption completely.
The leaves of the plants were in an atmosphere having 70 per cent relative
humidity and at 22° C. Tagawa (1934), using beans in culture solutions,
found that an OP of 1.94 atm. was sufficient to prevent absorption by ex-
cised roots (active absorption) whereas 14.68 atm. were required to pre-
vent absorption by whole plants. The relative humidity was 60 per cent
and the temperature 28° C.

In contrast to these findings with culture solution plants, Richards
and Weaver (1944), with soils from Southern California, found plants to
endure higher stress. At first permanent wilting, values (except for one
soil) reached 7.5 to 16 atm., of which 1 to 9.8 atm. represented OP of the
soil moisture. Total stress at the ultimate wilting point for 19 out of 24 of
these soils ranged from 17.7 to 32.9 atm. (tension 16 to 28.9 atm., OP, 1.8
to 7.8 atm.). One soil showed a total moisture stress of 60 atm. at ultimate
wilting, a value that seems unaccountably high.

Unfortunately, the above results can only be regarded as tentative be-

Grafts et al. — 154 — Water in Plants

cause of excessive sampling of the soils and the use of approximate methods
for determining osmotic pressure. Since the OP values usually found for
fertile irrigated soils range from 1 to 2 atm., it is apparent that the soils
reported above were actually saline. Crop production was poor in soils
having OP values of 10 or more atmospheres at PWP; growth was com-
pletely inhibited where OP values reached 47 atm. (M agist ad and
Reitemeier, 1943).

Water Movement: — Experimental work has proved rather conclusive-
ly that the rapid longitudinal movement of water in plants takes place
through the nonliving tracheal elements of the xylem. The forces involved
in such movement are not as clearly understood. Both vitalistic and physical
theories have been propounded. Copeland's (1902) review presents the
early concepts.

Vesque (1883), Strasburger (1891), and Dixon and Joly (1894)
showed that plants could conduct water through dead tissues, and physiol-
ogists have generally discounted purely vitalistic interpretations of water
movement; most, however, accept the view that living cells in some way
condition the xylem tubes, keeping them free of gum and obstructions.
MacDougal and Overton (1927) have pointed out that living cells in
contact with tracheal elements develop tyloses that plug them; anatomical
investigations, however, indicate that tylose formation occurs only in xylem
that is injured or that is approaching senility. The functioning of young
active xylem and the provision of new xylem through cambial activity de-
pend upon Hving cells ; this does not prove that such cells are actively en-
gaged in pumping water through tracheal elements.

Bose (1923) has been the chief advocate in recent years of water trans-
port by vital activity. He claims that by very delicate instruments he has
been able to detect a pulsation of the protoplasm of living cells throughout
the plant and proposes that this action of the cells promotes conduction by
injecting liquid into the xylem thus setting up an intra-vascular pressure
and producing a mechanical transport. Molisch (1928, 1929) supported
this theory but the experimental evidence has not been generally accepted
(Dixon, 1924; Benedict, 1927; MacDougal and Overton, 1927).

It has been proposed (Priestly, 1935) that water movement into the
expanding leaves is associated with the processes of growth and differentia-
tion and should be interpreted on an anatomical basis. The living xylem
elements are assigned a major role in conduction ; dead trachael elements are
thought to act chiefly as storage tissue. That the living cells of the xylem
are involved in the ascent of sap and that the dead elements are incidental
and only function as reservoirs was suggested again in 1939 by Handley.

Cohesion Theory: — Despite the objections cited, the cohesion theory
of water movement in plants has gained almost universal acceptance by plant
physiologists. This theory is based on the classical researches of Dixon
and Joly (1894), Askenasy (1895), and Dixon (1914, 1924). As gen-
erally accepted the cohesion theory proposes that water is able to move
through the xylem of plants, even to the tops of tall trees, by reason of its
continuity and cohesion, and that such movement results from differences in
diffusion pressure of water between the soil, the plant, and the aerial en-
vironment.

Cohesion and Adhesion: — Forces of cohesion between the water
molecules and of adhesion between water and the cell walls permit the main-

Chapter VIII — 155 — Uptake and Movement

tenance of continuity under conditions of greatly reduced pressure, and pre-
vent, by the action of surface tension, the penetration of undissolved gas
through the wet cellulose walls. Theoretical values for the cohesion force of
water are very great (cf. Table 2, Chapter II, internal pressure).

Harkens (1926) calculated the force of cohesion from the surface ten-
sion of water in the following manner. The surface tension of water at
20° C. = 72.8 dynes per cm. Assuming that a water column one square
centimeter in cross section is ruptured to produce two new surfaces, each of
one square centimeter, the energy required is equal to twice the free surface
energy or 145.6 ergs per cm-. The distance over which molecular attractive
forces are effective approximates molecular dimensions, and force diminishes
as a power of the distance. Assuming that the summation of molecular at-
tractive forces acts as a constant force through a distance of 10"^ cm., then,
since force = ^^^^ — , the force required to rupture the water column would

distance

be ^ = 1.456 X 101 0 dynes or 1.48 X 10^ grams per square cm. This
is equivalent to about 14,000 atmospheres. The force of cohesion as cal-
culated by van der Waals' equation is 11,000 atmospheres; by vaporiza-
tion studies it is 10,500 atmospheres (Shull, 1924) ; from internal pressure
calculations it is approximately 17,000 atmospheres. All of these represent
ideal values ; practically the conditions necessary for obtaining such co-
hesions are impossible of attainment.

Experimental values for the cohesive force of plant sap obtained by
measurement of the diffusion pressure deficit of water in the cells of fern
sporangia are about 316 atmospheres, sufficient to support a static column
around 10,000 feet in height, and entirely adequate to account for movement
through xylem ducts to the top of the tallest tree (Ursprung, 1915 ; Ren-
NER, 1915).

Dixon (1914) extracted sap from Ilex aqiiifolium and tested its tensile
strength by sealing it in a capillary tube, warming until the sap just filled
the tube and then cooling until the column was ruptured. From the thermal
expansion of glass and water he was able to calculate the tension at the
temperature of rupture ; values determined were between 133 and 207 atmos-
pheres.

In view of the fact that cellulose walls imbibe water and are thoroughly
wet, the forces of adhesion must exceed those of cohesion. Many centers
of hydration occur along the cellulose chains making up the walls. Conse-
quently, in the immediate spheres of influence of these coordinating centers
water must be held with such force that its molecules are oriented, the quasi-
lattice structure assuming a closer packed and more stable condition than in
the body of the liquid. At the same time, due to the rigidity of the walls,
and the excess of transpiration over uptake, water in the centers of the
lumina of conducting elements must be at times in a rarified or tensile condi-
tion. This would tend to expand the centers of abnormal coordination
(holes) reducing viscosity and facilitating flow.

Liquid Continuity in the Xylem: — The cohesion mechanism, to
function, depends upon continuous water columns. Evidence for and against
such a condition is cited by Copeland (1902), Dixon (1914, 1924, 1938a),
Maximov (1929a), Meyer (1938), and Strugger (1943).

When a gas bubble is present in a conducting element of the xylem,
that element is eliminated, for the time, as a conductor of liquid water under
tension. Many objections have been raised against the cohesion theorv

Grafts et al. — 156— Water in Plants

based on evidence that the tracheal elements of the xylem contain gas.
CoPELAND (1902), in his review of work on the rise of sap, concluded that
the existence of chains of bubbles in the xylem (Jamin's chains) precludes
the possibility of tensile columns. He postulated atmospheric pressure as
the cause of the rise of sap. Much of the early work on this problem, which
CoPELAND reviewed, is invalidated by the fact that early workers did not
realize the ease with which bubbles could be formed in a tensile water column
by manipulation.

Four principal methods have been used in modern studies on the state
of water in xylem ducts: (1) dye penetration; (2) freezing of the tissue;
(5) transpiration measurements; and (4) direct observation of the xylem
contents.

Dye Penetration: — On the basis of injection of dyes Priestley (1935)
concluded that the mature wood of trees acts as a reservoir ; only differentiat-
ing elements conduct water. He contended that water is drawn from the
non-living vessels by the differentiating elements, and by some unexplained
process is driven into the expanding tissues of the leaves. By isolating in
early summer a branch from a tree having diffuse porous wood, Priestley
found that upon removing the bark he could inject the tracheae all around
the branch, the injections proceeding in rapid succession around the stem.
At a loss to explain where the water originally present before injection
went, he concluded that the tracheae had contained some gas at low pres-
sure.

If the sap originally present in the branch was under tension, it would
be surprising if during the process of isolating the branch the tensile columns
had not broken. Probably much of the gas in the injected elements resulted
from the isolation treatment. Where, due to excessive stress, bubbles are
actually formed in stems, they may collapse during later periods when water
is more abundant. If water remains wanting, then such elements are iso-
lated by gum or tylose formation and new elements of smaller diameter may
be formed as a result of cambial activity. In ring porous woods there is
usually a direct correlation between the diameter of xylem elements and the
availability of water. Whether this is merely a reflection of the effect of
limiting water on turgor expansion during growth, or an adaptation on the
part of the plant to greater tensions in the xylem, the result is the same,
namely the production, as stress increases, of narrower and narrower ele-
ments that are less liable to bubble formation and more suitable for collapse
of bubbles once they are formed.

MacDougal, Overton, and Smith (1929) studied the direction and
path of dye movement in various woods and concluded that they were able
to introduce the dye into the closed system of the conducting tubes. This
was accomplished by forcing the dye under one or two atmospheres pres-
sure into a hole in the stem, tightly fitted with a metal tube. The dye moved
tangentially from the hole into elements of the xylem which had not been
injured. From these experiments they concluded that since different species
of trees showed different dye patterns, the patterns are determined by the re-
lation of gas-filled to water-filled elements. Radial movement of the dye
from one annual ring to another was not observed.

Baker and James (1933) criticized the results obtained by Mac-
Dougal, Overton, and Smith, and emphasized the fact that the introduc-
tion of a dye into the conducting system of a plant necessarily requires open-
mg it at some point with an alteration of the original conditions of tension.

Chapter IX — 157 — Uptake and Movement

The long time periods between injection and observation by the above work-
ers were believed by Baker and James to have led to error. Their own
work resulted in radial transfer of dye from one annual ring to another.
Because most dyes are poisonous to living plant cells, it seems possible that
MacDougal, Overton, and Smith might have caused injury to their
material, resulting in death of cells, invasion of air, and formation of tyloses.

It is commonly observed that when an intact stem of a transpiring plant
is cut beneath a dye solution, the dye rushes in, going both up and down the
stem. Arndt (1929) interpreted the downward movement of eosin solu-
tion applied to cut ends of Coffea arabica stems as indicating that the
descending sap in the xylem should provide an adequate mechanism for the
downward transport of food. Dixon (1924) had similarly interpreted
backward flow of eosin in potato shoots. Postulating that different xylem
tracts might be isolated, he proposed that release of tension within a leaf
might result in backward movement of foods in solution at the same time
that water and salts were moving upward in other isolated tracts. Dixon
later reversed his opinion concerning food movement (1933).

The immediate penetration of dye into a stem of an actively transpiring
plant that has been cut beneath the solution may result from several actions :

i) If the liquid is under tension, a strain is developed in the conducting elements
tending to reduce their diameter. Bode (1923) has shown by direct observation that
this may amount to a reduction of several per cent in the total diameter, and MacDougal
(1925) proved by dendrographic measurements that a contraction of up to one per
cent in the whole stem may be found during the day. Actual vessel contraction may
greatly exceed this value since vessels make up only a fraction of the woody tissue and
living parenchyma would probably shrink less. Immediately upon cutting a contracted
vessel there occurs an elastic expansion which causes a rapid inward flow of dye at
both cut surfaces.

2) Simultaneous with the rupture of a tensile water column in the xylem the
diffusion pressure of water within that column increases and water moves out of the
conducting elements into adjacent living cells which were under a saturation deficit, but
in approximate equilibrium with the tensile water columns before cutting. Even root
cells in which a saturation deficit occurred would contain water with a lower diffusion
pressure than that in the xylem after cutting. Because of this dye may be drawn down-
ward through a cut stem into the roots underground (Kennedy and Crafts, 1927). In
fact, this mechanism is employed for the destruction of deep-rooted perennial weeds by
the acid-arsenical method (Kennedy and Crafts, 1927; Crafts, 1933a, 19336, 1937).
In this method, instead of cutting, the foliage is sprayed with an acid solution con-
taining arsenic. The acid kills the foliage and renders it permeable, whereupon the sap
from the leaves and stems, plus the arsenic from the spray solution, is drawn down into
the roots. Root systems of wild morning-glory (Convolznilus arvensis) and Russian
knapweed (Centaiirea repens) have been killed to depths of six feet in some instances
and often as high as 95 per cent of a population may have its roots destroyed to a
depth of four feet. Sodium chlorate, ammonium thiocyanate and other chemicals may
be translocated by this same mechanism (Crafts, 1935; Robbins, Crafts, and Raynor,
1942). Individual plants may be treated by cutting off their tops under one of the
above solutions, or by simply immersing them in a container of solution and allowing
them to stand until the tops are killed and rendered permeable. This latter method is
more effective when used on individual tops of plants that are interconnected under-
ground. Here the undipped tops continue to transpire and thus draw solution through
the dipped plant into all plants that are attached through the root system. A detailed
discussion of the mechanics of this method, its limitations, and its advantages will be
found in Robbins, Crafts, and Raynor (1942).

2) If the xylem elements contain gas under subatmospheric pressure, the dye
solution is forced into them as the gas contracts under the pressure of the solution at
atmospheric pressure. If the gas is water vapor, it will contract and condense com-
pletely at ordinary temperatures.

4) Air will be slowly forced into solution by the additional pressure resulting from
capillary forces. Surface tension would cause water to be forced into a tube with a

Grafts et al. — 158— Water in Plants

radius of 0.1 mm. with a pressure of about 0.015 atmosphere. This is equivalent to a
rise of about 15 centimeters in an open tube. More important than the actual forcing
of air into solution is the movement of liquid resulting from the shifting balance of
capillary forces between tubes of different sizes. When a stem is cut vapor may be
formed in a number of elements of varying diameters. If the gas is pure water vapor it
will condense completely and dye will enter and move along all conducting elements.
If air gets in through the cut or comes out of solution from the xylem sap, competition
between capillaries will cause readjustment after the first influx of dye and bubbles
may be seen to be moving in opposite directions in different elements as liquid from the
larger ones flows into the smaller to satisfy capillary gradients.

5) A slight elastic expansion of the liquid itself occurs with the release of tension.
This is of a minor magnitude.

Preston (1938) discussed the above possibilities and concluded that the
last two are relatively unimportant. He developed equations expressing the
rate at which dye would move into the xylem under the first three conditions
basing these upon Poisouille's law. He assumed that resistance due to
irregularities of the walls would not be appreciable. This contrasts with
the view of Dixon and Ewart, who considered that resistance in vessels
would reduce flow to one-half the expected amount. Poisouille's law is
based on streamline flow. When one considers the contour of vessel walls
and visualizes the turbulence which the pits and thickenings might cause,
he is forced to the conclusion that Dixon and Ewart were probably nearer
the truth than Preston.

By fitting his experimental data to his formula, Preston concluded that
many vessels in the ash tree used were filled with gas under pressures rang-
ing between 0.4 and 0.9 atmospheres. Other vessels may have become in-
jected due to elastic expansion of vessels containing water under tension.
Gas-filled vessels are located deeper in the trunk than water-filled ones.

Preston's conclusions were probably correct but they do not prove that
the vessels contained gas in the intact trunk. The difficulty with this type
of experiment, as may be easily confirmed under the dissecting microscope,
is that when the columns are under high tension they may be disrupted by
deformation of the walls before the vessels are actually severed. Hence,
water vapor may occupy many vessels by the time the opening is made and
the dye solution flows in. If the gas in the elements is pure water vapor, its
pressure at 20° C. would be about 17.5 mm. of mercury or approximately
0.023 of an atmosphere. The pressure difference would be practically one
atmosphere (actually 0.977) and inward flow should be rapid. The differ-
ence between this value and those quoted from Preston probably repre-
sents the role played by resistance due to turbulent flow along the tubes.

When tensions in the xylem are extremely high it seems doubtful if
injection can ever be accomplished without some vapor bubble formation
as the very inertia of the dye solution would resist the extremely rapid
motion required to maintain liquid continuity as the elastic expansion
throughout the length of a stem caused recession of the sap from the cut. To
be convinced of this one has only to watch the formation of vapor in the
elements of a woody stem of a plant in a state of permanent wilting as the
bark is removed under a dissecting binocular. Although the stem will under-
go considerable bending and twisting, as soon as the tensile columns are
subjected to deformation by jarring, squeezing, or cutting the tubes, they
break and vapor occupies the vessels as the water columns recede from the
point of rupture. Many, often most, of the elements have vapor in them be-
fore the dye can flow in and make contact with the sap columns.

Baker and James (1933) report that in the majority of cases investi-

Chapter IX — 159 — Uptake and Movement

gated the upward movement of dye approximately equaled the downward
movement, and the ratio was independent of the saturation deficit in the
plant if the measurements were made very quickly after the injection. This
led to the conclusion that the first equal injections were the result of released
tension. Long time measurements of dye penetration show the amount of
movement as a function of the amount of water deficit in the plant parts
toward which the dye is flowing.

Kennedy and Crafts (1927) and Crafts and Kennedy (1930)
studied the rate of movement of eosin solution into the stems of wild morn-
ing-glory plants growing under varying conditions of temperature, humidity,
and soil moisture and which were cut under the solution. They concluded
that if the rate of transpiration exceeded absorption (causing tension in the
xylem) dye penetration was influenced chiefly by the amount of transpira-
tion before cutting, i.e., the saturation deficit. When transpiration was low
it varied directly with the amount of transpiration and inversely with soil
moisture. Brett (1943) has confirmed these results.

Plant injection has become a standard practice in the treatment of min-
eral deficiency diseases including chlorosis, zinc deficiency, etc. A variety
of methods have been developed depending upon the nature of the chem-
icals to be supplied and the reaction of the tissues to them. Some applica-
tions are made as liquid through cut branches and bore holes at the base of
the tree ; others involve use of pure chemicals in bore holes or slits in the
bark, and some chemicals are readily absorbed through the leaves so the
injection is not required.

Because Roach (1938, 1939) reviews much early work on this topic and
describes detailed methods used by him and his collaborators for diagnosing
and curing mineral deficiencies, only a few of the recent publications will be
mentioned. Bennett (1931), using solutions and dry salts, developed
methods for treating lime-induced chlorosis of pears and the adoption of
his dry-salt method has resulted in salvaging a large acreage of fruit trees
that were destined for removal. Over 75,000 trees had been treated by
1931 and the method has been widely used since.

Roach (1938) and Roach and Roberts (1945) stress the use of in-
jection for diagnosing mineral deficiencies. In the latter paper the test
solutions used in detecting deficiency of N, P, K, Ca, Mg, Fe, Mn, Zn, Cu,
Ni, and B are described and all necessary equipment is pictured. Injection
methods for curative purposes for both liquids and dry salts are mentioned
and apparatus is illustrated. These methods are finding wide use in treat-
ing fruit trees. Their success depends largely upon a thorough knowledge
of the physiology of sap movement as the transpiration stream is largely re-
sponsible for distributing the chemicals.

Much effort has been expended in attempts to inject insecticidal, fungi-
cidal, and bacteriocidal agents to better the welfare of plants. However, at
present, less success has attended this work than that on mineral deficien-
cies.

Freezing of Stems: — Peirce (1936a, 1936 b) froze portions of intact
buckeye and castor bean stems with liquid air under various environmental
conditions, and, after detaching, tested the conductivity of the frozen portion
to gas by blowing through it. Under conditions of water deficit there seemed
to be evidence that some of the elements were not filled with ice. Such a
procedure might be faulty, however, because contractions and expansions

Crafts et al. — 160 — Water in Plants

must occur in a stem subjected to such rapid cooling. Expansion during
the formation of ice in the outer part of a stem would cause local strains on
the vessels nearer the center causing rupture in the liquid. Furthermore,
disturbance of forces of cohesion at the liquid: crystal interface as ice is
formed might result in rupture.

Transpiration Experiments: — In 1914 Bakke performed a series of
experiments on sunflower plants that were lifted from the soil and brought
into the laboratory to wilt. He made continuous transpiration experiments
using cobalt chloride indicator paper and found a sharp rise in transpiration
rate just beyond the permanent wilting condition. He interpreted his re-
sults as indicating that wilting occurs at a definite point and permanent
wilting represents the most advanced stage of wilting possible without seri-
ous rupture of the water columns in the xylem. Continuing the work in
1918, Bakke found the stomatal movements not to be important during
advanced wilting. During progressive wilting there was a gradual drop in
transpiration, followed by a plateau of no change and then an abrupt rise in
rate. The plateau was considered to represent a state of equilibrium be-
tween the plant and its environment representing the maximum tension
that the plant could develop. The rise at the end of the permanent wilting
range represented serious rupture of the water columns. When this rup-
ture was complete the plant could not be expected to recover when moisture
was added to the soil.

From a priori reasoning Knight (1922) decided that as a plant passes
from a state of turgor to a wilted condition, there is no reason to expect that
at any particular stage in the wilting process the water columns would sud-
denly rupture. He pictured replacement of water by gas as a gradual
process during wilting, the water columns being severed one by one until
enough are broken to bring about failure in the supply to the leaves. Work-
ing with detached shoots in potometers. Knight made simultaneous meas-
urements of stomatal opening and transpiration during progressive wilting.
Wilting was induced by closing the stopcock of the potometer, thus stopping
the water supply. There was a sharp increase in stomatal opening accom-
panied by an increase in transpiration during early wilting; this was fol-
lowed by a gradual drop. The increase however did not correspond with
that observed by Bakke, as it occurred early in the wilting process. Ob-
jection could be made to the use of detached shoots in such experiments for
reasons mentioned earlier.

Direct Observation of Xylem : — Numerous direct observations have
been made on the xylem of plants to determine the presence or ab-
sence of gas in the conducting tracts. Among the first to use this method
were Vesque (1883) and his student Capus (1883). Unfortunately their
method of preparing their material by cutting and peeling undoubtedly
caused formation of vapor phase in every element whose water was under
tension. Their observations were largely responsible for the persistence of
the idea that there were liquid-gas chains (Jaminian chains) in xylem con-
ductors.

HoLLE (1915) and Bode (1923) made critical observations on the state
of water in the xylem of plants. They concluded that even under conditions
of extreme wilting there were continuous intact columns of water in the
xylem. Conclusions of earlier workers that gas was present were found
to be the result of faulty preparation and observation. Bode used a hori-

Chapter IX — 161 — Uptake and Movement

zontal microscope to observe the vessel contents of rooted shoots of
Cucurbita, Tradescantia, Impatiens, and Elatostemma. In some instances
the xylem could be observed directly through the transparent overlying tis-
sues. Removal of the outer collenchyma before observation was found not
to affect the state of v^^ater in the underlying xylem, if care was taken to
avoid jarring or destroying the ducts. The water threads remained con-
tinuous in spite of wilting that greatly increased the tension. Any slight
local disturbance was found to cause a rupture when a high state of tension
existed.

Wilting and drying of leaves of sunflower and squash plants begin with
the most basal leaves and progresses upward. Under these conditions there
is found a progressively increasing number of xylem elements which contain
gas, either as a result of penetration from the outside through hydathodes
or drying areas or due to a rupture of columns caused by the strains placed
upon them as a result of changes in tissue form and tensions, which ac-
company shrivelling of the leaves (Stocking, 1943). Nevertheless, in-
tact water-filled elements are found even in severely wilted leaves.

Hains (1935) combined direct observation with a dye penetration
method to study the state of water in the xylem of trees. By carefully peel-
ing off the bark and exposing the xylem he was able to detect gas in the
vessels, due to differences in the refraction of light by gas- and water-filled
elements. If no difference in appearance was noted the elements contained
all gas or all water. Puncturing the stem he could determine whether gas
or water was present and injection of dye indicated the existence of subat-
mospheric gas pressure or of tensions in the water columns.

Experiments were carried out on Alnus, Acer, Aesculus, Samhiicus, and
Fraxinus. Gas was not present in the stems during early spring but ap-
peared at the onset of hot dry weather ; it disappeared during the wet fall in
Sambucus and Fraxinus. All outer wood of Alnus, Acer, and Aesculus con-
tained gas in the fall and Hains considered this to be chiefly in nonconduct-
ing fibres.

From these observations, plus many that have been made on the maple
tree {Acer) which is noted for its bleeding in the spring, it seems that dur-
ing the hot, dry period of summer when water becomes deficient gas may
form in most of the larger xylem vessels. These may therefore serve as
reservoirs during the summer as well as conductors during the spring
when water is abundant and the flush of growth is on. During winter and
early spring many of these probably regain their liquid contents, but in
some trees gas apparently continues to occupy a fair portion of the wood for
it is the gas pressure formed upon warming of the trunk that causes the
rapid bleeding observed in maple. The original source of this bleeding sap
is the roots that actively absorb water as spring growth commences ; the
sugar results from hydrolysis of starch present in the wood parenchyma.
Detailed studies on this hydrostatic-pneumatic system in trees have been
made by MacDougal and his associates.

Crafts (1939o?), studying Ribes inerine plants that were in a state of
permanent wilting, found that the bark could be removed without rupturing
the water columns in the outer xylem vessels. However, a slight jarring of
the exposed vessels, as by tapping with a dull instrument (the back of a
scalpel) caused formation of vapor, and recession of the water columns
took place so rapidly that it could not be followed ; change in appearance
from the translucent water-filled to the glistening gas-filled condition ap-

Grafts et al. — 162 — Water in Plants

peared to be instantaneous. Working with plants having lower deficits and
observing xylem elements of different sizes, it was possible to produce
bubbles that expanded rapidly or slowly and, using plants with little or no
water deficit, bubbles could be formed that gradually contracted and disap-
peared. These experiments parallel those of Bode and illustrate the role
played by capillarity in the healing of broken water columns under condi-
tions of reduced stress in the xylem. The simple fact that vapor bubbles
may be formed in uncut xylem vessels and that these continue to expand
after being formed is proof that tension exists, for the vapor phase would
not continue to expand if the pressure were above that of saturated water
vapor at the temperature of the experiment, that is around 17.5 mm. of mer-
cury. The extremely rapid expansion observed in vessels of plants that
are permanently wilted proves that the tension is high for in so expanding
the water originally present, except for the small amount involved in forma-
tion of vapor, must move out at high rates, overcoming inertia and the resist-
ance to flow through capillaries.

Tensions Developed in the Xylem; Direct Measurements: —

Difficulties in technique have thus far prevented the direct measurement
of tensions that occur in the xylem sap. Use of manometers attached directly
to cut branches, stumps, and stems entails severing of the conducting tracts.
Tensions greater than one atmosphere cannot be developed within a sys-
tem containing gas for the gas will expand indefinitely under subatmos-
pheric pressure. The attachment of manometers to bulk tissue rather than
to individual vessels permits the drawing of air into the system through
the intercellular spaces with the result that only pressures above the zero
atmosphere level can be measured.

BoEHM (1892) and Thut (1932a) were able to demonstrate that if
branches were carefully prepared a rise of mercury above the 76 cm. level
could be obtained. The use of conifers that possess tightly packed tissue,
few intercellular spaces, and short elements of the tracheid type tends to
prevent the occurrence of gas movement out of the wood and into the
manometer. Boehm was able to obtain a height of 90.6 cm. and Thut
recorded a maximum rise of mercury of 101.8 cm. in one experiment with
Juniperus. To obtain such a rise the base of the stem was boiled to re-
move air. Such experiments, while clearly demonstrating the ability of
the leafy shoot to lift mercury above the barometric level, do not pretend
to measure the actual tensions that exist within the conducting tracts of
intact plants.

Calculations by Dixon (1914) on the resistance to flow through xylem
conductors indicate that a force at least twice that represented by the
weight of the transpiration columns would be required to move water through
stems at a normal rate of flow. If this approximation is valid there should
exist tensions of 20 to 24 atmospheres or more in the uppermost parts of
tall trees during periods of rapid transpiration.

Potometer Experiments: — Renner (1912) used the potometer to
study tensions in the xylem of a plant. Uninjured branches, tops of shoots,
or twigs were installed in simple potometers. Freshly cut surfaces provided
for water uptake, and the resistance of the stem was increased by the use of
a clamp, or by double notching. After the rate of water uptake was ob-
served the branch above the potometer was cut. The stump still in the
potometer was taken into the laboratory and the rate of water uptake under

Chapter IX — 163 — Uptake and Movement

a known pressure difference was observed by attaching a suction pump to
the stem. On the assumption that the resistance in the stump remained
constant and that the rate of water movement into the stem was proportional
to the pressure drop across the resistance, Renner cakulated that tensions
of 1.0 to 1.5 atmospheres were customary in herbaceous plants. He found
an extreme tension of 5 atmospheres in one experiment with ForsytJiia. The
soil around all of the experimental plants was well watered, which probably
explains the low tensions in the plants.

Earlier (1911a) Renner had used potometer methods to determine
tensions in rooted bean plants as well as in leafy shoots. He recorded pres-
sure deficits of wilted leaves of 10 to 20 atmospheres, while tensions of
culture solution plants were found to be about 1 atmosphere. Renner be-
lieved that the maximum tension was limited by the osmotic pressure of the
leaves.

Nordhausen (1916, 1917, 1921) was skeptical of Renner's results,
feeling that important changes occurred in the resistance of the stem caus-
ing a reduced flow under the force of the pump. This would result in the
tension values being too high. He modified Renner's method by intro-
ducing a porous clay disk of known resistance at the base of the cut stem.
By comparison of the rates of flow through this resistance under the action
of the stem, with the rates obtained with a known pressure drop, he cal-
culated tensions of 3 to 4 atmospheres in trees and 7 to 8 atmospheres in
cut shoots. The higher values in the cut shoots he explained as arising
from increased water deficits from restricted supply caused by cutting and
applying the clay resistance.

Dixon (1924) believed that Nordhausen's values represented mini-
mum tensions as the clay resistance would cause some plugging of the stem
and air would be introduced during the preparation of the experiment.

HoLLE (1915) pointed out that the vessels in wilted leaves of Alliaria
officinalis are full of water as shown by direct observation. Under this con-
dition the diffusion pressure deficit of water in the vessels should equal the
osmotic pressure of the mesophyll cells (since turgor equals zero). The
osmotic pressure of the xylem sap due to solutes would reduce this value
slightly.

A modification of Renner's method was used by Eaton (1941) in cal-
culating the overall tension in the xylem. A water culture plant was placed
with half of its roots growing in a high salt solution and the other half grow-
ing in a low salt solution. The roots were alternated between the solutions
so that for a given time period there was an equal area of absorbing sur-
face in each solution. Measurement was made of the amount of water ab-
sorbed from each solution for a given length of time. It was assumed that
the flow of solution into the roots was proportional to the pressure head
and that the resistance was the same in both cases. An average value for
thjs tension at the base of the shoot which satisfied both sets of water uptake
and osmotic values was found to be 3.55 atmospheres.

No consideration was given to the effect of the different solutions on the
permeability of the roots, and the influence of time and temperature on the
osmotic pressure of the solutions was neglected. As Eaton points out,
such a calculation represents at best only an overall figure, and a fluctua-
tion from perhaps positive pressures at night to relatively high tensions dur-
ing the periods of rapid transpiration is probably undergone by water in the
xylem.

Grafts et al.

— 164

Water in Plants

As previously mentioned Preston ( 1938) studied the rate at which dyes
were injected into cut ash stems. He calculated the tensions which must
have existed at zero time, to cause the rate of injection observed, to be in
two instances of the order of 3 atmospheres. This value represented very
approximately in his estimation the tension present in the vessels im-
mediately after cutting. It is questionable whether this can be taken to rep-
resent the tension in the vessel system before cutting since, as Preston
points out, the equations which he used are not valid at zero time.

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Fig. 43. — DPD gradient from the root up the stem of
Arthrophytum haloxylon. The vertical axis is DPD gradient in
atmospheres per meter, the horizontal axis centimeters. The
zero point represents the crown and all negative values are
measured downward along the root. From Arcichovskij and
Ossipov (19316).

"Suction Tension" Measurements : — If there is a direct water con-
tact between the liquid in the xylem and that of the neighboring cells, the
diffusion pressure of water in the cells should approach quite closely the
diffusion pressure of water in the nonliving xylem conductors. Calculation
of the tension in the xylem should be possible from a knowledge of the dif-
fusion pressure deficit of water in the living cells and the osmotic pressure
of the tracheal contents.

A large amount of work has been done in measuring the DPD (suction
tensions) of plant cells and tissues. Ursprung (1935) and Beck (1928)

Chapter IX

— 165 —

Uptake and Movement

have reviewed the results; Molz (1926) has made many determinations.
The method has been described in Chapter VII.

Ernest (1931, 1934&) and Oppenheimer (1930a) have severely criti-
cised the plasmolytic methods for determining DPD. Some of these criti-
cisms are given in Chapter VII. Though many of them may be vaUd, the
method has provided an approximate picture of the water balance in cells
and tissues that is extremely useful in explaining the behavior of plants.

Newer Methods of Estimating Tensions in Xylem: — Arcichov-
SKij and associates (1931), in a series of five papers, discuss the results of
investigations on suction pressure measurements in plants. Six methods
are considered, i.e., gravimetric, potometric, "Schlieren," optical, mechan-
ical, and electrical methods. Fundamentally all of these methods are based
on determining the concentration of a solution which has a DPD equal to
that of the test material.

!

^ /9

Fig. 44. — Diurnal fluctuation in transpiration and in DPD at diflferent heights
on the stem of Arthrophytum haloxylon. The heavy dashes represent transpiration,
the heavy lines DPD, the Hght dashes relative humidity and the light line tempera-
ture. From Arcichovskij and Ossipov (1931&).

The "Schlieren" method appears to be the most useful. When a twig
with a ring of exposed xylem is immersed in a sugar solution, water will
move into or out of the cells, depending on whether the immersion solution
has a lower or higher DPD than that of the tissue. This water movement
will be apparent in the solution due to the differences in the refractive in-
dices. If the solution has a lower DPD water will move into the stem and
will be indicated by concentration currents falling away from the twig. Con-
versely, water moving out of the twig into a solution of higher DPD will be
detected by the rising of the diluted solution. If the experimental twig is
horizontal and the test area is in a glass chamber containing a sugar solu-
tion, the streaking of the density currents in the solution can be observed
and a solution having an equivalent DPD determined.

Certain objections to this method immediately come to mind. Such
an exposure of xylem caused by removing the bark must expose much rup-
tured phloem tissue, and phloem exudation into the test solution would set

Crafts et al.

166 —

Water in Plants

up complicating density currents. Furthermore, such a method is depend-
ent on a differentially permeable membrane and the walls of dead xylem
elements are not differentially permeable although it is possible that a care-
ful removal of bark would leave a layer of living xylem cells which could
act like such a membrane.

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Fig. 45. — DPD measurements on the petioles of squash plants. Solu-
tions injected into the petioles tend to assume a concentration having the
same DPD as the tissue. DPD values in atmospheres are shown on the
vertical axis. (From Stocking, 1945).

Arcichovskij and Ossipov (1931&) used this method to determine the
suction pressure of the Central Asia desert shrub Arthrophytum haloxylon
Litiv. DPD's up to 142.9 atmospheres were recorded for this plant and a
DPD gradient of as much as 44 atmospheres per meter was observed. The
specific gravity of sap flowing from cut twigs was very low in relation to
that of the external sugar solution, indicating that the moisture deficit of
the tissue was due to high tension rather than osmotic pressure. Figure
43 shows the increasing gradient in DPD from the roots up the stem of such
a plant as measured by Arcichovskij and Ossipov (1931Z?). Figure 44
shows the diurnal fluctuation in transpiration and DPD of Arthrophytum
with data on temperature and relative humidity.

Plants with hollow petioles such as those of squash or pumpkin are con-
venient material to use in testing the DPD of the tissue (Stocking, 1945).

Chapter IX

167 — Uptake and Movement

Standard sucrose solutions of known OP can be injected directly into the
petioles with little injury to the leaf and at periodic intervals one or two
drops can be withdrawn with a micropipette and analyzed by use of a re-
fractometer. When such a technique is used it is found there is a very
rapid initial change in concentration of the injected solution because there
is a tendency for the DPD of the tissue to approach that of the solution.
Subsequent to this initial change there is a diurnal fluctuation in the OP
of the injected solution following the normal trend of the DPD of the
tissue when the plants are growing in the field on a normal day. Figure
45 gives some evidence for such a diurnal fluctuation representing changes
in the tissue of from less than 1 atmosphere DPD at night to more than
5 atmospheres during the period of greatest water deficit in the day. Cul-
ture solution plants also exhibit the same general trends.

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Fig. 46. — DPD values of solutions in the petioles of a squash plant in the labora-
tory. Placing the plant in the sun caused a rapid rise in DPD. Reinjection on the
second day indicated a DPD of about 3 atmospheres.

A graphic reflection of the influence of the environment on the DPD
of water in the intact plant is illustrated in the case of a plant growing in
culture solution in the moderate atmosphere of the laboratory. Figure 46
shows the OP of sugar solutions injected into the petioles. As a result of
being placed in the sun for 20 minutes at 2:10 p.m. an immediate jump in
the OP of the injected solution was observed which gradually returned to
the lower steady state upon return of the plant to the laboratory. In this case
leaf 1 was originally injected at 9 :00 a.m. on July 1 with a solution having an
OP of 2.4 atmospheres. On the following morning leaf 1 was reinjected
with a sugar solution of OP 1.8 and leaf 2 with a solution of OP 11.5.
There was a very rapid drop in concentration of the solution in leaf 2 and
only a slow increase in solution 1, indicating a DPD of less than 3 atmos-
pheres in the tissue.

Rate of Water Movement Through Plants : — Although our knowl-
edge of the movement of water in the xylem dates back, as does much of our

Grafts et al. — 168— Water in Plants

insight into plant physiological processes, to the time of Stephen Hales
(1738), nevertheless the rate at which materials are moved through the tis-
sues of transport in the plant has been an elusive problem. Confronted, in
higher plants, by an organism which possesses in contrast to the higher ani-
mals no specialized pumping organ nor large, easily recognized conducting
tubes, the plant physiologist has been slow to understand the mechanisms
of transport in the plant.

The rate of movement of the ascending "transpiration stream" has been
studied in three ways : 1 ) observation of quantity of material moved and
cross sectional area of the tissue of transport, 2) by the use of indicators, and
5) by direct observation {see discussion above). Attempts have been made
using these methods to study the movement of the "assimilation stream"
as well.

Quantity of Material Moved and Area of Transport : — It is

obvious that if the amount of material moving in a given time through a
conductor of known cross sectional area is determined, the rate of flow can
be calculated. This method has, until the recent introduction of radioactive
tracers, been the chief method of studying the movement of materials in the
phloem. Such a method, while improved with our increasing knowledge
of the anatomy of the conducting tissues, at best is limited to average values
over relatively long periods of time.

Indicators: — The introduction of indicators into the moving stream
has been the most widely used method of measuring the rate of flow in the
xylem. These indicating methods have employed the use of four different
types of indicators: 1) salts, 2) dyes, 3) radioactive material, and 4) heat.
Some results of early work with indicators are presented in Table 44.

Salts. — While salt absorption, as often pointed out (Hoagland, 1944),
may be more dependent on climatic factors determining carbon fixation,
respiration, and other biochemical processes than on the amount of water
absorbed, still the transpiration stream provides an efficient and rapid means
of transporting to the rest of the plant body materials absorbed by the roots.
Salt movement may not necessarily follow exactly the flow of water, but in
the xylem elements where water movement is usually many times in excess
of diffusion rates, salt movement may approximate that of the water.

Sachs early pointed out the fallacy in using cut branches or plants with
injured roots in experiments on sap movement in plants, criticizing the re-
sults of early workers (McNab, 1871 ; and Pfitzer, 1878). His determina-
tions were made by use of lithium salts applied directly to the roots in solu-
tion or to soil. The rate of movement of the lithium through the plant was
tested by means of the typical lithium flame test.

Dyes. — While the use of dyes facilitated the detection of the tracer
materials, the possibility of adsorption and consequent reduced rate of move-
ment made such determinations open to criticism, particularly if strongly
adsorbed dyes were used. Strugger (1943) and others have used fluo-
rescent materials in studying the movement of the transpiration stream.
Special emphasis was placed by Strugger on the movement of the fluo-
rescent material via the cell walls from the i^ascular bundles through the
intervening tissue to the evaporating surface. While stressing the fallacy
of Sachs' imbibitional theory in respect to intravascular movement of wa-
ter, he places great emphasis on imbibition as a factor in the extravascular
movement. Materials passed rapidly through the walls of the mesophyll
and palisade tissue without entering the living cells.

Chapter IX

169

Uptake and Movement

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firmed Strugger's results on water migration in the walls of parenchyma-
tous cells.

Radioactive tracers. — While radioactive indicators have proven ex-
tremely useful in clarifying the path of mineral transport (Stout and
HoAGLAND, 1939), and while such tracers are being extensively used in
investigations on food manufacture and transport in plants, relatively few
experimenters have employed them in the study of the velocity of water
movement in the xylem. Huber (1932), together with his student Baum-
GARTNER (1934) uscd Thorium B in studying the movement of the xylem
contents in some conifers and in Sanchezia. Their results are given in

Grafts et al.

170 —

Water in Plants

Table 44. In this early work the electroscope and photoplates were used
as analytical tools. Apparently analyses with the Geiger counter of the
movement of radioactive phosphorus in the phloem and xylem of an intact
plant are useful only when there are large concentrations of radioactive
material in a given portion of the plant (Colwell, 1942).

Heat. — The ingenious thermoelectric method first used by Rein (1928,
1929) for determining the rate of flow of blood and developed by Huber
(1932) for measuring plant stream velocities, allows determinations to be
made on intact plants with a minimum of injury. Repeated and localized
measurements are possible on the same plant; this is a distinct advantage
over any method previously used. The method consists essentially of the
brief local application of moderate heat and of measurement by means of
thermocouples of the rate of transfer upward or downward. In rapidly mov-
ing streams the thermocouples are placed at a distance sufficient to prevent
interference by radiation or conduction. This is from 4 to 25 cm. from the
heating element.

Fig. 47. — Arrangement of heating element and thermocouples used by Huber and
Schmidt (1937) for measuring slow rates of movement of sap streams. Cu — Cu
represents the thermocouple points.

Measurement of velocities in more slowly moving streams, i.e., in conifers
or in the phloem, is more difficult. Dixon (1937) modified the thermo-
electric method by placing the thermoindicators 1 cm. apart and the near
junction 1 cm. from the heater. Both the junctions and the heater were
placed on the undisturbed bark. Determinations took from 'one-half to a
whole hour. Huber and Schmidt (1937) modified the thermoelectric
method by bringing the thermocouples closer to the source of heat. They
placed one thermocouple on either side of the heat source with the lower ele-
ment somewhat closer than the upper, i.e., 16 and 20 mm. By measuring
the rate of heat transfer both in a normally transpiring plant, and a control
in which, due to very humid conditions, there was supposedly no movement
of the transpiration stream, they were able to measure rates of sap move-
ment in the neighborhood of 5 cm. per hour. Figure 47 shows the ar-
rangement of the heating element and thermocouples used by Huber and
Schmidt.

Chapter IX

171

Uptake and Movement

Variations in the Rate of Sap Flow: — Use of the thermoelectric
method permitted the measurement of velocity of sap flow in roots, stem
bases, and tips. A striking lessening of velocity was observed by Huber
and Schmidt (1936) in going from base to tip of trees. This is explained
on the relationship of number of conducting elements to number of leaves
drawing water through these elements. Baumgartner (1934) found that
the slender branches of birch exhibited the opposite state, i.e., the rate in-
creased rapidly toward the tip. In this case the sum of the effective cross
section of the branches must be less than that in the stem. Roots are
especially adapted to rapid flow of material in the xylem as pointed out by
RiEDL (1937).

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40

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6a/n.

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Fig. 48. — Diurnal variation in rate of sap movement in Larix, Picea, and Fagus.
The vertical axis is rate in centimeters per hour. The solid line through the center
represents the null point readings on an excised shoot. From Huber and Schmidt
(1937).

Huber and Schmidt (1937) investigated the velocity of the transpira-
tion stream in a number of trees and classified them into three groups : 1 )
oak-type, in which the velocity diminishes toward the tip, being greatest at
the base of thick limbs; 2) birch-type, where the velocity increases toward
the tip and from stem to bough to twig; 3) the normal type, where the
differences in velocity between different parts of the tree were absent. This
last type, according to these authors, was postulated by the Leonardo-
Jaccard theory of constant water conductivity as being a normal situation.
The majority of the trees investigated fell into the oak-type.

Ring porous trees such as oak, ash, and locust have their xylem-con-
ductive area confined in general to a relatively small portion of the outer
part of the annual rings. According to Huber and Schmidt, this is a re-
sult of air entering the wide vessels during the course of vegetative develop-

Grafts et al. — 172 — Water in Plants

ment. Consequently the rate of sap movement is in this case high (up to
43.6 meters per hour), being about ten times that found in diffuse porous
trees such as beech, maple, linden, etc., where a greater conducting area is

available.

In the case of conifers, the velocity of sap movement is usually less than

half a meter per hour.

Diurnal variations in rate of flow, as would be expected, follow fairly
closely the transpirational fluctuations. From a relatively quiet transpira-
tion stream at night, movement is initiated in the morning in the upper
branches and gradually reaches the lower parts of the stem and lastly the
roots. At night the reverse takes place as water moves into the roots and
stem after the tip has ceased to transpire. This movement continues as
long as water deficits exist within the plant. Figure 48 shows the diurnal
variation in the rate of sap movement in Larix, Picea, and Fagus. The
maximum velocity of the transpiration stream is reached several hours after
the peak of transpiration.

Annual variations in transpiration rates are reflected in annual cycles
in the movement of water through the plant. Baumgartner (1934), using
birch, found such a trend. Any factor causing a reduction in absorption or
transpiration will correspondingly affect the rate of movement of water
through the plant.

Water Balance and Redistribution: — During conditions of high
transpiration a shrinkage of stem diameter is a characteristic phenomenon.
Bode observed an expansion of vessels upon cutting and explained this as
being due to the release of high tensions in the stem. MacDougal, Over-
ton, and Smith (1929) report that their dendrographic measurements
"confirm the contention of several investigators that the tensions set up by
the transpiring leaves may amount to as much as two hundred atmospheres."
Undoubtedly the greatest strain on the cohesive water columns in an indi-
vidual plant must occur during the most advanced stages of wilting as long
as liquid continuity is maintained.

Meschazeff (1882) is mentioned by Delf (1912) as being the first to
call attention to the ecological importance of internal redistribution of water
in plants during periods of water deficits. Pringsheim (1906), Chand-
ler (1914), Bartholomew (1926), Savastano (1934), and Furr and
Taylor (1935) have emphasized that certain plant organs may act as res-
ervoirs from which others may draw water. Young leaves of some plants
are able to draw water from older leaves and remain turgid longer during
progressive wilting. Fruits may act as reservoirs from which leaves can
draw water during wilting.

Pringsheim (1906) and Chandler (1914) found a correlation be-
tween the osmotic pressure of the sap expressed from an organ and its ability
to absorb water. Beck (1928), however, repeated some of Pringsheim's
work and reported that water did not move along an osmotic gradient but
along a "suction tension" gradient which was maintained by reason of dif-
ferences in the elasticity of the cell walls of young and old leaves.

While it is obvious that if direct water contact exists between two
organs the water will flow along a gradient in diffusion pressure of water,
it is not necessarily true that such a gradient is maintained only by differ-
ences in osmotic pressures and wall pressures of the various cells. Imbibi-
tional forces as well as osmotic forces influence the diffusion pressure of
water, and as has been discussed earHer, certain recent evidence has been

Chapter IX — 173 — Uptake and Movement

presented to support the theory that the vital activity of the plant cells also
influences the diffusion pressure of water (cf. Chapter VIII) . Although the
amount of colloid in a highly vacuolated parenchyma cell may be small and
perhaps can be neglected in studying the water relations of the individual
cell, imbibitional forces associated with the colloids in less vacuolated cells
may be very important.

Stocking (1943) was unable to find a correlation between the osmotic
pressure of the expressed saps of squash leaves and the sequence of wilting
except in the case of shaded leaves where the osmotic pressure falls to an
extremely low value. Wilting in this case occurs first in the shaded leaves.
In squash the tip leaves may continue to grow and expand even when the
plant is removed from the culture solution and allowed to wilt in a mod-
erately shaded condition. The flower buds may continue to open and bloom
while the basal leaves are dying. It seems probable that the greater im-
bibitional pressures of the slightly vacuolated, highly protoplasmic, expand-
ing cells of the young leaves could account for the lowered diffusion pres-
sure of the water in their sap. Thus imbibition enables them to maintain
their turgidity in the presence of the wilted lower leaves ; otherwise these
cells must actively secrete water into their vacuoles. Kerr and Anderson
(1944) have recently come to this conclusion in regard to the ability of
young cotton seeds to absorb water.

The water reserves of plants in general are not equally distributed
among the different organs but the lower leaves and shade leaves are usually
richer in water and lower in osmotically active solutes than the upper or
sun leaves (Marsh, 1941 ; Maximov and K. Maximov, 1924). During the
wilting of sunflower this difference disappears as a result of withdrawal of
water from lower leaves by the upper. When such wilted plants were
watered, Maximov and K. Maximov found that the older leaves were un-
able to recover but continued to dry out. However, if these leaves were
cut and placed in water they were able to again recover their turgor. The
Maximovs explain this as being due to the greater absorbing power of
the young leaves and hence their ability to deprive the older leaves of water
even after the wilted plant was well watered.

Direct observation of the xylem contents led Stocking (1943) to con-
clude that the lower leaves of severely wilted sunflower plants contain many
air filled xylem elements. Such a discontinuity of the water in the xylem
would lead to a high resistance to water movement. He believed that
whereas the young leaves were able to obtain water during the wilting of
the plant at the expense of the older leaves, the application of water to the
soil should cause the upper leaves to regain their turgor and failure of any
leaves to recover could in part be traced to a disruption of the xylem con-
tents so that the rate of water absorption could not reach that of water
loss. The recovery of the basal wilted leaves when placed with cut petioles
directly into water as found by Maximov tends to substantiate this con-
clusion.

Chandler (1914) felt that the internal redistribution of water in the
plants which he investigated occurred at such slow rates that movement
would not have to occur through the non-living xylem conductors but could
occur through the living cells of the stem.

The effects of drought upon the various functions of plants are im-
portant in agriculture and forestry. Work in this field is considered in
Chapter X.

Grafts et al. — 174 — Water in Plants

Translocation of Solutes :— Work by Maskell and Mason (1929) and Clements
and Engard (1938) on inorganic solute movement, and by Stout and Hoagland
(1939), and Bennett and Snell (1939) using radioactive indicators shows that the
major primary movement and distribution of mineral nutrients in plants takes place in
the xylem in conjunction with the transpiration stream. Hoagland (1944) has em-
phasized that absorption of water and salts are largely independent, and the selective
uptake of nutrient ions by living cells during their upward passage through the xylem
further indicates their independent behavior. However, while they occur together to
make up the xylem sap there seems to be no reason for assuming an independence of
movement; once started on their way through the xylem tubes, water and salts move
along together, and, with the exception of those absorbed along the route they reach
their final destination in the leaves where the bulk of the water is lost by transpira-
tion.

Much of the salt absorbed by roots is used in the building of leaves. However,
when these have accumulated sufficient to meet their needs for growth and the osmotic
retention of water, there is evidence (Mason and Maskell, 1931) that the surplus is
moved out via the phloem in conjunction with carbohydrates.

Concerning food movement in plants, there is general agreement that gradients in
concentration are intimately involved (Mason and Maskell, 1928a, 19286; Curtis,
1935; LooMis, 1935, 1945; Engard, 1939a; Huber, Schmidt, and Jahnel, 1937). On
the mechanism of movement there are many theories ; these may be grouped into two
categories : 1 ) those postulating movement of solute molecules in, through, or upon
the surface of the sieve-tube protoplasm. By this mechanism solutes are pictured as
moving along gradients but independently of each other and of the solvent water; 2)
the mass-flow or convectional-flow theory that visualizes a flow of solution through
the phloem along positive gradients of hydrostatic pressure developed osmotically. The
first category includes several mechanisms including diffusion accelerated by proto-
plasmic streaming (Curtis, 1935), "activated diffusion" a term implying an ac-
celerated diffusion carried on by the protoplasm with the utilization of metabolic energy
(Mason and Maskell, 1937), and diffusion or some analogous process accounting
for rapid attainment of equilibrium by molecules adsorbed on the surface of the sieve-
tube protoplasm (Loomis, 1935, 1945; Clements, 1934a, 1940). All these call upon
the active protoplasm for the required energy and their advocates picture the function-
ing sieve tube as an active element, rich in protoplasm, and capable of expending
metabolic energy at a high rate.

The second category includes only one mechanism but presents two views of the
sieve-tube function. That of Munch (1930), introducing the symplast concept,
visualizes the interconnected protoplasts of the plant as constituting a continuous sys-
tem, relatively impermeable on its outer surface but highly permeable throughout its
mass. The sieve tubes are pictured as making up a specialized mechanism of the
symplast where water, absorbed osmotically, brings about high turgor in the regions
of synthesis. This turgor promotes a rapid mass-flow of solution through tubular con-
nections that traverse the sieve plates to regions of utilization where assimilation, res-
piration, and condensation for storage reduce the osmotic activity of the solutes allow-
ing the water to return to the xylem. Crafts (1938, 1939a), on the basis of anatomical
studies, contends that the mature, functioning sieve tubes become permeable, allow-
ing a ready passage of solutes in solution from cell to cell. Leakage to the outside is
prevented by a limiting layer of living cells around each sieve-tube strand. Movement
from mesophyll to sieve tube is by diffusion, accelerated by protoplasmic streaming via
the symplast. Removal of assimilates from the sieve tubes is pictured as an active
absorption by living parenchyma cells of the phloem followed by symplastic move-
ment to points of utilization or storage. The mass-flow mechanism involves a recircula-
tion of water; Munch calculates that around 5 per cent of the total water used by
the plant is involved in this recirculation. He performed experiments wherein this
water as it flowed from the cambium of trees was collected and measured.

The protoplasmic theories are supported by evidence that under certain conditions
solutes appear to move independently in the phloem, that the sieve tubes are rich in
proteinaceous materials indicating a high protoplasmic content, and that radioactive
phosphorus absorbed into the phloem of leaves moves both upward and downward in
the stem whereas mass flow should be predominantly in one or the other direction. The
greatest weakness of these theories is that no exact physical mechanism is known or
has been pictured that will account for rapid transport of the quantities of material
found to move either along the protoplasm or through the lumen independent of the

Chapter IX — 175 — Uptake and Movement

water. From the pictures that have been presented in the preceding chapters of the
molecular structure of water, carbohydrates, amino acids, and proteins, it seems that the
mutual attraction of these substances, as indicated by the various points available for
coordination through hydrogen bonds, would resist the movement of food substances
either along the surface of the protoplasm or through static water. Those molecules
that were held in the protoplasm would be removed only with difficulty. Those in the
solution occupying the lumina of sieve tubes would diffuse independently over short
distances to satisfy local gradients ; their rapid movement over great distances inde-
pendent of the solvent is not possible by any known physical mechanism.

Although the rapid migration of hydrogen and hydroxyl ions has been pictured as
involving a proton jump along chains of coordinated water molecules, a similar move-
ment of large molecules seems improbable.

The mass-flow mechanism is supported by evidence for a permeable condition of
the functioning sieve tube, the presence of a concentration gradient of osmotically active
materials in the phloem and the phenomenon of phloem exudation that seems, at least
in some instances, to account for normal rates of flow. It receives substantiation from
studies on the movement of virus, auxin, and radioactive indicators that depict a simul-
taneous transport of foods and indicators, depending largely upon the establishment of
source and sink by regulating assimilation or hydrolysis at the source and utilization
or storage at the sink. Weaknesses of the mass-flow theories include a lack of
knowledge relative to the resistance offered by cross walls in the phloem, need for more
convincing evidence for the passive function of sieve tubes, a fundamental discrepancy
in the composition of phloem exudate from cucurbits as contrasted with composition of
their fruits, and a general lack of analytical data on the sieve-tube contents of a variety
of plants. Cucurbits which have been employed so much have proved atypic (Crafts
and LoRENZ, 19446) and should be used with caution. Future work, to be of value,
should make use of viruses, auxin, and radioactive isotopes that can be injected with
little or no injury and detected by very delicate and accurate methods.

Reviews on translocation cover the literature quite thoroughly up to about 1940;
Curtis (1935) and Mason and Phillis (1937) emphasize the viewpoint of the
protoplasmic theories; Crafts (1938, 19396) supports the mass-flow hypothesis. More
recent publications on the relation of the structure of the sieve tubes to their function
(Crafts, 1939a, c) extends the view that these elements become permeable at maturity
allowing ready flow of nutrients in solution through the phloem. Observations on
phloem exudation are extended. Clements (1940) and Cooil (1941) question the evi-
dence for the lowered activity of mature sieve tubes and the attendant high permeability.
Crafts and Lorenz (1944a, 6) found translocation into the fruits of cucurbits to
be very rapid but concluded that phloem exudation in these plants could not be accepted
as a manifestation of normal phloem transport. The carbon : nitrogen ratio of the
exudate was not compatible with that of the fruits.

Analyses of the stems and leaves of raspberry by Engard (1939a, 6) support the
view that transport of organic materials follows a diffusion pattern, gradients being
present where translocation occurs. Major carbohydrate movement is in the form
of sucrose and occurs in the phloem. Nitrogen absorbed by the roots moves upward
freely in the xylem and is available for reduction and elaboration to amino acids and
protein in all living cells of the plant.

LooMis (1945) from many studies on carbohydrate metabolism in maize con-
cludes that sucrose is the principal compound involved in translocation. He considers
that neither the protoplasmic nor mass-flow mechanism can explain carbohydrate trans-
port in maize and proposes a mechanism involving polarized movement, a secretory
type of movement that commonly takes places against gradients in concentration.
Went and Carter (1945) show that injury of the petioles of tomato plants hinders
sugar transport through the adjacent stem.

Indicators of various types have been employed in translocation studies. Rouschal
(1941) used fluorescein and found that it tends to accumulate in young active cells and
in the protoplasm of mature sieve tubes but not in their lumina. The dye streams
along with assimilates in the phloem and this flow can be reversed, hence he pictures a
mass-flow mechanism. He found that whereas the main flow of the assimilate stream
from a leaf occurred in the large sieve tubes of the petiole, independent and opposed
streaming may take place in extrafascicular sieve tubes. Thus movement both in and
out of a given leaf might occur by mass flow. Such movement might also take place
in opposite directions in the stem in different vascular bundles.

Radiophosphorus has been used as an indicator. Biddulph (1940) found this ele-

Crafts et al. — 176 — Water in Plants

ment to be rapidly absorbed by bean roots and carried upward in the transpiration
stream. In further experiments (1941) he found radiosposphorus injected into bean
leaves to migrate out, the initial movement being predominately downv^^ard, a fair por-
tion reaching the roots during the evening following injection. A small amount moved
upward. Such movement might take place by the mechanism mentioned by Rouschal.
CoLWELL (1942) used radiophosphorus for translocation studies on squash plants.
His results emphasize the care needed to restrict movement to the phloem. When the
tracer is so restricted, its movement is correlated with food movement in the plant.

BiDDULPH and Markle (1944) studied the distribution of radiophosphorus in cot-
ton plants following injection into leaves. They found movement out of the leaf and
down the stem to occur at a rate of better than 21 cm. per hour. Though movement
followed a diffusion pattern, rates indicated an active mechanism. Upward move-
ment in the stem was variable. Movement from the phloem into the xylem occurred,
resulting in a recirculation within the plant. Rabideau and Burr (1945) used radio-
carbon as a tracer for transport studies. Exposing leaves to CO2 containing C^^,
photosynthesis resulted in formation of carbohydrates that moved both upward and
downward in the plants. Radiophosphorus applied to roots moved readily past killed
areas of the stem but photosynthate containing radioactive carbon failed to pass such
regions. They conclude that the latter materials move exclusively through the phloem.
Studies on translocation of the reproductive stimulus in plants have shown that
flower induction is caused by a definite substance that may act locally or may be
moved. Loehwing (1938) using a slit panel, so that the tops and bases of soybean
plants could be subjected to different photoperiods, showed that whereas flower induc-
tion is normally localized in these plants, defoliation brought about transfer from donor
to receptor regions. He concluded that flower initiation was not caused by photo-
synthates but by some hormone-like substance. Flower initiation on a receptor region
subject to long-day treatment could be brought about by defoliation if a donor region
consisting of another portion of the plant was given short-day treatment.

Hamner and Bonner (1938), using Xanthinm pennsylvanicum, found that though
the initial perception of the floral stimulus is by mature leaves subject to a short photo-
period, the stimulus may be transported from these to other portions of the same plant
or that it might move through a graft or across a diffusion contact to another receptor
plant. Defoliation is not required in Xanthinm as the stimulus moves normally both up
and down the stem to donor regions that have not received induction. They attribute
floral initiation to a definite hormone-like substance.

WiTHROW and Withrow (1943) studied floral induction in the same plant and
found translocation between an induced donor plant and a receptor only when tissue
union was established as a result of uninterrupted tissue contact for four days or more.
The floral stimulus failed to translocate downward through a killed petiole or through
functional xylem. It also failed to cross a ring. They concluded that it moved only
in the bark, a result in harmony with tliose of Moshkov (1939). Stout (1945), study-
ing translocation of the floral stimulus in sugar beet, found that it would move down
a donor shoot and into a darkened shoot but not into an illuminated shoot^ where all
three shoots came from the same beetroot. This substantiates the defoliation results
of Loehwing and indicates that the flower-inducing substance like curly-top virus
(Bennett, 1937, 1940a, b) is translocated along with food material.

Further studies on virus movement (Bennett, 1940a, b; Hildebrand, 1942;
Hildebrand and Curtis, 1942; Bennett, Carsner, Coons, and Brandes, 1946)
confirm Bennett's earlier convictions (Crafts, 1939b) that rapid transport of virus
takes place in the phloem and is correlated with food movement. If this is true,^ and
much evidence now indicates that it is, there seem to be only two interpretations:
first, that virus particles are bonded to food molecules and that they move together
along concentration gradients of food, or, second, that they all move together by mass
flow of the assimilate stream. The first possibility seems doubtful in view of the ac-
cumulation of curly-top virus in the seed coats of sugar beet seed during storage of
carbohydrates in the seeds. There seems to be no logical reason for their separation
at this point if they are really tightly bonded. That Bennett favors the second
alternative is indicated in his review article (Bennett, 1940&).

Studies on transport of thiamin in tomato by Bonner (1942) show that this
material is synthesized in mature leaves and transported to the young developing leaves,
where it concentrates to a high level, and to the roots. It accumulated above a girdle at
the second node of the plants and below one made by steaming the stem in a region
between the mature leaves and the young expanding ones. These tests indicate a

Chapter IX — 177 — Uptake and Movement

movement in the direction which food materials might be expected to move. Studies
on pantothenic acid and riboflavin (Bonner and Borland, 1943) indicated that distri-
bution of these compounds follows a pattern similar to that of thiamin. Pantothenic
acid accumulated at girdles in the same way as thiamin; riboflavin failed to accumu-
late.

In a third series, Bonner (1944) tested large lots of tomato plants by girdling
some and leaving others ungirdled and measuring concentrations initially and finally
present in both girdled and ungirdled plants. He found that thiamin, pyridoxine, panto-
thenic acid, riboflavin, sucrose, total nitrogen, and non-protein nitrogen all accumu-
lated above the girdle. The extents and rates of accumulation varied and Bonner
questions the value of such evidence as it relates to the mechanism of mass flow. In
view of the possible differences in rate of synthesis, rate of transport, degree of accumu-
lation, utilization in growth, destruction, and possibly leakage into the xylem, it seems
unlikely that uniform rates and ratios of accumulation could be expected. The evi-
dence, such as it is, indicates a simultaneous movement of these various materials in
the tomato, and such movement can best be explained by mass flow.

Recent work on translocation of plant hormones has concerned the effects of
dichlorophenoxyacetic acid on weeds. In 1944, Beal found that the roots of sweet pea
produced nodule-like swellings and that the stems bent and grew abnormally following
application of 4-chlorophenoxyacetic acid to the stem or leaflets. Evidently this material
moved within the plant.

Ferri (1945), studying translocation of synthetic growth substances, showed that
they could be absorbed by roots from the soil and moved upward through the xylem.
Such movement was independent of living cells. Similar experiments were performed
by Skoog in 1938.

Recent work on absorption and translocation of 2,4-dichlorophenoxyacetic acid
by Mitchell and Brown (1946) shows that movement of the stimulus resulting from
treatment is correlated with movement of organic foods in bean plants. When the
2,4-D was applied to roots the resulting stimulus was translocated through non-living
cells of the stem, indicating that it traveled in the transpiration stream in the xylem.
Weaver and De Rose (1946) obtained similar results with bean plants. In alfalfa,
basipetal movement was most rapid in young active tissues. Figures 1 and 2 of their
paper give excellent evidence for upward movement in the xylem past a killed region
and subsequent accumulation in the phloem. This is reminiscent of the results of
Maskell and Mason for nitrogen and of Skoog for auxin.

From this review it is evident that the tendency is toward use of non-toxic and
naturally occurring indicators in translocation studies. The more recent work sup-
ports the view that such indicators, when in the phloem, accompany any food materials
in their movement through the plant. The simplest and most compatible mechanism
to account for such flow would seem to be mass flow along hydrostatic gradients de-
veloped osmotically. Such a mechanism involves a recirculation of water and entails
a close correlation between the functioning of xylem and phloem. A thorough con-
sideration of this mechanism in all of its ramifications brings out the fact that practically
every plant function is in some way tied in with processes of solute and water move-
ment. Water is absolutely essential to the welfare of higher plants and as such plants
have left their moist habitats and invaded the valleys, the mountains, and the deserts,
they have developed intricate absorption and translocation mechanisms and marvelous
protective coatings. By means of these they have maintained their aqueous sur-
roundings to a remarkable degree ; that is, they have taken their environment with
them. And if we, as agriculturists, wish to further their welfare and so protect our-
selves, we must, to the limit of our abilities, improve the environments of our crop
plants. We must do to a greater degree what the plant has so largely done for itself,
that is, we must simulate the perfect environment for plant growth and production by
supplying, along with minerals and a favorable soil, an adequate and unfailing supply
of water.

Summary: — Simple plants having no vascular tissue are limited to moist habitats;
higher plants have developed tissues for rapid uptake and transport of water ; protective
layers restrict water loss; these plants survive dry habitats whilst their living cells
have an aqueous medium. The soil acts as a reservoir from which the plant can absorb
so much water.

Water may be absorbed actively from soils near their field capacity. In a saturated
atmosphere such absorption causes guttation. Under conditions of high transpiration
water loss exceeds uptake and a passive absorption by the roots takes place.

Grafts et al. — 178— Water in Plants

Water movement takes place in the non-living xylem elements along a hydrostatic
gradient developed by transpiration pull. The water columns are in a state of tension
much of the time, a state made possible by the forces of cohesion (internal pressure)
between the water molecules and adhesion between water and the cellulose walls. When
such columns break, bubbles of water vapor form and the elements occupied by them
are rendered ineffective in water transport. When tension is reduced, such bubbles
may shrink and collapse, mending the columns.

Studies on the xylem have been made using dyes, freezing, transpiration measure-
ments, and direct microscopic observation. These studies largely confirm the co-
hesion mechanism. Many methods have been used to estimate tensions in the xylem;
potometer methods, measurement of "suction tension", and observation of differences
in refractive indices have proved useful. Periodic measurement of the concentration
of solutions injected into the petioles of squash leaves has given a view of changes in
water status of the plants.

Many methods have been used to study rates of water movement in plants ; com-
parison of the weight of water lost with the transverse area of conducting elements
gives an over-all average rate. Indicators, including salts, dyes, radioactive materials,
and heat have proved valuable; rates up to several meters per hour have been meas-
ured. Rates of flow vary with water supply, transpiration, and with structural varia-
tions in the plant.

Water balance in plants varies and with it water distribution. Redistribution be-
tween leaves, fruits, trunk, and roots takes place under fluctuating stress. Young
leaves usually exceed old in their ability to absorb and hold water ; sun leaves exceed
shade leaves.

Translocation of water and solutes in the plant are correlated. Minerals absorbed
from the soil travel upward in the xylem with the transpiration stream. Foods syn-
thesized in the leaves move downward to the roots and upward to growing parts and to
developing fruits through the phloem. Excess minerals, with the possible exception of
calcium, may move out of leaves via the phloem.

Two main theories on the mechanism of food movement are 1) the protoplasmic
theories postulating movement in, on, or along the surface of the sieve-tube protoplasm
independent of water and accelerated by metabolic energy; 2) mass flow, picturing
a simultaneous movement of foods and water in solution through the lumina of the
sieve tubes along a gradient of hydrostatic pressure developed osmotically. Sieve tube
elements are assumed to have a ready permeability for such flow and the solution under
pressure is maintained in phloem strands by sheaths of living parenchyma cells.

Current research on translocation indicates that sugars, nitrogen compounds,
fluorescein, radiophosphorus, the flower-inducing hormone, phloem-limited viruses,
thiamin, pyridoxine, pantothenic acid, riboflavin, auxin, and 2,4-dichlorophenoxyacetic
acid may all be transported in the phloem and some experiments indicate that two or
more are commonly carried along together. The simplest explanation for such move-
ment is the mass-flow mechanism which involves a recirculation of water and entails
a close correlation between the functioning of the xylem and phloem.

Chapter X
WATER LOSS AND WATER RETENTION

Introduction : — The maintenance of a favorable balance between water
loss and water absorption is essential in the economy of a plant, for growth
and development depend upon an adequate water supply. Favorable, as
used here, does not imply a positive hydrostatic pressure within the xylem
conductors, nor does it necessarily mean a positive supply in the soil. It
has been shown that a plant may absorb water from the soil against a dififu-
sion pressure deficit variously estimated to be 4 to 16 atmospheres, and it is
obvious that tensions within the xylem may attain much higher values in
plants that are developing normally. The inference is that there are definite
limits of DPD for water in the soil, and water in the plant, above which the
plant cannot operate normally. And because of these limits, water loss
must be eventually counterbalanced by an equal uptake if the plant is to
grow and develop and maintain life throughout a normal span. Because
the larger vessels of the xylem, and many parenchyma cells of xylem,
phloem, and cortex may act temporarily as reservoirs, the plant may survive
diurnal periods when loss exceeds uptake. However, such deficits must be
replenished, at least during periods of active growth, if the plant is to thrive.
And because of the interrelation between the various processes of absorp-
tion, movement, and loss, an effect on one will cause a correlated effect on
the others. The term water balance is commonly used by plant physiologists
to describe these processes as they relate to the water economy of plants.

Transpiration, that is, loss of water from plants in the vapor form, has
been termed both beneficial (Clements, 1934&) and a necessary evil
(Barnes, 1902; Curtis, 1926). Probably both viewpoints have some
justification. Certainly the maintenance of moist wall surfaces within the
stomatal chambers of leaves, for absorption of CO2 during rapid photo-
synthesis, results inevitably in loss of water by evaporation, and much
evidence is at hand to prove that such evaporation is usually far in excess
of any requirement, because plant growth in tropical regions where the air
approaches water saturation is very luxuriant.

Although the work of Hasselbring (1914), Kiesselbach (1916),
MuENSCHER (1922), and Mendiola (1922) indicates that different rates
of transpiration may occur without materially affecting mineral salt absorp-
tion, experiments on translocation of minerals within the xylem by Clem-
ents and Engard (1938), Wright (1939), and Stout and Hoagland
(1939) prove that transpiration facilitates the distribution of minerals
within the plant. More recent studies by Hoagland ( 1944) show that the
previous salt status of the plant is extremely important in such studies. It
seems therefore that some loss of water by transpiration is necessary; on
the other hand, transpiration in most terrestrial environments is far in
excess of requirements for purposes of translocation. Hence, conserva-
tion of water by the plant is important both economically, where the supply
may fail and so limit growth, and essentially for xerophytes, where the
water supply may determine survival.

Most studies on transpiration emphasize water loss and the relation of
the various physical mechanisms and physiological processes to it. From

Grafts et al. — 180— Water in Plants

the ecological viewpoint water retention or the prevention of loss is of the
greatest importance. From this viewpoint it becomes obvious that the pri-
mary function of the above mechanisms and processes in the plant is water
conservation. Opening of stomata is necessary for the gaseous exchange
essential to photosynthesis ; stomatal closure is just as necessary for the
conservation of water, particularly in arid regions. While stomatal open-
ing is of general occurrence and most plants photosynthesize, differences in
time and extent of stomatal closure are extreme. Many important ecologi-
cal relations of plants are related to this process as it functions in water
conservation and the adaptation of plants to the supply of water in their
environments.

Under conditions of rapid absorption and low transpiration, water may
be lost from plants by guttation. These conditions may prevail in fields of
young grain and in lawns (Curtis, 1944) during the night and early morn-
ing hours in the spring; they have been observed in vegetable and forage
crops and they are prevalent under the humid environment of the tropics.
They are favored by warm soils, cool saturated atmosphere and adequate
but not excessive soil moisture.

In contrast to the above situation, it is widely recognized that most
plants live the greater part of their lives in a state of negative water bal-
ance; that is, transpiration often exceeds absorption and pressure in the
xylem is subatmospheric. Transpiration is termed stomatal, cuticular, or
lenticular, depending upon the structures through which the water vapor
passes, and all three types may occur at the same time.

Stomatal transpiration is normally the predominant form in leafy plants
and it consists of -Z ) evaporation of water from wet cell walls of the stomatal
chambers, 2) diffusion from the humid atmosphere of these chambers
through the stomata into the external atmosphere, and 3) movement of the
vapor from the surface of the leaf where under still conditions it tends to
form a molecular cloud.

Laws of Evaporation : — Although many publications describe the loss
of water from plants, soils, and free water surfaces (Leick, 1939), few
formulae have been presented that express the nature of this loss in meas-
urable terms. Such formulae as exist apply only to relatively simple sys-
tems and they prescribe the most rigid experimental conditions.

Even the simplest case — the evaporation of water from a free water
surface — is sufficiently complex so that only a few conditions governing the
loss can be exactly expressed mathematically. Nevertheless, certain general
principles of evaporation are demonstrable, and apply not only to loss from
free water surfaces but, with modification, to transpiration as well.

Because of the kinetic movement of liquid molecules and the statistical
rules governing energy distribution among them, there is a certain escape
of high energy individuals from any gas-liquid interface. Since this escape
represents a loss of kinetic energy from the hquid cooling occurs, and, if
the evaporation rate is to be maintained, heat must flow into it.

Molecules entering the gas phase partake of the nature of gas mole-
cules— that is, they have unordered motion. Consequently, molecular
movement at the interface is omnidirectional and condensation as well as
evaporation is taking place. When the gas and liquid are at the same tem-
perature and in a restricted space, an equilibrium attains ; the gas is saturated
and the net loss or gain of water by the liquid is zero. Although evapora-
tion and condensation under these conditions have real and equal values.

Chapter X — 181 — Loss and Retention

in the more general use of the term meaning net loss, evaporation would be
considered as having a value of zero. Because of the ease of measuring
net water loss, the term is more conveniently used in this sense.

Evaporation from a Free Water Surface: — Dalton in 1801 made
several important observations on evaporation ; he has been credited with a
formula for calculating the rate of evaporation from extensive areas of
water. Humphreys (1940) doubts the authenticity of the formula at-
tributed to Dalton. The following statements by Dalton indicate the
state of knowledge of the subject up to Dalton's time.

"Some fluids evaporate more quickly than others. The quantity evapo-
rated is in direct proportion to the surface exposed, all other circumstances
alike. An increase of temperature in the liquid is attended with an increase
of evaporation, not directly proportional. Evaporation is greater where
there is a stream of air than where the air is stagnant. Evaporation from
water is greater the less the humidity previously existing in the atmosphere,
all other circumstances the same."

Dalton's observations lead him to conclude that " . . . . the evaporat-
ing force must be universally equal to that of the temperature of the water
[vapor pressure of water at saturation at surface temperature] diminished
by that already existing in the atmosphere [vapor pressure of water in
air]." He further concluded that, "The quantity of any liquid evaporated
in the open air is directly as the force of steam from such liquid [vapor
pressure] at its temperature, all other circumstances being the same."

Since, in studying transpiration, we are dealing with evaporation from
relatively small areas, Stefan's (1881) analysis of the rate of evaporation
from flush circular areas is of interest. Stefan found that such evapora-
tion into still air could be expressed mathematically by the formula

P — oo

V = 4 a k log ^ , where (i)

ir — p^

V = amount of water evaporated per unit area,
a = radius of the evaporational area.

k =z the diflfusion coefficient.

P = atmospheric pressure.

pO z= the vapor pressure of the liquid in air.

pi = the vapor pressure of the liquid at saturation at the surface temperature.

The coefficient of diffusion of water vapor into air has the dimensions of
mass diffused per unit area per unit time for a unit vapor-pressure gradient.

H pO and p^ are small in comparison with P, the equation becomes ap-
proximately

V = 4 a k AP PJ (2)

P

The importance of Stefan's relation is that evaporation from small
areas is shown to be proportional to the linear dimensions of the evaporating
surface and not to the area as is suggested by Dalton for large areas.

Evaporation from Tubes: — When water evaporates from a surface
that is not flush with the edge of the container molecules escaping near the
periphery of the liquid cannot diffuse laterally but are confined to the tube
immediately above the fluid and hence contribute to the total vapor pressure
above the liquid. Evaporation under these conditions is reduced. Stefan
(1873) calculated the rate of evaporation in this case to be

Grafts et al. — 182 — Water In Plants

„ kA , P — p" , ,,.

V = — — log — , where (J)

h P — p

V := rate of total evaporation.
A = area of the tube.

h = height of the tube from the liquid surface to the free air space,
p" = partial pressure of vapor at the free end of the tube,
p' = partial pressure of vapor at the evaporating surface.

The equation becomes V = — — - — ■ (4)

when p' p" are small in comparison to P.

Factors Affecting Evaporation from Free Water Surfaces of Uni-
form Shape and Area : — Since evaporation involves the escape of mole-
cules from a body of fluid against internal forces of attraction and forces
resident in the surface film as well, many factors relating to intermolecular
forces in the liquid affect evaporation rates. The more important factors
will be discussed.

a) Solutes. — Any solute dissolved in a liquid reduces the diffusion
pressure and hence the rate of evaporation of the liquid. Because they
dissociate, salts in aqueous solution are particularly effective in lowering
the evaporation rate of water. For example sea water evaporates about 5
per cent less rapidly than fresh water under similar conditions.

b) Temperature. — Evaporation of a liquid increases with increased
temperature in approximately the same ratio as the vapor pressure at satura-
tion increases with temperature. When air temperature is above that of
water the latter will cease to evaporate before the air is saturated ; if the
temperature relations are reversed, evaporation will continue into the satu-
rated atmosphere and dew may be deposited.

c) Dryness of the air. — The evaporation rate of a liquid depends on
the concentration of liquid molecules in the vapor layer in contact with the
liquid surface, and it is the gradient of concentration above, that determines
diffusion away. In still air evaporation is approximately equal to the diff-
erences in absolute dryness of the air rather than to relative humidity as is
shown by equations (2) and (4). That is,

V = k P'-P" , where (5)

V = amount of water evaporated per unit area, the area being large and the water

flush with the surface,
k = the diffusion coefficient.

pi = the vapor pressure of the liquid at saturation at the surface temperature.
pO = the vapor pressure of the liquid in air.
P = atmospheric pressure.

Vapor pressure deficit is defined as the difference between the actual
vapor pressure at a given temperature and the maximum possible vapor
pressure (saturated) at the same temperature. It should be understood
that the above equation holds only where the temperatures of the evaporat-
ing surface and the air are the same. Leighly (1937) discusses vapor
pressure deficits stressing the fact that the temperature equality mentioned
above represents a special case seldom attained.

d) Atmospheric pressure. — Evaporation rate changes approximately
in inverse proportion to the total barometric pressure (equation 5). This
follows from the kinetic law of gases.

e) Wind velocity. — Evaporation increases with wind velocity due to
eddy diffusion of vapor near the liquid surface (Jeffreys, 1918; Van

Chapter X — 183 — Loss and Retention

Orstrand and Dewey, 1915). According to Leighly (1937), air mov-
ing over an evaporating surface has two effects: -Z) a turbulence or eddy
diffusion at some small distance from the surface and 2) a laminar move-
ment of air very near the surface in which region the velocity decreases to
zero at the surface layer. In this boundary layer the gradients of vapor
concentration, air velocity, and temperature are linear.

The thickness of this boundary layer of even simple evaporimeters is a
function of at least five factors, namely: 1) wind velocity in free air, 2)
length of evaporating surface in the direction of the wind, 3) density of the
air, 4) molecular viscosity of the air, and 5) length of evaporating surface
at right angles to the wind (Martin, 1943). In the case of evaporation
from leaves the qualitative character of the surface must be added. Cor-
rugation, creasing, occurrence of hairs, and close proximity of adjacent
leaves all tend to increase the thickness of the boundary layer.

Leighly's formula for evaporation into moving air, modified by Mar-
tin to include the length of the evaporating surface at right angles to the
direction of wind flow is as follows,

V = c k (p— p") R-O-2 L-0.3 w+O-5 where (6)

V = amount of water evaporated per unit area,
c = a proportionality constant.

k = the diffusion coefficient.

p' = vapor pressure at the surface.

p" = vapor pressure at the edge of the boundary layer.

R = length of the evaporating surface at right angles to the wind direction.

L = length of the evaporating surface parallel to the wind direction.

W = wind velocity.

The exponents for the factors R, L, and W were determined experi-
mentally using blotting-paper evaporimeters and are in good agreement
with data reported by Renner (1910, 1911&), Thomas and Ferguson
(I917a,b), Jeffreys (1918), Gallenkamp (1919), Sierp and Seybold
(1927), and Seybold (1929).

The above equation may be summarized by stating that evaporation from
small areas varies as the square root of the wind velocity and inversely as
the 0.3 and 0.2 powers of the length and breadth of the area. Certain pre-
sumed variations of this law are, according to Martin, probably due to
failure to determine the actual temperature of the evaporating surface (see
Walter, 1925).

It should be noted that this expression of the rate of evaporation into
moving air is very different from the formula given above expressing the
rate of evaporation into still air where diffusion is molecular and "eddy
diffusion" is not involved. The first holds approximately where a steady
horizontal wind is present and cannot be reduced to the second which ap-
plies only in the absence of air movement.

Transpiration: — Because of the complex nature of the process of
evaporation and the added complexities of leaf structure, transpiration is an
intricate process that presents many experimental difiiculties. On the other
hand gross measurements of the rate and volume of water loss by plants
may be readily made and many methods for studying water relations of
plants both in the greenhouse and in the field have been devised. But hav-
ing made measurements on water loss, the experimenter often meets his
most difficult task in attempting to interpret their meaning, and unless a
rigid control has been exercised over most or all of the external factors,
their explanation may be difficult or even impossible. Of the immense

Grafts etal. —184— Water in Plants

literature on transpiration it is obvious that only a small portion can be
treated here.

Methods of Measuring Transpiration: — The methods which have
been extensively utilized in determining the rate of water loss by a transpir-
ing plant logically fall into three groups: determinations of 1) the loss of
weight by whole plants or plant parts, 2) the amount of water vapor given
off, and 3) the rate of water absorption. Detailed considerations of the
various techniques employed with descriptions of the types of apparatus
used and the numerous modifications suggested are given by Burgerstein
(1920), Grafe (1924), Maximov (1929a), and Leick (1939).

While it has the advantage that the transpiring organs need not be en-
closed in an artificial atmosphere which would modify the rate of water
loss, the unwieldiness of the loss in weight method has somewhat restricted
its use particularly when ecological experiments with numerous large plants
have been desired.

If the surfaces of the culture vessel in which a plant is growing are
sealed in a way such that water loss is restricted to the plant itself, changes
in weight are brought about by transpiration (lenticular, cuticular, and
stomatal) combined with the processes of metabolism (catabolic and ana-
bolic). Under normal conditions of even moderate or reduced transpira-
tion, the loss of water vapor completely overshadows any changes due to
other physiological processes in the plant. Periodic weighings coupled with
accurate determinations of leaf area, or green or dry weight enables the
investigator to express the rate of transpiration per unit of plant area or
weight.

Several problems arise, however, which make the analysis difficult.
These are chiefly concerned with abnormalities in response of culture
plants compared with those growing undisturbed in the field. Obviously,
injury to the root system of the experimental plant through transplanting
to a culture vessel immediately before making observations on transpira-
tion rate is to be avoided. Not so apparent, however, are the modifications
which may arise in plants grown continually in pots or large containers.
Control "of nutrition, water supply, soil pU, and air supply to the roots are
among the most troublesome problems. These may be appreciated when
one realizes the volume of soil which the roots of single herbaceous plants
may permeate under natural conditions. Not only may the water supply
be abnormal in potted plants but stomatal response (because of variation in
water supply or due to some other cause) may be different in potted as com-
pared with field grown plants. Obviously the transpiration rate from such
an experimental plant would be far from comparable with that of its field
grown complement.

Nevertheless, the loss in weight method when employed critically has
been one of the most useful, particularly for laboratory and greenhouse
experiments, as well as for certain ecological and irrigation studies such as
those carried out by Briggs and Shantz (1913, 1916), Kiesselbach
(1916), Veihmeyer (1927), and Veihmeyer and Hendrickson (1927).
Various automatic recording devices have been developed to obtain con-
tinuous records of weight loss (Briggs and Shantz, 1915; Brauner,
1931 ; ScHRATZ, 1931 ; and others).

The unwieldiness of using whole plants in the weight method has been
circumvented by modifications whereby plant parts are removed and their
loss in weight while standing in water recorded. Such a drastic treatment

Chapter X — 185 — Loss and Retention

may so alter the water supply (either by increasing the resistance due to
plugging of the tracheae with air, latex, or mucilage, or by decreasing the
resistance through the removal of the roots and much of the conduction
resistance) that completely abnormal results may be obtained (Table 45).
Cutting under water at a time of day when little or no tension exists should
avoid the plugging of xylem conductors with air and hence provide experi-
mental plant material most like that of the normal plant. A dramatic illus-
tration of the plugging action of latex can easily be observed on Poinsettia.
The flowers of this plant, when cut and placed in water, often begin to wilt
within a few hours. If the shoots are removed from the water and im-
mediately held with the lower 1 to 2 inches of their bases in a flame, the
heat and generated steam cause the latex plugs in the xylem to be blown
out. If then the ends, while still hot, are placed in water, a very rapid
recovery from wilting is often observed. The recovery may be so rapid, if
wilting has not progressed too far, that the leaves will move visibly, and
the drooping tip becomes erect within a few minutes. A similar effect
upon a wilted shoot plugged with air may be shown by sealing its base in
a bottle of water and aspirating it (Stocking, 1948).

Table 45. — Transpiration of Solidago virga aurea in milligrams per gram fresh weight
per minute. Cut surface renewed hourly {data of Leick, 1939) : —

Rooted plant Adjacent cut plant Difference in
Time of day richly watered standing in water per cent

10:30 A.M.

61.5

35.8

42

11:00 A.M.

50.7

41.2

19

11:30 A.M.

53.7

46.5

13

12:00M.

54.3

53.2

2

1 : 00 P.M.

63.1

54.7

13

2:00 P.M.

60.4

50.0

17

3 : 00 P.M.

49.4

45.3

12

A further modification of the loss in weight method is one in which the
cut part is immediately weighed without placing in water and rapid sub-
sequent weighings made. Under favorable conditions the initial loss in
weight of the cut piece approaches that from the piece before cutting.
Studies have been made by this method which has proven fairly successful
for plants that transpire slowly (Huber, 1923; Ivanov, 1924, 1928) but
not for rapidly transpiring plants ( Maximov, 1929a) ,

Recently Weinmann and le Roux (1945) have made a critical analy-
sis of the rates of transpiration of barley, maize, oats, wheat, and tall
fescue plants before and after cutting. The transpiration of the intact
plant was measured in each case by weighing on a torsion balance and im-
mediately afterwards under identical atmospheric conditions the transpira-
tion of the cut shoot was determined. These experiments were carried out
on relatively young plants and agreement in the rate of water loss for three
minutes before and after cutting was poor. The average discrepancies
ranged from 49 per cent in maize to 106 per cent in barley. In only ap-
proximately 35 per cent of the experiments with wheat and only 10 per cent
of those with the other plants did the transpiration rates agree within 20
per cent. This further emphasizes the fact that extreme caution must be
used in interpreting the results of transpiration experiments on detached
plant parts.

Grafts et al.

186

Water in Plants

Quantitatively the amount of water transpired by a known leaf area
may be most accurately determined by drawing air of known moisture con-
tent over the leaf and subsequently through an absorbent such as anhydrous
calcium chloride or magnesium perchlorate. The increased gain in weight
of the absorbent over the gain found when an equal volume of air of the
same original humidity is drawn through the absorbent gives the weight of
water transpired. Such a method used by Freeman ( 1908) is a modifica-
tion of the methods of E. and J. Verschaffelt (1890) and Geneau de
Lamarliere (1892) and has been further modified by Minckler (1936)
and HiESEY (1940). The latter investigators used a vacuum pump to draw
air over the plant part and flow meters to determine the volume of air mov-
ing. The main objection to this method is that an artificial atmosphere
exists around the experimental part.

Qualitative and semi-quantitative investigations have been carried out
using cobalt chloride paper, hygrometers (of horn, cellophane, and Yucca),
and calcium chloride-containing vessels attached directly to the transpir-
ing leaf. Of these only the cobalt
chloride method has been used exten-
sively and it has been standardized as
a quantitative method. Livingston
and his students have been especially
responsible for the development of this
method (Livingston, 1913; and Liv-
ingston and Shreve, 1916).

Indirectly the amount of water ab-
sorbed can be used as an indicator of
water lost by the plant. Potometers
for cut and intact plants are especially
good for class demonstrations and,
when combined with the weight meth-
od, yield valuable data on the lag be-
tween water loss by transpiration and
water absorption. In this way the
fluctuation in the water content of a
plant can be studied. Such an appara-
tus is shown in Figure 49. The artifi-
cial environment surrounding the ab-
sorbing area is again a source of error.
In summary, it may be said that
extreme difficulty has been encountered
in devising a method of studying trans-
piration which is accurate but does not significantly affect the environment
of the experimental plant. The gravimetric and potometric methods on
whole plants are most likely to alter the action of absorbing organs and in-
directly the stomata while the volumetric method may alter the environ-
ment of the leaves. Any method utilizing cut parts must be employed
with extreme caution and only after adequately checking the various pos-
sible effects of cutting. Choice among the methods available must be made
after carefully considering and, if possible, measuring the effects that the
method will have on the plant itself.

Expression of Results: — In order to express the amount of water
transpired on an area basis exact measurement of leaves is important. The

\^

Fig. 49. — Potometer with grow-
ing plant for study of water absorption
and loss. Redrawn from Meyer and
Anderson (1939).

Chapter X — 187 — Loss and Retention

numerous methods include weighing of cut out leaf tracings on paper of
known weight per unit area, calculating area by photoelectric means, leaf
matching, and other methods. Miller (1938) discusses these various
techniques at some length.

Three general methods of recording water loss have been used: i) by
expressing the weight of water lost per unit time per plant unit, such as
unit of area, green weight, or dry weight, 2) by comparing the water lost
by unit plant area with that lost from a similar area of water, and J) by
expressing the total amount of water lost over an extended period of time
per unit weight of dry substance produced by the plant for the same time
period. These three means of expressing transpiration have been termed
respectively : 1 ) intensity of transpiration, rate of transpiration, absolute
transpiration, 2) relative transpiration, transpiration capacity, transpiring
power, transpiration resistance, and 3) water requirement, efficiency of
transpiration, coefificient of transpiration,

Guticular Transpiration : — Not only the thickness but also the com-
position of the cuticle on the surface of epidermal cells varies considerably
with species, age, position, and condition of the plant organ. An example
of the influence of the environment on cuticle formation may be found in
seedlings of citrus grown under humid conditions. Such plants were sub-
ject to sun scorch due to the little cuticular protection afforded and to poor
stomatal response (Nigam, 1934). In fruit the cutin may so completely
permeate the epidermal and hypodermal layer as to completely imbed the
epidermal cells. Cuticular transpiration though a function of the per-
meability may not be directly correlated with cuticular thickness. Cutin
is a heterogeneous substance composed of several wax-and-oil-like frac-
tions. Markley and Sando (1931) studying the wax-like coating on the
surface of apples separated the petroleum ether soluble fraction from the
one that was petroleum ether insoluble. The former was composed chiefly
of the hydrocarbon triacontane (C30H62) with small amounts of hepta-
cosanol (C27H56O) and traces of true oils, while the latter was identified
as ursolic acid C30H48O3. These two fractions increased with age and
development of the fruit but not uniformly. The oily petroleum ether solu-
ble fraction increased faster rendering the layer more impervious to water.

PiENiAZEK (1944) found that when lenticular transpiration, which
amounted to from 8 to 25 per cent of the total water loss, was excluded,
by covering each lentical with paraffin, the rate of transpiration from sev-
eral varieties of apples was not directly related to thickness of the cuticle
itself but that varieties with a large wax deposit on the surface of the
cuticle lost water most slowly. This wax deposit on apples has different
chemical and physical characteristics from the cuticle (Markley and
Sando, 1931).

Diurnal fluctuations have been observed in the physical properties of
leaf cuticle. By measuring the contact angle of water drops on leaves of
Brassica sinapis and Triticum vulgar e, Fogg (1944) studied the wettabil-
ity of the cuticle. A diurnal change in the contact angle was observed
(Figure 50) . This would imply that there is a diurnal fluctuation in cuticu-
lar transpiration in normal plants. Wilting caused a marked reversible
rise in contact angle which was not, according to Fogg, a mechanical
stretching effect.

Cuticular transpiration is most conveniently studied in hypostomatal
leaves where the stomata can be covered by vaseline or other water im-

Grafts et al.

188

Water in Plants

pervious coatings, thus limiting water loss to that which passes through the
cuticle of the upper leaf surface. Using this method and comparing trans-
piration from wilted leaves and leaves with the stomata sealed, Agamov
(1927) showed that even with wilted leaves having apparently completely-
closed stomata, cuticular transpiration was slightly augmented by stomatal
transpiration.

/Voo/7

A^/dn/a/?/'

-20

G.M.T

Fig. so. — Diurnal fluctuation in the wettability of leaf cuticle as shown by change in contact
angle of water drops. The solid line with circles represents contact angle; the vertical bars show
significant differences for the points. The dashed line represents temperature; the light solid line,
relative humidity. Data of Fogg (1944).

Under certain abnormal conditions such as wilting, or with age, the
cuticle of leaves may develop fine cracks through which water loss is in-
creased. HoLLE (1915) describes such cuticular fissures in wilted leaves
of Rochea falcata, and Kamp (1930) found that the cuticle of old leaves
split into fine fissures. Although no observation of such cuticular cracks
were made by Mendel (1945), he felt that such cracking of the normally
very slightly permeable cuticle of citrus (Bartholomew, 1931; Oppen-
HEiMER and Mendel, 1939) might explain an observed anomolous trans-
piration rate following irrigation of certain trees which had suffered from
low soil moisture. The transpiration rate from these trees failed to reflect
stomatal movement.

That cuticular transpiration may reach appreciable values is shown by
the work of Rudolph (1925), who found that cuticle on the leaves of
many plants permits a considerable water loss. As much as 30 per cent
of the total water loss was found to be cuticular. In contrast, desert suc-
culents may survive for months and even years with no water uptake, be-
cause cuticular transpiration is reduced to a negligible value.

An extensive discussion of the effects of environmental factors on the
rate of cuticular transpiration has been presented by Gaumann (1942)
with a theoretical consideration of the possible effects of the structural

Chapter X

189 —

Loss and Retention

resistance of the cuticle, protoplasmic resistance, self-generated convection
currents, air humidity, leaf cooling, and wind velocity.

Fluctuations in the Rate of Transpiration: — The rate at which
water escapes from the colloidal walls of the mesophyll is a function of the
complex interaction of internal and external forces and their influence upon
the DPD of the water in the wall and in the intercellular spaces. Under
conditions of adequate water supply the normal daily fluctuations in me-
teorological conditions are reflected in the daily march of transpiration.

*3.3^

1

•

1

1

^/zoo

0

1

<5 SOO
3/M

1

1

/

1

/

J

/

/

/

/

/

^

y

lOO 90 ao 70 60 ,50 40 30 10 10 Q

Relative Jiumidity in percent

Fig. 51.— DPD of the atmosphere at 20° C as re-
lated to relative humidity. Data of table 18, Chapter V.

For, although equilibrium is never attained, the movement of water into,
through, and out of the plant responds to the tendency to establish equilib-
rium of water in the soil, plant, and atmosphere. In the final analysis, it is
the diffusion pressure deficit of the water vapor in the atmosphere which
most profoundly affects this movement. In Table 18 of Chapter V, fig-
ures are given for the DPD of the atmosphere at constant temperature and
changing relative humidity. Figure 51 shows a graph of this relation.
Shull (1939) has pointed out that the influence of temperature at con-
stant relative humidity is very small. He did not, however, point out that

Crafts et al.

190

Water in Plants

the capacity of a given volume of air to hold water is greatly afifected by
the temperature. For example, as shown in Figure 52, a change in tem-
perature from 40° F. to 50° F. may cause a change in relative humidity
of from 76 to 54 per cent, or a change in DPD of from 350 to 810 at-
mospheres when the air contains 0.004 pound of water per pound of dry
air.

JOO

30 -40 SO 60 70 SO SO 100 110 IZO

Temperature -^F

Fig. 52. — Relation between atmospheric temperature
and relative humidity for an atmosphere containing .004
pounds of water per pound of dry air.

As a result of humidity and temperature changes there is a diurnal fluc-
tuation in DPD of the atmosphere, as shown in Figure 53. Coupled with
this fluctuation is the influence of light upon transpiration. This may act
either directly to increase the temperature of the leaves above that of the
atmosphere or physiologically to alter the permeability of the protoplasm
or the movement of stomata. Diurnal fluctuations in transpiration rates

I

J

Chapter X

191

Loss and Retention

have often been measured (Briggs and Shantz, 1916; Sayre, 1920;
Maximov, 1929a; Chung, 1935; Miller, 1938; and others). A more
critical analysis of the individual effects of various factors on the rate of
transpiration has been made through use of control cabinets where tem-
perature, humidity, light, and air flow are rigidly controlled (Davis and
Hoagland, 1928; Arthur and Stewart, 1933; Biale, 1941; Monter-
Moso and Davis, 1942; and Martin, 1943).

2000

/750 -

23

^■C

25

Days - u'une /9J2

Fig. 53. — Diurnal fluctuations in DPD of the atmosphere at a station in Wash-
ington, D. C, during a 10-day period in June 1932. Data of Shull (1939).

Rhythmic fluctuations in transpiration independent of environmental
conditions have been known for a long time (Kohl, 1886; Curtis, 1902;
Lloyd, 1908). Experimenting with Fouquieria splendens in a dark room
and as near constant temperature as possible, Lloyd found a variation in
transpiration rate. He felt that stomatal opening after midnight might
explain a rise observed in the transpiration at that time but that such
stomatal movement could not cause a lowering in transpiration rate after
the maximum was reached. The occurrence of an early morning maximum
in plants kept in total darkness was observed. The potometer method, us-
ing cut shoots, was employed, hence the fluctuations appear to be inde-
pendent of root pressure rhythm which Hofmeister (1862), and Grossen-
BACHER (1938) found.

Using lemon cuttings and working in control cabinets with constant
darkness, temperature, and humidity, Biale (1941) verified the existence
of a rhythmic fluctuation in transpiration. Similarly, Montermoso and
Davis (1942) found such a rhythm in rooted Coleus leaves grown under
controlled conditions (Figure 54). The rigid control used in the cham-
bers permits no doubt as to the existence of this rhythm. By altering the
light and dark periods before placing the experimental material under con-
stant dark conditions, these authors were able to invert the maximum and
minimum points on the curve as shown in the figure.

The work of Montermoso and Davis has not been carried to such a
point that a complete explanation of the phenomenon may be made. Un-
fortunately, no data on stomatal movement were presented. Two possible

Grafts et al.

192 —

Water in Plants

explanations appear. The most obvious would involve a rhythmic fluctua-
tion in stomatal movement in the dark, such as has been observed in some
plants. Sayre (1926) found a diurnal opening of stomata in the dark for
two days. After this length of time the degree of opening was reduced.
The curves presented by Montermoso and Davis appear to show a decline
after about this same period of time. Tagawa ( 1936) observed continued
stomatal movements in beans for several days when kept in constant dark-
ness or when exposed to constant light. The second possible explanation
would involve a diurnal fluctuation in root resistance. Such changes in
root pressure have been discussed in Chapter IX.

.30

o- — -o £^pt E

M/}.

/^oon

Mn.

A/oon

Mn.

A/oon

Fig. 54. — Diurnal fluctuations in water loss from rooted Coleus leaves under controlled condi-
tions. The leaves were in the dark during the above tests and temperature and humidity were con-
stant. The solid line represents plants preconditioned under normal light-dark conditions, the
dashed line plants preconditioned under reversed light-dark cycle. From Montermoso and Davis
(1942).

Internal Factors Affecting Transpiration: — The anatomical and
physico-chemical differences among species, and between individuals of a
single species causing variation in transpiration rate are difficult to differ-
entiate and evaluate. However, they have received considerable study. It
is convenient to group them into two sets (a) those affecting the diffusion
pressure of water in the walls directly, and (&) those influencing the rate
of removal of water from the sub-stomatal chambers. In the first group
are i) wall structure, 2) physical resistance to water movement (friction),
and 5) osmotic properties of cells. In the second group are 1) internal
exposed surface, 2) stomatal aperture, 5) cuticle, and 4) morphological
modifications.

Wall Structure: — The cell walls of plants are made up largely of carbohydrates
and polyuronides. The organic portions of these compounds have the generalized ap-
proximate formula Cn H[2i.-2: Ocn-i: where n has a value of 5 or 6 or some simple
multiple thereof. From this formula it is evident that, of the organic portion of the
molecules, around 25 per cent of the atoms are oxygen. And whereas each of these
oxygens has a covalence of 2 that is ordinarily satisfied in the molecular constitution
of the compound, an equal number of secondary or residual valences exist, each of
which is capable of holding one water molecule through a hydrogen bond. Further-
more, the cellulose and uronide chains making up the walls are so spaced in the micelles
or fibrils that they form a microcapillary matrix capable of holding much water by
microcapillary forces.

When the hydrostatic pressure of the water in the xylem approaches atmospheric

Chapter X — 193 — Loss and Retention

pressure in value, all walls are highly hydrated and it has been assumed that the outer
surfaces constitute virtually free water surfaces. Little energy is required to move
water up to and through cell walls under these conditions for movement is simply a
process of exchange. When the water balance shifts far to the negative side some
water is withdrawn from the walls, the constituent molecules come closer together, and
the capillary structure becomes more dense. The walls perform two essential functions
in relation to transpiration by leaves in addition to their usual structural service ; by
virtue of their colloidal and microcapillary nature they act as conductors; because of
their imbibitional properties they store water during periods of abundance against
deficits that develop during rapid water loss.

Bangham and Lewis (1937), using cut strips of Ficus elastica leaves, found that
water did not readily enter the intercellular passages by capillarity. Less polar com-
pounds (paraffin oil, benzene, chloroform, ether, and essential oil) did enter. The
writers concluded that transpiration does not take place from a liquid film of water on
the cell walls of the mesophyll.

Lewis (1945) has performed experiments indicating that the mesophyll walls in
leaves are hydrophobic in nature : that is, they are not readily wet, the interfacial ten-
sion between walls and water being low. The effect of such a condition would be that
water would remain deep within the walls and that the surfaces would remain rela-
tively dry. Lewis' experiments indicate this to be true. Earlier Hausermann (1944)
had demonstrated that such a lyophilic liquid as butyl alcohol which has some hydro-
philic properties best wets the inter-cellular surfaces of Dianthus barbatus leaves.
From this observation he proposed that such surfaces are composed of cutin.

Transpiration from such surfaces could not approach that from free water sur-
faces of equal area. This may explain the low ratio of transpiration to evaporation
from free water surfaces found by Turrell in experiments to be described. Release
of fatty substances by living cells has been postulated to explain deposition of cutin
and suberin in the endodermis and cuticle of plants. Possibly this same type of ma-
terial may escape from mesophyll cells in minute quantities and impregnate the outer
exposed wall surfaces rendering them hydrophobic.

Another characteristic of the cell wall which may greatly influence the water hold-
ing capacity of the cell is the degree of elasticity which it possesses. The cell walls of
some xerophytes exhibit little or no elasticity. In this connection, it has been pointed
out (Pringsheim, 1931; Ernest, 1934c) that there is essentially no change in volume
from the water-saturated state to limiting plasmolysis. Hence, the DPD can increase
rapidly and considerably with the loss of a very small amount of water. This is be-
lieved to enable plants with inelastic walls to adapt themselves to drought conditions.
These cells may rapidly change from a condition of complete turgor to zero turgor and
hence from zero DPD to a DPD equal to the OP of the cell sap with little correspond-
ing change in the volume of water lost. Such a condition would greatly enhance the
plant's resistance to water loss.

Physical Resistance to Water Movement: — As mentioned above the
volume of the cell walls varies with water balance and as the walls become
thin during periods of high deficits resistance to longitudinal movement
parallel to the cell surface must be appreciably increased. Covalent forces
that space the carbohydrate molecules along the chains, however, are little
affected by water content and so lateral movements across walls as occur in
movement from cell to cell are not greatly changed. Hence dehydration of
cell walls probably does not become a major factor of resistance to trans-
piration until a point is reached where air begins to replace water in the
capillary structure. This does not occur until other disturbances have
severely affected the cells. Study of the walls of living mesophyll by means
of the microscope shows that they remain plastic, elastic, and normal in
appearance throughout the normal stages of wilting and in fact until the
cells are almost completely collapsed. Probably cell wall structure has
little direct effect upon water movement and loss in leaves, at least until
they reach a stage of permanent wilting.

Nevertheless, the resistance offered by even the minor veins of the
leaves is extremely small compared to that offered by the cell (Wylie,

Crafts et al. — 194— Water in Plants

1938; Mer, 1940). Root pruning and stem incisions have shown that over
50 per cent of the cross section of the root as well as of the stem may be
prevented from functioning without severe injury to young citrus trees, and
ready lateral transfer of water may be observed (Elazari-Volcani, 1936).
The protoplast particularly exhibits a high resistance to rapid water move-
ment which may be increased or decreased by the presence of specific ions,
by changes in temperature, or by light intensity, as will be discussed in a
subsequent section.

Osmotic Properties: — Just as imbibitional forces enable cell walls to
maintain their form and water content, so do osmotic forces through the
development of turgor make possible the normal unwilted stature of plants.
And in proportion to the efifect of such form upon evaporation from leaves
does osmosis affect transpiration. The direct effects of solute concentra-
tion upon vapor pressure of water are shown by Table 18 to be almost
negligible at concentrations that occur in plants. Furthermore, as explained
in Chapter V, turgor increases the vapor pressure of water in the cell so
that at full turgor the water-vapor pressure of the cell sap is equal to that
of free water of the same temperature and pressure.

Shreve (1931) found that free cell sap with osmotic pressures of 4.9,
11.7, and 20.0 atmospheres respectively evaporated in the ratios 1.011:-
1.005 : 1.000. This reflects the relatively minor influence of small additions
of osmotically active solutes upon vapor pressure. When sugar solutions
were allowed to evaporate through semi-permeable membranes the reduc-
tion in rate was much greater, but in this case the solutes were probably
able to accumulate in the solution immediately in contact with the membrane
or actually within the pores of the membrane, a phenomenon that does not
occur in leaves.

Boon-Long (1941) similarly found that the presence of a collodion
membrane reduced by 10 per cent the rate of evaporation of a sucrose solu-
tion over that brought about by osmotic concentration alone. Increase in
osmotic pressure of leaf cell sap by exposure to light, scalding of petioles,
or direct introduction of glucose caused a decrease in the permeability of the
tissues to water as determined by the plasmolytic method. According to
Boon-Long this lowering of permeability is chiefly responsible for reduc-
tion in the transpiration rates which he found in plant organs subjected to
these treatments. However, the hardening of cabbage tissues during ex-
posure for seven days to 5° C. caused an increase in permeability which
more than counterbalanced a simultaneous increase in osmotic pressure.
Consequently the transpiration was greater for the hardened than for the
non-hardened plants.

Internal Surface: — Because evaporation is a function of surface it
follows that, when transpiration is high, the exposed intercellular surface
of the leaf might become a limiting factor. Most investigators have fol-
lowed the development of air spaces in leaves by determining their volume
by infiltration methods. In the leaves of vascular plants the volume of
space differs not only among plants but also as a response to environmental
conditions.

SiFTON (1945) in a review of the subject reports that Unger (1854)
found in 41 species a minimum air space of 77 parts per 1000 in leaves of
Camphora officinalis and a maximum of 713 parts per 1000 in Pistia
texeris. Shade leaves with more spongy parenchyma have a greater vol-

Chapter X — 195 — Loss and Retention

ume of air space than sun leaves. Air space varies with water supply but
not uniformly among plants, and evergreens have a smaller volume than
deciduous plants.

TuRRELL (1936) attempted to determine the area of internally exposed
cell walls and to compare this with total leaf area. A wide variation was
found in different species, and a comparison of leaves in high and low light
showed differences within a species. In general the palisade parenchyma
exposes much more surface per unit vokime than does spongy tissue.
Ratios of internally exposed tissue to external surface are given in Table
46.

Table 46. — Ratios betiveen internally exposed cell walls and external leaf surface

{data of TuRRELL, 1936) : —

Ratio
of internal to
Plant Type external surface

Bryophyllum calicynum succulent sun 7.9

Syringa vulgaris mesomorphic shade 6.8

Syringa vulgaris mesomorphic sun 13.2

Ricinus communis mesomorphic shade 12.7

Vitis viilpina mesomorphic sun 11.6

Catalpa speciosa mesomorphic sun 19.2

Gaultheria shallon xeromorphic shade 8.2

Berberis nervosa xeromorphic shade 9.9

Citrus grandis xeromorphic sun 17.2

Eucalyptus globulus xeromorphic sun 31.3

TuRRELL (1944) concluded that leaves having the largest internal ex-
posed surface should also have the highest transpiration rate, other condi-
tions being similar. To test this assumption he studied Nerium oleander
and Vinca rosea (periwinkle) grown under conditions of low and high
light intensity. These two genera with different stomatal distributions
were chosen in order to minimize the effects of stomatal control over trans-
piration. Stomata of periwinkle were considered 2.5 times too close to-
gether for maximum diffusion into still air; those of oleander 1.49 times
considering each stomatal pit as a unit and 5.7 times for the stomata within
a pit. The stomatal pits of oleander occur only on the dorsal surface,
whereas the stomata of periwinkle occupy both leaf surfaces.

The xeromorphic leaves of both species, grown under high light in-
tensity, had greater internal : external surface ratios than the mesomorphic
ones developed in lower light. In spite of the fact that the internal transpir-
ing leaf surface averaged 8.1 times the external for periwinkle, and 15.6
times for oleander, the ratio of transpiration per unit internal leaf surface
to evaporation from a unit free water surface was only 0.012 for periwinkle
and 0.027 for oleander. These figures indicate that factors other than
internal evaporating surface are concerned in controlling the rate of trans-
piration from xeromorphic leaves.

Stomatal Number and Distribution: — In spite of conflicting opinions {see Miller,
1938), the weight of evidence indicates that a genetic factor is involved in determining
the numbers and distribution of stomata in leaves. Any factor that limits the expansion
of a leaf increases the ratio of stomatal number to leaf area and also the pore space per
unit area (Salisbury, 1927).

Maximov (1929a) gives a detailed consideration of Zalenski's (1904) investiga-
tions on the anatomical differences of leaves of the same plant. Zalenski, v^forking on

Crafts et al. — 196— Water in Plants

the relation of structure to position of leaves, found that small apical leaves, having
numerous stomata and thick cuticle, were more xerophytic than broad basal ones.
Smith (1941) found a similar condition in bean. Stomatal frequency increased from
base to apex of the lamina, and from base to top of the plant. Salisbury (1927) found
that variations in stomatal number on an area basis depended upon the size of the
epidermal cells according to the formula :

S
I r= E ■ s X 100 where (7)

I = the stomatal index that is constant for any species.

S =: number of stomata per unit area.

E =: number of epidermal cells in the same unit area.

Leaves or portions of leaves which, because of position on the plant or because of
xerophytic condition, are reduced in size, show high stomatal frequencies (number of
stomata per unit area). A similar condition is shown by exposed upper leaves, sun
leaves, apices and margins of leaves, and by leaves of woody as compared with herba-
ceous plants, and leaves of marginal vegetation as compared with shade species. All of
these differences, however, are due chiefly to variations in growth of the epidermal
cells, that is, to the spacing of the stomata and not the number developed. Under con-
ditions of high humidity there does, however, appear to be a reduction in the proportion
of stomata to epidermal cells formed. Aquatics have lower stomatal indices than land
plants. Poor nutritional conditions appear in some instances to reduce the stomatal
index.

Methods of Measuring Stomatal Openings : — Both direct and indirect methods
have been utilized in studying the degree of stomatal opening. These methods have
been discussed by Stalfelt (1929) and Nadel (1938). The latter author critically
examined the alcohol fixation method of Lloyd which has been used extensively and at
times without due caution.

It is obvious that where the conditions of the experiment and the nature of the
leaf permit, the direct microscopical observation of intact, untreated leaves is the most
reliable means of determining the degree of stomatal opening. Often, however, condi-
tions are such that this is not possible and resort must be taken to the microscopical
examination of treated material or to one of the more indirect methods such as porome-
try, infiltration, or measurement of transpiration.

Of the various treatments which have been used to prepare the epidermis for
microscopical observation (drying, placing in water, and chemical treatment to pre-
vent stomatal change) only the alcohol fixation method proposed by Lloyd (1908) has
been used extensively. This method consists of stripping the epidermis from the leaf
and quickly immersing it in absolute alcohol. There is an immediate, rapid dehydra-
tion and hardening of the cellulose walls before the alcohol penetrates the cells appre-
ciably. Obviously, any turgor changes which might occur during the stripping and
immersion would tend to invalidate the method. Loftfield (1921) used the alcohol
method in studying some sixty dififerent species of plants. He checked the method
against direct observation of undisturbed leaves of alfalfa and several other species,
finding good agreement between the two methods. Rapid stripping and transfer of the
epidermis into the alcohol was found, however, to be essential to prevent dimensional
changes.

A more critical analysis of the method (Nadel, 1938) showed it to be valid only
for plants with easily detachable epidermis while plants with adhering or partially ad-
hering epidermis were not found suitable. Even in the first group, if mesophyll cells
are left attached to the epidermis, changes in stomatal opening may take place in alcohol.

AsHBY (1931) has compared the porometer method with Lloyd's alcohol fixation
method with good agreement in Geranium and Verbena. The well known porometer
method first introduced by Darwin and Pertz (1911) measures the rate at which air
can be drawn through the leaf. Thus it gives an average or statistical value for the
diffusion capacity rather than an absolute measure of pore size. This measure is a
function of mesophyll resistance, viscosity of the air, pressure difference employed, as
well as being subject to the mechanical difficulties of attaching the porometer cup to
the leaf by lightly clamping the leaf between a glass plate and a greased (beeswax-
vaseline) washer. Nevertheless the method has been used with apparent success (Op-
penheimer, 1926; Hartsuijker, 1935).

Few attempts have been made to calculate the actual aperture of the stomata from
figures obtained by use of the porometer. These have usually been of an empirical

Chapter X — 197 — Loss and Retention

nature where porometer figures have been plotted against observed stomatal areas ob-
tained either directly by use of a microscope on the living leaf or indirectly by Lloyd's
alcohol fixation method. The most recent attempt to calibrate the porometer is that
of Heath (1941) using the resistance porometer described by Gregory and Pearse
(1934). This instrument employs standard capillaries in series with leaf resistance.
In contrast to the older flow porometers, it can be used to calculate independently of
the pressure drop the resistance or conductance of the leaf area through which the
air flows. The conditions for the valid use of methods of calculating flow resistance
and the theoretical limitations involved can be found in Heath's paper.

It has been observed (Williams, 1947) that the behavior of stomata under a
porometer cup cannot be regarded as normal at least so far as shock response is con-
cerned and consequently doubt is thrown upon observations made by use of the porom-
eter on stomatal response to light-dark stimuli. Further clarification of this problem
is needed.

A very simple and rapid semiquantitative method which is well adapted to use in
the field where rough determinations are to be made is found in the infiltration method
(Molisch, 1912; Stein, 1912). If a drop of liquid of low surface tension and viscosity
is placed on a leaf with open stomata, it will penetrate through the stomata, filling the
intercellular spaces and giving a water-logged appearance. If a series of liquids of
differing surface tensions and viscosities is used, an approximate estimation of the
degree of stomatal opening can be made. In place of the series of liquids proposed by
Molisch (absolute alcohol, benzol, and xylol), other authors have used different
liquids. Stahl (1894) used paraffin oil, petroleum, and petroleum ether; Dietrich
(1925), xylol, petroleum, castor oil plus turpentine, and parafiin oil; Schorn (1929),
varying proportions of isobutyl alcohol and ethylene glycol. Weber (1923) employed
ammonium vapors which penetrate the leaf killing the cells and turning them brown.

Certain other gross qualitative methods have been used to determine the opening
of stomata such as the use of cobalt chloride paper as a measure of transpiration, and
techniques in which the curling of strips of cellophane are used as a measure of humid-
ity. These methods are, however, of little value in studies of stomatal opening.

Genetic Factors: — Shafer (1942), experimenting with excised leaves
of several inbred and hybrid corn seedhngs was unable to demonstrate any
differences in water loss between the various strains. Gyorffy (1941)
concluded from a comparative study of the osmotic values of a number of
plants that polyploids are more variable in osmotic pressure and hence
more adaptable to dry conditions than diploids. In grafts an advantageous
increase of osmotic value beyond the 2n level was observed in tetraploids.
Clark, Hecht, Curtis, and Shafer (1941) found that the stomata of
high yielding varieties of inbred and hybrid corn open earlier and close
later than those of low yielding strains.

Stomatal Regulation of Transpiration: — The role of stomatal closure
in the control of transpiration has proved difficult to determine. Early
concepts following the demonstration of stomatal movement pictured
stomatal control as being complete except for cuticular transpiration.
Lloyd's (1908) work indicated that stomatal movement and transpiration
are not closely related. Loftfield (1921) claims these results to be in-
valid because of faulty technic. In work on alfalfa and several other plants,
Loftfield showed that stomatal movement and transpiration from cut
shoots paralleled each other quite closely. Stomatal movement in cut
shoots differed markedly from that in intact plants, hence Lloyd's compari-
son of stomatal opening on intact plants with transpiration of cut shoots
was hardly valid (see also Lloyd, 1912, 1913). Renner (1910) also
criticised Lloyd's conclusions.

According to current opinion, it seems that when stomata are wide open,
transpiration rate depends upon factors governing evaporation. Among
these the diffusion pressure deficit of water as determined by tension in

Crafts et al.

198 —

Water in Plants

the xylem and f fictional resistance to rapid flow plays a major role. These
factors continue to be dominant until the stomata are at least half closed ;
from then on, stomatal opening apparently determines water loss quite ac-
curately.

Effect of Mineral Nutrients on Transpiration: — The presence of
osmotically active solutes reduces the diffusion pressure of water; other
things being equal, evaporation from a solution is a function of solute con-
centration. Some effects of the presence of salt in the soil solution are:
osmotic influences upon water uptake ; increased equilibrium concentration
within plant cells; and secondary effects, specific for the particular salt,
upon permeability, guard cell movement, leaf abscission, etc.

The immediate effect of adding a salt to a soil of low salt content is to
increase the diffusion pressure deficit of soil water, reducing the gradient
between the soil solution and the root cells (Magistad, 1945). This re-
duces water absorption, less water is available to the leaves, and transpira-
tion is lowered. If, however, the added salt concentration is low and the
soil used is of low salt content, certain ions may act specifically causing an
increase or decrease in transpiration depending upon the nature of the ion
and not on its colligative properties (Harter, 1908; Reed, 1910; Bouyou-
cous, 1911). Similar results were obtained with the use of single salt
solutions (Hansteen-Cranner, 1914; Kisser, 1927).

In short time studies on the addition of salt to low salt soils, Meyer
(1931) found a reduction in transpiration for all concentrations used. The
specific effects of low salt concentrations were not noted; apparently the
slight increases in the transpiration of plants in solutions of very low con-
centration were obscured by the concentration of salts naturally present in
the soil employed. The differences in retarding effect between different
salts could in this case be explained on the basis of their colligative proper-
ties.

Secondary effects of salts were observed by Iljin (1922a), who found
sodium and potassium to stimulate opening of guard cells ; calcium checked
the opening. Sodium and calcium in high concentrations induced leaf ab-
scission. The water requirement decreased rapidly with increase in salt
concentration (Meyer, 1931), while CaCl2 caused, first, an increase, and
then a decrease. There was an increased succulence of tops where NaCl
and CaQ2 were added to the soil.

Influence of Disease, Sprays, Dusts, and Waxes on Transpiration

Rate: — Numerous workers have recently concerned themselves with the

Table 47. — Water requirement in Zea as affected by nutrition
(data of Desai, 1937) : —

Fresh weight

Top

ROOT

ratio

Amount of

water LOST

IN 3 weeks

Water re-
quirement per
unit fresh wt.

Treatment

Top

Root

Top

Root

Full nutrient

gm.

. . 81.60
. . 68.33
.. 11.82
. . 7.27

gm.

24.38

36.13

9.60

6.43

3.34
1.88
1.22
1.13

gm.

377.33
413.00
145.66
181.00

4.624

6.044

12.323

24.896

15.47

Potassium — deficient . .
Phosphorus — deficient .
Nitrogen — deficient . . .

11.43

15.17
28.15

Chapter X — 199 — Loss and Retention

effect of disease and the appHcation of sprays, dusts, and wax dips on the
rate of water loss from plants.

Studies of the effect of nutrient deficiency (lack of N, P, and K) on
stomatal behavior of a number of plants, including bean, corn, and tobacco
have indicated that a disruption of the general metabolism of the plant is
reflected in sluggish stomatal response to changing environment. Sub-
normal stomatal behavior is accompanied by an increased water require-
ment and a decreased yield and size (Table 47) (Desai, 1937). This re-
sult is in agreement with observations that application of fertilizers to in-
fertile soils reduces the water requirements of plants growing on them
(Kiesselbach, 1916).

Increased water requirements and rates of transpiration are usually
observed in plants suffering from fungus diseases. This is not always
true (Reed and Cooley, 1913), as the fungus may occasionally actually
obstruct water loss from the leaf. Frequently the effect of the fungus is
most strongly manifested through a disruption of the normal diurnal
transpiration curve (Graf-Marin, 1934; Yarwood, 1936; Gassner and
GoEZE, 1936; and Johnston, 1940). Rupture of the cuticle or altered
stomatal response may cause a greatly increased rate of transpiration, espe-
cially at night when the cuticular influence would normally be most mani-
fest, and the amount of water lost directly through the mycelium of the
fungus may be considerable. Alteration in permeability caused by secretory
products of the fungus has also been suggested as a cause of accelerated
water loss (Weaver, 1916).

Although many conflicting results have been obtained on the effect of
insecticides on transpiration, it seems certain that insecticides may alter the
rate of transpiration, either mechanically or chemically. The mechanical
action may cause a reduction in transpiration through physical interference
with water vapor diffusion from the leaf, or it may cause an increase due
to the presence of minute dust particles wedging in the stomata preventing
closure. This latter effect was observed by Beasley (1942) when fine
inert dusts were applied to the stomatal bearing surfaces of leaves; night
water loss was increased.

Chemically, the specific effects of the insecticides may alter the permea-
bility of the cuticle, or stomatal sensitivity. Copper sprays have been shown
to increase transpiration by increasing cuticular transpiration (Wilson and
Runnels, 1933 ; Krausche and Gilbert, 1937 ; Foster and Tatman,
1940) ; other investigators have been unable to find significant effects on
transpiration by treatment with Bordeaux sprays (Childers, 1936; Mil-
ler, 1938; Loustalot, 1944).

Excessive water loss during transplanting and storage of nursery stock
coupled with the great reduction of absorbing roots left on the plant largely
determines the production of new roots and the plant's survival. Seven
common practices Hsted by Erickson (1945) are employed to keep the
water loss below the absorption. These are 1) hardening of the plants,
2) retention of as many roots as possible, 3) removal of part of the top,
4) avoidance of drying between digging and planting, 5) planting in moist
soil and watering immediately, 6) protection from wind and sunshine, and
7) planting during cool, humid weather.

The work of Morris (1921), Neilson (1928, 1931), Tukey and
Brase (1931), and Maney (1931), has been particularly influential in
making commercially practicable the use of melted wax in the treatment

Grafts et al. — 200 — Water in Plants

of nursery stock to prevent excessive water loss during transplanting and
storage. Wax emulsions, because of their ease of application, are now
largely replacing the earlier melted wax treatment (Emerson and Hil-
DRETH, 1933; Miller, Nielson, and Bandemer, 1937). A technique
of evaluation, through use of controlled environment and infra-red pho-
tography, of foliar injury caused by wax and oil emulsions has been de-
scribed by Comar and Barr (1944).

Physiological Effects of Water Deficits:— A condition of water
deficit in the plant, whether brought about by low soil moisture, high solute
concentration in the soil solution, or desiccating atmospheric conditions,
greatly modifies the transpi rational, photosynthetic, assimilatory, respira-
tional, and reproductive activities of the plant. A more thorough study of
the changes which take place in the colloidal properties of cell protoplasm
upon the withdrawal of water as a consequence of freezing or desiccation
is necessary in order to understand the physicochemical and biochemical
reactions that take place during drought and freezing, and to learn why in
some cells such changes lead to irreversible coagulation of the protoplasm
and death, whereas other cells are able to withstand such changes.

Emphasis of early work on drought resistance was laid particularly on
an ecological consideration of xeromorphism and is adequately reviewed
in Maximov's work (1929a). Xeromorphic structure is characterized
anatomically hy 1) a decrease in dimensions of cells including guard cells,
2) a greater number of stomata per unit area, J) a denser network of
veins, 4) a thickening of the cuticle, and 5) a greater density of hair cov-
ering. All or part of these anatomical features may be induced or inten-
sified by continued mild exposure to drought conditions. Physiologically,
a xeromorphic condition is characterized by i) a more intense assimila-
tion ; the chloroplasts function only part time because of a restriction of
their activity during periods of water deficit, and 2) an increase in stomatal
transpiration but a decrease in cuticular transpiration. In spite of Sey-
bold's (1929) attempt to revive the earlier view of Schimper that xero-
phytes have a lower transpiration due to their structure, it is generally
agreed that this is not the case when the stomata are open and, in fact, un-
der these conditions xeromorphy is often characterized by more intense
transpiration (Maximov, 1931; Vassiliev, 1930).

Recently Bennet-Clark (1945) has suggested that the anatomical
features which had formerly been considered as helping to reduce the trans-
piration rates of xerophytes (i.e., hairs, sunken stomata, and leaf roll)
actually benefit the plant, tending to prevent stomatal closure by maintain-
ing a higher humidity at the leaf surface. This increases the duration of
active photosynthesis by allowing gaseous exchange to continue.

Current studies stress the effects of drought on biochemical reactions.
Maximov (1941) has reviewed the course of hydrolytic enzyme reactions
that take place under dry conditions. He has noted their effect on photo-
synthesis and has discussed theories of water-supply relations and starva-
tion with special reference to transpiration, respiration, and dry matter
production. The effects of withholding water as a means of hardening
plants to withstand drought is also discussed.

Enzymatic activity is greatly affected by the water balance of the plant
cell. Catalase and reductase activity is high during all phases of water
deficit as is also the velocity of the hydrolytic activity of amylase. The

Chapter X

— 201 —

Loss and Retention

content of reducing sugars was found under these conditions also to be
high (GoLOViNA, 1939). Loss of 30 to 40% of the water from leaves de-
creases the synthetic and increases the hydrolytic power of phosphatase.
Monophosphatase and diphosphatase do not, however, respond identically
to the same water deficit (Sisakian and Kobiakova, 1940).

The net rate of protein formation from amino acids decreases with re-
duced water content or in other words the net rate of hydrolyses increases
(Petrie and Wood, 1938). While the synthetic activity of invertase and
protease is particularly low in drought-susceptible varieties (Sisakian,
1939, 1940; Golovina, 1939; Kursanov, 1941). The periodicity in the
rhythm of invertase and protease activity is lost in non-drought-resistant
wheat on wilting (Sisakian and Kobiakova, 1941). Generally, then, the
hydrolytic processes in the cell are accelerated and the synthetic process de-
creased during drought. This usually results in injury. Sisakian believes
that such disturbances in the enzymic equilibrium are the principal causes
of the death of plants from drought.

Co/7centrono/7

Fig. 55. — The effect of DPD of CUmella cells upon photo-
synthesis. The cells were immersed in sucrose solutions of
the indicated molal concentrations. Photosynthetic activity
was greatly reduced as the concentration increased above 0.4
mol. Data of Greenfield (1942).

The effect of desiccation on photosynthesis cannot be attributed directly
to the fact that water is a reactant in the process but must be due to the
physico-chemical changes which occur in protoplasm, the altered action
of enzymes, and the stomatal response which takes place with changing
water supply. Protoplasmic dehydration may be brought about either di-
rectly by drying or indirectly by the use of hypertonic solutions. The re-
sults of these two types of experiments are not always the same (Chre-
LASHVILI, 1941).

Investigations using hypertonic solutions have shown that photosynthe-
sis is very sensitive to changes in the colloidal state of protoplasm ; perhaps
more so than respiration (Walter, 1928, 1929; Rabinowitch, 1945).

Studies on the relation of water supply to photosynthetic activity show
that desiccation brought about by growing Chlorella cells in solutions of

Grafts et al.

202 —

Water in Plants

varying osmotic concentration may affect the process through the dark or
enzymatic reaction rather than through the Hght reaction (Greenfield,
1941, 1942). Desiccation had Httle effect in subdued Hght but profoundly
affected photosynthesis in strong light where the dark reaction is limiting.
Figure 55 from Greenfield (1942) shows the effect of such physiological
desiccation on photosynthesis.

Plasmolysis inhibits the production of chlorophyll, and soil moisture
content may affect pigment production (Furlinger, 1938; Beck, 1942).

Slight dehydration may cause an increase in photosynthesis. Maximum
photosynthesis was found by Chrelashvili (1941) to take place in all
plants with slight dehydration but this action varied in different plants and
with different conditions. In Allimn, for instance, the photosynthetic ac-
tivity increases up to a certain sugar concentration ; in Primula no such
upper limit was observed under the experimental conditions. In Zea an
increase in carbohydrates caused a depression in photosynthesis and an
increase in respiration (Brilliant, 1924; Alexeev, 1935; Chrelash-
vili, 1941 ) . Here, again, the effect appears to be found only in relatively
high light intensities (Brilliant and Chrelashvili, 1941). With con-
siderable dehydration, photosynthetic activity decreases and finally ceases
in both strong and weak light (Dastur, 1924, 1925; Vassiliev, 1927;
Tumanov, 1929; Skvortzov, 1931 ; Dastur and Desai, 1933; Heinicke
and Childers, 1935 ; Brilliant and Chrelashvili, 1941 ; Loustalot,
1945).

An excellent treatment of the carbohydrate changes which occur in
wheat during wilting can be found in the work of Vassiliev (1931) and
Vassiliev and Vassiliev (1936). The dynamics of sugar transformations
depend to a high degree on the water supply; hydrolysis predominates
during water deficit, condensation when the water supply improves. When
a wheat plant undergoes wilting, four stages can be noted : i ) a decrease in
the amount of monosaccharides and sucrose due to reduced photosynthesis
before wilting is evident; 2) on wilting of the lower leaves there is an in-
tense hydrolysis of insoluble carbohydrates and an accumulation of sucrose.
This process gradually spreads to the upper leaves, stem, and fruit; 3) be-
ginning again at the lower leaves, sucrose decreases and monosaccharides
accumulate; 4) finally, with the disappearance of sucrose and monosac-
charides, the plant is well on its way to complete desiccation. The lower
leaves are the first to lose sugars. Here the role of sugars in drought seems
to be the same as in freezing ; in both cases sugars protect the plants against
adverse desiccation. A similar trend in carbohydrates was observed in
lucerne by Henrici (1943, 1945) ; starch was the dominant reserve carbo-
hydrate instead of hemicellulose, as found in wheat.

Investigations on the acceleration of starch dissolution due to a decrease

Table 48. — The effect of decrease in water content on the amylolytic activity and

starch dissolution of sunflozver leaves kept in the dark for 18 hours

(data of Spoehr and Milner, 1939) : —

% Change

IN relative

% Starch

Original

Final

amylolytic

Conditions

DECREASE

PH

pn

activity

Leaves in water, 22° C

17

6.83

6.90

— 4.8

Leaves without water, 22° C.

69

6.85

6.87

-^44.8

Chapter X — 203 — Loss and Retention

in water content of leaves have also been carried out by Rywosch (1908),
MoLiscH (1921), ScHROEDER and Horn (1922), Tollenaard (1925),
and Collorio (1928). That this conversion of starch is associated with
the increase of amylolytic activity during water deficit and apparently not
with a pH shift was shown by the quantitative investigations of Spoehr
and MiLNER (1939), Table 48.

In spite of adequate soil moisture, the desiccating effects of hot dry
winds may halt the accumulation of dry substance due at least in part to
the closure of the stomata. The previous conditions under which a plant
has been grown will influence this response. Thus stomatal closure in
wheat grown in soil at 70% of its water-retaining capacity was evidenced
when water reached 40 to 50 per cent of this value. Stomata of wheat
grown in soil at 40 per cent of its water-retaining capacity were always
open (Vassiliev, 1929; Kondo, 1931). The difficulty of maintaining a
soil at any moisture content below field capacity throws doubt upon the
results of such experiments (Veihmeyer, 1927). Kransosselsky-Maxi-
Mov (1930) found redistribution of water from the inflorescence to leaves
in plants subjected to artificial dry wind even when they were growing in
well watered soil. Stomatal closure also occurred.

Several workers have found that water deficit per se tends to cause
stomatal closure (Iljin, 1923; Stalfelt, 1932; Scarth, 1932). In the
guard cells dehydration tends to cause conversion of sugar to starch, ac-
cording to the work of Iljin (19226) and Steinberger (1922). This is
in harmony with the observations of Spoehr on the carbohydrate ratio in
cacti but contrary to the general trend of increased hydrolytic amylolytic
activity during drying found in most plants. It appears, however, that
turgor loss induces a pH reduction in guard cells, which condition is con-
ducive to the sugar-to-starch transformation.

Huber (1937) has emphasized the profound influence that water has
on the life processes of plants. In the field of genetics he refers to the
work of Oehlkers (1937), and Kisch (1937). Oehlkers and his co-
workers have shown that water deficiency during the conjugating phase
of reduction division decreases the number of the terminal junctions and
chiasmata, as shown in Table 49.

Table 49. — Influence of zcater absorption on vieiosis in Oenothera {data of Kisch

and Oehlkers after Huber, 1937) : —

Osmotic value

of inflorescence decrease in chiasma

Treatment atm. frequence in %

Normal 6.5 4.71

3 days dry 9.4 4.60

5 days dry 9.7 13.42

7 days dry 12.6 20.31

Huber also points out that at least part of the effect of low temperature
in inhibiting conjugation may be attributed to water deficiency as the effect
vanishes when water is supplied in sufficient quantities.

Subaqueous Transpiration : — It is commonly agreed that water move-
ment through the xylem system results either from root pressure or trans-
piration pull. In a normally transpiring plant water loss causes a diffusion
pressure deficit in the wall colloids of the mesophyll, sufficient to explain

Grafts et al.

— 204-

Water in Plants

water movement to the highest tree tops. Such movement is not generally
considered to utilize respirational energy except as it is required to maintain
the normal structure of the cells.

The first demonstration of vi^ater movement through submerged plants
v^as made by Unger in 1862 (Thut, 1932b). In this experiment a Pota-
mogeton crispus plant was placed with its roots in one vessel and its tops
in another with both submerged. In eight days the vessel containing the
tops had gained 1.6 grams. Similar experiments on water movement in
submerged plants were performed by Snell (1886), Sauvageau (1891),
HocHREUTiNER (1896), DixoN (1898), Pond (1903), and Thoday and
Sykes (1909). Such experiments utilized rooted plants, and cut shoots.

Experiments on rooted plants (Unger, 1862; Thut, 1932&) need not
be considered here for, as Thut has explained, root pressure may account
for the water movement. Observations of water movement through sub-
merged cut shoots are of great interest here for such movement cannot be
caused by root pressure nor by transpiration pull.

Dixon, the champion of the cohesion
theory through many stormy years, was
forced to resort to water secretion to explain
subaqueous transpiration (Dixon, 1898,
1914, 1938&; Dixon and Barlee, 1940).
Secretion was called on to account for the
rise of eosin through the xylem in the ex-
periment illustrated in Figure 56 (Dixon,
1938a).

Thoday and Sykes (1909) attached
small glass bulbs containing eosin to the cut
ends of submerged branches of Potamoge-
ton luccns and found a rapid rise (5.7 to
9.5 cm. per minute) of eosin in the stems.
When the apex of the branch was removed,
movement slowed down and when the leaves
were removed little or no movement oc-
curred. From these observations they con-
cluded that the leaves were the organs responsible for movement.

Smith, Dustman, and Shull (1931) criticised the early experiments
of Dixon and others, maintaining that proper precautions were not taken
to ensure complete water saturation. They argued that the ascent of the
transpiration stream is caused by evaporation from cell wall colloids of the
mesophyll and is not dependent upon secretion or respirational energy.
When they took proper precautions to completely saturate their experi-
mental plant materials they found no rise of eosin into completely sub-
merged shoots.

In a later paper, Shull (1939) answers van der Paauw's (1935)
criticism, that adsorption of eosin might have been responsible for Smith,
Dustman, and Shull's negative results, by the postulation that slow solu-
tion of gases might explain water movement in his (van der Paauw's)
experiments. Neither advanced any support for Dixon's secretion theory.
Nor was Wilson (1947) able to demonstrate the existence of continued
movement of the material in a transpiration stream in completely sub-
merged cut shoots of Ranunculus fluitans. Some short time absorption of

Fig. 56. — Experimental con-
ditions for demonstrating sub-
aqueous transpiration. Redrawn
from Dixon (1938a).

Chapter X — 205 — Loss and Retention

eosin by these shoots was explained on the basis of internal water adjust-
ments and certain observed stimulative effects of added salts seem best
explainable on their action on the osmotic nature of the cells of the shoot.

In later work, Dixon (1938^7) and Dixon and Barlee (1940) satu-
rated test plants by submersion for 24 hours and still found that eosin rose
from 15 to 69 centimeters. Oxygen and light favored the movement which
was very slight in the dark.

Crafts (1939t/) confirmed the fact that subaqueous transpiration oc-
curs only in the light and suggested that rise of water results from osmotic
uptake by the phloem according to the mechanism of Munch (1930). In
the light photosynthesis in leaves and utilization of foods in the growing
and respiring or storing cells causes a transport of osmotically active solutes
via the phloem. By mass flow this would be accompanied by a certain move-
ment of water. When one branch of a forked Syringa vulgaris stem was
ringed and the whole shoot submerged, upward movement of eosin was
more rapid in the xylem of the unringed branch in which, presumably, some
phloem transport was taking place.

The importance of this work would seem to justify more research, for
if subaqueous transpiration can be satisfactorily explained without resort
to a secretory mechanism, upward movement of water through the xylem
may be accounted for on a physical basis. On the other hand, if water
secretion is involved, experimentation in this field is greatly complicated,
for such an activity is difficult to measure and control. For anyone inter-
ested in this work, it is suggested that chlorotic or albino plants may prove
useful in an experimental approach to the problem.

Conclusions : — In the tremendous literature on transpiration, only a
portion of which has been presented here, the emphasis has been largely
on the effect of purely physical factors upon rate and volume of water loss.
And, though physiological processes have been studied in relation to trans-
piration, the active functions of plant cells have received little attention.
Likewise the structure and unique nature of water have been neglected.

The high surface tension of water and the hydrophilic nature of cell
walls and protoplasm are essential to the development of transpiration pull
in leaves. The strong coordinating forces that cause water to adhere to the
conducting elements are vital to the maintenance of the continuous water
columns. The strong bonding forces between water molecules and solutes
enable the cells to retain their aqueous contents in competition with desiccat-
ing factors of the soil and atmosphere. It seems that the very ability of
plants to leave their aquatic environments and invade the land depends not
alone upon the adaptive nature of the plants but also upon the peculiar char-
acter of the water too.

The active control of water by plants is a fairly new subject of in-
vestigation. Though the "vital" forces involved may elude direct study,
they must all be explainable eventually in terms of physical and chemical
laws. Because of the intimate relation between such forces and the proces-
ses of metabolism, studies on cell energy relations should offer an advan-
tageous point of attack. In this relation it is encouraging that many cur-
rent researches in plant physiology are concerned with active processes of
water and salt absorption.

The essential role of water involves its contribution to the stature and
elemental composition of the plant, its place in metabolism, its function in
the congregation of solutes at the absorbing surfaces of the roots, and its

Grafts et al. — 206 — Water in Plants

aid in the transport of these solutes throughout the vascular system and in
their final distribution by various mechanisms of translocation, both pri-
mary and secondary.

From results presented, it is evident that water deficits may have pro-
found effects upon plants. Studies on energy absorption by leaves indicate
there is no lack of available energy for photosynthesis and the transport of
water, particularly during full sunlight. Work on the state of water in the
conducting tracts indicates furthermore that the mechanism of transloca-
tion breaks down only after the occurrence of severe wilting. The problem
of the plant's water supply then seems to resolve itself into a question of the
relation of the rate of absorption by roots to the rate of loss from the
leaves. Though certain leaf structures may prevent the loss of the vapor
cloud from the leaf surface and hence conserve moisture, most leaves dur-
ing still weather and all leaves during wind lose large amounts of water
if the stomates are open so that photosynthesis may go on. Consequently,
most plants require large amounts of water from the soil reservoir. In
many soils around one half of the water held against the force of gravity
is available for plants and in light to medium textured soils the movement
of such moisture through the soil to the roots and the growth of roots
through the soil are rapid enough so that plants do not suffer from lack
of moisture until the permanent wilting percentage is approached. In a
heavy soil, on the other hand, Aldrich, et al. (1940) found that pear trees
began to suffer when only one half of the so-called available moisture had
been used. Apparently movement of water through such a soil was so
slow that even though the average moisture content was well above the
PWP the soil immediately around the roots became dry during the day
and was not replenished during the night and root growth was not suffi-
cient to counter-balance this effect.

Plants exhibit many means for meeting conditions of drought. Leaf
and stem modifications have been mentioned as have biochemical trans-
formations within the plant. Diverse root forms, tap roots for deep soils,
fibrous roots for shallow or claypan soils, branching roots, and root hairs
have all been resorted to to enable the plant to obtain adequate water
from the soil. However, there is a limit to root extension. Carbohydrate
supply, the concentrations and proportions of mineral nutrients, soil oxy-
gen, and even genetic factors control the size and form of roots of a given
plant. And in plant populations, particularly crops, competition may
severely limit the extent to which any one root system may profitably grow
into its water supply. For these various reasons there is a definite limit
determined by immutable physical laws that regulates the production of
plants in a given environment. This limit is related to the ability of the
plant to adjust itself to a low water supply and at the same time absorb
CO2 for photosynthesis.

Certain drought-resistant plants may endure long periods of permanent
wilting but though they survive they grow very little while wilted. Suc-
culent plants may close their stomata and carry on an internal economy
that enables them to survive drought but they, too, grow very little in the
process. Other plants, such as the bunch grasses, space themselves so that
they have enough moisture to carry them through the dry periods and still
others dry up and die, leaving only seeds to carry on when moisture is
again available.

Many studies have pointed out the relations of plant distribution to

Chapter X _ 207 — Loss and Retention

moisture supply and the almost innumerable adaptations by which plants
have left their original aquatic environment and moved across valleys and
over mountains and into the remotest wastes of the deserts and frozen arctic
regions. One wonders if during the ages of development, plants, by the
many ways described in these pages, have not about exhausted the possible
means of adaptation. This would impose a definite limit on the extent to
which man may be able by plant breeding or other technical means, to in-
crease production in the absence of available moisture. Though plant ex-
ploration may find plants better able to survive drought, and hybridization
may result in plants with more extensive root systems, it seems that the
most promise lies in the possibility of conserving and utilizing our existing
supplies of water more fully. As forests become depleted, oil reserves
fail, and agricultural lands run short, it may become profitable to harvest
cellulose and other plant products from the vast desert regions that still
remain relatively unoccupied. Production on such areas, however, must
remain slow. More hopeful seems to be the efificient utilization of our pro-
ductive agricultural lands through control of pests, effective use of fertili-
zers, advanced methods of culture and harvest and, at least in our semi-
arid regions where irrigation is practiced, the most effective possible use of
our water resources.

In many lands the impounding and distribution of flood waters and
conservation of all available supplies keynote the current developments in
agriculture.

Summary: — A favorable water balance is essential to growth and development
of plants. Considerable energy may be expended in the absorption of water from the
soil ; however, there are definite limits of DPD of water in the plant above which the
plant cannot operate normally.

Transpiration may be essential in the transport and distribution of mineral nutrients
throughout the plant ; it usually exceeds by far the proportions required for their func-
tion; it has been termed a necessary evil for it is an inevitable consequence of the
gaseous exchange necessary for photosynthesis. Water conservation is important in
the economy of most plants and many of the mechanisms considered in studies on
transpiration serve a vital role in water retention.

Transpiration may be stomatal or cuticular. Much of the regulation of water loss
is effected by stomatal movements.

Studies on evaporation and factors controlling it have aided in an understanding of
transpiration. Evaporation follows definite physical laws and is affected by osmotic
concentration of the solution, temperature, dryness of the air, atmospheric pressure,
and wind velocity.

Many methods have been devised for measuring transpiration such as loss of
weight, water absorption, and various measurements on water vapor given off. All
of these involve restriction of some type upon the natural activity of the plant or the
creation of artificial conditions. The greatest problem in such studies is interpretation
of the results.

The cuticle of plants is a wax-like coating that may vary widely in composition
and thickness. Cuticular transpiration may make up an appreciable portion of the total
water loss of some shade plants. It may be negligible in desert succulents.

Fluctuations in transpiration may be related to wall structure, physical resistance
of the root system to absorption, osmotic properties of cells, internal surface, stomatal
number and distribution, and other internal factors as well as the many external factors
of the environment that govern evaporation.

Opening and closing of stomata have been studied by many methods. Such studies
show that the stomatal mechanism plays an essential role in regulating transpiration
but that the laws are complex and difficult to interpret. Mineral nutrients may affect
transpiration through their osmotic effects upon the DPD of the soil solution, their
tendency to increase the concentration within plant cells, and through secondary effects
such as prevention of deficiency diseases, water permeability, stomatal movement, leaf
abscission, and the like.

Crafts et al. — 208 — Water in Plants

Disease may disrupt metabolism and result in sluggish stomatal response to chang-
ing environment; increased transpiration may result. Insecticides may reduce trans-
piration by interfering with diffusion, or increase it by wedging the stomata open.
Copper sprays increase transpiration by increasing the permeability of the cuticle.

Excessive water loss by nursery stock may be prevented by hardening the plants,
coating with wax, and avoiding any conditions tending to enhance rapid evaporation.

Water deficits affect the physiology of plants by stimulating hydrolysis and in-
hibiting condensation reactions. These responses are brought about by the effect of
water balance on enzymatic activity. Starch dissolution during wilting is a common
reaction of this type. Water deficit may also affect reproductive processes.

Subaqueous transpiration has been explained as due to faulty experimental technic,
water secretion by leaf cells, and the functioning of the mass-flow mechanism of phloem
transport.

Transpiration is important in the physiology of plants as it relates to movement of
mineral and organic nutrients, to the supply of water for metabolic needs, and to its
secondary effects upon growth and development as these integrate the complex rela-
tions of the plants' physical environment. Under many conditions the health of plants,
and hence the well-being of man, depends upon the efficient utilization of the available
supply of water.

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— , and L. P. Gould 1934: The hydrols (Ibid. 38: 197-211).

Bangham, D. H., and F. J. Lewis 1937: Wettability of the cellulose walls of the mesophyll in the
leaf (Nature 139: 1107-1108).

Barger, G. 1904: A microscopical method of determining molecular weights (Jour. Chem. Soc. 85:
286-324).

1924: Eine mikroskopische Methode zur Bestimmung des Molekulargewichtes (Handb. d.

biolog. Arb. Meth. von E. Abderhalden, Abt. Ill, Teil a 1, Heft 4, p. 729).

Barnes, C. R. 1902: The significance of transpiration (Science 15: 460).

Barnes, T. C, and T. L. Jahn 1934: Properties of water of biological interest (Quart. Rev. Biol.

9; 292-341).
Bartell, F. E. 1923: Membrane potentials and their relation to anomalous osmose (Colloid Sympos.

Monograph 1: 120-144).
Bartholomew, E. T. 1926: Internal decline of lemons. III. Water deficit in lemon fruit caused

by excessive leaf evaporation (Amer. Jour. Bot. 13: 102-117).

1931: Certain phases of citrus leaf transpiration (Ibid. 18: 765-783).

Baumgartner, a. 1934: Thermoelektrische Untersuchungen uber die Geschwindigkeit des Trans-

pirationsstromes (Zeitschr. Bot. 28: 81-136).
Beadle, L. C. 1934: Osmotic regulation in Gunda ulvae (Jour. Exp. Biol. 11: 382-396).
Beal, J. M. 1944: Some telemorphic effects induced in sweet pea by application of 4-chloropheno-

xyacetic acid (Bot. Gaz. 105: 471-474).

y

Crafts etal. —210— Water in Plants

Beasley, E. W. 1942: Effects of some chemically inert dusts upon the transpiration rate of yellow

Cole'us plants (Plant Physiol. 17: 101-108).
Beck, W. A. 1927: Cane sugar and potassium nitrate as plasmolysing agents (Protoplasma 1:

15-72).
■ 1928: Osmotic pressure, osmotic value, and suction tension (Plant Physiol. 3: 413-440).

1942: Effect of drought on the production of plant pigments {Ibid. 17: 487-491).

Beck, W. A. and B. Andrus 1943: The osmotic quantities of the cells in the hypocotyl of Helian-

thus annuus seedlings (Bull. Torrey Bot. Club 70: 563-598).
Benedict, H. M. 1927: Application of Bose's theory of sap rise to ten species of trees (Amer.

Jour. 'Bot. 14: 623).
Bennett, C. W. 1937: Correlation between movement of the curly top virus and translocation of

food in tobacco and sugar beet (Jour. Agr. Res. 54: 479-502).
1940o: Relation of food translocation to movement of virus of tobacco mosaic (Jour. Agr. Res.

60: 361-390).

1940&: The relation of viruses to plant tissues (Bot. Rev. 6: 427-473).

, E. Carsner, G. H. Coons, and E. W. Brandes 1946: The Argentine curly top of sugar beet

(Jour. Agr. Res. 72: 19-48). ^ ,-. . ^

Bennett, J. P. 1931: The treatment of lime-induced chlorosis with iron salts (Calif. Agr. Expt.
Sta. 'Circ. S21: 1-12).

, and A. Snell 1939: Transport and distribution of radioactive potassium in the pear tree

(Mss. Private communication).

Bennet-Clark, T. a. 1945: Adaptation to drought (London, Royal College of Science, Scientific
Journal 15: 99-102).

, and D. Bexon 1940: Water relations of plant cells, II (New Phytol. 39: 337-361).

'and 1943: Ibid., III. The respiration of plasmolysed tissues (Ibid. 42: 65-92).

[ and 1946: Ibid., IV. Diffusion effects observed in plasmolysed tissues (Ibid. 45: 5-17).

' A. D. Greenwood, and J. W. Barker 1936: Water relations and osmotic pressures of plant

cells (Ibid. 35: 277-291).

Berkeley, Earl of, and E. G. J. Hartley 1906: On the osmotic pressure of some concentrated
aqueous solutions (Phil. Trans. Roy. Soc. London A206: 481-507).

^ and 1916: Further determinations of direct osmotic pressures (Proc. Roy. Soc. London

'a92: 477-492).

^ and C. V. Burton 1919: On osmotic pressures derived from vapor-pressure measure-
ments:' aqueous solutions of cane sugar and methyl glucoside (Phil. Trans. Roy. Soc. London
A2n: 295-349). . ,. ,

Berl, E., O. Hefter, F. Rau, G. S. Diang, and H. Umstatter 1940: Uber eiiie Abanderunj? der
Molekulargewichtbestimmungsmethode nach Barger (Liebig's Ann. d. Chem. 478: 235-246).

Bernal, J. D. 1937: An attempt at a molecular theory of liquid structure (Trans. Far. Soc. 33:
27-40).

1940: Structural units in cellular physiology (A.A.A.S. The Cell and Protoplasm. Publica-
tion No. 14: 189-205. Science Press, Lancaster, Pa.)

and R. H. Fowler 1933: A theory of water and ionic solution, with particular reference to

hydrogen and hydroxyl ions (Jour. Chem. Phys. 1: 515-548). '
BiALE, J. B. 1941: Periodicity in transpiration of lemon cuttings under constant environmental

conditions (Proc. Amer. Soc. Hort. Sci. 38: 70-74).
BiDDULPH, O. 1940: Absorption and movement of radiophosphorus in bean seedlings (Plant Physiol.

15: 131-136). -r x,
1941: Diurnal migration of injected radiophosphorus from bean leaves (Amer. Jour. Bot.

28: 348-352).
^ and J. Markle 1944: Translocation of radiophosphorus in the phloem of the cotton plant

(Amer. Jour. Bot. 31: 65-70).
Blackman, V. H. 1921: Osmotic pressure, root pressure, and exudation (New Phytol. 20: 106-

Blanchard, K. C. 1940: Water, free and bound (Cold Spring Harbor Symposia on Quant. Biol.

»•• 1-8)'
Blegen, E., and P. Brandt Rehberg 1938: Method for the determination of the osmotic pressure

of biological fluids (Skand. Arch. Physiol. 80: 40-45).
Bode. H. R. 1923: Beitrage zur Dynamik der Wasserbewegung in den Gefasspflanzen (Jahrb. wiss.

Bot. 62: 92-127).
BoEHM, J. A. 1892: Ueber einer eigenthumlichen Stammdruck (Ber. deut. bot. Ges. 10: 539-544).
Bonner, J. 1942: Transport of thiamin in the tomato plant (Amer. Jour. Bot. 29: 136-142).
. 1944: Accumulation of various substances in girdled stem of tomato plants (Ibid. 31: 551-

555).

and R. Dorland 1943: Some observations concerning riboflavin and pantothenic acid in

tomato plants (Ibid. 30: 414-418).

Boon-Long, T. S. 1941 : Transpiration as influenced by osmotic concentration and cell permeability
(Ibid. 28: 333-343).

BosE, J. C. 1923: The physiology of the ascent of sap, 277p. (Longmans, Green and Co., London).

1928: The motor mechanism of plants, 429 p. (Longmans, Green and Co., London).

BouiLLENNE, R. 1932: Contribution a I'etude des phenomenes d'osmose dans les cellules vegetales
(Archives de I'lnstitut de Botanique de I'Universite de Liege 9: 1017-1026).

BouRDiLLON, J. 1939: An apparatus for the rapid and accurate determination of low osmotic pres-
sures (Jour. Biol. Chem. 127: 617-625).

BousFiELD, W. R. 1914: Note on osmotic pressure (Proc. Roy. Soc. London A90: 41-5).

• 1917: Osmotic pressure in relation to the constitution of water and the hydration of the

solute (Trans. Far. Soc. 13: 141-155).

, and T. M. Lowry 1910: Liquid water a ternary mixture; solution volumes in aqueous solu-
tions (Ibid. 6: 85-104).

BouYoucous, G. J. 1911: Transpiration of wheat seedlings as affected by soils, by solutions of
different densities, and by various chemical compounds (Jour. Amer. Soc. Agron. 3: 130-191).

Brauner, L. 1931: Fine neue automatische Transpirationswage (Jahrb. wiss. Bot. 75: 295-303).

*

Water in Plants — 21 1 — Bibliography

1932: Das kleine pflanzenphysiologische Praktikum, II. Die physikalische Chemie der

Pflanzenzelle (Fischer, Jena).

■, and M. Brauner 1940: Further studies of the influence of light upon the water intake and

output of living plant cells (New Phytol. 39: 104-128).

, and 1943o: The relations between water intake and oxybiosis in living plant-tissues, II.

The tensility of the cell wall (Istanbul Universitesi fen Faciiltesi Mecmuasi. B/8: 30-75).

, and 1943b: Studies in the relations between water permeability and electric charge in

membrane models and in living plant cells {Ibid. B/8: 264-310).

, and M. Hasman 1946: Untersuchungen iiber die anomale Komponents des osmotischen Po-
tentials lebender Pflanzenzellen (Ibid. B/11: 1-37).

, and 1947: Weitere Untersuchungen iiber die anomale Komponente des osmotischen Po-
tentials lebender Pflanzenzellen (Ibid. B/12: 210-254).

Brett, P. G. C. 1943: The downward movement of sap produced by rapid killing of the leaves
(So. African Jour. Sci. 39: 126-138).

Briggs, D. R. 1932: Water relations in colloids, II. Bound water in colloids (Jour. Phys. Chem.
36: 367-386).

Briggs, L. J., and H. L. Shantz 1912: The wilting coefficient for different plants and its indirect
determination (U. S. Dept. Agri. Bur. Plant Ind. Bull. 230: 1-83).

, and 1913: The water requirement of plants, I-II (U. S. Dept. Agri. Bur. Plant Ind.

Bulls. 284: 1-49; 285: 1-96).

, and 1915: An automatic transpiration scale of large capacity for use with freely ex-
posed plants (Jour. Agr. Res. 5; 117-132).
-, and 1916: Daily transpiration during the normal growth period and its correlation

with the weather (Ibid. 7 : 155-213).
Brilliant, B. 1924: La teneur en eau dans les feuilles et I'energie assimilatrice (Compt. Rend.

178: 2122-2125).
Brilliant, V. A., and M. N. Chrelashvilli 1941: The relation of photosynthesis in light and

darkness to the degree of dehydration of the assimilating tissues (Trudy Botan. Inst. Acad. Sci.

USSR, Ser. IV (Exptl. Botany) 5; 88-100).
Brooks, S. C. 1929: The accumulation of ions in living cells — a non-equilibriura condition (Proto-

plasma 8: 389-412).

— 1938: The penetration of radioactive potassium chloride into living cells (Jour. Cell. Comp.
Physiol. 11: 247-252).

■ 1940: The standardization of osmotic pressure as a term (Science 92: 428-429).

■ 1945: Permeability (Ann. Rev. Physiol. 7: 1-33).

, and M. M. Brooks. 1941: The permeability of living cells, 395 p. (Borntraeger, Berlin).

Brown, A. J. 1909: The selective permeability of the coverings of the seeds of Hordeum vulgare

(Proc. Roy. Soc. London B.81: 82-93).
Broyer, T. C. 1939: Methods of tissue preparation for analysis in physiological studies with

plants (Bot. Rev. 5: 531-545).

1946: The movement of materials into plants. Part I. Osmosis and the movement of water

in plants (Bot. Rev. 13: 1-58).

, and A. H. Furnstal 1941: A press for recovery of fluids from plant tissues (Plant Physiol.

16: 419-421).
Buhmann, a. 1935: Kritische Untersuchungen iiber vergleichende plasmolytische und kryosko-

pische Bestimmungen des osmotischen Wertes bei Pflanzen (Protoplasma 23: 579-612).
Bull, H. B. 1943: Physical biochemistry, 347 p. (Wiley and Sons, New York)
BiJNNiNG, E. 1935: Zellphysiologische Studien an Meeresalgen (Protoplasma 22: 444-456).
Burcerstein, a. 1920: Die Transpiration der Pflanzen, zweiter Teil (Fischer, Jena).
BuRiAN, R., and K. Drucker 1909: Gefrierpunktsmessungen an kleinen Fliissigkeitsmengen (Zen-

tralbl. Physiol. 23: 772-777).

Callendar, H. L. 1908: On the vapor pressure and osmotic pressure of concentrated solutions

(Proc. Roy. Soc. London A80: 466-500).
Capus, G. 1883: Sur I'observation directe du mouvement de I'eau dans les plantes (Compt. Rend.

97: 1087).
Carrick, D. B. 1930: Some cold-storage and freezing studies on the fruit of the Vinifera grape

(Cornell Univ. Agr. Expt. Sta. Memoir 131: 1-37).
Chadwell, H. M. 1927: The molecular structure of water (Chem. Rev. 4: 375-398).
Chambers, R. 1944: Some physical properties of protoplasm (In Alexander, J. ed. Colloid Chem-
istry 5: 864-875. Reinhold, New York).
Chandler, W. H. 1914: Sap studies with horticultural plants (Missouri Agr. Expt. Sta. Res. Bull.

14, pp. 491-552).
Chandler, R. C. 1941: Nature of bound water in colloidal systems (Plant Physiol. 16: 273-291).
Chatfield, C, and G. Adams 1940: Proximate composition of American food materials (U. S.

Dept. Agric. Circ. 549: 1-91).
Chibnall, a. C. 1923: A new method for the separate extraction of vacuole and protoplasmic

material from leaf cells (Jour. Biol. Chem. 55: 333-342).
Childers, N. F. 1936: Some effects of sprays on the growth and transpiration of tomatoes (Proc.

Amer. Soc. Hort. Sci. 33: 532-535).
Chrelashvili, M. N. 1941: The influence of water content and carbohydrate accumulation on the

energy of photosynthesis and respiration (Trudy Botan. Inst. Acad. Sci. USSR, Ser. IV

[Exptl. Botany] 5; 101-137, p. 136-37 Eng. sum.).
Chu, Chien-Ren 1936: Der Einfluss des Wassergehaltes der Blatter der Waldbaume auf ihre

Lebensfahigkeit, ihre Saugkrafte und ihren Turgor (Flora 130: 384-437).
Chung, C. H. 1935: A study of certain aspects of the phenomenon of transpiration periodicity

(Ph.D. Dissert., The Ohio State University).
Clark, D. G., H. Hecht, O. F. Curtis, and J. I. Shafer, Jr. 1941: Stomatal behavior in inbred

and hybrid maize (Amer. Jour. Bot. 28: 537-541).
Clements, H. F. 1934o: Translocation of solutes in plants (Northwest Sci. 8(4): 9-21).
• 1934b; Significance of transpiration (Plant Physiol. 9: 165-172).

Crafts etal. —212— Water in Plants

— — 1940: Movement of organic solutes in the sausage tree, Kigelia africana (Plant Physiol. 15:

689-700).
, and C. J. Engard 1938: Upward movement of inorganic solutes as aflFected by a girdle

(Plant Physiol. 13: 103-122).
CoLLANDER, R. 1939: Permeabilitatsstudien an Characeen, III. Die Aufnahme und Abgabe von

Kationen (Protoplasma 33: 215-257).
, and H. Barlund 1933: Permeabilitatsstudien an Chara ceratophylla, II. Die Permeabilitat

fur Nichtelektrolyte (Acta Bot. Fenn. No. 11: 1-114).
CoLLORio, H. M. 1928: Untersuchungen iiber die Beziehungen zwischen der Wasserabgabe der

Pflanzen und ihrer Atmungsgrosse (Planta 5: 1-27).
CoLWELL, R. N. 1942: The use of radioactive phosphorus in translocation studies (Amer. Jour. Bot.

29: 798-807).
CoHN, E. J. 1936: Influence of the dielectric constant in biochemical systems (Chem. Rev. 19:

241-273).
Comar, C. L., and C. G. Barr 1944: Evaluation of foliage injury and water loss in connection

with use of wax and oil emulsions (Plant Physiol. 19: 90-104).
Commoner, B., S. Fogel, and W. H. Muller 1943: The mechanism of auxin action. The effect

of auxin on water absorption by potato tuber tissue (Amer. Jour. Bot. 30: 23-28).
, and K. V. Thimann 1941: On the relation of growth and respiration in the Avena coleoptile

(Jour. Gen. Physiol. 24: 279-296).
Conrad, J. P., and F. J. Veihmeyer 1929: Root development and soil moisture (Hilgardia 4:

113-134).
CooiL, B. J. 1941: Significance of phloem exudate of Cucurbita pepo with reference to transloca-
tion of organic materials (Plant Physiol. 16: 61-84).
CoPELAND, E. B. 1902: The rise of the transpiration stream: an historical and critical discussion

(Bot. Gaz. 34: 161-193, 260-283).
Crafts, A. S. 1931: Movement of organic material in plants (Plant Physiol. 6: 1-42).

1932: Phloem anatomy, exudation, and transport of organic nutrients in cucurbits (Ibid. 7:

183-225).

■ 1933o: The use of arsenical compounds in the control of deep-rooted perennial weeds (Hil-
gardia 7: 361-372).

1933&: Sulfuric acid as a penetrating agent in arsenical sprays for weed control (Ibtd. 8:

125-147).
■ 1935: Physiological problems connected with the use of sodium chlorate in weed control

(Plant Physiol. 10: 699-711).

■ 1936: Further studies on exudation in cucurbits (Ibid. 11: 63-79).

— — 1937: The acid-arsenical method in weed control (Jour. Amer. Soc. Agron. 29: 934-943).

- 1938: Translocation in plants (Plant Physiol. 13: 791-814).

■ 1939(7: The relation between structure and function of the phloem (Amer. Jour. Bot. 26;

172-177).

19396: Movement of viruses, auxins, and chemical indicators in plants (Bot. Rev. 5; 471-504).

1939c: The protoplasmic properties of sieve tubes (Protoplasma 33: 389-398).

• 1939rf: Solute transport in plants (Science 90: 337-338).

, and T. C. Broyer 1938: Migration of salts and water into xylem of the roots of higher

plants (Amer. Jour. Bot. 25: 529-535).

^ and P. B. Kennedy 1930: The physiology of Convolvulus arvensis (morning glory or bind-
weed) in relation to its control by chemical sprays (Plant Physiol. 5; 329-344).

, and O. A. Lorenz 1944a: Fruit growth and food transport in cucurbits (Ibid. 19: 131-138).

— — ^ and 19446: Composition of fruits and phloem exudate of cucurbits (Ibid. 19: 326-337).

Currier, H. B. 1943: Water relations of root cells of Beta vulgaris L. (Ph.D. Dissert. Univ. of
Calif.).

1944a: Water relations of root cells of Beta vulgaris (Amer. Jour. Bot. 31: 378-387).

19446: Cryoscopy of small amounts of expressed tissue sap (Plant Physiol. 19: 544-550).

Curtis, C. C. 1902: Some observations on transpiration (Bull. Torrey Bot. Club 29: 360-373).
Curtis, L. C. 1944: The exudation of glutamine from lawn grass (Plant Physiol. 19: 1-5).
Curtis, O. F. 1926: What is the significance of transpiration? (Science 63: 267-271).

• 1935: The translocation of solutes in plants (McGraw-Hill, New York).

, and H. T. Scofield 1933: A comparison of osmotic concentrations of supplying and re-
ceiving tissues and its bearing on the Munch hypothesis of the translocation mechanism (Amer.
Jour. Bot. 20: 502-512).

Dalton, J. 1802: Experimental essays on the constitution of mixed gases; on the force of steam
or vapor from water and other liquids in different temperatures, both in a Torricellian vacuum
and in air; on evaporation; and on the expansion of gases by heat (Mem. Manchester Lit. Phil.
Soc. V: 535-602).

Darwin, F., and D. F. M. Pertz 1911: On a new method of estimating the aperture of stomata
(Proc. Roy. Soc. London B84: 136-154).

Dastur, R. H. 1924: Water content, a factor in photosynthesis (Ann. Bot. 38: 779-788).

1925: The relation between water content and photosynthesis (Ann. Bot. 39: 769-786).

, and B. L. Desai 1933: The relation between water-content, chlorophyll content, and the

rate of photosynthesis in some tropical plants at different temperatures (Ann. Bot. 47: 69-88).

Davis, A. R., and D. R. Hoagland 1928: An apparatus for the growth of plants in a controlled
environment (Plant Physiol. 3: 277-292).

Davis, C. H. 1940: Absorption of soil moisture by maize roots (Bot. Gaz. 101: 791-805).

Davson, H., and J. F. Danielli 1943: The permeability of natural membranes, 361 p. (Cam-
bridge. Univ. Press, Cambridge, Eng.).

DeBye, p. 1936: Dielectric properties of pure liquids (Chem.'Rev. 19: 171-182).

Delf, E. M. 1912: Transpiration in succulent plants by measurement of rate of shrinkage of
turgid tissues (Ann. Bot. 26: 409-442).

■ 1916: Studies of protoplasmic permeability (Ann. Bot. 30: 283-310).

Denny, F. E. 1917: Permeability of certain plant membranes to water (Bot. Gaz. 63: 373-397).

l<

Water in Plants — 213 — Bibliography

Desai, M. C. 1937: Effect of certain nutrient deficiencies on stomatal behavior (Plant Physiol.

12: 253-283).
Dietrich, M. 1925: Die Transpiration der Schatten- und Sonnen-pflanzen in ihren Beziehungen

zum Standort (Jahrb. f. wiss. Bot. 65: 98-194).
Dixon, H. H. 1898: Transpiration into a saturated atmosphere (Proc. Roy. Irish Acad. Ill, 4:

627-635).

1911: A thermo-electric method of cryoscopy (Proc. Roy. Dubl. Soc. XIII: 49-63).

' 1914: Transpiration and the ascent of sap in plants, 216 p. (Macmillan, New York).

■ 1924: The transpiration stream, 80 p. (Univ. of London Press, Ltd., London).

1933: Bast-sap (Sci. Proc. Roy. Dublin Soc. 20: 487-494).

1937: The convection of heat and materials in the stem of a tree (Notes from the Botanical

School, Trinity College, Dublin, 4: 269-278).

• 1938o: Transportation of substances in plants {Ibid. 4: 279-304).

1938&: Subaqueous transpiration (Sci. Proc. Roy. Dublin Soc. 22: 55-57).

, and W. R. G. Atkins 1912: Changes in the osmotic pressure of the sap of the developing

leaves of Syringa vulgaris {Ibid. 13: 219-222).

, and 1913: Osmotic pressures in plants, I. Methods of extracting sap from plant

organs (Ibid. 13: 422-433).

, and 1916: Osmotic pressure in plants, VI. On the composition of the sap in the con-
ducting tracts of trees at different levels and at different seasons of the year {Ibid. 15: 51-62).

, and J. S. Barlee 1940: Further experiments on transpiration into a saturated atmosphere

(Ibid. 22: 211-222).
-, and J. JoLY 1894: On the ascent of sap (Ann. Bot. 8: 468-470).

DoNEEN, L. D. 1934: Method for the preparation of green plant material for the extraction of

juices (Plant Physiol. 9: 839-843).
DoRSEY, N. E. 1940: Properties of ordinary water-substance, 673 p. (Reinhold, New York).
Drucker, C, and E. ScfaREiNER 1913: Mikrokryoskopische Versuche (Biolog. Zentralbl. 33:

99-103).
Dutrochet, R. J. H. 1827: Nouvelles observations sur I'endosmose, et sur la cause de ce double

phenomene (Annales Chim. Phys. 35: 393-400).

Eaton, F. M. 1941. Water uptake and root growth as influenced by inequalities in the concen-
tration of the substrate (Plant Physiol. 16: 545-564).

1943: The osmotic and vitalistic interpretations of exudation (Amer. Jour. Bot. 30: 663-674).

Edlefsen, N. E. 1941 : Some thermodynamic aspects of the use of soil-moisture by plants (Amer.

Geophys. Union Trans, of 1941, part III, 22: 917-926).

, and A. B. C. Anderson 1943: Thermodynamics of soil moisture (Hilgardia 15: 31-298).

Elazari-Volcani, T. 1936: The influence of a partial interruption of the transpiration stream by

root pruning and stem incisions on the turgor of citrus trees (Palestine Jour. Bot. Hort. Sci.

1: 94-96).
Eley, D. D. 1944: The structure of water in aqueous solutions (Trans. Far. Soc. 40: 184-194).
Elford, W. J. 1937: Principles governing the preparation of membranes having graded porosities.

The properties of "Gradocol" membranes as ultrafilters (Ibid. 33: 1094-1104).
Emerson, J. L., and A. C. Hildreth 1933: Preliminary report on reducing transpiration of

transplanted evergreens (Science 77: 433-434).
Engard, C. J. 1939a: Translocation of carbohydrates in the Cuthbert raspberry (Bot. Gaz. 100: 439-

464).

1939&: Translocation of nitrogenous substances in the Cuthbert raspberry (Ibid. 101: 1-34).

Erickson, L. C. 1945: The water factor in transplanting guayule (Amer. Jour. Bot. 32: 634-643).
Ernest, E. CM. 1931: Suction-pressure gradients and the measurement of suction pressure (Ann.

Bot. 45: 717-731).

1934a: Studies in the suction pressure of plant cells, II (Ibid. 48: 293-305).

1934&: The effect of intercellular pressure on the suction pressure of cells (Ibid. 48: 915-918).

1934c: The water relations of the plant cell (Jour. Linn. Soc. [London] Bot. 49: 495-502).

1935. Factors rendering the plasmolytic method inapplicable in the estimation of osmotic

values of plant cells (Plant Physiol. 10: 553-558).

Ewell, R. H., and H. Eyring 1937: Theory of the viscosity of liquids as a function of temperature

and pressure (Jour. Chem. Phys. 5: 726-736).
Eyster, H. C. 1940: A standardization of osmotic pressure as a term (Science 92: 171-172).

1943: Osmosis and osmotic pressure (Bot. Rev. 9; 311-324).

Farr, W. K. 1944: Plant cell membranes (In Alexander, J., Colloid Chemistry 5: 611-667. Chemi-
cal Catalog Co., New York).

Ferri, M. G. 1945: Preliminary observations on the translocation of synthetic growth substances
(Contrib. Boyce Thompson Inst. 14: 51-68).

FiNBAK, C. and H. Viervoll 1943: The structure of liquids, II. The structure of liquid water
(Tids. Kjemi, Bergvesen Met. 3: 36-40).

FiNDLAY, A. 1919: Osmotic pressure, 116 p. (Longmans, Green and Co., London).

Fischer, M. H. 1921: Oedema and nephritis, 3rd. ed., 209 p. (Wiley, New York).

1923: On the theory of the lyophilic colloids and the behavior of protoplasm (Colloid Sympos.

Monograph 1: 244-264).

Fitting, H. 1917: Untersuchungen iiber isotonische Koeffizienten und ihren Nutzen fiir Permeabili-

tatsbestimmung (Jahrb. wiss. Bot. 57: 553-612).
Fogg, G. E. 1944: Diurnal fluctuation in a physical property of leaf cuticle (Nature 154: 515).
Foster, A. C, and E. C. Tatman 1940: Effect of certain fungicides and environmental factors on

the rate of transpiration of tomato plants (Jour. Agr. Res. 61: 721-735).
Frazer, J. C. W., and R. T. Myrick 1916: The osmotic pressure of sucrose solutions at 30°

(Jour. Amer. Chem. Soc. 38: 1907-1922).
Freeman, G. F. 1908: A method for the quantitative determination of transpiration in plants (Bot.

Gaz. 46: 118-129).
Frey-Wyssling, a. 1938: Submikroskopische Morphologie des Protoplasmas und seiner Derivate,

317 p. (Borntraeger, Berlin).

Crafts et al. —214— Water in Plants

■ 1939: The submicroscopic structure of cell walls (Science Progress 34: 249-262).

1940: The submicroscopic structure of the cytoplasm (J. Roy. Microscop. Soc. 60: 128-139).

Fritsch, F. E. 1922: The moisture relations of terrestrial algae, I. Some general observations and
experiments (Ann. Bot. 36: 1-20).

FuRLiNGER, H. 1938: Plasmolyse verhindert ErgrQnen (Protoplasma 30: 328-333).

FuRR, J. R., and E. S. Degman 1932: Relation of moisture supply to stomatal behavior of the
apple (Proc. Amer. Soc. Hort. Sci. 28: 547-551).

, and J. O. Reeve 1945: Range of soil-moisture percentages through which plants undergo

permanent wilting in some soils from semiarid irrigated areas (Jour. Agr. Res. 71: 149-170).

, and C. A. Taylor 1935: The apparent growth rate of lemon fruits as an index of the mois-
ture supply of the tree (Amer. Soc. Hort. Sci. Proc. 33: 70).

, and 1939: Growth of lemon fruits in relation to moisture content of the soil (U. S.

Dept. Agr. Tech. Bull. 640: 1-72).

Gail, F. W. 1926: Osmotic pressure of cell sap and its possible relation to winter killing and leaf

fall (Bot. Gaz. 81: 434-445).
— — , and W. H. Cone 1929: Osmotic pressure pU measurements on cell sap of Pinus ponderosa

(Bot. Gaz. 88: 437-441).
Gallenkamp, W. 1919: Versuche fiber den Zusammenhang von Verdunstungsmenge und Grosse

der verdunstenden Flachen (Meteorol. Zeitschr. 36: 16-22).
Gasser, R. 1942: Zur Kenntnis der Aenderung der Saugkraft bei Grenzplasmolyse durch Was-

serunter- und -uberbilanz (Ber. schweiz. botan. Ges. 52: 47-110).
Gassner, G., and G. Goeze 1936: Einige Versuche fiber die Physiologische Leistungsfahigkeit

rostinfizierter Getreideblatter (Phytopath. Zeitschr. 9; 371-386).
Gaumann, E. 1942: Uber die pflanzliche Transpiration. 1. Die kutikulare Transpiration (Zeit-

schrift fur Botanik 38: 225-327).
Gehenio, p. M. 1941: The freezing points of living and dead myxomycetes (Biodynamica 3:

348-352).
Gi;NEAU de LAMARLiiRE, L. 1892: Recherches physiologiques sur les feuilles, developpees a I'ombre

et au soleil, VI. Transpiration (Rev. Gen. Bot. 4: 529).
Getman, F. H., and F. Daniels 1943: Outlines of Theoretical Chemistry, 691 p. (John Wiley

and Sons, New York).
GiBBS, R. D. 1935: Studies of wood, II. The water content of certain Canadian trees, and changes

in the water-gas system during seasoning and flotation (Canad. Jour. Res. 12: 727-760).
GiCKLHORN, J., and F. Weber 1926: Ober Vakuolenkontraktion und Plasmolyseform (Protoplasma

1: 427-432).
Glasstone, S. 1940: Textbook of physical chemistry, 1289 p. (D. Van Nostrand, New York).
GoDLEWSKi, E. 1884: Zur Theorie der Wasserbewegung in den Pflanzen (Jahrb. wiss. Bot. 15:

569-630).
Goldsmith, G. W., and J. H. C. Smith 1926: Some physico-chemical properties of spruce sap and

their seasonal and altitudinal variation (Colorado Col. Pub. Sci. Ser. 13: 13-71).
Golovina, a. S. 1939: The effect of drought on the biochemical processes of plants (Voronezh.

Gosudarst. Univ., Nauch. Raboty Studentov [1939]: 51-58).
GoRTNER, R. A. 1938: Outlines of biochemistry, 1017 p. (Wiley and Sons, New York).
, and J. A. Harris 1914: Notes on the technique of the determination of the depression of the

freezing point of vegetable saps (Plant World 17: 49-53).
, J. V. Lawrence, and J. A. Harris 1916: The extraction of sap from plant tissues by pres-
sure (Biochem. Bull. 5: 139-142).
Grafe, V. 1924: Messung der Gas- und Wasserbewegung (Handbuch der biolog. Arb. Meth. von

E. Abderhalden, Abt. XI, Teil 2: 105-186).
Graf-Marin, a. 1934: Studies on powdery mildew of cereals (N. Y. [Cornell] Agr. Expt. Sta.

Mem. 157: 1-48).
Greenfield, S. S. 1941: Differential inhibition of photochemical and dark reactions m photo-
synthesis by inorganic compounds (Science 93: 550-551).
1942: Inhibitory effects of inorganic compounds on photosynthesis in Chlorelta (Amer. Jour.

Bot. 29: 121-131).
Gregory, F. G., and Pearse, H. L. 1934: The resistance porometer and its application to the study

of stomatal movement (Proc. Roy. Soc. B.114: 477-493).
Gross, F. 1934: Zur Biologie und Entwicklungsgeschichte von Noctiluca miliaris (Arch. Protistenk.

83: 178-196).
1940: The osmotic relations of the plankton diatom Ditylum brighttvelti (West.) (Jour. Mar.

Biol. Assn. U. K. 24: 381-415).
Grossenbacher, K. A. 1938: Diurnal fluctuation in root pressure (Plant Physiol. 13: 669-676).

1939: Autonomic cycle of rate of exudation of plants (Amer. Jour. Bot. 26: 107-109).

GuiLLiERMOND, A. 1941: The cytoplasm of the plant cell, 247 p. (Chronica Botanica Co., Waltham,

Mass.).
GuYE, P. A. 1910: The chemical nature of molecular association (Trans. Far. Soc. 6: 78-85).
Gyorffy, B. 1941: Untersuchungen fiber den osmotischen Wert polyploider Pflanzen (Planta 32:

15-37).

Haan, I. DE 1933: Protoplasmaquellung und Wasserpermeabilitat (Recueil des Travaux botaniques

Neerlandais 30: 234-335).
Haberlandt, G. 1914: Physiological plant anatomy. Translation from the 4th German edition by M.

Drummond, 777 p. (Macmillan and Co., New York).
Haines, F. M. 1935: Observations on the occurrence of air In conducting tracts (Ann. Bot. 49:

367-379).
Haldane, J. S. 1918: The extension of the gas laws to liquids and solids (Biochem. Jour. 12:

464-498).
Hales, S. 1738: Vegetable staticks, Vol. 1, 3rd ed., 376 p. (London).
Halket, a. C. 1913: On various methods for determining osmotic pressures (New Phytol. 12:

164-176).

Water In Plants —215— Bibliography

Hall, W. H. 1940: Suggestions regarding a proposed standardization of osmotic pressure as a

term (Science 92: 334).
Halma, F. F., and A. R. C. Haas 1928: Effect of sunlight on sap concentration of citrus leaves

(Bot. Gaz. 86: 102-106).
Hamner, K. C, and J. Bonner 1938: Photopetiodism in relation to hormones as factors in floral

initiation and development {Ibid. 100: 388-431).
Handley, W. R. C. 1939: The effect of prolonged chilling on water movement and radial growth

in trees (Ann. Bot. 2: 803-813).
Hannig, E. 1912: Untersuchungen iiber die Verteilung des osmotischen Druckes in der Pflanze in

Hinsicht auf die Wasserleitung (Ber. deut. bot. Ges. 20: 194-204).
Hansteen-Cranner, B. 1914: Uber das Verhalten der Kulturpflanzen zu den Bodensalzen, III

(Jahrb. wiss. Bot. 52: 536-602).
Harkens, W. D. 1926: Surface energy and surface tension {In Alexander, J., Colloid Chemistry

1: 192-264. Chemical Catalog Company, New York).
Harned, H. S., and B. B. Owen 1943: The physical chemistry of electrolytic solutions, 611 p.

(Reinhold, New York).
Harris, J. A. 1915: An extension to 5.99° of tables to determine the osmotic pressure of expressed

vegetable saps from the depression of the freezing point (Amer. Jour. Bot. 2: 418-419).

1934: The physico-chemical properties of plant saps in relation to phytogeography, 339 p.

(Univ. Minnesota Press, Minneapolis).

— — , and R. A. Gortner 1914: Notes on the calculation of the osmotic pressure of expressed

vegetable saps from the depression of the freezing point, with a table for the values of P for A

= 0.001° to A = 2.999° (Amer. Jour. Bot. 1: 75-78).
- — ■ — , J. V. Lawrence, and R. A. Gortner 1916: The cryoscopic constants of expressed vegetable

saps as related to local environmental conditions in the Arizona deserts (Physiol. Res. 2: 1-49).
Harter, L. L. 1908: The influence of a mixture of soluble salts, principally sodium chlorid, upon

the leaf structure and transpiration of wheat, oats, and barley (U. S. Dept. Agric. Bur. Plant

Ind. Bull. 124: 1-22).
Hartmair, V. 1937: tJber Vakuolenkontraktion in Pflanzenzellen (Protoplasma 2S: 582-592).
Hartsuijker, K. 1935: Kritische Bemerkungen ueber einige der wichtigsten Methoden zur Ermitt-

lung des Oeffnungszustandes der Spaltoeffnungen (Rec. trav. botan. Neerland. 22: 516-542).
Harvey, E. B. 1917: A physiological study of specific gravity and of luminescence in Noctiluca,

with special reference to anesthesia (Publ. Carnegie Inst. 11, p. 237-253).
Hausermann, E. 1944: The amount of wetting of mesophylHc intercellular spaces (Ber. schwez.

boton. Ges. 54: 541-578).
Hasman, M. 1943: A study of the shape of the determinant curve in measurements of the suction

potential in plant tissues (Istanbul Universitesi fen Facultesi Mecmuasi. B/8: 167-200).
Hasselbring, H. 1914: The relation between the transpiration stream and the absorption of salts

(Bot. Gaz. 57: 72-73).
Hayward, H. E., and W. B. Spurr 1944: Effects of isosmotic concentrations of inorganic and or-
ganic substrates on entry of water into corn roots (Bot. Gaz. 106: 131-139).
Heath, O. V. S. 1941: Experimental studies of the relation between carbon assimilation and

stomatal movement, II. The use of the resistance porometer in estimating stomatal aperture

and diffuse resistance. (Ann. Bot. N.S. 5: 455-500).
Heinicke, a. J., and N. F. Childers 1935: The influence of water deficiency in photosynthesis and

transpiration of apple leaves (Proc. Amer. Soc. Hort. Sci. 22: 155-159).
Henderson, L. J. 1924: The fitness of the environment, 317 p. (Macmillan, New York).
Hendrickson, a. H., and F. J. Veihmeyer 1941: Moisture distribution in soil containers (Plant

Physiol. 16: 821-826).
, and 1942: Irrigation experiments with pears and apples (Calif. Agr. Exp. Sta. Bull.

667).
^ and 1945: Permanent wilting percentages of soils obtained from field and laboratory

trials (Plant Physiol. 20: 517-539).
Henrici, M. 1943: Data on the sugar and starch content of some fodder plants under different

physiological conditions (So. African Jour. Sci. 40: 157-161).

1945 : The effect of wilting on the direct assimilates of lucerne and other fodder plants {Ibid.

41: 204-212).

Hermans, P. H., and A. Weidinger 1946: The hydrates of cellulose (Jour. Colloid Sci. 1: 185-

193).
Herrick, E. M. 1933: Seasonal and diurnal variations in the osmotic values and suction tension

values in the aerial portions of Ambrosia trifida (Amer. Jour. Bot. 20: 18-34).

1934: A three wire thermocouple system for use in cryoscopic investigations {Ibid. 21: 673-

687).

Heuser, E. 1944: The chemistry of cellulose, 660 p. (Wiley, New York).

Heyn, a. N. J. 1940: The physiology of cell elongation (Bot. Rev. 6: 515-574).

Hibbard, R. p., and O. E. Harrington 1913: Depression of the freezing point in triturated plant

tissues and the magnitude of this depression as related to soil moisture (Physiol. Res. 1: 441-

454).
Hiesey, W. M. 1940: A critical study of transpiration rates of alpine and lowland forms of Poten-

tilla gracilis (Ph.D. Dissert., Univ. of California).
Hildebrand, E. M. 1942: Rapid transmission techniques for stone-fruit viruses (Science 95: 52).
, and O. F. Curtis 1942: A darkening technique for inducing virus symptoms in mature as well

as in growing leaves (Science 95: 390).
Hildebrand, J. H. 1924: Solubility (Amer. Chem. Soc. Monograph Ser., 206 p., Chem. Catalog.

Co., New York).

1936: Solubility of non-electrolytes, 2nd ed., 303 p. (Reinhold, New York).

1937: Intermolecular forces in solutions (Trans. Far. Soc. 22: 144-151).

Hill, A. V. 1930: A thermal method of measuring the vapour pressure of aqueous solutions (Proc.

Roy, Soc. London A127: 9-19).
HiRSCHFELDER, J., D. Stevenson, and H. Eyring 1937: A theory of liquid structure (Jour. Chem.

Physics 5: 896-912).

Crafts et al. — 216 — Water in Plants

HoAGLAND, D. R. 1944: Lectures on the inorganic nutrition of plants, 226 p. (Chronica Botanica
Co., Waltham, Mass.)

, and T. C. Broyer 1936: General nature of the process of salt accumulation by roots with

description of experimental methods (Plant Physiol. 11: 471-507).

^ and 1942: Accumulation of salt and permeability in plant cells (Jour. Gen. Physiol.

25: 865-880).

^ and A. R. Davis 1923a: The composition of the cell sap of the plant in relation to the absorp-
tion of ions {Ihid. S: 629-646).

^ and 1923fc: Further experiments on the absorption of ions by plants, including observa-
tions on the effect of light (,Ibid. 6: 47-62).

^ and 1929: The intake and accumulation of electrolytes by plant cells (Protoplasma 6:

610-626).

HoBER, R. 1945: Physical chemistry of cells and tissues, 676 p. (Blakiston, Philadelphia).

HocHREUTiNER, G. 1896: Physiologic des plantes aquatiques du Rhone et du Port de Geneve (Rev.
Gen. Bot. 8: 158-167).

Hoffmann, C. 1932: Zur Frage der osmotischen Zustandsgrossen bei Meeresalgen (Planta 17: 80S-
809).

HoFLER, K. 1917: Die plasmolytisch-volumetrische Methode und ihre Anwendbarkeit zur Messung
des osmotischen Wertes lebender Pflanzenzellen (Ber. deut. bot. Ges. 35; 706-726).

1920: Ein Schema fur die osmotische Leistung der Pflanzenzelle (Ibid. 38: 288-298).

1930: Das Plasmolyse-Verhalten der Rotalgen (Zeitschr. f. Bot. 23: 570-588).

1931: Hypotonietod und osmotische Resistenz einiger Rotalgen (Oesterr. Bot. Zeitschr. 80:

51-71).

1939: Kappenplasmolyse und lonenantagonismus (Protoplasma 33: 545-578).

HoFMEisTER, L. 1940a: Mikrurgische Studien an Borraginoideen-Zellen, I. Mikrodissektion (Pro-
toplasma 35: 65-94).

1940b: Ibid., II. Mikroinjektion und mikrochemische Untersuchung Ubid. 35: 161-186).

HoFMEiSTER, W. 1862: Uber Spannung, Ausflussmenge und Ausflussgeschwindigkeit von Saften

lebender Pflanzen (Flora 45: 97-108).
HoLLE, H. 1915: Untersuchungen ueber Welken, Vertrocknen und Wiederstraffwerden (l<lora,

N.' F. 8: 73-126). . , .

HoMAN, C, T. F. Young, and C. A. Shull 1934: Evaporation in its thermodynamic relation to

the cohesion theory and to imbibitional and osmotic pressures of cell constituents (Plant Physiol.

9: 653-661).
HousKA, H. 1941: Beitrage zur Kenntnis der Kappenplasmolyse (Protoplasma 36: 11-51).
HuBER, B. 1923: Transpiration in verschiedener Stammhohe, I. Sequoia gigantea (Zeitschr. f. Bot.

1932: Beobachtung und Messung pflanzlicher Saftstrome (Ber. deut. bot. Ges. 50: 89-109).

1933: Beitrage zur Kenntnis der Wasserpermeabilitat des Protoplasmas (Ibid. 51: 53-64).

1937: Wasserumsatz und Stoffbewegungen (Fortschitte der Botanik 7: 197-207).

, and K. H5fler 1930: Die Wasserpermeabilitat des Protoplasmas (Jahrb. f. wlss. Bot. 73:

351-511). , ^ . ^.

^ and E. Schmidt 1936: Weitere thermoelektrische Untersuchungen uber den Transpirations-

strom der Baiime (Tharandter Forstl. Jahrb. 87: 369-412).

and 1937: Eine Kompensationsmethode zur thermoelektnschen Messung langsamer Saft-
strome (Ber. deut. bot. Ges. 55: 514-529). _

and H. Jahnel 1937: Untersuchungen tiber den Assimilatstrom, I (Tharandter J^orsti.

Jahrb. 88: 1017-1050).
Humphreys, W. J. 1940: Physics of the air, 676 p. (McGraw-Hill, New York).

Iljin, W. S. 1922o: Wirkung der Kationen von Salzen auf den Zerfall und die Bildung von

Starke in den Pflanzen (Biochem. Zeitschr. 132: 494-510).
19226: Ueber den Einfluss des Welkens der Pflanzen auf die Regulierung der Spaltoffnungen

(Jahrb. f. wiss. Bot. 61: 670-697, 698-712).
1923: Der Einfluss des Wassermangels auf die Kohlenstoffassimilation durch die Pflanzen

(Flora 116: 360-378).
International Critical Tables 1933 (McGraw-Hill, New York).
IVANOV, L. A. 1924: Ueber die Transpiration der Holzgewiichse im Winter, I u. II (Ber. deut. bot.

Ges. 42: 44-49, 210-218).
1928: Zur Methodik der Transpirationsbestimmung am Standort (Ibid. 46: 306-310).

Jaccard, p., and A. Frey-Wyssling 1934: Uber Versuche zur Bestimmung der Zellsaftkonzentra-
tion in 'der Kambialzone beim exzentrischen Dickenwachstum, I (Jahrb. f. wiss. Bot. 79: 655-

680).
Janse, J. M. 1913: Die Wirkung des Protoplasten in den Zellen welche bei der Wasserbewegung

beteiligt sind (Jahrb. f. wiss. Bot. 52: 603-621).
Jeffreys, H. 1918: Some problems of evaporation (Phil. Mag. 35: 270-280).
Jenny, H., and R. Overstreet 1939: Cation interchange between plant roots and soil colloids (Soil

Science 47: 257-272). . .

Johnston, C. O. 1940: Modification of diurnal transpiration in wheat by infections of Pucama

triticina (Jour. Agr. Res. 61: 427-444).

Kaiserlehner, E. 1939: Uber Kappenplasmolyse und Entmischungsvorgange im Kappenplasma
(Protoplasma 33: 579-601).

Kamp, H. 1930: Untersuchungen iiber Kutikularbau und kutikulSre Transpiration von Blattern
(Jahrb. f. wiss. Bot. 72: 403-465).

Katzoff, S. 1934: X-ray studies on the molecular arrangement in liquids (Jour. Chem. Physics 2:
841-851).

Kendall, J. 1937: Pure liquids and liquid mixtures (Trans. Far. Soc. 33: 2-7).

Kennedy, P. B., and A. S. Crafts 1927: The application of physiological methods to weed con-
trol (Plant Physiol. 2: 503-506).

Water in Plants — 217 — Bibliography

, and 1931: The anatomy of Convolvulus arvensis, wild morning glory or field bindweed

(Hilgardia 5; 591-622).
Kenny, I. J., and D. R. Porter 1941: Relative rind toughness among watermelon varieties (Proc

Amer. Soc. Hort. Sci. 38: 537-540).
Kerr, T., and D. B. Anderson 1944: Osmotic quantities in growing cotton bolls (Plant Physiol

19: 338-349).
Kessler, W., and W. Ruhland 1938: Weitere Untersuchungen uber die inneren Ursachen der

Kalteresistenz (Planta 28: 159-204).
Keys, A. 1932: The mechanism of adaption to varying salinity in the common eel and the general

problem of osmotic regulation in fishes (Proc. Roy. Soc. London B112: 184-199).
KiESSELBACH, T. A. 1916: Transpiration as a factor in crop production (Nebraska Agr. Exp Sta

Res. Bull. 6: 1-214).
KiNCAiD, J. F., H. Eyring, and A. E. Stearn 1941: The theory of absolute reaction rates and its

application to viscosity and diffusion in the liquid state (Chem. Rev. 28: 301-365).
KiRKwooD, J. G. 1936: Statistical mechanics of liquid solutions (Chem. Rev. 19: 275-307).
KiscH, R. 1937: Die Bedeutung der Wasserversorgung fiir den Ablauf der Meiosis (Tahrb. f. wiss

Bot. 85: 450-484).
Kisser, J. 1927: Untersuchungen fiber den Einfluss der Nahrsalze auf die Wasserabgabe, Wasser-

aufnahme, relative Spross- und Wurzelmasse und die Blattstruktur (Planta 3: 562-577).
KiTCHiNG, J. A. 1938: Contractile vacuoles (Biol. Rev. 13: 403-444).
Knight, R. C. 1922: Further observations on the transpiration, stomata, leaf water-content, and

wilting of plants (Ann. Bot. 36: 361-383).
Knight, T. A. 1801: Account of some experiments on the ascent of sap in trees (Phil. Trans. Roy.

Soc. London 91: Z^Z-Zli).
Kohl, F. G. 1886: Die Transpiration der Pflanzen und ihre Einwirkung auf die Ausbildung

pflanzlicher Gewebe, 124 p. (H. Braunschweig, Brunn).
Kondo, I. N. 1931: The influence of external factors as well as of the stages of development on the

resistance of plants to dehydration (Bull. Applied Bot. of Genetics and Plant Breeding, Trudy

27: 129-156).
KoRSTiAN, C. F. 1924: Density of cell sap in relation to environmental conditions in the Wasatch

mountains of Utah (Jour. Agr. Res. 28: 845-907).
KoTTE, H. 1915: Turgor und Membranquellung bei Meeresalgen (Wiss. Meeresunters , Abt Kiel

N. F. Bd. 17: 115-167).
Kramer, P. J. 1933: The intake of water through dead root systems and its relation to the problem

of absorption by transpiring plants (Amer. Jour. Bot. 20: 481-492).

1939: The forces concerned in the intake of water by transpiring plants {Ihid. 26: 784-791).

1944: Soil moisture in relation to plant growth (Bot. Rev. 10: 525-559).

■ 1945: Absorption of water by plants (Bot. Rev. 11: 310-355).

Krasnosselsky-Maximov, T. A. 1931: Physiological analysis of windburn by means of artificial

dry wind (Bull. Applied Bot. of Genetics and Plant Breeding, Trudy 25: 3-44).
Krausche, K. K., and B. E. Gilbert 1937: Increase of transpiration rates of tomato leaves due

to copper sprays (Plant Physiol. 12: 853-860).
Krogh, a. 1939: Osmotic regulation in aquatic animals, 242 p. (Cambridge Univ. Press).

1946: The active and passive exchange of inorganic ions through the surfaces of living cells

and through living membranes generally (Proc. Roy. Soc. London B133: 140-200).

Kursanov, a. L. 1941: The physiological role of enzymes in plants (Collection of papers on plant
physiology in memory of K. A. Timiryazev, Acad. Sci. USSR, Inst. Plant Physiol, in the
name of K. A. Timiryazev 1941, 131-169).

Kuster, E. 1928: Uber die Gewinnung nackter Protoplasten (Protoplasma 3: 223-233).

1929: Beobachtungen an verwundeten Zellen (^Ibid. 7: 150-170).

1935: Die Pflanzenzelle, 672 p. (Gustav Fischer, Jena).

1940: Neue Objekte fur die Untersuchung der Vakuolenkontraktion (Ber. deut bot Ges 58-

413-416). ■ ■

Latshaw, W. L., and E. C. Miller 1924: Elemental composition of the corn plant (Tour Aer Res
27: 845-859). vj • s ■

Leick, E. 1939: Bestimmung der Transpiration und Evaporation mit Rucksicht auf die Bedurfnisse
der Okologie (Handb. d. biolog. Arb. Meth. von E. Abderhalden, Abt. XI, Teil 4: 1573-1735).

Leighly, J. 1937: A note on evaporation (Ecology 18: 180-198).

Leitch, J. L. 1934: The water exchanges of living cells, II. Non solvent volume determinations
from swelling and analytical data (Jour. Cell. Comp. Physiol. 4: 457-473).

Lepeschkin, W. W. 1936: The "Vitaids." A theory of the fundamental substances of living
matter (Biodynamica 1 [No. 19]: 1-6).

1937: Zell-Nekrobiose und Protoplasma-Tod, 198 p. (Borntraeger, Berlin).

1938: Kolloidchemie des Protoplasraas, 244 p. (Steinkopff, Dresden).

Levitt, J. 1941: Frost killing and hardiness of plants, 211 p. (Burgess Publ. Co., Minneapolis,
Minn.).

1946,: Osmotic pressure determinations with isolated protoplasmic proteins (Plant Physiol

21: 562-572).

• 1947: The thermodynamics of active (non-osmotic) water absorption {Ibid. 22: 514-525).

, and G. W. Scarth 1936: Frost hardening studies with living cells (Canad. Jour Res C14-

267-305).

. . and R. D. Gibbs 1936: Water permeability of isolated protoplasts in relation to volume

change (Protoplasma 26: 237-248).

Lewis, F. J. 1945: Physical conditions of the surface of the mesophyll cell walls of the leaf (Na-
ture 156: 407-490).

, and G. M. Tuttle 1920: Osmotic properties of some plant cells at low temperatures (Ann

Bot. 34: 405-416).
Lewis, G. N. 1908: The osmotic pressure of concentrated solutions, and the laws of the perfect

solution (Jour. Amer. Chem. Soc. 30: 668-683).
1916: The atom and the molecule (.Ibid. 38: 762-785).

Crafts etal. —218— Water in Plants

, and M. Randall 1923: Thermodynamics and the free energy of chemical substances, 653 p.

(McGraw-Hill, New York).
Li, T. T. 1929: Effect of climatic factors on suction force (Quart. Rev. Biol. 4: 401-414).
Livingston, B. E. 1903: The rSle of diffusion and osmotic pressure in plants, 149 p. (Univ. of

Chicago Press, Chicago).

1906: The relation of desert plants to soil moisture and to evaporation (Carnegie Inst, of

Washington Publ. No. 50: 1-78).

1913: The resistance offered by leaves to transpirational water loss (Plant World 16: 1-35).

, and E. B. Shreve 1916: Improvements in the method for determining the transpiring power

of plant surfaces by hygrometric paper {Ibid. 19: 287-309).
Livingston, L. C, and B. M. Duggar 1934: Experimental procedures in a study of the location

and concentration within the host cell of the virus of Tobacco Mosaic (Biol. Bull. 67: 504-

512).
Lloyd, D. J., and W. B. Pleass 1927: The absorption of water by gelatin (Biochem. Jour. 21:

1352-1367).
Lloyd, F. E. 1908: The physiology of stomata (Carnegie Inst. Wash. Publ. 82: 1-142).

1912: The relation of transpiration and stomatal movements to the water content of the leaves

of Fouquieria splendens (Plant World 15: 1-14).

1913: Leaf water and stomatal movement in Gossypium and a method of direct visual observa-
tion of stomata in situ (Bull. Torrey Bot. Club. 40: 1-26).

LOEiHWiNG, W. F. 1938: Locus and physiology of photoperiodic perception in plants (Proc. Soc.

Expt. Biol. Med. 37: 631-634).
Loftfield, J. V. G. 1921: The behavior of stomata (Publ. Carnegie Inst. Wash. No. 314: 1-104).
London, F. 1937: A general theory of molecular forces (Trans. Far. Soc. 33: 8-26).

1942: On centers of van der Waals attraction (Jour. Phys. Chem. 46: 305-316).

Longinescu, G. G. 1929: Molecular association (Chem. Rev. 6: 381-418).

LooMis, W. E. 1935: The translocation of carbohydrates in maize (Iowa State Col. Jour. Sci. 9:
509-520).

1945: Translocation of carbohydrates in maize (Science 101: 398-400).

LousTALOT, A. J. 1944: Apparent photosynthesis and transpiration of pecan leaves treated with
Bordeaux mixture and lead arsenate (Jour. Agr. Res. 68: 11-20).

1945: Influence of soil moisture conditions on apparent photosynthesis and transpiration of

pecan leaves (Jour. Agr. Res. 71: 519-532).

LucKfe, B., and M. McCutcheon 1932: The living cell as an osmotic system and its permeability

to water (Physiol. Rev. 12: 68-139).
Lundegardh, H. 1940: Investigations as to the absorption and accumulation of inorganic ions

(Reprint from The Annals of the Agricultural College of Sweden 8: 233-404).

1946: Transport of water and salts through plant tissues (Nature 157: 575-577).

LuTMAN, B. F. 1919: Osmotic pressures in the potato plant at various stages of growth (Amer.

Jour. Bot. 6: 181-202).
LuYET, B. J., and P. M. Gehenio 1937: The double freezing point of living tissues (Biodynamica

1 (30): 1-23).
^ and 1939: The physical states of protoplasm at low temperatures. Review and critical

study (Ibid. 2(48): 1-128).
, and G. Galos 1940: The effect of the rate of cooling on the freezing point of living tissue

(Ibid. 3(65): 157-169).
Lyon, C. J. 1936: Analysis of osmotic relations by extending the simplified method (Plant Physiol.

11: 167-172).

1940: Improvements in the simplified method for osmotic measurements (Ibid. 15: 561-562).

1941: Osmotic pressure for the plant physiologist (Science 93: 374-375).

1942: A non-osmotic force in the water relations of potato tubers during storage (Plant Physiol.

17: 250-266).

MacDougal, D. T. 1925: Reversible variations in volume, pressure, and movement of sap in trees
(Carnegie Inst. Wash. Publ. 365).

1926: The hydrostatic systems of trees (Ibid. 373: 1-125).

, and J. B. Overton 1927: Sap flow and pressure in trees (Science 65: 189-190).

, , and G. B. Smith 1929: The hydrostatic-pneumatic system of certain trees: movement

of liquids and gases (Carnegie Inst. Wash. Publ. No. 397: 1-99).
MacLeod, D. B. 1937: The compressibility of liquids and a method of obtaining the compressibility

of molecules (Trans. Far. Soc. 33: 694-707).
Magistad, O. C. 1945: Plant growth relations on saline and alkali soils (Bot. Rev. 11: 181-230).
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591-620).
Mallery, T. D. 1934: Comparison of the heating and freezing methods of killing plant material

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Marsh, F. L. 1940: Water content and osmotic pressure of certain prairie plants in relation to

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Martens, P. 1931: Phenomenes cuticulaires et phenomenes osmotiques dans les poils staminaux de

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Water in Plants — 219 — Bibliography

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■ • 1931: Effects of mineral salts upon the transpiration and water requirement of the cotton

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— — 1929: The movement of sap in plants (Science 69: 217-218).

Mole, F. J. 1926: A study of suction force by the simplified method, I. Effect of external factors.

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Grafts et al. — 220 — Water in Plants

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Pflanz'en 24: 113-137). . ^ u /r, j

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bot. Ges. 58: 29-40).
MosHKOV B. S. 1939: Transfer of photoperiodic reaction from leaves to growing points (Compt.

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Jour. Bot. 9: 311-329).
Muller-Thurgau, H. 1880: t)ber das Gefrieren und Erfrieren der Pflanzen (Landw. Jahrb. 9:

133-189).

• 1886: Ihid. 15: 453-610.

Munch, E. 1930: Die Stoffbewegvingen in der Pflanze, 234 p. (Fischer, Jena).

Nadel, M. 1938: Sur la mesure de I'ouverture des stomates (Thesis, University Paris).

Neilson, J. A. 1928: Parafine wax — an aid to growth in transplanted trees and shrubs (Proc.

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■ 1931: Reducing storage and transplanting losses in nursery stock (Florida Exch. and Hort.

Trade World 78: 27, 35).
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Agric. Sci. 14: 178-191). ^ ., ^ ^ .
, W. R. Brown, and W. M. Martin 1926: The extraction of plant tissue fluids and their

utility in physiological studies Plant Physiol. 1: 57-65).
NiGAM, B. S. 1934: Effect of excessive humidity on the resistance of citrus plant to sun scorch

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des Sciences Memoires, p. 57-104).
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619-639). ,-, , u

1917: Zur Kenntnis der Saugkraft und der Wasserversorgung transpirierender Sprosse (Jahrb.

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■ 1921: Weitere Beitrage zum Saftsteigeproblem (Jahrb. f. wiss. Bot. 60: 307-353).

Northern, H. T. 1942: Relationship of dissociation of cellular proteins by auxins to growth (Bot.

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Oehlkers, F. 1937: Neue Versuche iiber zytologisch-genetische Probleme (Biol. Zbl. 57: 126-149).
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19306: Dehnbarkeit und Turgordehnung der Zellmembran {Ibid. 48: 192-206).

. 1932a: Uber Zuverliissigkeit und Anwendungsgrenzen der iiblichsten Methoden zur Bestim-

mung der osmotischen Konzentration pflanzlicher Zellsiifte (Planta 16: 467-517).

1932b: Untersuchungen zur Kritik der Saugkraftmessungen (Planta 18: 525-549).

1936: Remarks on two recent critical contributions concerning methods used in plant physiol-
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, and K. Mendel 1939: Orange leaf transpiration under orchard conditions (Agric. Res. Sta.,

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Osterhout, W. J. V. 1913: Protoplasmic contractions resembling plasmolysis which are caused by
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. 1922: Some aspects of selective absorption (Jour. Gen. Physiol. 5; 225-230).

1936: The absorption of electrolytes in large plant cells (Bot. Rev. 2: 283-315).

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1945: Water relations in the cell {Ibid. 29: 73-78).

1947a: The absorption of electrolytes in large plant cells, II. (Bot. Rev. 13: 194-215).

19476: Some aspects of secretion, I. Secretion of water (Jour. Gen. Physiol. 30: 439-447.

— -, and E. S. Harris 1927: Protoplasmic asymmetry in Nitella as shown by bioelectric measure

ments (Jour. Gen. Physiol. 11: 391-406).
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365-390). , . , c •.

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Palva, p. 1939: Die Wasserpermeabilitat der Zellen von Tolypellopsis Stelhgera (Protoplasma 32:

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19366: The state of water in ducts and tracheids (Plant Physiol. 11: 623-628).

Pennycuick, S. W. 1928: The structure of water (Jour. Phys. Chem. 32: 1681-1696).

Water in Plants — 221 — Bibliography

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1931: L^ntersuchungen iiber Turgordehnung und Membrangeschaffenheit {Ibid. 74: 749-796).

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• 1929: Die Thermo-Stromuhr, II. Mitteilung der Arbeitsbedingungen und Arbeitsmoglichkeiten

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■ 1942: Intake of water by parenchymatic tissue (Rec. trav. botan. Neerland. 39: 1-140).

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1911fc: Zur Physik der Transpiration (Ber. deut. bot. Ges. 29: 125-132).

• • 1912: Versuche zur Mechanik der Wasserversorgung, I. Der Druck in den Leitungsbahnen

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1915b: Theoretisches und Experimentelles zur Kohasionstheorie der Wasserbewegung (Jahrb.

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• 1932: Zur Kenntnis des Wasserhaushalts javanischer Kleinepiphyten (Planta 18: 215-287).

Rkuhr, B. 1935: Hydrations- und Permeabilitatsstudien an unbefruchteten Fucus-Eiern {Fucus

vesictilosjis L.) (Protoplasma 24: 531-586).
Richards, L. A. 1941: A pressure-membrane extraction apparatus for soil solution (Soil Sci. 51:

377-386).
, and L. R. Weaver 1944: Moisture retention by some irrigated soils as related to soil-moisture

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2302).
, and S. Palitzsch 1919: Compressibility of aqueous solutions, especially of urethane, and the

polymerization of water (Jour. Amer. Chem. Soc. 41: 59-69).
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Plant Crops Tech. Comm. 10: 1-78).

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, and W. O. Roberts 1945: Further work on plant injection for diagnostic and curative pur-
poses (Imperial Bur. Hort. and Plant Crops Tech. Comm. 16: 1-12).

Grafts et al. — 222 — Water in Plants

RoBBiNs, W. W., A. S. Crafts, and R. N. Raynor 1942: Weed control (McGraw-Hill, New

York).
Roberts, O., and S. A. Styles 1939: An apparent connection between the presence of colloids

and the osmotic pressures of conifer leaves (Sci. Proc. Roy. Dublin Soc. 22: 119-125).
RoEPKE, R. R. 1942: The thermoelectric method of measuring vapor pressure (J. Phys. Chem. 46:

359-366).
Rontgen, W. C. 1892: Ueber die Constitution des fliissigen Wassers (Ann. Physik. u. Chemie.

N. F. 45: 91-97).
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by means of the osmotic pressure of the external solution (Amer. Jour. Bot. 2S : 402-410).
RouscHAL, E. 1941: Untersuchungen iiber die Protoplasmatik und Funktion der Siebrohren (Flora

i5: 135-200).
Rudolph, K. 1925: Epidermis und epidermale Transpiration (Bot. Arch. P; 49-94).
Rywosch, S. 1908: Zur Stoffwanderung im Chlorophyllgewebe (Bot. Zeitung 66: 121-130).

Sabinin, D. a. 1925: On the root system as an osmotic apparatus (Bull. Inst. Rech. Biol. Univ.
Perm 4: [suppl. 2] 1-136).

Salisbury, E. J. 1927: On the causes and ecological significance of stomatal frequency, with spe-
cial reference to the woodland flora (Phil. Trans. Roy. Soc. B216: 1-65).

Sauvageau, C. 1891: Sur les feuilles de quelques monocotyledones aquatiques (Ann. Sci. Nat. Bot.
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Savastano, G. 1934: Ricerche fisiologiche sul raggrinzimento delle drupe dell 'olivo (Boll. R. Staz.
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Sayre, J. D. 1926: Physiology of stomata of Riune.r paticntia (The Ohio Jour, of Sci. 26: 233-266).

, and V. H. Morris 1932: Use of expressed sap in determining the composition of corn tissue

(Plant Physiol. 7: 261-272).

Scarth, G. W. 1923: Adhesion of protoplasm to cell wall and the agents which cause it (Transact.
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1932: Mechanism of the action of light and other factors on stomatal movement (Plant Phvsiol.

7: 481-504).

1941: Dehydration injury and resistance {Ibid. 16: 171-179).

1944: Cell physiological studies of frost resistance: a review (New Phytol. 4i: 1-12).

ScATCHARD, G. 1936: Concentrated solutions of strong electrolytes (Chem. Rev. 19: 309-327).

, and L. F. Epstein 1942: The calculation of the thermodynamic properties and the association

of electrolyte solutions (Chem. Rev. 30: 211-226).
Schmidt, C. L. A. 1938: The chemistry of the amino acids and proteins, 1031 p. (C. C. Thomas,

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Schneider, G. W., and N. F. Childers 1941: Influence of soil moisture on photosynthesis, respira-
tion, and transpiration of apple leaves (Plant Physiol. 16: 565-583).
ScHRATZ, E. 1931: Vergleichende Untersuchungen iiber den Wasserhaushalt von Pflanzen im

Trockengebiete des siidlichen Arizona (Jahrb. f. wiss. Bot. 74: 153-290).
ScHROEDER, H., and T. Horn 1922: Das gegenseitige Mengenverhaltnis der Kohlenhydrate im

Laubblatt in seiner Abhangigkeit vom Wassergehalt (Biochem. Zeit. 130: 165-198).
ScHORN, M. 1929: Untersuchungen ueber die Verwendbarkeit der Alkoholfixierungs- und Infiltra-

tionsmethode zur Messung von Spaltoeffnungs-weiten (Jahrb. f. wiss. Bot. 71: 783-840).
ScoFiELD, H. T., and L. E. Yarman 1943: Some investigations of the water relations of lichens

(Ohio Jour. Sci. 43: 139-146).
Seifriz, W. 1928: New material for microdissection (Protoplasma 3: 191-196).
■ 1938: The physiology of plants, 315 p. (Wiley, New York).

1942: A symposium on the structure of protoplasm, 283 p. (Iowa State College Press, Ames).

Seybold, A. 1929: Die physikalische Komponente der pflanzlichen Transpiration, 214 p. (Monogr.

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Shaker, J. 1942: Water loss from excised leaves (Amer. Jour. Bot. 29: 89-91).
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1931: The role of sap concentration in transpiration (Carnegie Inst. Wash. Yearbook 30: 262-

263).

Shull, C. a. 1913: Semipermeability of seed coats (Bot. Gaz. 56: 169-199).

1924: Imbibition in relation to absorption and transportation of water in plants (Ecology

5; 230-240).

• 1939: Atmospheric humidity and temperature in relation to the water system of plants and

soils (Plant Physiol. 14: 401-422).
SiDGWicK, N. V. 1936: Dipole moment and molecular structure (Chem. Rev. 19: 183-194).
SiERP, H., and A. Seybold 1927: Untersuchungen zur Physik der Transpiration (Planta 3: 115-

168).
Sifton, H. B. 1945: Air-space tissue in plants (Bot. Rev. 11: 108-143).
Signer, R. 1930: Uber eine Abanderung der Moleculargewichtsbestimmungsmethode nach Barger

Justus Liebig's Ann. Chem. 478: 246-266).
Sisakian, N. M. 1939: The biochemical characteristics of the drought resistance of plants (Sbornik

Akad. Nauk, Prezidentu Akad. Nauk SSSR Akad. V. L. Komarovw (1939): 707-719).

1940: The character of enzyme action in connection with the drought resistance of plants

(Trudy Moskov. Doma Uchenykhi Inst. Biokhira. Akad. Nauk SSSR (1940), No. 4: 29-38).

, and A. Kobiakova 1940: The behavior of enzymes as an index of drought resistance in crop

plants, IV. The effect of wilting upon the trend of esterification and hydrolysis of phosphoric

esters in plants (Biokhimiia 5: 225-233).
— ■ — , and 1941: The prevailing direction of enzyme action as an index of drought resistance

in cultivated plants, V. The diurnal rhythm of the trend of enzyme action in withering (Ibid.

6: 103-112).
Skoog, F. 1938: Absorption and translocation of auxin (Amer. Jour. Bot. 25: 361-372).
, T. C. Broyer, and K. A. Grossenbacher 1938: Effects of auxin on rates, periodicity, and

osmotic relations in exudation (Amer. Jour. Bot. 25: 749-759).

J

Water in Plants — 223 — Bibliography

Skvortzov, S. S. 1931: On the influence of desiccating winds on photosynthesis (Bull. Applied
Bot. of Genetics and Plant Breeding, Trudy. 25, No. 3: 45-68).

Smith, F., R. B. Dustman, and C. A. Shull 1931: Ascent of sap in plants (Bot. Gaz. 91: 395-
410).

Smith, H. B. 1941: Variation and correlation of stomatal frequency and transpiration rate in
Phaseolus vulgaris (Amer. Jour. Bot. 28: 722-725).

Snell, H. 1886: Untersuchungen uber die Nahrungsaufnahme der Wasserpfianzen (Flora 98: 213-
249).

SoLLNER, K. 1930: Zur erklarung der abnormen Osmose an nicht quellbaren Membranen, I. Teil
(Ztschr. f. Elektrochemie und angewandte physikalische Chemie 36: 36-47).

1930: Zur erklarung der abnormen Osmose an nicht quellbaren Membranen, II. Teil {Ibid.

36: 234-241).

, I. Abrams, and C. W. Carr 1941: The structure of the collodion membrane and its electrical

behavior, II. The activated collodion membrane (Jour. Gen. Physiol. 25: 7-27).

, and A. Grollman 1932: Zur erklarung der abnormen osmose an nichtquellbaren membranen,

III. Teil (Ztschr. f. Elektrochemie und angewandte physikalische Chemie 3S : 274-282).

Spoehr, H. a., and H. W. Milner 1939. Starch solution and amylolytic activity in leaves (Proc.
Amer. Phil. Soc. 81: 37-78).

Sponsler, O. L. 1940: Molecular structure in protoplasm (/n A. A. A. S. The Cell and Proto-
plasm. Publication No. 14: 166-187. Science Press, Lancaster, Pa.).

, and J. D. Bath 1942: Molecular structure in protoplasm {In Seifriz, W. 1942: The Struc-
ture of Protoplasm. Iowa State College Press, Ames, pp. 41-79).

and J. W. Ellis 1940: Water bound to gelatin as shown by molecular structure studies

(Jour. Phys. Chem. 44: 996-1006).
Stahl, E. 1894: Einige Versuche ueber Transpiration und Assimilation (Bot. Zeit. 52: 117-145).
Stalfelt, M. G. 1929: Neuere Methoden zur Ermittlung des OfFnungszustandes der Stomata

(Handb. d. biolog. Arb. Meth. von E. Abderhalden, Abt. XI, Teil 4: 167-192).

1932: Der stomatare Regulator in der pflanzlichen Transpiration (Planta 17: 22-85).

Stamm, a. J. 1936: Colloid chemistry of cellulosic materials (U. S. Dept. Agric. Misc. Publ. 240:

1-90).

1944: Surface properties of cellulosic materials {In Wise, L. E., ed. : Wood Chemistry, pp.

449-550. Reinhold, New York).

Stefan, J. 1873: Versuche uber die Verdunstung (Sitzungsber. Akad. Wiss. Wien 68: 385-428).

1881: Uber die Verdarapfung aus einem kreisformig odor elliptisch begrenzten Becken {Ibid.

83: 943-954).

Stein, E. 1912: Bemerkungen zu der Arbeit von Molisch: Das Offen- und Geschlossensein der
Spaltoeffnungen veranschaulicht durch eine neue Methode (Ber. deut. bot. Ges. 30: 66-68).

Steinberger, a. L. 1922: Uber Regulation des osmotischen Wertes in den Schliesszellen von Luft-
und Wasserspalten (Biol. Centralb. 42: 405-419).

Stern, K. 1933: Pflanzenthermodynamik (Springer, Berlin).

Steward, F. C. 1928: The maintenance of semi-permeability in the plant cell during leaching experi-
ments (Proc. Leeds Phil. Soc. 1: 258-270).

— — 1932: The absorption and accumulation of solutes by living plant cells, I. Experimental con-
ditions which determine salt absorption by storage tissue (Protoplasma 15: 29-58).

, and J. A. Harrison 1939: Ibid., IX. The absorption of Rubidium bromide by potato discs

(Ann. Bot. 3: 427-453).

, W. E. Berry, C. Preston, and T. K. Ramamurti 1943: Ibid., X. Time and temperature

effects on salt uptake by potato discs and the influence of storage conditions of the tubers on
metabolism and other properties (Ann. Bot. 7: 221-260).

, P. R. Stout, and C. Preston 1940: The balance sheet of metabolites for potato discs showing

the effect of salts and dissolved oxygen on metabolism at 23° C. (Plant Physiol. 15: 409-447).

Stewart, G. W. 1939: The variation in the structure of water in ionic solutions (Jour. Chem. Phys.
7: 869-877).

1943: Ibid. II (Jour. Chem. Phys. 11: 72-74).

1944: Physical concepts of ionic and other aqueous solutions (Amer. Jour. Physics 12: 321-

323).

Stiles, W. 1937: The constitution of plant cell membranes (Trans. Far. Soc. 33: 923-930).

, and I. Jorgensen 1917: Studies in permeability, V. The swelling of plant tissue in water

and its relation to temperature and various dissolved substances (Ann. Bot. 31: 415-434).
Stocking, C. R. 1943: Continuity, tensions, and redistribution of water in plants (Ph.D. Dissert.

Univ. of California).
1945: The calculation of tensions in Cucurbita pepo (Amer. Jour. Bot. 32: 126-134).

1948: Recovery of turgor by cut shoots after wilting (Plant Physiol. 23: 152-155).

Stoddart, L. a. 1935: Osmotic pressure and water content of prairie plants (Plant Physiol. 10:

661-680).
Stout, M. 1945: Translocation of the reproductive stimulus in sugar beets (Bot. Gaz. 107: 86-95).
Stout, P. R., and D. R. Hoagland 1939: Upward and lateral movement of salt in certain plants

as indicated by radioactive isotopes of potassium, sodium and phosphorus absorbed by roots

(Amer. Jour. Bot. 26: 320-324).
Strasburger, E. 1891: Ueber den Bau und die Verrichtungen der Leitungsbahnen in den Pflanzen,

Vol. 3 (Gustav Fischer, Jena).
Strugger, S. 1934: Beitrage zur Physiologic des Wachstums, I. Zur protoplasma physiologischen

Kausalanalyse des Streckungs-Wachstums (Jahrb. f. wiss. Bot. 79: 406-471).

1935: Praktikum der Zell- und Gewebephysiologie der Pflanze (Borntraeger, Berlin).

1943: Der aufsteigende Saftstrom in der Pflanze (Naturwiss. 31: 181-192).

Sutherland, W. 1900: The molecular constitution of water (Phil. Mag. 5th Ser. 50: 460-489).

1902: The electric origin of molecular attraction (Phil. Mag. [6] 4: 625-645).

1910: The constitution of water (Trans. Far. Soc. 6: 105-116).

Tagawa, T. 1934: The relation between the absorption of water by plant root and the concentra-
tion and nature of the surrounding solution (Jap. Jour. Bot. 7 : 33-60).

1936: The influence of light on the stomatal opening (Jap. Jour. Bot. 8: 95-112).

Grafts et al. — 224 — Water in Plants

Tamiya, H. 1938: Zur Theorie der Turgordehnung und iiber den funktionellen Zusammenhang

zwischen den einzelncn osmotischen Zustandsgrossen (Cytologia 8: 542-562).
Thoday, D. 1918: On turgescence and the absorption of water by the cells of plants (New Phytol.

17: 108-113).
, and M. G. Sykes 1909: Preliminary observations on the transpiration current in submerged

water-plants (Ann. Bot. 23: 635-637).
Thomas, N., and A. Ferguson 191 7o: On the reduction of transpiration observations (Ann. Bot.

31: 241-255).

■ , and • 1917b: On evaporation from a circular water surface (Phil. Mag. 34: 30S-321).

Thren, R. 1934: Jahreszeitliche Schwankungen des osmotischen Wertes verschiedener okologischer

Typen in der Umgebung von Heidelberg. Mit einem Beitrag zur Methodik der Pressaftunter-

suchung (Zeitschr. f. Bot. 26: 449-526).
Thut, H. F. 1932a: Demonstrating the lifting power of transpiration (Amer. Jour. Bot. 19:

358-364).
— — • 1932b: The movement of water through some submerged plants {Ibid. 19: 693-709).
Tinker, F. 1917: The colloidal membrane: its properties and its function in the osmotic system

(Trans. Far. Soc. 13: 133-140).
Tollenaard, D. 1925: Omzettingen van Koolhydratenin het Blad van Nicotiana tabacum L. (Dis-
sert., H. Veenman and Zonen, Wageningen).
Traube, M. 1867: Experimente zur Theorie der Zellbildung und Endosmose (Archiv. Anat.

Physiol, und wiss. Medicin, p. 87-165).
TuMANOV, I. I. 1929: Wilting and drought resistance (Bull, of Applied Bot. of Genetics and Plant

Breeding, Trudy 22. No. 1: 107-146).
TuKEY, H. B., and K. Brase 1931: The effect of paraffining, pruning and other storage treatments

upon the growth of roses and cherry trees (Proc. Amer. Soc. Hort. Sci. 28: 489-495).
TuRRELL, F. M. 1936: The area of the internal exposed surface of dicotyledon leaves (Amer. Jour.

Bot. 23: 255-264).
• 1944: Correlation between internal surface and transpiration rate in mesomorphic and xero-

morphic leaves grown under artificial light (Bot. Gaz. 105: 413-425).

Unger, F. 1854: Beitrage zur Physiologic der Pflanzen, I. Bestimmung der in den Interzellular-

gangen der Pflanzen enthaltenen Luftmenge (Kon. Akad. Wiss. Wien Math.-Natw. 12: 367-

378).
• 1862: Neue Untersuchungen fiber die Transpiration der Pflanzen (Sitzb. K. Akad. Wissensch.

[Wien] 44: 181-217).
Urquhart, a. R. 1929: The mechanism of the adsorption of water by cotton (Jour. Text. Inst. 20:

T125-T132).
Ursprung, a. 1915: t)ber die Kohasion des Wassers im Farnannulus (Ber. deut. bot. Ges. 33:

153-162).
1923: Zur Kenntnis der Saugkraft, VII. Eine neue vereinfachte Methode zur Messung der

Saugkraft (Ibid. 41: 338-343).
■ 1935: Osmotic quantities of plant cells in given phases (Plant Physiol. 10: 115-133).

1938: Die Messung der osmotischen Zustandsgrossen pflanzlicher Zellen und Gewebe (Handb.

d. biolog. Arb. Meth. von E. Abderhalden, Abt. XI, Teil 4, Heft 7, pp. 1109-1572).

, and G. Blum 1916a: Uber die Verteilung des osmotischen Wertes in der Pflanze (Ber. deut.

bot. Ges. 34: 88-104).

^ and 1916&: Zur Methode der Saugkraftmessung {Ibid. 34: 525-539).

, and 1927: Eine Methode zur Messung der Saugkraft von Hartlaub (Jahrb. wiss. f. Bot.

67: 334-348).
, and 1930: Zwei neue Saugkraft-Messmethoden (Ihid. 72: 254-334).

van Laar, J. J. 1917: Communication to the symposium on osmotic pressure (Trans. Far. Soc. 13:

171-173).
VAN Orstrand, C. E., and F. P. Dewey 1915: Preliminary report on the diffusion of solids (U. S.

Geol. Survey Profess. Paper 95-G: 83-96).
VAN OvERBEEK, J. 1942: Water uptake by excised root systems of the tomato due to non-osmotic

forces (Amer. Jour. Bot. 29: 677-683).

1944: Auxin, water uptake and osmotic pressure in potato tissue (Ibid. 31: 265-269).

VAN DER Paauw, F. 1935: The entrance of water into cut leafy shoots under conditions which pre-
vent transpiration (Rec. trav. bot. Neerl. 32: 293-310).

van't Hoff, J. H. 1887: Die Rolle des osmotischen Drucke in der Analogic zwischen Losungen und
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. 1888: The function of osmotic pressure in the analogy between solutions and gases (Phil.

Mag. Ser. 5, 26: 81-105).

Vassihev, I. M. 1929: The investigation of drought-resistance in wheat (Bull. Applied Bot. of
Genetics and Plant Breeding, Trudy 22: No. 1: 147-218).

1930: The water relations of plants of the sand desert South Eastern Kara-Kum (Ibid. 25,

No. 3: 185-272).

1931: Effect of drought on the carbohydrate transformation in wheats (Ibid. 27, No. 5:

47-69).

1932: Plant physiology in relation to drought control (Soc. Zern. Khoz. 1-2: 10-12).

, and M. G. Vassiliev 1936: Change in carbohydrate content of wheat plants during the process

of hardening for drought resistance (Plant Physiol. 11: 115-125).
Veihmeyer, F. J. 1927: Some factors affecting the irrigation requirements of deciduous orchards

(Hilgardia 2: 125-284).

1938: Effect of organic matter on the infiltration of water into soils (Amer. Geophysical

Union Trans. 19, Pt. 1: 326-342).

, and N. E. Edlefsen 1936: Water in soils and its movement (Union Geodesique et Geoph.

Internat. Assoc. Internat. d'Hydr. Sci. Bull. 22: 355-365).
, and — ■ — 1937: Interpretation of soil moisture problems by means of energy changes (Amer.

Geophysical Union Trans. 18, Pt. 2: 302-308).

Water in Plants — 225 — Bibliography

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to plants (Plant Physiol. 18: 66-78).
, and A. H. Hendrickson 1927: The relation of soil moisture to cultivation and plant growth

(Proc. First Int. Cong. Soil Sci. Wash. 3: 498-513).
^ and 1934: Some plant and soil moisture relations (Amer. Soil Survey Assoc. Bull. 15:

76-80). .^ ^ ^
^ and - 1938: Soil moisture as an indication of root distribution in deciduous orchards

(Plant Physiol. 13: 169-177).
^ and 1943: Essentials of irrigation and cultivation of orchards (Calif. Agr. Ext. Serv.

Circ. 50).
^ and 1946: Soil density as a factor in determining the permanent wilting percentage

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Vernon, H. M. 1891: On the maximum density of water (Phil. Mag. Sth Ser. 31: 387-392).
Verschaffelt, E., and J. Verschaffelt 1890: De transpiratie der planten in koolzuurvrije lucht

(Bot. Jaarboek, witgegeven door het Kruidkundig Genootschap Dodonaea te Gent, 2: 305).
Vesque, J. 1883: Sur I'interpretation d'une experience de Hales concernant le role des vaisseaux

Compt. Rend. 97: 1085-1087).
Vries, H. de 1884: Eine Methode zur Analyse der Turgorkraft (Jahrb. wiss. Bot. 14: 427-601).

1888: Osmotische Versuche mit lebenden Membranen (Zeitschr. f. Physik. Chem. 2: 415).

1918: Opera e periodicis coUata v. 1-2. Collected works of Hugo de Vries published in

Utrecht by A. Oosthoek.

Wadleigh, C. H., and A. D. Ayers 1945: Growth and biochemical composition of bean plants as
conditioned by soil moisture tension and salt concentration (Plant Physiol. 20: 106-132).

, H. G. Gauch, and V. Davies 1943: The trend of starch reserves in bean plants before and

after irrigation of a saline soil (Proc. Amer. Soc. Hort. Sci. 43: 201-209).

Walker, J. 1910: Summary of symposium (Trans. Far. Soc. 6: 122-123).

Walter, H. 1925: Die Verdunstung von Wasser in bewegter Luft und ihre Abhiingigkeit von der
Grosse der Oberflache (Ztschr. f. Bot. 18: 1-47).

1928: Die Bedeutung des Wassersattigungszustandes fur die CO2- Assimilation der Pflanzen

(Ber. deut. bot. Ges. 46: 530-539).

1929: Plasmaquellung und Assimilation (Protoplasma 6: 113-156).

• 1931a: Die kryoskopische Bestimmung des osmotischen Wertes bei Pflanzen (Handb. d. biolog.

Arb. Meth. von E. Abderhalden, Abt. XI, Teil 4, Heft 2, pp. 353-371).

1931fe: Die Hydratur der Pflanze, 174 p. (Fischer, Jena).

, and O. Weismann 1935: tJber die Gefrierpunkte und osmotischen Werte lebender und toter

pflanzlicher Gewebe (Jahrb. f. wiss. Bot. 82: 273-310).

Wann, F. B. 1943: Water relations of the plant cell (Mimeographed correspondence).

Washburn, E. W. 1921: An introduction to the principles of physical chemistry, 516 p. (McGraw-
Hill, New York).

Weaver, J. E. 1916: The eflfect of certain rusts upon the transpiration of their hosts (Minnesota
Bot. Studies 4: 379-406).

Weaver, R. J., and H. R. DeRose 1946: Absorption and translocation of 2, 4-dichlorophenoxyacetic
acid (Bot. Gaz. 107: 509-521).

Weber, F. 1923: Zur Physiologic der Spaltoeffnungsbewegungen (Osterr. bot. Zeitschr. 72: 43-57).

1924: Plasmolyseform und Protoplasmaviskositat (Ibid. 73: 261-266).

1925: Plasmolyseform und Kernform funktionierender Schliesszellen (Jahrb. f. wiss. Bot. 64:

687-701).

1929a: Plasmolysezeit und Lichtwirkung (Protoplasma 7: 256-258).

■ • 1929b: Plasmolyse in verdunntem Gewebesaft (Ibid. 8: 437-439).

Weidlich, H. 1930: Die Bewegungsmechanik der Variationsgelenke (Bot. Archiv. 28: 219-254).

Weinmann, H., and M. le Roux 1945: A critical study of the torsion balance for measuring trans-
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Weismann, Otto 1938: Eine Theoretische und Experimentelle Kritik der "Bound Water-Theorie"
(Protoplasma 31: 27-68).

Went, F. W. 1944: Plant growth under controlled conditions. III. Correlation between various
physiological processes and growth in the tomato plant (Amer. Jour. Bot. 31: 597-618).

, and M. Carter 1945: Wounding and sugar translocation (Plant Physiol. 20: 457-460).

White, P. R. 1938: Cultivation of excised roots of dicotyledonous plants (Amer. Jour. Bot. 25:
348-356).

1942: "Vegetable Staticks" (Amer. Scientist 30: 119-136).

Whiting, H. 1884: Theory of cohesion (Harvard University, Cambridge, Mass.).

Whitman, W. C. 1941: Seasonal changes in bound water content of some prairie grasses (Bot.

Gaz. 103: 38-63).
Wildervanck, L. S. 1932: Osmotic adaptation of Nitella translucens Agardh (Rec. trav. bot.

Neerl. 29: 227-378).
Williams, W. T. 1947: Shock-induced stomatal movements (Nature 160: 364-365).
Wilson, J. D., and H. A. Runnels 1933: Some effects of Bordeaux mixture on transpiration

(Ohio Agr. Exp. Sta. Bimonthly Bull. 165: 147-151).
Wilson, K. 1947: Water movement in submerged aquatic plants, with special reference to cut

shoots of Ranunculus fluitans (Ann. Bot. N. S. 9: 91-122).
Wise, L. E. ed. 1944: Wood chemistry, 900 p. (Reinhold Publ. Co., New York).
Withrow, a. p., and R. B. Withrow 1943: Translocation of the floral stimulus in Xanthium

(Bot. Gaz. 104: 409-416).
Wright, K. E. 1939: Transpiration and the absorption of mineral salts (Plant Physiol. 14: 171-

174).
Wylie, R. B. 1938: Concerning the conductive capacity of the minor veins of foliage leaves (Amer.

Jour. Bot. 25: 567-572).
Wyman, J. 1936: The dielectric constant of solutions of dipolar ions (Chem. Rev. 19: 213-239).

Yabwood, C. E. 1936: The diurnal cycle of the powdery mildew Erysiphe polygani (Jour. Agr.
Res. 52: 645-657).

Grafts et al. — 226 — Water in Plants

Zalenski, v. 1904: Materials for the study of the quantitative anatomy of different leaves of the

same plant (Mem. Polytech. Inst. Kiev. 4: 1-203).
Zeeuw, J. DE 1939: On water exosmosis in Chaetomorpha linum (Miill.) Kiitz. (Rec. trav. botan.

Neerland. S6: 268-346).
ZiEGENSPECK, H. 1945: Fluoroskopische Versuche an Blattern, uber Leitung, Transpiration und

Abscheidung von Wasser (Biol. Gen. [Vienna] 18: 254-326).
ZiRKLE, C. 1937: The plant vacuole (Bot. Rev. 3: 1-30).

INDICES

AUTHOR INDEX

A. A. A. S., 2

Aamodt, O. S., 2

Abrams, I., 79

Adams, F., 209

Adams, G., 62

Adolph, E. F., 137, 141

Agamov, S., 188

Akerman, A., 125

Aldrich, W. W., 152, 206

Alexeev, A. M., 202

Alter, W., 15

Anderson, A. B. C, 2, 29, 76

Anderson, D. B., 33, 44, 45, 50, 53, 56, 73, 79,

90, 105, 132, 148, 173, 186
Andrus, B., 106
Arcichovskaja, N., 105
Arcichovskij, V., 105, 164, 165, 166
Arens, K., 107, 125
Armstrong, H. E., 5, 26
Arndt, C. H., 157, 169
Arthur, J. M., 191
Ashby, E., 104, 196
Askenasy, E., 154
Atkins, W. R. G., 90, 100
Ayers, A. D., 153

Babbitt, J. D., 24, 26, 29, 64

Bailey, I. W., 63

Baker, H., 156, 157, 158

Bakke, A. L., 160

Balazs, E., 85

Baldes, E. J., 97

Bancroft, W. D., 1, 7, 34, 40

Bandemer, S. L., 200

Bangham, D. H., 193

Barger, G., 95, 96

Barker, J. W., 2, 70, 83, 85, 112, 116, 117, 123

Barlee, J. S., 140, 204, 205

Barlund, H., 79, 83

Barnes, C. R., 179

Barnes, T. C, 6, 17

Barr, C. G., 200

Bartell, F. E., 142

Bartholomew, E. T., 172, 188

Bath, J. D., 2, 66

Baumgartner, A., 169, 171, 172

Beadle. L. C., 137

Beal, J. M., 176

Beasley, E. W., 199

Beck, W. A., 2, 53, 7i, 82, 106, 164, 172, 202

Benedict, H. M., 154

Bennett. C. W., 176

Bennett, J. P., 159, 174

Bennet-Clark, T. A., 2, 70, 83, 85, 89, 108, 112,

116, 117, 118, 119, 123, 124, 126, 131, 137,

143, 144, 200
Berkeley, Earl of, 1, 36, 37, 38, 41, 53, 85
Berl, E., 97
Bernal, J. D., 3, 11, 12, 13, 14, 15, 25, 27,

29, 30, 67
Berry, W. E., 223
Bexon, D., 2, 89, 108, 112, 118, 119, 123, 124,

143, 144
Biale, J. B., 191
Biddulph, O., 175, 176
Blackman, V. H., 140, 141
Blanchard, K. C, 17
Blegen, E., 97

Blum, G., 96, 97, 99, 100, 102, 105, 108, 113
Bode, H. R., 1, 157, 160, 162, 172
Boehm, J. A., 162
Bonner, J., 176, 177
Boon-Long, T. S., 194
Bose, J. C, 111, 140, 154
Bouillenne, R., 105
Bourdillon, J., 97

Bousfield, W. R., 6, 47, S3

Bouyoucous, G. J., 198

Brandes, E. W., 176

Erase, K., 199

Brauner, L., 2, 80, 81, 85, 131, 140, 144. 184

Brauner, M., 80, 85, 140

Brett, P. G. C, 159

Briggs, D. R., 29

Briggs, L. J., 2, 184, 191

Brilliant, B., 202

Brilliant, V. A., 202

Brooks, M. M., 2, 28, 68, 77, 136

Brooks, S. C, 2, 28, 52, 68, IT, 79, 136, 139

Brown, A. J., 81

Brown, J. W., 176

Brown, L. N., 209

Brown, W. R., 90

Broyer, T. C., 2, 75, 76, 91, 109, 138, 139, 149,

150
Buhmann, A., 82. 83. 84, 99, 113, 114, 115
Bull, H. B., 2, 28, 30, 64
Biinning, E., 85
Burgerstein, A., 184
Burian, R., 91
Burr, G. O., 176
Burton, C. V., 1, 38, 41, 86

Callendar, H. L., 33, 54

Capus, G., 160

Carr, C. W., 79

Carrick, D. B., 132

Carsner, E., 176

Cartellieri, E., 61, 105

Carter, M., 175

Chadwell, H. M., 7, 11

Chambers, R., 69, 78

Chandler, R. C, 26, 30

Chandler, W. H., 101, 172, 173

Chatfield, C, 62

Chibnall, A. C, 70, 91

Childers, N. F., 152, 199, 202

Chrelashvili, M. N., 201, 202

Chu, Chien-Ren, 105

Chung, C. H., 191

Clark, D. G., 197

Clements, H. F., 174, 175, 179

Cohn, E. J., 31

Collander, R., 69, 79, 82

Collorio, H. M., 203

Colwell, R. N., 170, 176

Comar, C. L., 200

Commoner, B. S., 124, 127, 128, 129

Cone, W. H., 100

Conrad, J. P., 152

Cooil, B. J., 175

Cooley, J. S., 199

Coons, G. H., 176

Copeland, E. B., 154, 155, 156

Crafts, A. S., 65, 100, 109, 149, 150, 151, 157,

159, 161, 174, 175, 176, 205
Currier, H. B., 83, 84, 85, 90, 91, 94, 98, 104,

113, 115, 122, 123, 126, 133
Curtis, C. C, 191
Curtis, L. C, 180
Curtis, O. F., 132, 174, 175, 176, 179, 197

Dalton, J., 181

Danielli, J. F., 2, 78, 79

Daniels, F., 1

Darwin, F., 196

Dastur, R., 202

Davies, V., 152

Davis, A. R., 69, 79, 138, 191, 192

Davis, C. H., 152

Davis, H. L., 1, 34, 40

Davson, H., 2, 78, 79

Water in Plants

— 229

Author Index

DeBye, P., 31

Degman, E. S., 152

Delf, E. M., 85, 172

Denny, F. E., 81

De Rose, H. R., 176

Desai, B. L., 202

Desai, M. C, 198, 199

Dewey, F. P., 183

Dietrich, M., 197

Dixon, H. H., 1, 90. 91, 100, 120, 123, 140, 154,

155, 157, 158, 162, 163, 170, 204, 205
Djang, G. S., 210
Doneen, L. D., 91
Dorland, R., 177
Dorsey, N. E., 17
Drucker, K., 91
Dugger, B. M., 70, 87
Dustman, R. B., 204
Dutrochet, R. J. H., 1, 52

Eaton, F. M., 126, 141, 163

Edlefsen, N. E., 2, 23, 29, 76, 152

Elazari-Volcani, T., 194

Eley, D. D., 16, 17, 23, 29

Elford, W. J., 79

Ellis, J. W., 2, 66

Emerson, J. L., 200

Engard, C. J., 174, 175, 179

Epstein, L. F., 31

Erickson, L. C, 199

Ernest, E. C. M., 75, 82, 83, 102, 123, 165, 193

Ewell, R. H., 16, 22

Eyring, H., 15, 16, 22, 31, 67

Eyster, H. C, 44, 52

Farr, W. K., 63

Ferguson, A., 183

Ferri, M. G., 176

Finbak, C., 16

Findlay, A., 1, 18, 27, 35, 36, 52

Fischer, M. H., 68, 78, 137

Fitting, H., 86, 87

Fogel, S., 127, 128, 129

Fogg, G. E., 187, 188

Foster, A. C, 199

Fowler, R. H., 3, 11, 12, 13, 14, 25, 27, 29, 30

Frazer, J. C. W., 1, 34, Zl , 38, 41, 54, 55, 86

Freeman, G. F., 186

Frey-Wyssling, A., 2, 63, 67, 68, 90, 132, 133.

145
Fritsch, F. E., 61
Fiirlinger, H., 202
Furnstal, A. H., 91
Furr, J. R., 152, 172

Gail, F. W., 100, 101

Gallenkamp, W., 183

Galos, G., 133

Gasser, R., 85, 101, 127

Gassner, G., 199

Gauch, H. G., 152

Gaiimann, E., 188

Gehenio, P. M., 132, 133

Geneau de Lamarliere, L., 186

Getman, F. H., 1

Gibbs, R. D., 61, 80

Gicklhorn, J., 69

Gilbert, B. E., 199

Glasgow, A. R., 92, 95

Glasstone, S., 1, 33, 41

Godlewski, E., Ill, 140

Goeze, G., 199

Goldsmith, G. W., 91

Golovina, A. S., 201

Gortner, R. A., 2, 24, 25, 26, 29, 90, 91, 93,

94, 137
Gould, L. P., 7
Grafe, V., 184
Graf-Marin, A., 199
Greenfield, S. S., 201, 202

Greenwood, A. D., 2, 70, 83, 85, 112, 116, 117,

123
Gregory, F. G., 197
Grollman, A., 142
Gross, F., 89, 124, 137
Grossenbacher, K. A., 150, 191
Guilliermond, A., 2, 69
Guye, P. A., 5
Gyorffy, B., 197

Haan, I. de, 81

Haas, A. R. C, 100

Haberlandt, G., 1

Haines, F. M., 161

Haldane, J. S., 1, 27. 37, 38, 44, 49, 50, 57

Hales, S., 1, 111, 168

Halket, A. C, 95, 96

Hall, W. H., 44

Halma, F. F., 100

Hamner, K. C, 176

Handley, W. R. C, 154

Hannig, E., 100

Hansteen-Cranner, B., 198

Harkens, W. D., 155

Harned, H. S., 16, 31

Harrington, O. E., 90

Harris, E. S., 78

Harris, J. A., 2, 90, 91, 93, 94, 99, 101

Harrison, J. A., 138

Harter, L. L., 198

Hartley, E. G., 1, Z(>, 37, 38, 41, 86

Hartmair, V., 125

Hartsuijker, K., 196

Harvey, E. B., 137

Hasman, M., 75, 131, 144

Hasselbring, H., 179

Hausermann. E., 193

Hayward, H. E., 153

Heath, O. V. S., 197

Hecht, H., 197

Hefter, O., 210

Heinicke, A. J., 202

Henderson, L. J., 3

Hendrickson, A. H., 2, 152, 153, 184

Henrici, M., 202

Hermans, P. H., 64

Herrick, E. M., 91, 106

Heuser, E., 2, 28, 29

Heyn, A. N. J., 84, 85, 140

Hibbard, R. P., 90

Hiesey, W. M., 186

Hildebrand, E. M., 176

Hildebrand, J. H., 7, 14, 15, 19, 20, 40, 46, 47

Hildreth, A. C., 200

Hill, A. v., 97

Hirschfelder, J. D., 15, 67

Hoagland, D. R., 2, 69, 79, 138, 139, 168, 169,

174, 179, 191
H6ber, R., 2, 20, 21, 31, 79, 81, 138, 142, 145
Hochreutinger, G., 204
Hoffman, C., 85

Hofler, K., 2, 73, 75, 80, 82, 83, 85, 88, 89, 125
Hofmeister, L., 69
Hofmeister, W., 191
Holle, H., 160, 163, 188
Homan, C., 56
Horn, T., 203
Houska, H., 125
Huber, B., 80, 82, 83, 169, 170, 171, 174, 185,

203
Humphreys, W. J., 181

Il/in, W. S., 198, 203
International Critical Tables, 94
Ivanov, L. A., 185

Jaccard, P., 90, 132, 133

Jahn, T. L., 6, 17

Jahnel, H., 174

James, W. O., 156, 157, 158

Crafts et al.

230 —

Water in Plants

Janse, J. M., Ill, 140
Jeffreys, H., 182, 183
Jenny, H., 79
Johnson, A. F., 97
Johnson, C. O., 99
Johnson, W. H., 2
Joly, J., 154
Jorgenson, I., 127

Kaiserlehner, E., 125

Kamp, H., 188

Katzoff, S., 14

Kendall, J., 14, 47

Kennedy, P. B., 151, 157, 159

Kenny, I. J., 107

Kerr, T., 90, 105, 132, 173

Kessler, W., 135

Keys, A., 137

Kiesselbach, T. A., 179, 184, 199

Kincaid, J. F., 31

Kirkwood, J. G., 31

Kisch, R., 203

Kisselew, N., 209

Kisser, J., 198

Kitching, J. A., 137

Knight, R. C, 160

Knight, T. A., 1, 111

Kobiakova, A., 201

Kohl, F. G., 191

Kondo, I. N., 202

Korstian, C. F., 100

Kotte, H., 85

Kramer, P. J., 148, 151

Krassnosselsky-Maximov, T. A., 173, 203

Krassulin, H., 209

Krausche, K. K., 199

Krogh, A., 2, 71, 136, 137

Kursanov, A. L., 201 "

Kiister, E., 2, 78, 81, 124, 125

Latshaw, W. L., 61

Lauro, F. de, 125

Lawrence, J. V., 90, 101

Leick, E., 180, 184, 185

Leighly, J., 182, 183

Leitch, J. L., 136

Lepeschkin, W. W., 68, 78, 118

Le Roux, M. de, 185

Levitt, J., 2, 80, 90, 97, 117, 122, 132, 133,

134, 135, 146
Lewis, F. J., 132, 193
Lewis, G. N., 1, 19, 22, ii, 34, 35, 38, 39,

40, 93
Lewis, M. R., 152
Li, T. T., 106

Livingston, B. E., 1, 105, 186
Livingston, L. G., 70
Lloyd, D. J., 145
Lloyd, F. E., 191, 196, 197
Loehwing, W. F., 176
Loftfield, J. V. G., 196, 197
Lominadze, T., 113
London, F., 14, 21, 23
Longinescu, G. G., 8, 9, 14
Loomis, W. E., 174, 175
Lorenz, O. A., 175
Loustalot, A. J., 199, 202
Lowry, T. M., 6, 47
Lucke, B., 82, 136

Lundegardh, H., 69, 79, 139, 145, 150
Lutman, B. F., 100
Luyet, B. J., 132, 133
Lyon, C. J., 2, 52, 103, 106, 131

MacDougal, D. T., 1, 154, 156, 157, 161. 169,

172
MacLeod, D. B., 14
Magistad, O. C, 2, 148, 154, 198
Mair, B. J., 92, 95
Mallery, T. D., 91, 101

Malpighi, M., 1

Maney, T. J., 199

Mark, H., 63

Markle, J., 176

Markle, K. S., 187

Marsh, F. L., 101, 173

Martens, P., 85

Martin, E. V., 183, 191

Martin, J. H., 2, 122

Martin, W. M., 90

Maskell, E. J., 174, 177

Mason, T. G., 2, 68, 70, 117, 118, 119, 145,

174, 175, 177
Maxiniov, N. A., 1, 2, 80, 100, 101, 113, 132,

155, 173, 184, 185, 191, 195, 200
McCutcheon, M., 82, 136
McDermott, J. J., 150
McNab, W. R., 168, 169
Mendel, K., 188
Mendiola, N. B., 179
Menjinshaja, E., 209
Mer, C. L., 194

Meyer, B. S., 1, 2, 33, 43, 44, 45, 50, 52,
53, 55, 56, 71, li, TJ , 79, 83, 90, 91, 101,
102, 103, 104, 106, 108, 109, 120, 131, 148,
155, 186, 198
Migahid, A. M., 135

Miller, E. C, 61, 148, 187, 191, 195, 199
Miller, E. J., 200
Milner, H. W., 202, 203

Minckler, L. S., 186

Mitchell, J. W., 176
Molisch, H., 154, 197, 203

Molz, F. J., 86, 106, 165

Montemartini, L., 1

Montermoso, J. C, 191, 192

Morgan, J., 15, 17, 29

Morris, R. T., 199

Morris, V. H., 91

Morse, H. N., 1, 34, 35, 36, i7, 41, 47, 53, 55,
79, 86

Mosebach, G., 91, 126, 127

Moshkov, B. S., 176

Mozhaeva, L. V., 80

Muenscher, W. C., 179

Muller, W. H., 127, 128, 129

Miiller-Thurgau, H., 132

Munch, E., 61, 174, 205

Murray, J. W., 141, 143

Myrick, R. T., 1, 34, i7, 38, 41, 54, 55, 86

Nadel, M., 196
Neilson, J. A., 199, 200
Nernst, W., 6

Newton, R., 90, 120, 121, 122
Nigam, B. S., 187
Nollet, M. I'Abbe, 32, 42
Nordhausen, M., 105, 163
Northern, H. T., 145

Oehlkers, F., 203

Oppenheimer, J. D., 82, 83, 102, 113, 114, 165,

188, 196
Ossipov, A., 105, 164, 165, 166
Osterhout, W. J. V., 71, 78, 124, 138, 141, 143
Overbeck, F., 107
Overstreet, R., 79
Overton, E., 79

Overton, J. B., 154, 156, 157, 172
Owen, B. B., 16, 31

Pagliani, S., 7

Palitzsch, S., 7

Palladin, V. I., 52

Palva, P., 80

Pauling, L., 13, 79

Pearse, H. L., 197

Peirce, G. J., 159

Pennycuick, S. W., 8, 9, 10, 12, 27

Pertz, D. F. M., 196

Water in Plants

231 —

Subject Index

Peters, J. P., 82
Petrie, A. H. K., 201

Pfeflfer, W. F. P., 1, 18, 32, 42, 81, 140

Pfitzer, E., 168, 169

Phillis, E., 2, 68, 70, 117, 118, 119, 145. 195

Picken, L. E. R., 138

Pieniazek, S. A., 187

Pisek. A., 61, 105

Pleass, W. B., 145

Pond, R. H., 204

Ponder, E., 136

Porter, A. W., 35, Zl

Porter, D. R., 107

Prat, S., 123, 124

Preston, C, 128, 139

Preston, R. D., 158, 164

Priestley, J. H., 109, 149, 154, 156

Pringsheim, E. G., 84, 85, 172, 193

Rabideau, G. S., 176

Rabinovich, A. I., 7

Rabinowitch, E. I., 201

Ramamurti, T, K., 223

Randall, M., 1

Rao, I. R., 7

Raoult, F. M., 4, 7, 14, 18, 19, 34, 41, 42, 48,

92
Rau, F., 210
Raynor, R. N., 157
Reed, H. S., 2, 198, 199
Reeve, J. O., 152
Rehberg, P. Brandt, 97
Reimer, F. C, 152
Rein, H., 170

Reinders, D. E., 127, 129, 145, 146
Reitemeier, R. F., 154
Remick, A. E.. 22, 31, 79

Renner, O., 1, 105, 148, 155, 162, 163, 183, 197
Resiihr, B., 80
Richards, L. A., 151, 153
Richards, T. W., 7, 10
Riedl, H., 171
Roach, W. A., 159
Robbins, W. W., 157
Roberts, O., 69, 90, 113
Roberts, W. O., 159
Roepke, R. R., 97
Rontgen, W. C, 4, 5
Rosene, H. F., 153
Rossini, F. D., 92, 95
Rouschal, E., 175, 176
Rudolph, K., 188
Ruhland, W., 135
Runnels, H. A., 199
Ryall, A. L., 152
Rywosch, S., 203

Sabinin, D. a., 104, 150

Salisbury, E. J., 195, 196

Sando, C. E., 187

Sauvageau, C, 204

Savastano, G., 172

Sayre, J. D., 91, 191, 192

Scarth, G. W., 2, 80, 83, 89, 90, 134, 135, 203

Scatchard, G., 31

Schmidt, C. L. A., 2, 29, 30

Schmidt, E., 170, 171, 174

Schneider, G. W., 152

Schorn, M., 197

Schratz, E., 184

Schreiner, E., 91

Schroeder, H., 203

Scofield, H. T., 61, 132

Seifriz, W., 2, 78

Seybold, A., 183, 200

Shafer, J., 197

Shafer, J. I. Jr., 197

Shantz, H. L., 2, 152, 184, 191

Shreve, E. B., 186, 194

Shull. C. A., 56, 81, 155, 189, 191, 204

Sidgwick, N. V., 31

Sierp, H., 183

Sifton, H. B.. 194

Signer, R., ^1

Sisakian, N. M., 201

Skoog, F., 150, 176, 177

Skvortzov, S. S., 202

Smith, F., 204

Smith, G. B., 156, 157, 172

Smith, H. B., 196

Smith, J. H. C., 91

Snell, A., 174

Snell, H., 204

Sollner, K., 79, 142

Spoehr, H. A., 202, 203

Sponsler, O. L., 2, 66, 67, 145

Spurr, W. B., 153

Stahl, E., 197

Stalfelt, M. G., 196, 203

Stamm, A. J., 2, 64

Steam, A. E., 31

Stefan, J., 181

Stein, E., 197

Steinberger, A. L., 28, 203

Stern, K., 2

Stevenson, J. D., 15, 67

Steward, F. C., T), 83, 128, 138, 139, 145

Stewart, G. W., 16

Stewart, W. D., 191

Stiles. W.. 78, 127

Stocking, C. R., 100, 104, 161, 166, 173, 185

Stoddart, L. A., 101

Stout, M., 176

Stout, P. R., 128, 139, 169, 174, 179

Strasburger, E., 1, 154, 169

Strugger, S., 2, 81, 88, 89, 90, 108, 140, 155,

168, 169
Styles, S. A., 69, 90, 113
Sutherland, W., 4, 5, 6, 8
Sykes, M. G., 204

Tagawa, T., 153, 192
Tamiya, H., 75
Tatman, E. C., 199
Taylor, C. A., 152, 172
Thimann, K. V., 124
Thoday, D., 2, 1Z, 204
Thomas, N., 183
Thren, R., 120
Thut, H. F., 162, 204
Tinker, F., 79
Tollenaard, D., 203
Traube, M., 32, 42
Tukey, H. B., 199
Tumanov, I. I., 2, 202
Turrell, F. M., 193, 195
Tuttle, G. M., 132

Umstatter, H., 210
Unger, P., 194, 204
Urquhart, A. R., 64

Ursprung, A., 1, 2, 73, 81, 85, 86, 96, 97, 99,
100, 102, 103, 105, 107, 108, 113, 155, 164

Van Laar, J. J., 36, 44

Van Ostrand, C. E., 182

Van Overbeek, J., 2, 70, 83, 100, 104, 124,

129, 130, 140, 150
Van der Paauw, F., 204
Van't HofT, J. H., 1, 6, 7, 18, 32, II, 34, 37,

39, 42, 52, 53, 54, 55
Vassiliev, I. M., 2, 200, 202, 203
Vassiliev, M. G., 2, 202

Veihmeyer, F. J., 2, 23, 29, 152, 153, 184, 203
Vernon, H. M., 4
Verschaflfelt, E., 186
Verschaffelt, J., 186
Vesque, J., 154, 160
Viervoll, H., 16
Vries, H. de, 1, 18, 33, 81, 86, 87, 102, 105

Crafts et al.

— 232 —

Water in Plants

Wadleigh, C. H., 152, 153

Walker, T., 6

Wallace, A. M., 83, 102, 103, 104, 106

Walter, H., 1, 2, 90, 91, 94, 119, 120, 132, 133,

183, 201
Wann, F. B., 52
Warren, B. E., 15, 17, 29
Washburn, E. W., 1
Weaver, J. E., 199
Weaver, L. R., 151, 153
Weaver, R. J., 177
Weber, F., 69, 80. 87, 124, 197
Weidinger, A., 64
Weidlich, H., 140
Weinmann, H., 185

Weismann, O., 90, 91, 122, 132, 133, 135
Went, F. W., 113, 175
White, P. R., 105, 140, 149
Whiting, H., 4
Whitman, W. C, 135
Wildervanck, L. S., 97, 113, 127

Williams, W. T., 197
Wilson, J. D., 199
Wilson, K., 204
Wise, L. E., 2
Withrow, A. P., 176
Withrow, R. B., 176
Wolf, R., 104
Wood, J. G., 201
Work, W. A., 152
Wright, K. E., 179
Wylie, R. B., 193
Wyman, J., 31

Yarman, L. E., 61
Yarwood, C. E., 199
Young, T. E., 56

Zalenski, v., 195
Zeeuw, J. de, 65, 80, 85
Ziegenspeck, H., 169
Zirkle, C, 78

G^iq. 22.

SUBJECT INDEX

A (see Intercellular pressure), 107
Absorption, forces involved, 138
Absorption of water, 148-154

active, 149-151

at permanent wilting, 152

"induced," 138

passive, 151

"primary," 138
Accumulation, solute, 79, 126, 138

water (see Active water relations)
Acer, gas in stem of, 161
Acid-arsenical method, of weed control, 157
"Activated diffusion," theory of translocation,

174
"Active pressure," 129
Active water relations, 111-147

diffusion pressure deficits and, 131-132

evidence from animal physiology, 136-138

evidence from frost and drought resistance,
132-136

mechanisms proposed for, 141-146

solute accumulation, relation to, 138-139
Active water uptake, auxin and, 127-131
Activity, defn., 41, 77

freezing pt. detn. of, 93
Adhesion, cytoplasm to wall, 83-85, 114-115

water movement and, 154-155
Aesculus, gas in stem of, 161
Alfalfa, 2,4-D movement in, 177
Algae, osmotic detns., 85, 127

vacuolar sap of, 70

vacuolization in, 68
Alkalinity, effect on growth, 148
Alliaria officinalis, water continuity in, 163
Allium (see also Onion), photosyn. and sug.

cone, 201
Alnus, gas in stem of, 161
Aloe, expressed sap of, 119
Amino acids, protein constituents, 66

in protein synthesis, 129
Ammonia, thermal properties of, 4
Ammonium thiocyanate, translocation of, 157
Amylase, hydrolytic activity of, 200, 202, 203
Anaphoresis, 141

Anatomy, root structure and absorption, 149
Anguilla vulgaris, water regulation in, 137
Animal physiology, evidence for active water

relations, 136-138
Anion accumulation, 139
Anomalous osmosis, 141, 142, 143
Anomalous plasmolytic behavior, 124
Anthocyanin, as indicator of cell injury, 84,

118, 125
Apple, water content of fruit, 62
Aquatic animals, osmoregulatory processes in,

136
Arsenic, weed control and, 157
Arthrophytum hatoxylon, DPD of, 166

DP gradient in, 164, 165

transpiration of, 165
Ash tree, gas in, 157
Association factor, of water, 37
Atmospheric moisture, relative humidity, 56

vapor pressure, 56

DPD of, 56
Atmospheric pressure, DPD base line, 48

and sap rise, 156

effect on evaporation, 181-182
Atoms, forces between, 20
Atriplex confertifolia, OP sap of, 99
Auxin, active water relations and, 127-131

effect on cell, 140

effect on exudation, 150

effect on respiration, 124

translocation of, 175

Barger-Halket method, OP by, 96
Barley, water content of, 62
Bean, water content of, 62

2,4-D movement in, 177

water absorption by, 153
Beet (see Beta)

Begonia rex, plas.-cryos. discrepancy in, 112
Bellis perennis, DPD of, 106
Benzene, phys. properties of, 4

stomatal detns. with, 197

wettability of cell wall by, 193
Berberis nervosa, internal leaf surface in, 195
Bergenia cordifolia, adhesion pressure, 83

OP values of, 114-115
Beta vulgaris, adhesion in, 84

flower stimulation in, 176

freezing pt. of sap of, 112, 115, 116, 118,
120, 126, 134

plas.-cryosc. discrep. in, 112, 115, 118

plastic stretching of walls, 84
Bioelectric forces, in plant cells, 144
Birch, rate of sap flow in, 171
Blood, thermoelectric detn. of rate of movement,

170
Bombardment theory, of OP, 33
Bonds (see also Forces), and hydration, 29

hydrogen, 22, 29

ionic, 22, 29
Boraginaceae, colloids in vacuoles of, 69
Bordeaux spray, effect on transpiration, 199
Bound water, 24-26

calorimetric method for, 121, 122 '

definitions of, 24

frost resistance and, 135
Boundary layer, evaporation and, 183
Boyle's law, 32
Brassica napobrassica, plas.-cryos. discrepancy,

112
Brassica sinapis, wettability of cuticle, 187
Bryophyllum calicynum, internal leaf surface of,

195
Buckeye, gas in stem, 159
Butyl alcohol, leaf penetration by, 193
Buxus sempervirens, expressed sap of, 120

Cabbage, water content of, 62
Caladium bicolcrr, plas.-cryos. discrepancy, 112
Calcium, salt effect on leaves, 198
Calcium chloride, electroosmosis and, 131, 144
moisture detn. with, 186
plasmolyzing agent, 83
water requirement and, 198
Calcium ferrocyanide, OP of solutions, 38, 39
Callithamnion roseum, cell walls of, 85
Calorimetric method, 121, 122
Cambium, OP of, 100

water movement and activity of, 154, 156
Camphora officinalis, air space in leaves of, 194
Capillarity, and liquid in xylem, 162
Capillary force, and injection into xylem, 157
Capillary tubes, in OP detns., 96, 97
Carbohydrates, translocation of, 175
Carbon: nitrogen ratio, in phloem exudate, 175
Carbon dioxide, physical properties, 4
Carbon disulfide, physical properties, 4
Carbon tetrachloride, physical properties, 4
Carrot, water content of, 62
Castor bean, gas in stem of, 159
Castor oil, in infiltration method, 197
Catalase activity, water deficit and, 200
Catalpa, protoplasmic volume in, 135
Catalpa speciosa, internal leaf surface of, 195
Cell method for diffusion pressure deficit, 102-

103
Cell sap, (see also Sap) cryoscopic value of en-
tire, 118

Grafts et al.

234 —

Water in Plants

Cells, anatomy of, 59-61

hardened, 134

minimum volume of, 98

osmotic properties and transpiration, 194

osmotic quantities of, 73-110

plasmolytic behavior of, 122-126
Cell walls, as cellular components, 59-61

chemical composition of, 62-65

dehydration of, 193

elastic nature, 85, 172, 193

imbibition by, 107, 193

inelastic stretching, 114

intercellular substance of, 62

internal leaf surface and, 195

permeability of, 81

plastic behavior, 85, 101, 140

structure of, 62-65, 192-193

water in, 62-65, 155
Cell water relations {see Water relations)
Cellophane, use in stomatal studies, 197

membrane, permeability of, 81
Cellular proteins, dissociation of, 145
Cellulose, hydration of, 24, 63-65, 155
Centaurea repcns, herbicide movement in, 157
Chaetomorpha linutn, water exosmosis from, 80
Chamacrops humilis, sap expression from, 120,

123
Chara, solute accumulation in, 38
Chelate ring, 22

Chiasmata, water deficit and, 203
Chlorella, water deficit and photosynthesis, 201
Chloride ion, membrane permeability to, 144
Chloroform, leaf permeability to, 193
Chlorosis, injection treatment of, 159
Chodat-Hecht filaments, 85
Citrus grandis, internal leaf surface of, 195
Citrus limonum, freez. pt. of sap, 120
Coacervate formation, 69
Cobalt chloride paper, transpiration detn. with,

186, 197
Coefficient of diffusion, evaporation and, 181,

182, 183
Coffea, rate of sap flow in, 169

arabica, dye movement in stem of, 157
Cohesion, hydration and, 138

calculated, for water, 46, 155

theory of water movement, 154, 204
Cohesive force, of plant sap, 155
Cold hardiness, 117, 120, 121, 134
Coleus, rhythmic fluctuations in transpiration,

191
Colloidally bound water (.see Bound water)
Colloid osmotic pressure, 97
Colloids, vacuolar, 69, 113
Companion cells, OP of, 99, 100
Conducting elements, cone, of sap in, 100
Conifers, rate of sap flow in, 169, 172
Convolvulus arvcnsis, herbicidal translocation

in, 157
Corn, water absorption by, 153

water content of, 62

water loss from hybrid, 197
Cortex, OP of, 100
Cortical rays, OP of, 100
Cotton, water uptake by, 24
Covalent bonds, 22, 29, 63, 138, 192, 193
Crocus, intercellular pressure in, 102
Cryoscope, 91-92

calibration curve for, 94, 95
Cryoscopic values of OP, 112, 114, 115, 118,

127
Cryoscopy, 90-95, 126

expression of sap for, 90-91

freezing point detn. in, 93-95

of tissues vs. bathing solns., 126-127

osmotic pressure detns. by, 90-95, 99

physical principles, 92-93
Cucurbita (see also Squash), translocation in,
175

water continuity in, 161
Curly-top virus, translocation of, 176
Cuticle, and transpiration, 192

composition of, 187, 193

fissures in, 188

thickness of, 187

wettability of, 187, 188
Cytoplasm (see also Protoplasm), adhesion of,
82, 83

hydration of, 69, 125
Cystosira barbata, cryoscopy of sap, 127

Debye-Huckel theory, 7
Dehydration, of ectoplasm, 134

(see also Water deficit)
Dendrograph, 157
Deplasmolysis, 83, 84, 89, 114

injury, 134

values, 116
Dianthus barbatus, wettability of cell walls of,

193
Dichlorophenoxyacetic acid, translocation of, 177
Dielectric capacity, 6
Dielectric constant, 4, 25, 31
Differential ion migration, 142
Differentially permeable membrane, 43, 49, 79-

81
Differentiation, water conduction and, 156
Diffuse porous trees, rate of sap flow in, 171
Diffusion, capacity, 196

coefficient, 182, 183

constant, 80
Diffusion pressure, defined, 43-44

effect of cutting xylem on, 157

energy reduction and, 29

evaluation of, 55

evaporation and, 182

factors affecting, 151

internal water movement and, 172

lowering, 48

of water, 192, 198

of solvent, 55-57

water movement and, 154
Diffusion pressure deficit, and active water re-
lations, 111-113, 131-132

as an osmotic quantity of plant cells, 71-77

concept of, in osmotic systems, 50-52

defined, 44, 71

electrokinetic component of, 144

freezing of tissue and, 133

gradient, 108-109, 164

growth and, 153

in root, 164

in stem, 165

magnitude and fluctuation of, 105-107, 165

methods of measuring, 98, 101-105

of atmosphere, 189, 190, 191

other terms for, SO

passive absorption and, 149

transpiration and, 193, 197

water absorption and, 153

wilting and, 163
Dihydrol, 5, 13
Dihydrone, 5
Diraer, 22
Dipole, 20, 21

Disease, effect on transpiration, 198-200
Ditylum brightwellii, stimulation plasmolysis in,

124
Diurnal fluctuations, in root pressure, 150

in transpiration, 189, 190, 191

in stomatal opening, 192
DOg, (OP by deplasmolysis), 115, 116
DPD (see Diffusion pressure deficit)
DPDu (Diffusion pressure deficit, normal), 131
Drosera rotundifolia, OP in tentacles of, 125
Drought resistance, evidence for active water

relations, 132-136
Drought resistant plants, 206

Water in Plants

— 235

Subject Index

Drought susceptible varieties, 201
Dusts, effect on transpiration, 198-200
Dyes, downward movement in xylem, 157, 159
explanation of penetration, 157
indicators of water movement, 168-169
movement and rate of sap flow, 156, 169
penetration and water continuity, 156-159
water soluble, 69

Ectoderm, 137

Ectoplasm, permeability of, 135

refractive index of, 134

rupture of, 89
Ectoplast, 78, 87

Elatostemma, water continuity in, 161
Electrokinetic activity, 81, 142, 143, 144
Electrolytes, electrokinetic potential and, 142
Electrolytic solutions, thermodynamics of, 31
Electronegativity, and hydrogen bond, 22
Electroosmosis, 141, 145
Electrostatic forces, 21, 67
Elodea, 60, 124, 125

Endodermis, deposition of cutin and suberin in,
193

role in water absorption, 149
Endosperm, sap expressed from, 132
Enzyme activity, and water deficit, 200-203
Eosin, movement in stems, 204, 205
Epidermis, OP in, 99, 125
Erythrocytes, osmotic behavior of, 136
Escaping tendency, 38, 41, 44, 76, 93
Essential oils, wettability of intercellular sur-
faces with, 193
Ethyl alcohol, physical properties of, 4
Ethylene glycol, stomatal studies with, 197
Ethyl ether, wettability of intercellular surfaces

with, 193
Eucalyptus, rate of sap flow in, 169
Eucalyptus globulus, internal leaf surface of,

195
Evaporating surface, internal, 195
Evaporation, atmospheric pressure and, 182

factors affecting, 182-183

from free water surface, 181

from tubes, 181-182

laws of, 180-183

rate of, 182
Exudation, 104, 139

auxin effect, 150

from roots, 150

Fagus, rate of sap flow in, 171, 172
sylvatica, OP in, 99, 118
water content of leaves of, 61

Faraday Symposium, 5-6, 14-15

Fibrils, 63, 64, 192

Ficus elastica, wettability of intercellular sur-
faces of, 193

Field capacity, 23

Fissures, cuticular, 188
protoplasmic, 117, 118

Flower initiation, translocation of stimulus of,
176

Fluorescein, indicator of translocation, 175

Fluorescent materials, use in sap movement ex-
periments, 168, 169

Foods, mechanism of movement, 174

Force fields, 76

Forces between atoms and molecules, 20-24, 182
coordination, 29

Forsythia, tensions in, 163

Fouquieria splendens, transpiration in, 191

Fraxinus, gas in stem of, 161

Free energy, 29, 30, 76-77

Freezing point (see also Cryoscopy), detn. of,
93-95
of tissue, 132-136
thermometers, 92
values, 120, 134

Frost hardening, 145

Frost resistance, evidence for active water re-
lations, 132-136

Fruits, as reservoirs of water, 172

Fucus, permeability of eggs of, 80

Fugacity, definition, 41, Tl
gradient, 77

Fumarate, active water relations and, 129

g (limiting plasmolysis), 98
Gas constant R, 32

Gas in xylem, experimental techniques and,
155-157, 160-161

field demonstration of, 161

wilting and, 160
Gas law, ideal, 19

Gaultheria shallon, internal leaf surface of, 195
Gay Lussac's law, 32

Girdling, translocation studies by, 176-177
Glandular secretion, 140
Glucose residues, 63
Gossypium barbadense, OP of, 118
Growth, soil moisture influence on, 148

water movement and, 154
Gum, 69, 154, 156

Gunda ulvae, water relations of, 137
Guttation, 148, 150

Haldane's equation, 37

HalocysHs, solute accumulation in, 138

Hard leaf method, of DPD detn., 105

Hardening, 135, 199

Heat capacity, 4

Heat method, in detn. of rate of water move-
ment, 170

Heat of fusion, 4

Heat of vaporization, 4

Hedera helix, expressed sap of, 120

Helianthus annuus, osmotic quantities of seed-
lings of, 106-107

Helianthus tuberosus, active water relations in,
146

Helleborus foetidus, OP in, 99

Hemicellulose, 202

Heptacosanol, in cutin, 187

Heteroauxin (see Auxin)

Hexahydrate, of sucrose, 27, 37

Hexane, physical properties of, 4

Hookeria tucens, adhesion in, 83

Hormones, translocation of, 176-177

Hydration, 24-30, 135, 141
foliar, 145
heats of, 25
number, 35, 36, 37
of cellulose, 24, 63-65, 155
of ions, 27
of protoplasm, 65-69, 71, 107, 122, 134-135

Hydrogen, thermal properties of, 4

Hydrogen bonds, 15, 22, 29, 67, 175

Hydrogen ion, high migration velocity, 13, 30

Hydrogen sulfide, thermal properties of, 4

Hydrols, 5, 13

later work on theory of, 6-7

Hydrolytic enzyme reactions, 200

Hydrones, 5

Hydrophilic colloid, 62, 113, 137

Hydrophily, 134, 135

Hydrophobic wall surfaces, 193

Hydrophyte, 99

Hydropot cells, 125

Hydrostatic-pneumatic system, in trees, 161

Hydrostatic pressure, 44, 45, 144, 192
minimum, 108

Hydroxyl ion, high migration velocity of, 13, 40

Hygrometers, 186

Hypertonic solution, 81, 126

Hypostomatal leaves, 187

Hypotonic environment, 124, 125, 126

Hysteresis, 64

Crafts et al.

236

Water in Plants

Ice, dielectric constant of, 13

entropy of, 13

latent heat of fusion of, 5

lattice structure of, 12
Ilex aquifolium, sap expression from, 120

tensile strength of sap, 155
Imbibant, 29
Imbibition, 27, 28

diffusion pressure and, 172, 173

extravascular water movement and, 168
Imbibition pressure, 28, 125, 140, 145
Imbibitional forces, 62, 118, 132, 194
Impatiens, water continuity in, 161
Impatiens balsimina, OP of xylem sap, 100
Incipient plasmolysis, 81, 83
Indicators, and water movement, 168-170

use in translocation studies, 175-176
Indoleacetic acid {see Auxin)
Infiltration method, for stomatal opening detn.,

197
Injection, deficiency diseases and, 159

method of DPD detn., 104

methods of, 159

of dyes into stem, 156-159

of radiophosphorus into leaves, 176

saturation deficit and, 159
Insecticides, effect on transpiration, 199
Intercellular pressure (A), 107
Internal leaf surface, effect on transpiration, 194-

195
Internal pressure, 4, 41, 46, 155
Intrabilitat, 78, 87
Invert sugar, 33

Invertase, drought and activity of, 201
Ionic bonds, 22, 29
Ionic mobility, 6
Ions, colligative properties, 198

electric fields of, 20-21, 29

hydration of, 13

mobility of, 30-31

role in electroosmosis, 144
Iris, DPD of, 102

gertnanica, freezing point of sap of, 120
Isobutyl alcohol, stomatal measurements with,

197
Isotonic coefficient, 86
Isotonic sap, 123
Isotonic solutions, 33, 81, 97, 141

Jamin's chains, 156, 160
Juniperus, tension in xylem of, 162

Kidney, water relations of, 137

Laminar air movement, 183

Larix, rate of sap flow in, 171, 172

Leaf, detn. of area, 187

internal surface of, 192, 194-195

wettability of, 187-188
Lettuce, water content of, 62
Lever method of DPD detn., 105
Lianas, rate of sap flow in, 169
Ligustrum vulgare, vacuolar contraction in, 89
Limiting plasmolysis, 81, 83, 97, 98, 99, 105,

126, 193
Linaria cymbalaria, OP of sap of, 120
Lipoids, 69, 78
Lipoid-sieve theory, 79
Liquid-junction potential, 142
Liquid state, 21, 47, 48
Liquids, association of, 10, 15

internal pressure of, 41

properties and structure of, 14-16
Lithium, use in sap movement expts., 168-169

Magnesium chloride, electroosmosis and, 144

perchlorate, transpiration meas. with, 186
Majanthemum, permeability of, 83
Malate, electroosmotic flow and, 143
Malate effect, active water relations and, 123

Mannitol, as plasmolyzing substance, 82

auxin action and, 129

use in root pressure expts., 104
Manometer, tension measurement with, 162
Mass flow theory, 174-175
Meiosis, water deficit and, 203
Membrane potential, 142
Membranes, copper ferrocyanide, 32

permeability of, 43, 77-81
Mercury, physical properties of, 4
Meristematic cell, 60
Mesomorphic leaves, 195
Mesophyll resistance, 196
Mesophyll walls, hydrophobic nature, 193
Methyl glucoside, OP of solns. of, 38, 39
Micelles, in cell walls, 63-64, 192
Microcapillary forces, in cell walls, 192
Micrurgical methods, 69, 70, 78
Mimosa pudica, turgor response in, 140
Mineral deficiencies, injection and cure, 159
Moisture stress, soil moisture and, 152, 153
Molecular, concentration, 8-9

interaction, 20, 24

viscosity of air, 183

volume, 21

weight, large molecules, 42
Molecules, elec. properties of, 20-21

forces between, 20

migration of, 30
Mol fraction, 33
Moments, electric, 21
Monohydrol, 7

Monosaccharides, effect of wilting on, 202
Morse's equation, 35
Movement of solutes, 174-177
Mucilages, 69
Mucilaginous colloids, 113
Myxomycete, freezing point of, 133

Naphthaleneacetic acid, 129, 130

Nerium oleander, internal leaf surface in, 195

Net osmotic pressure, 50

Net water loss, 181

Neutral red, 69, 125

Nitella, cell sap of, 70

extensibility of wall of, 75

OP of, 113

osmotic properties of, 141

solute accumulation in, 138

turgor pressure of, 107
Nitella transluccns, OP of, 97, 127
Nitrogen, thermal properties of, 4

translocation of, 175, 177
Non-ideal behavior of solutions, 41-42
Non-solvent space, 134
Non-solvent water {see Bound water)
Noctiluca miliaris, specific gravity of, 136
"Normal" and "Polar" liquids, 7-8

O {see Osmotic pressure), 98
Oak, rate of sap flow in, 171
Oenothera, water deficit and meiosis in, 203
Og {see Osmotic pressure at limiting plasmoly-
sis)

values, 84, 99
On {see Osmotic pressure at normal cell vol-
ume)
Onion, epidermis, 125

water absorption by, 153

water content of, 62
OP {see Osmotic pressure)
Opuntia phaeacantha, OP of sap, 119

versicolor, OP of sap, 119
Organic acids, formation and water absorption,

149
Osmoregulation, 101, 127, 136, 137
Osmometer, method for detg. OP, 97
Osmosis, anomalous, 141, 142, 143

calculation of by indirect methods, 54-55

defined, 43

Water in Plants

— 237 —

Subject Index

in liquid systems, 46-48
kinetic basis of, 45-46
mechanism of, 43-58
osmotic pressure and, 32-42

role of, 57

"solvent" and "solute" pressure theories of,
52-54
Osmotic adaptation, 127
Osmotically-inactive material, 136
Osmotic diflFusion pressure, SO
Osmotic pressure, at limiting plasmolysis, 89,
101, 112, 116, 123, 126, 131

at normal cell volume, 112, 115, 116, 126, 131

Barger-Halket method, 96, 113

Bombardment, theory of, 33

by replasmolysis (ROg), 116

capillarimetric method, 96-97, 113

cryoscopic method, 90-95, 99, 116, 129-130

defined, 43

discrepancies in methods of detg., 111-117,
118

DPD and, SO, 51, 52, 105, 106, 163

effect on dark reaction, 201

effect on transpiration, 194

equation of, 18, 32, 33, 34

influence of atmosphere on, 48

in soils, 1S3-1S4

internal water movement and, 172, 173

law, refinement of, 33-40

magnitude and variation in plants, 99-101

manometric detn. of, 146

methods of measuring, 81-98

minimum cell volume method, 97

osmometric method, 32-39, 97

plasmolytic method, 81-89, 99, 116

plasmometric method, 89-90

simplified method, 97, 98, 99

soil, and water absorption, 149

"solute pressure" theory of, 33, 53

"solvent pressure" theory of, 33, 54

turgor pressure and, 107, 108

vapor pressure equation, 28, 44

vapor pressure methods, 95-97

vapor pressure theory of, 33
Osmotic pressure values,

of plants, 99-134, 203

of solutions, 32-39, 100

of xylem and phloem exudates, 100
Osmotic quantities of plant cells, 73-110
Osmotic value, 100
Osmotic work, 124, 135
"Oxidized collodion," 144
Oxygen, root pressure and, 149-150

subaqueous transpiration and, 205

thermal properties of, 4

Palladium, as a membrane, 46
Pantothenic acid, translocation of, 177
Paraffin oil, in DPD detn., 102

leaf wall wettability with, 193

stomatal studies with, 197
Parenchyma, OP of, 99, 100
Parthcnocissus qu in qui f alius, OP of, 118
Partial molal volume, 23

PCD isee Plasmolytic-cryoscopic discrepancy)
Peanut, moisture content of, 62
Pectin, in vacuoles, 69
Pentahydrate, 27, 37
Peptide linkages, 66-67
Permanent wilting, 153

and transpiration, 160
Permanent wilting percentage, 206

water absorption at, 148
Permanent wilting point, defined, 152

soil texture and, 152
Permeability, membranes and, 77-81

of cell walls, 81

water absorption and, 149
Petroleum, stomatal studies with, 197
Petroleum ether, stomatal studies with, 197

pH, and sugar-to-starch transformation, 202,

203
Phaeophyceae, plasmolytic studies of, 85
Phaseolus, cryoscopy of, 132

rate of sap flow in, 169
Phenoxyacetic acid, 4-chloro-, 177

2,4-dichloro-, 177
Phloem, exudate, 100, 175

exudation and Schlieren method, 165
translocation in, 174-177
Phosphatase, water deficit and, 201
Phosphorus, use in sap flow expts., 169
Photosynthesis, water deficit and, 201, 206
Picea, rate of sap flow in, 171, 172
Pigment production, 201
Pinus laricio, adhesion pressure in, 83

plas.-cryos. discrepancy, 115
Pinus rigida, cryoscopy of, 91

cold hardiness in, 120, 121
Pistia texeris, internal air space in, 194
Pith, OP of, 100
Plasmalemma, 78
Plasmolysis, anomalous, 124

chlorophyll production and, 201

forms, 87-88, 125

incipient, 82

limitations of method, 82-85

limiting, 82

stimulative, 88, 124
Plasmolytic-cryoscopic discrepancy, 111-117,

123, 126, 134
Plasmolytic method, exptl. procedure, 85-87

limitations of, 82-85

measurement of OP, 81-89

uses other than for OP, 87-89
Plasmolyzing agents, 82-83
Plasmometric method, measurement of OP by,

89-90
Plasmoptysis, 108

Plastic stretching of cell wall, 84, 140
Pleurococcus, 61

POg (OP at the first lim. plasmol.), 115, 116
Poinsettia, wilting of, 185
Poisouille's law, 157
Polar liquids, 7-8

Polar molecules, properties of, 7-8
Polar movement, translocation and, 175
Polarizability, 21
Polygonum officinale, 124
Polypeptide chains, 66-67
Polyuronides, 79, 192
Porometer, 196-197
Porter's equation, 35

Potamogeton crispus, water movement in, 203
Potamogeton lucens, water movement in, 204
Potassium, bromide, and water absorption, 128

chloride, effect on cell water relations, 144

fumarate, in cell water relations, 128

in cell water relations, 128

ion, membrane permeability to, 144

nitrate, as plasmolyzing agent, 82

OP values of solutions of, 100

sulfate, 131
Potato, auxin and water uptake, 127-128

DPD of tuber of, 104, 131

solute accumulation in, 139

volume changes in, 98

water content of tuber, 62

water in phloem walls, 65
Potometer, method of DPD detn., 104-105

pressure difference measured by, 163

tension measurement with, 162-163

transpiration detn. with, 186
Pressure, adhesion, 82-84, 114-116

diffusion (see Diffusion pressure)

equation of state, components of, 19-20

functional, 50

gas, 32, 33

intercellular, 102

Grafts et al.

238 —

Water in Plants

hydrostatic, 44, 48

osmotic {see Osmotic pressure)

turgor {see Turgor pressure)

vapor {see Vapor pressure)
Pressure-dehydration tests, 122
Pressure-membrane, moisture tension detn. with,

151
Primary wall, 63
Primula, water deficit in, 201
Protease, water deficit and, 201
Protein, in plasma membranes, 78

in protoplasmic structure, 66-67

in vacuoles, 69

metabolism, 139

synthesis, 129
Proton, 13, 30
Protoplasm, coagulation of, 89

constituents of, 61

contractile nature of, 71, 126

differential permeability of, 67

fissures in, 119

function in translocation, 174

hydration of, 65-69, 71, 107, 122, 134-135,
145-146

imbibition pressure of, 140

pulsations of, 154

streaming of, 68, 125

structure of, 65-69

structural viscosity of, 145

volume of, in cells, 135

water in, 65-69
Protoplasmic theory of translocation, 174, 175
Protoplast, active contraction of, 125, 140
Pulvinus, 140

Pyridoxine, translocation of, 177
Pyrus tnalus, freez. pt. of sap, 120
PWP {see Permanent wilting percentage)

Radioactive carbon, indicator of translocation,

176
Radioactive tracers, in phloem, 168

rate of water movement and, 169-170
Radiophosphorus, indicator of translocation, 175-

176
Radish, water content of root, 62
Raffinose, mol. wt. detn. of, 87
Ranunculus fluitans, water movement in, 204
Raoult's law, 8, 14, 19, 34, 41, 48, 92
Reductase activity, water deficit and, 200
Relative humidity, 23, 24, 189, 190
Replasmolysis, 83-84, 114-116
Reserve carbohydrates, water deficit and, 202
Respiration, activity, 124, 132

inhibitors of, 132

rate of, and water uptake, 89, 129, 149-150
Rheum rhaponticum, PCD values in, 112
Rhodophyceae, 85

Rhoeo discolor, adhesion pressure of leaf cells,
83

OP of sap, 119

plasmolytic studies of, 82, 87
Rihcs inertne, tensions in xylem of, 161
Riboflavin, translocation of, 177
Ricinus communis, internal leaf surface of, 195
Ring porous trees, rate of sap flow in, 156, 171
Rochea falcata, cuticular fissures in, 188
ROg {see Osmotic pressure by replasmolysis),

116
Root pressure, as a non-osmotic force, 104, 150

concentration of sap and, 104, 150

method of DPD detn., 104

subaqueous transpiration and, 203-204
Rytiphlaea iinctora, OP of sap of, 127

Sagittaria, vacuolar contraction in, 125
Salt, {see also Solute)

indicator of water movement, 168

translocation of, 174
Salvinia auriculata, permeability of leaf cells, 80
Samhucus, gas in xylem of, 161
Sanchezia, rate of sap flow in, 169

Sap, elastic expansion of, 157

expressed, 126

expression of, 90-91

expression data, interpretation of, 117-122

injury, 118

protoplasmic, 70

vacuolar, 70
Sap movement, methods for determining rate of,
168-170

rate of, 167-172
Sargassum linifolium, sap of, 126
Saturation deficit, and dye movement in xylem,

159
"Schlieren" method of DPD detn., 105, 165
Secondary wall, 63
Sedum album, OP of sap, 120
Sedum nicaense, vol. changes in, 84, 98
Sedum reflexum, OP of sap, 120
Sieve-tubes, structure and function, 174

translocation in, 174-175
Simplified method of DPD detn., 98, 103-104
Sodium chlorate, translocation of, 157
Sodium chloride, as plasmolyzing subst., 83

influence on transpiration, 198
Soil, OP of, and plant growth, 154

saline, 153, 154
Soil moisture, availability, 152

classification, 151

DPD at field capacity, 152

influence on absorption and growth, 148

permanent wilting and, 152

stress, 153

tension, 151
Solanum, tonoplasts in, 78

tuberosum, OP of sap, 120
water relations of, 127
{see also Potato)
Solidago virga aurea, transpiration of, 185
Solute absorption, water absorption and, 149,

168
Solute accumulation, 79, 126, 138

active water relations and, 138-139
Solute theory of osmosis, 52-54
Solutes, translocation, 174-177
Solution volume, 6, 47
Solutions, colligative properties, 18

forces between atoms and molecules in, 20-
24

hydration and, 26-30

ideal, 18-19

interrelations of properties of, 18-20

mobility of ions in, 30-31

molecular interaction and water binding, 24-
26

non-ideal behavior of, 41-42

osmotic pressure of {see Osmotic pressure)

properties of, 18-31
Solvent, association of, 36

diffusion pressure of, 55-57

free energy of, 39

theory of osmosis, 52-54
Sprays, effect on transpiration, 198-200
Spruce wood, water absorption by, 24
Spyridia filamentosa, cryoscopy of sap, 127
Squash, tension in, 166

translocation in, 176

wilting of, 173
Squirting cucumber, 107
Staminal hairs, 60, 85
Starch, conversion of, 202
Stem, contraction, 157
Stimulative plasmolysis, 124, 125, 145
Stomata, methods of measuring, 196-197

number and distribution, 195-196

regulation of transpiration by, 180, 197-198

rhythmic fluctuations, 192

wilting and, 160

xeromorphism and, 200
Strip method of DPD detn., 103-104
Subaqueous transpiration, 203-205
Sucrose, effect of wilting of plants on, 202

OP of solns. of, 32-39

Water in Plants

239

Subject Index

translocation of, 175, 177

as plasmolyzing subst., 82
Suction force (see also DPD), 50
Suction pressure (see also DPD), SO
Suction tension (see also DPD), 50

internal water movement and, 172
Sulfur, thermal properties of, 4
Sulfur dioxide, thermal properties of, 4
Sunflower, wilting of, 173
Surface tension, 4

and injection of xylem, 157

role in water movement, 155
Sweet pea, 4-chlorophenoxyacetic acid effect on,

177
Swelling pressure (see also Imbibition pressure),

28, 29
Symphytum officinale, vacuolar sap of, 69
Symplast, 61, 174

role in water absorption, 149
Syneresis, 69

Syringa oblata, DPD in, 106
Syringa vulgaris, internal leaf surface, 195

subaqueous transpiration in, 205

Tactoids, 67
Tannins, 69

Taxus baccata, expressed sap of, 119, 120
Tensile strength (see Cohesion)
Tension, direct measurement of, 162
effect on tracheae, 157
fluctuations in, 148
gas formation and, 156
injection method of determining, 166-167
in xylem, 162-167
manometric measurement of, 162
newer methods of estimating, 165-167
potometric measurement, 162-163
"suction tension" measurements, 164-165
water absorption and, 151
xylem shrinkage and, 172
Tetrahydrone, 5

Thermal properties of substances, 3-5
Thermoelectric detns., of OP, 91, 97

of rate of sap movement, 170
Thiamin, translocation of, 176-177
Thorium B, use in sap movement expts., 169
Tissue tension method of DPD detn., 105
Tissue, volume changes of, 112, 126, 131
Tobacco, phloem parenchyma cell of, 60

rate of sap flow in, 169
Tolypellopsis stelligera, water permeability of,

80
Tomato, translocation in, 177

water content of fruit of, 62
Tonoplast, 78, 87, 125, 143
TP (see Turgor pressure)

TP„ (see Turgor pressure at normal cell vol-
ume), 131
TP, (see Turgor pressure at water saturation),

131
Tradescantia, hair cells of, 60, 85

xylem contents of, 161
Translocation, of herbicides, 157

of solutes, 174-177
Transpiration, 183-200
cuticular, 184, 187-189
effect of disease on, 198-200
effect of dusts, sprays, waxes on, 198-200
effect of osmotic pressure on, 104
expression of results, 186-187
fluctuations in rate, 165, 189-192
genetic factors and, 197
indicators for study of, 175-176
internal factors affecting, 192-198
internal surface effect on, 194-195
lenticular, 184, 187
methods of measuring, 184-186
mineral nutrient effect on, 198
rate of, 179, 187, 188
resistance to water movement and, 193-194,

198
stomatal, 184, 199

stomatal regulation of, 197-198

subaqueous, 203-205

wall structure affecting, 192-193

water absorption and, 151

water continuity and, 160

wilting effects on, 160
Trentepohlia aurea, OP of, 101
Triacontane, 187
Tridymite, 12
Trihydrone, 5
Trihydrol, 4, 5, 13
Triticum vulgare, leaf cuticle of, 187
Turgor, deficit, 50

defined, 44-45

of plant cells, 107

measurement of, 107-108

role of, 107, 140
Turgor pressure, and the mechanics of osmosis,
48

at different cellular states, 73-75, 98, 131

defined, 45

relationship to OP and DPD, 50-82, 73-75, 98,
102, 107, 131
Turpentine, 197
Tyloses, 154, 156, 157
2,4-D (see Phenoxyacetic acid), 117

Ultimate wilting, 154
Umbilicaria, water content of, 61
Uronide chains, in cell wall, 192
Ursolic acid, in cutin, 187
Urtica dioica, OP of, 99

V (see Tissue, volume changes of), 98
Vacuolar, contraction, 71, 88, 125, 145

"envelopes," 78

sap, 69-71, 117-119 ^

Vacuoles, solutes in, 69

water in, 69-71
Vacuolization, 68
Valence, bonds, 22
Vallisneria, permeability of, 80
Valonia, sap of, 70, 138

macrophysa, 127

urticularis, 127
van der Waals', equation, 19, 40

surface tension and, 155

forces, 14, 21, 23, 67
Van't Hoff's law, 18, 32, 34
Vaporization, calc. of surface tension by, 155
Vapor pressure, 41, 54

deficit, 182

equation, 28, 33

gradient, 181

liquids, 181, 182

methods for detg. DPD, lOS

methods for detg. OP, 95-97

solvent, 38
Vicia faba, guard cells of, 87

rate of sap flow in, 169
Vinca minor, sap of, 120
Vinca rosea, internal leaf surface of, 195
Virus, movement of, 175
Viscous flow, activation energy of, 15, 16
"Vitaids", 68

Vitis vinifera, sap of, 120
Vitis vulpina, int. leaf surface of, 195
Volume measurement, immersion method, 104

Wall (see Cell wall)

Water I, II, III, 12, 13, 27

Water, absorption of (see Water absorption)

active absorption of, 104-105, 149-150

active relations, of cells, 111-147

anomalous properties of, 9

as a plant component, 59-72

association of, 5, 6, 8, 36

availability, methods of estimating, 151-154

binding forces, 24, 25, 26, 61-62, 63, 64

capillary condensed, 64

cavities in, 17

cohesion of, 154-155

continuity and movement of, 154-162

Grafts et al.

240

Water in Plants

coordination of, 13, 16, 29

diffusion pressure of {see Diffusion pressure)

dipole character of, 13

dipole moment of, 23

equilibrium in cell, 71-72

free energy of, 76-77

inexpressible, 121

intracellular distribution, 59-72

intercellular movement of, 108-109

loss and retention, 179-208

movement in xylem, 154-177

non-solvent, 25

of cell walls, 62-65

orientation in xylem elements, 155

packing of and tension, 155

partition, methods for determining, 70-71

passive absorption of, 151

physical properties of, 3-6

plant organs as reservoirs of, 172

protoplasmic, 65-69

quasi-lattice structure, 29

redistribution, 172-174, 203

self ionization of, 10, 11

structure of, 2-6, 3-17
early theories, 4-7
modern studies, 9-15
current research, 15-17

thermal properties of, 3, 4, 29

valence forces in, 9

viscosity of, 16

X-ray analysis of, 11-14, 15
Water absorption, active {see also Active water
relations), 149-151

active mechanism of, 104-105, 149-150

by roots, 148-154

DPD influence on, 153

passive, 149

root structure and, 148-149

salt absorption, relation to, 174

solute accumulation influence on, 149
Water-absorptive power, 50
Water balance, 172-174, 193, 200
Water conduction {see Water movement)
Water content of plants, 61-62
Water continuity, direct observation of, 160-162

dye penetration and, 156-159

freezing stems and, 159-160

in xylem, 155-162

methods of study of, 156

transpiration and, 160
Water deficit, physiological effects, 200-203

plant response to, 152-154
Water drops, contact angle of, 188
Water equilibrium, 52
Water in vacuoles, 69-70

Water loss {see Transpiration, and Guttation)
Water movement, adhesion and, 154-155

conducting tissue, 154

diffusion pressure and, 154

effect on transpiration, 193-194

physical theories of, 154

quantity of material moved, 168

rate, 167-172

resistance to, 162

surface tension and, 155

vitalistic theories of, 154
Water of constitution, 64
Water of crystallization, 29
Water relations, thermodynamic treatment of,

76-77
Water requirement, 187, 198, 199
Water saturation, 98, 102

Water secretion, 111-113, 116, 117, 118, 126,
143

active water absorption compared with, 149
Water storage cells, 59
Water vapor, coordination of, 22
Watermelon, water content, 62
Wax treatment effect on transpiration, 198-200
Weight method of DPD detn., 104
Wilting, effect on stomata, 160

sequence of, in plants, 152, 173

soil moisture and, 152-153
Wind velocity and transpiration, 183
Wood rays, OP of, 100

Xanthium pennsylvanicum, flower stimulation in,
176

strumariutn, OP of xylem exudate, 100
Xeromorphic condition, 200
Xeromorphic leaves, 195
Xerophyte, 135
Xylem, as a conductor, 154

as a reservoir, 161

contraction in, 157

exudate, 100

food transport in, 157

liquid continuity in, 155-156

parenchyma, OP of, 99

plugging of, 185

resistance in, 162

tension values in the, 104, 162-167
Xylol, in infiltration method, 197

Yield, DPD and, 153

Yucca, use as hygrometer, 186

Zea, water deficit and photosynthesis, 201

water requirement of, 198
Zea Mays, OP of xylem exudate, 100
Zostera marina, stimulative plasmolysis, 124

Pfeffer's original apparatus to determine
the osmotic pressure of solutions. — From
his "Osmotischen Untersuchungen" (1877).

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