ADVANCES IN
AGRONOMY VOLUME 30
CONTRIBUTORS TO THIS VOLUME
LINDOJ . BARTELLI DICKD . DAVIS E. C. DOLL
F. E. KHASAW...
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ADVANCES IN
AGRONOMY VOLUME 30
CONTRIBUTORS TO THIS VOLUME
LINDOJ . BARTELLI DICKD . DAVIS E. C. DOLL
F. E. KHASAWNEH BETTYKLEPPER
JAY D. MANN
R. L. PARFITT PETERL. STEPONKUS
H. M. TAYLOR
ADVANCES IN
AGRONOMY Prepared under the Auspices of the
AMERICAN SOCIETY
OF
AGRONOMY
VOLUME 30
Edited by N. C. BRADY International Rice Research Institute Manila, Philippines
ADVISORY BOARD
H.J. GORZ,CHAIRMAN K.M. KIM G.R. BLAKE R . B . GROSSMAN E.A. WERNSMAN
M. STELLY,EX
T.M. STARLING OFFICIO,
ASA Headquarters
I978
ACADEMIC PRESS New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich, Publishers
COPYRIGHT @ 1978, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC.
111 Fifth Avenue, New York, New York 10003
United Kingdom Edition published by ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London N W l
LIBRARY OF CONGRESS
7DX
CATALOG CARD
NUMBER:50-5598
!ISBN 0-12-000730-4 PRINTED IN THE UNITED STATES OF AMERICA
CONTENTS
CONTRIBUTORS TO VOLUME 30 ..................................
PREFACE....................................................
ix xi
ANION ADSORPTION BY SOILS AND SOIL MATERIALS
R. L . P d i t t I. I1. I11. IV . V. VI . VII .
Introduction ............................................ Techniques ............................................ Determination of Adsorption Sites on Mineral Surfaces . . . . . . . . . Adsorption Mechanisms .................................. Identification of Adsorption Sites in Soils . . . . . . . . . . . . . . . . . . . Adsorption by Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and Conclusions ................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
2 7 12 24 26 41 42
COLD HARDINESS AND FREEZING INJURY OF AGRONOMIC CROPS
Peter L . Steponkus I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Characterization of the Freezing Process and Freezing Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Environmental Factors Affecting Cold Hardiness . . . . . . . . . . . . . IV . Effect of Developmental Stage on Cold Hardiness . . . . . . . . . . . . V . Physiological and Biochemical Aspects of Cold Acclimation .......................................... VI . Screening and Stress Procedures for Determining Cold Hardiness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Summary and Conclusions ................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51 54 65 71 73 88 92 93
THE ROLE OF ROOTING CHARACTERISTICS IN THE SUPPLY OF WATER TO PLANTS
H . M . Taylor and Betty Klepper I . Introduction ............................................ 99 100 I1. A Model of Water Uptake by Roots ........................ I11. Diurnal Water Potentials in the Soil-Plant System . . . . . . . . . . . . 105
CONTENTS
vi
Axial Resistances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resistances in the Absorption Pathway ..................... Rooting Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Real World ........................................ Factors that Man Can Control ............................. A Final Thought . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV . V. VI . VII . VIII . IX .
106 i12 116 120 122 125 125
HYBRID COTTON: SPECIFIC PROBLEMS AND POTENTIALS
Dick D . Davis
I . Introduction ............................................ I1. Heterotic Expression of Cotton Hybrids .....................
111. IV . V. VI . VII. VIII . IX . X.
XI .
Effects of Heterosis on Phenology ......................... Plant Type and Harvest Efficiency ......................... Pest Resistance Potential for Hybrids ....................... Fiber Properties of Hybrids ............................... Breeding Hybrids with Marketable Fiber Properties . . . . . . . . . . . The Production of Hybrid Seed ............................ The Association of Heterosis and Plant Pubescence ........... Breeding Methodology ................................... Summary .............................................. References .............................................
130 131 134
138 140
142 144 147 150 152 153 153
THE USE OF PHOSPHATE ROCK FOR DIRECT APPLICATION TO SOILS
F . E . Khasawneh and E . C . Doll I . Introduction ............................................
............... I11. Reactions of Phosphate Rocks in Soils ......................
159 161 166
IV . Agronomic Evaluation of Phosphate Rock for Direct Application .......................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
183 204
I1. Mineralogy and Chemistry of Phosphate Rock
PRODUCTION
OF SOLASODINE FOR THE PHARMACEUTICAL INDUSTRY
Jay D . Mann
I . Perspective ............................................ 207 11. Solasodine-Containing Species of Solanurn .................. 209
CONTENTS
111. Chemistry
IV . V. VI . VII . VIII .
.............................................
Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation and Determination of Solasodine . . . . . . . . . . . . . . . . . . . Agronomy of Poroporo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Medical Aspects of the Glycoalkaloids ...................... Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vii
217 221 226 229 238 240 240
TECHNICAL CLASSIFICATION SYSTEM FOR SOIL SURVEY INTERPRETATION
Lindo J . Bartelli
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Principles of Technical Classification .......................
247 249 253 261 265
Systems for Organizing Soil Survey Interpretations . . . . . . . . . . . . Plant Suitability Evaluation Systems ........................ Systems to Evaluate Engineering Properties . . . . . . . . . . . . . . . . . . The Application of Technical Classification to Soil Surveys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 VII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 111. IV . V. VI .
SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
291
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CO NTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin.
LINDO J. BARTELLI (247), School of Forestry and Wood Products, Michigan Technological University, Houghton, Michigan 49931 DICK D. DAVIS (129), Department of Agronomy, New Mexico State University, Las Cruces, New Mexico 88001 E. C. DOLL (159), Division of Agricultural Development, Tennessee Valley Authority, Muscle Shoals, Alabama 35660 F . E . KHASAWNEH (159), Division of Agricultural Development, Tennessee Valley Authority, Muscle Shoals, Alabama 35660 BETTY KLEPPER (99), USDA, Science and Education Administration, Columbia Plateau Conservation Research Center, Pendleton, Oregon 97801 JAY D. MANN (207), Applied Biochemistry Division, Department of Scientific and Industrial Research, Christchurch, New Zealand R . L. PARFI'IT ( l ) , Soil Bureau, Department of Scientific and Industrial Research, Lower Hutt, New Zealand PETER L. STEPONKUS (5 l ) , Department of Agronomy, Cornell University, Ithaca, New York 14853 H. M . TAYLOR (99), USDA, Science and Education Administration, Ames, Iowa 50010
ix
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PREFACE The genetic yield potential of most crop plants is severalfold greater than the average yields actually achieved by farmers. Even the best performing cultivars commonly yield in the field no more than about half the level that under ideal conditions we know they can yield. Obviously in nature yields are constrained and often by factors of direct concern to agronomists. One of the primary responsibilities of crop and soil scientists is to identify the agronomic constraints on yield and then through research to find ways of removing these constraints. The contributions in this volume help us understand some of the constraints on crop yields and performance. Two reviews deal with variables related to climate. The effects of low temperatures on plants and the response of agronomic crops to these temperatures is the subject of one review. The nature of the roots in relation to the plants’ ability to meet its water supply needs is covered in a second review. Modern sensitivity to chemical hazards is expressed in this volume in keeping with a similar concern in the last few volumes. Two reviews deal with anions commonly found in soils. One gives a general coverage of the reactions and behavior of anions in different soils. The second focuses on a very important supplier of one of the most significant of these anions from a nutritional viewpoint-the phosphates. Rising costs of sulfur needed to manufacture superphosphates make it essential that rock phosphates be examined critically as sources of direct application for this important element. The potential of hybrid cotton along with the problems encountered with attempts to realize this potential are covered in this volume. The final review concerns soil survey interpretations, not only for agricultural purposes but for other uses as well. The authors who prepared these contributions have helped maintain the internationality of Advances in Agronomy. Likewise, the subjects they review are of considerable significance in most parts of the world. We are indebted to them for their efforts. N . C. BRADY
xi
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ADVANCES IN AGRONOMY, VOL. 30
ANION ADSORPTION BY SOILS AND SOIL MATERIALS
R. L. Parfitt Soil Bureau, D.S.I.R.,Lower Hutt, New Zealand
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Techniques.
................
.
1 2
A. Crystal Structure . . . . . . . . . . . . . . . B. Infrared Spectroscopy . . . . . . . . . . . . . . C. . . . . . . . ................... ..... C. Kinetics Surface Charge.. D. Ligand Exchange . . . . . . . . . . . . . . . . . . Adsorption Mechanisms . . . . . . . . . . . . . . . . A. Goethite (a-FeOOH) . . . . . . . . . . . . . . . C. . . .. .. .. ... . . . . . . . . . . . B . Surface HematiteCharge.. (a-Fe203) C. Other Iron Oxides . . . . . . . . . . . . . . . . . D. Gibbsite and Other Aluminum Surfaces E. Amorphous Hydroxides . . . . . . . . . . . . . F. Clay Minerals.. ................... G. Calcite ( C a C 0 3 ) .. . . . . . . . . . . . . . . . . .
............ ................................................. Calcite (CaC03) . . . . . . . ....
20 21 23 24 24 24 25 25 26
........ Arsenate .................... Molybdate ......................................................... ......................... D. Selenite.. . . . . . . . . . . . . E. Sulfate ... ...................... F. Boric Acid ............................................... G. Silicic Acid ...............................
34 34 35 36 36
E.
Amorphous Hydroxides ......................
G. V. Identification of Adsorption Si ............................. A. Surface Iron and Aluminum ........................................... B. Organometallic Complexes ............................................ C. Extraction of Phosphate.. .. ........ VI. Adsorption by S o i l s . . ......................................... B.
C.
............................................... .........................
Halides . . . . . . . . . . . . . . J . Nitrate.. . . . . . . . . . . . . . . . . . VII. Summary and Conclusions . . References .................................... I.
38
39 40
I. Introduction
In recent years there has been an increasing volume of literature dealing with the topic of anion adsorption by soils and soil materials, including the important I
Copyright @ 1978 by Academic Ress, Inc. All rights of reproduction in any form r e ~ e ~ e d .
ISBN-0-12-MX)730.4
2
R. L. PARFITT
iron and aluminum sesquioxide components. This has been due, in part, to the increased research effort going into tropical and subtropical areas, where many soils contain significant amounts of hydrous iron oxides and aluminum hydroxides. However, these reactive iron and aluminum surfaces are also common in many temperate soils (Mitchell et al., 1964; Wada and Harward, 1974). In particular, volcanic ash soils (andepts) often contain large amounts of reactive aluminum and iron gels, which can cause severe deficiencies of phosphate and sulfate in plants. In .4ustralia, Posner, Quirk, and co-workers have used iron and aluminum oxides with well-defined surfaces to study ion adsorption. This has stimulated much further research and has led to the development of a general model for ion adsorption on hydrous oxide surfaces, which takes account of the pH-dependent charge on the surface and the charge on the adsorbing ion (Bowden et al., 1977). It has become apparent that the adsorption behavior of many anions, in particular the important nutrients phosphate, sulfate, and molybdate. is very similar (Barrow, 1970). Work with synthetic iron and aluminum oxides has shown that fluoride, selenite, silicate, arsenate, carbonate, and other anions, including organic anions, are also adsorbed by similar mechanisms. This article attempts to review the work with soils, and with hydrous oxides of iron and aluminum, with particular reference to mechanisms and structural aspects of anion adsorption. Anion adsorption on clay minerals and calcium carbonate is also considered. II. Techniques
A. ADSORPTION ISOTHERMS
The determination of adsorption isotherms is one of the most useful experimental procedures in the study of the interaction of anions with hydrous oxides or soils. Most of the adsorption isotherms reported in the literature belong either to the Langmuir (L) type or to the high-affinity (H)type, as defined by Giles et al. (1960). Some examples are given in Fig. 1. The isotherms for phosphate adsorption on pure hydrous oxides are usually of the H type, indicating a large negative free energy, AG, of adsorption. On soils L-type isotherms can also be obtained, but the shape of the isotherm will depend on other factors such as the amount of native phosphate in the soil, the presence of fertilizer phosphate, and the influence of organic matter (Ryden and Syers, 1977). Several models have been used to describe anion adsorption, with most workers favoring the Langmuir model, which is described by the following equation:
3
ANION ADSORPTION BY SOILS
where c = solution concentration ;0 = XI&, where x = amount of ion adsorbed and x, = the sorption maximum; and K is a constant related to the adsorption energy. The equation may be written in the linear form: -c = - c x xlrl
+ Kxn, -1
and a plot of c/x against c should give a straight line of slope lh,,,, from which x, may be calculated and the constant K obtained from the intercept (Fig. 2). For anion adsorption the plot of clx against c does not give a straight line over a wide concentration range, and the simple Langmuir equation is not obeyed (Fig. 2). In the Langmuir model derived for the adsorption of gases onto solid surfaces, it is assumed that adsorption is restricted to a monolayer and that the energy of adsorption does not vary with surface coverage. Thus, the sites must be isolated and uniform, and the adsorbed molecules must not interact with each other.
100
0.2
0.4
OC? C
0.04 C
FIG. 1. Adsorption isotherms showing examples of high-affinity (H-type) isotherms for phosphate adsorption on goethite (a-FeOOH) in 0.1 M NaCl, pH 3.5,over different concentration ranges (A, B);Langmuir-typeisotherm for phosphate adsorption by soil (C); and an isotherm of an intermediate nature for phosphate adsorption on amorphous A1 (OH), in 0.1 M NaCI, pH 6.0 (D).x = anion adsorbed; c = final solution concentration.
R. L. PARFITT
4
Slope
1
=
qi /
/
C
FIG. 2. Langmuir plot of clx against c for phosphate adsorption on soil.
The failure of the simple Langmuir model to describe anion adsorption at solid-solution interfaces has led to the use of two- or three-term Langmuir equations, which give much better descriptions of experimental data (Muljadi et al., 1966a; Holford et al., 1974; Rajan, 1975c; Ryden et al., 1977b). These models assume that there are two or three sites for adsorption, with the energy of adsorption constant at each site. An equation similar to the Langmuir equation can be derived from considering adsorption equilibria (Graham, 1953). For adsorption: Anions "vacant sites" occupied sites desorbed ligands. If all sites are identical, and if there is no interaction between adsorbed anions, and if the activity coefficients of the occupied and unoccupied sites are the same, then
+
+
where K = the equilibrium constant, 8 = the fraction of the surface covered, and c A and c L = activities of the anion and desorbed ligand. If c L = 1 , the equation can be rewritten
which is identical to the Langmuir equation. If the activity coefficient is assumed to be unity, K may be determined from Langmuir plots and the standard free energy of adsorption AGO = -RT In K. Then -AGO gives a measure of the energy of adsorption due to bonding energy (AH") and entropy changes (AS"), where AGO = AH" - TAS". Ryden et al. (1977b) have used this approach to calculate AGO for three regions of the isotherm for phosphate adsorption on soils over a
ANION ADSORPTION BY SOILS
5
wide concentration range. The procedure was less successful for adsorption on Fe(OH), , where initial adsorption was of the high-affinity type. Attempts to use the Langmuir equation for phosphate adsorption on synthetic goethite have not been successful (Bowden el al., 1974), although synthetic goethite crystals have only one site available for anion adsorption (Parfht et al., 1976). This suggests that the assumptions made in using the Langmuir equation are not valid and that results obtained with this approach should be treated with caution. The Langmuir model is also inadequate when adsorption conditions such as pH or salt concentration are changed, because these factors affect both x , and K (Bowden et al., 1977). The inadequacy of the simple Langmuir model is perhaps hardly surprising. Being formulated for solid-gas systems, it takes no account of the charge on the anion being adsorbed nor of the surface charge. New models have recently been developed that are general and take account of charges during adsorption of anions and cations on amphoteric oxide surfaces (Yates et al., 1974; Bowden et al., 1977). In the approach used by Bowden el al. (1977), the free energy of adsorption (AGadB)is separated into three components: AGcoul,the coulombic or electrostatic component; hGchem,the chemical component; and AGint, the interaction component, which gives specificity to the coulombic binding. Thus,
Indifferent ions, such as nitrate, are adsorbed only on a positively charged surface, and AGcoul is significant, but hGchem is low. Adsorption is due to electrostatic interaction between the ion and the electric field of the surface. If an ion is adsorbed out of proportion to its activity in solution because of the size and polarizability of the ion, then AGlnt is also important. Potential-determiningions and ligand-exchanged ions can adsorb on a surface of like charge or zero charge, and AGchemis significant because of a specific interaction. Potential-determining ions are ions such as H30+ and OH-, which give their charge to a surface. Ions that are ligand-exchanged or specifically adsorbed are ions, such as phosphate, that are not present in the oxide lattice but that can also affect the surface charge on adsorption. Although these general models treat the surface as a continuum of sites, in energy terms, they are not inconsistent with the concept of discrete sites identified by infrared spectroscopy, since other factors such as proton adsorption and the presence of counter-ions may give sites of the same geometry a distribution of energy levels. The strength of the general model is that it describes experimental data obtained over a wide range of conditions (Bowden et al., 1973, 1974). Other simple equations that have been used to describe anion adsorption include the empirical Freundlich equation and the Temkin equation (Bache and
6
R. L. PARFITT
Williams, 1971). The Temkin equation includes the condition that the energy of adsorption decreases linearly with surface coverage. This model can be used over a wide concentration range, but the plot of x against log c is not linear over the whole range. Nevertheless, an equation that takes account of the continuous change in energy of adsorption with surface coverage during anion adsorption (Bache, 1964) should give a better description than the Langmuir equation. Adsorption isotherms can sometimes be used to give an indication of different adsorption mechanisms. The adsorption of oxalic acid on goethite (a-FeOOH) is an example. The shape of the isotherm has three distinct regions, suggesting that there are three different adsorption mechanisms (Parfin et al., 1977a). Infrared spectroscopy confirmed that oxalate was adsorbed as a bridging complex in Region I, and as a monodentate complex in Region 11, and it was suggested that dissolution occurred in Region 111. The isotherm for benzoic acid adsorption had a lower slope than that for oxalic acid adsorption, indicating a lower adsorption energy (Bache, 1964). Subsequently it was shown that this was due to monodentate adsorption of the benzoate, compared with bridging adsorption by the oxalate ( P d i t t et al., 1977a). Other examples have been described by Aylmore et al. (1967) and Theng (1971). Some other techniques that are related to adsorption isotherms include desorption studies. The rate of desorption of an adsorbed anion will give an indication of the energy of adsorption. Desorption is usually carried out by washing the adsorption complex with the same indifferent electrolyte used for adsorption and using the same pH conditions. With this method Hingston et al. (1974) were able to show that small amounts of phosphate could be desorbed from goethite, whereas fluoride could be desorbed almost completely. The desorption of OH ions during anion adsorption has also been studied, but it is usually difficult to interpret these results unambiguously (Rajan, 1975a). However, it has been possible to show that phosphate will displace silicate during adsorption on allophane clay, whereas selenite does not cause silicate desorption. This suggests that phosphate can be sorbed by displacing silicate (Rajan and Watkinson, 1976).
B. INFRARED SPECTROSCOPY Infrared spectroscopy can be used to study both the nature of adsorbing surfaces and the mechanism of adsorption on those surfaces (Little, 1966; Hair, 1967). Sample preparation is very important, since good films are required to minimize losses of radiation by scattering. Self-supporting films are ideal, but films that are evaporated onto infrared windows can also be used. Water molecules are usually adsorbed on hydrous oxide surfaces, and it is necessary to remove the water by evacuation in a vacuum cell, which prevents
ANION ADSORPTION BY SOILS
7
rehydration while the spectrum is being run. If the surface OH groups are discrete and if they represent a reasonable proportion of the total OH, including OH within the bulk of the oxides, then good resolution should be possible. Improved resolution is usually obtained by exchanging OH with b0 to give OD groups. Information on the coordination of anions to surfaces and their mechanism of adsorption can be obtained by comparing the spectra of the OH and OD groups before and after anion exchange. If the adsorbing ion itself has a spectrum in a range not obscured by lattice vibrations, then additional data are available on the coordination of the ion to the surface. It may also be possible to study the form of bonding in the presence of solvent as well as under vacuum. Thus, for phosphate adsorption on goethite it was possible to identify the mechanism of adsorption from the spectrum of the goethite surface groups and the spectrum of the adsorbed phosphate ion (Pditt et d , 1976; P d i t t and Atkinson, 1976). Further details on techniques used in infrared spectroscopy are available in a review by Russell (1974).
C. KINETICS
A study of the kinetics of anion adsorption can be used to gain information about the mechanism of the adsorption reaction. The data are usually presented to show the change in adsorption with time or the change in anion concentration with time. These basic experiments have indicated that the adsorption reaction is followed by a slow reaction on some systems (Chen et al., 1973b). A study of the effect of pH, temperature (which enables the activation energy to be calculated), or successive additions of anion may give further useful information about reaction mechanisms or rate-limiting steps, but generally it is difficult to interpret kinetic data without some reasonable theory. Studies on the kinetics of isotopic exchange of adsorbed anions have provided useful information about adsorption mechanisms. When Elovich kinetics have been applied, concentration-independent rate constants can be determined and comparisons made with other systems. This led Atkinson et al. (1972) to postulate that phosphate formed a bridging complex on goethite. Experiments on rates of isotopic exchange of phosphate have also provided useful information about the nature of labile phosphate in soils (Mattingly, 1975). 111. Determination of Adsorption Sites on Mineral Surfaces
The surfaces of iron and aluminum oxides and hydrous oxides usually consist of mixtures of OH- ions and water molecules, which &e coordinated to Fe3+ or
8
R. L. PARFITT
FIG. 3. Structure of the (100) face of goethite (a-FebOH) in plan a and section b. A, B, and C are hydroxyl groups, and C’ is an oxide ion.
A13+ ions directly below the surface. In the ideal structure the surface is assumed to have no net charge, and therefore at a freshly cleaved surface OH- ions and water molecules replace the structural 02-and OH- ions, which become exposed. The OH- ions may be in one, two, or three coordination to the metal ions below. From work with goethite (Fig. 3), it appears that only the one-coordinated OH- ions (A type) are able to take part in ligand exchange reactions (Parfitt er al., 1976). Under acid conditions they adsorb protons, giving positively charged OH+ ions, which are readily exchanged with other ligands. Lewis acid sites occur where water molecules are coordinated to metal ions exposed at a surface. In the presence of OH- ions a proton may be lost from these M*O& sites to give M-OH-at the surface (Mott, 1970). A. CRYSTAL STRUCTURE
The crystal structures of most minerals that occur in the clay fraction of soils are now known. If the adsorbent is well crystallized, then the crystal structure can be used as the starting point for determining the structure of the crystal faces. For goethite, electron diffraction has been used to show that the (100) face
9
ANION ADSORPTION BY SOILS
predominated, the other exposed faces being the (010) and (001) faces. The arrangement of the exposed surface ions could then be deduced (Atkinson, 1969; Russell et al., 1974), and the number of sites could be estimated from the size of the crystals. It was predicted that there were three types of OH groups (A, B, and C) on the (100) face, and in addition there were Lewis acid sites (Fe*OH2)on the (010) face (Fig. 3). On a high-surface-area goethite it was estimated that there were 410 pmol g-' A, B,and C-type OH groups, and 220 pmol g-I of Lewis acid sites (Pditt et al., 1976). These results were consistent with adsorption isotherm data and infrared spectra.
B. INFRARED SPECTROSCOPY Surface sites can often be identified on hydrous oxides by infrared spectroscopy after the adsorbed water has been removed. Figure 4 gives an example of the bands obtained for the OH sites on geothite. These bands are more clearly
3800
I '
I
3400 I
3ooo
I
I
2600
I
I
I
2200 mi' I
FIG. 4. Infrared spectra of goethite showing surface OH bands, which exchange with 40, giving OD bands.
10
R. L. PARFIT
resolved after exchange with 40, giving OD vibrations at 2584 cm-' and 2701 cm-'. The main band at 3 150 cm-' is due to the structural OH groups within the goethite crystals. The A-, B-, and C-type surface OD (OH) vibrations are shown in Fig. 5a. The B- and C-type OD vibrations are clearly resolved near 2700 cm-' after the 410pmol A-type OD at 2584 cm-' have been exchanged with 410pmol F- or 205 p mol HPOi- (Fig. 5 , b, c) (Parfitt et al., 1976). Lewis acid sites (M-OH,) can also be identified from infrared spectra by adsorbing ammonia or pyridine and examining the spectrum of the adsorbed ligand. On goethite, the pyridine bands occurred at 1608 cm-' and 1592 cm-', indicating that pyridine was held on Lewis acid sites (1608 cm-') and by hydrogen bonding (1592 cm-'). No band near 1540 cm-' was observed, implying that Bronsted acid sites were absent (Parfin et al., 1976).
FIG.5. Infrared spectra of goethite surface OD groups showing A-, B-, and C-type OD (a); showing A-type OH replaced by F and HPQ-, and the B- and C-type OD clearly resolved in the 2700-cm-' region (b, c).
ANION ADSORPTION BY SOILS
C . SURFACE CHARGE
The important sites for adsorption are the M*OHand M*OH2sites, which may become charged in the presence of excess H+ or OH- ions. Since the charge on oxide surfaces is pH-dependent, potentiometric titrations in indifferent electrolytes (for example, NaN03) are used to give information on the charge characteristic of these surfaces. The pH where the titration curves cross over is referred to as the pH of the point of zero charge (pzc). The pzc occurs between pH 7.5 and 9 for iron and aluminum oxides (Hingston ef al., 1972).
On the acid side of the pzc, H+ ions are adsorbed on the oxide surface at the first layer of coordinated OH or oxide ions (inner Helmholtz plane), and the counter-ions (for example NO,-) are adsorbed in the diffuse double layer. On the basic side of the pzc, H+ ions are desorbed from the first coordination shell, and the counter-ions (Na+ or K+) are in the diffuse double layer. For goethite and hematite the uptake of H+ or OH-, measured by potentiometric titration, did not reach any maximum value within the pH range 3.5-1 1. The largest uptake of protons in KCl solution occurred in 1 M solution at pH 3.5, giving one excess proton per 0.34 nm2 and 0.43 nm2 for hematite and goethite, respectively, assuming uniform distribution over the BET area (Atkinson et al., 1967; Hingston et al.. 1972). The calculated charge on goethite is one positive charge per 0.30 n d on the (100) face, assuming that there is one proton per unit cell on the (100) face, and one negative charge per 0.14 n d on the (010) face, assuming that there is one Lewis acid site per unit cell on the (010) face. This corresponds with positive and negative charges of one per 0.37 n d and 0.70 nm', respectively, where the total surface area is used in the calculation, which agrees with the experimental results of Atkinson et al. (1967). Breeuwsma and Lyklema (1971, 1973) measured the surface charge of hematite with a range of electrolytes and found that divalent cations were more strongly adsorbed than monovalent cations, which were weakly adsorbed. The maximum negative charge occurred with Mg2+ ions, where one proton desorbed per 0.20 n d . They concluded that the results could be explained satisfactorily in terms of the Gouy-Stern theory. The surface charge can also be determined by measuring the adsorption of indifferent ions at a range of pH values (Bolland et al., 1976). Hingston et al. (1972) found that the positive charge on a synthetic gibbsite in 0.005 M NaCl increased from 20 peq g-I at pH 7 to 50 p eq g-' at pH 4, using both C1 adsorption and potentiometric titrations. The sites that accept protons are modified when ligand exchange occurs, and the pzc usually shifts to lower pH as surfaces become more acidic and therefore accept fewer protons at any one pH. The negative charge added to the surface
12
R. L. P A W
when ligand exchange takes place on goethite was -0.38 eq mol-' of silicate, -0.7 eq mol-I of selenite, and -1.0 eq mol-I of phosphate (Hingston et al., 1968b, 1972; Parfitt and Atkinson, 1976).
D. LIGAND EXCHANGE
Ligand exchange can also be used as a method of estimating the number of surface sites. Phosphate has been used for this purpose, since it is very strongly adsorbed, replaces a very large proportion of the reactive OH, and often has a well-defined adsorption maximum (Atkinson et al., 1974). Fluoride adsorption and the determination of OH- ions released during fluoride adsorption has recently been used on a quantitative basis as a method for estimating reactive hydrous oxides in soils, and it may be possible to develop this method further so that specific sites can be identified (Perrott er al., 1976a,b). IV. Adsorption Mechanisms
A. GOETHITE (a-Fe00H)
Synthetic goethite can be prepared with a very well defined crystal surface. The predominant (001) face consists of rows of one-, two-, and three-coordinated OH (A, C, and B types, respectively) separated from the next three rows by a groove where oxide ions are exposed (Figs. 3 and 6) (Atkinson, 1969; Russell et al., 1974). Parfht et al. (1976) showed that a high-surface-area preparation of goethite had 410 pmol g-' of A-, B-, and C-type OH and C-type oxide ions exposed on the (100) face. The A-type OH were the only groups involved in ligand exchange, but the B- and C-type OH could form hydrogen bonds with adsorbing ligands. The (010) face was shown to be made up of 220pmol g-'.of Lewis acid sites and 220pmol g-' of two coordinated (C-type) OH ions. The surface area of this goethite preparation was calculated to be 90 d 8'. The mechanism of phosphate adsorption on goethite has been studied in more detail than has any other similar reaction. Atkinson et al. (1972) showed that the rate of exchange of 32Pon phosphated goethite was relatively slow, suggesting the formation of a binuclear bridging Fe OP(0) ,O * Fe surface complex rather than a monodentate Fe*OP(O), complex. This mechanism is consistent with the strong adsorption that has been observed in adsorption studies (Atkinson, 1969; Yates and Healy, 1975) and with the observation that phosphate could not be desorbed from goethite by washing at low pH (Hingston et al., 1974). Infrared studies have provided direct evidence for the formation of a bridging complex on goethite over the whole range of surface coverage (Atkinson et al.,
ANION ADSORPTION BY SOILS
13
FIG. 6. Ideal structure of goethite (a-FeOOH) showing surface OH (white), A-type OH (gray), and Lewis acid sites (black).
1974; P d i t t er af., 1976; P d i t t and Atkinson, 1976; Parftt and Russell, 1977), and it has been shown that the evacuated bridging complex is present as (FeOhPOOH rather than as (FeOkPOO- (Russell et al., 1975; P d i t t et al., 1976). Russell et al. (1974) showed that phosphate replaced A-type OH, and they suggested that the POH group formed a hydrogen bond to surface C'-type F e . 0 groups in the grooves on the (100) face (Russell et af., 1975) (Fig. 7). The evacuated bridging complex appears to retain a proton even at high pH (Parfht et al., 1976). Recent infrared and potentiometric titration results with goethite in suspension have shown that the bridging complex is ionized at high pH and protonated at low pH (Parfitt and Atkinson, 1976). The pH of net zero charge depends on the level of adsorbed phosphate. At half-coverage (100 pmol g-' phosphate) this pH is 5.1, compared with 8.1 for nonphosphated goethite in 0.01 M NaC1. The equations for phosphate adsorption at pH 3.6, 5.1, and 8.1 are
+
100
HPO:
+
-
FeOIOOLeo
p-O
-I
+ 100 OH- + 100 H 2 0
14
R. L. PARFITT
FIG. 7. Model of goethite with HPO, adsorbed on (100) face.
Hingston et al. (1968a, 1971, 1972, 1974) studied the adsorption of SO:-, YSiOL, H,POr, Moq-', S e a - , H,AsO;, and F on goethite and gibbsite and showed that these anions were selectively adsorbed in the presence of Cl-. This indicated that specific adsorption was taking place. It was suggested that these anions were adsorbed on goethite and gibbsite by ligand exchange with surface OH or OH: groups. Anions of fully dissociated acids are adsorbed if the surface carries a positive charge; thus, S 4 - is adsorbed only on the acid side of the pzc. However, anions of incompletely dissociated acids can also be adsorbed on the alkaline side of the pzc, since they can provide a proton, which is needed for removal of a surface OH at the adsorption site. Maximum adsorption occurs at a pH close to the pK of the acid, where the surface potential and the concentration of the adsorbing hydrolyzable ion are optimal (Bowden et al., 1974). Both C1- and NO; are adsorbed only at low pH when the surface is positively charged. This is consistent with chemical data showing that OH- and H,O are more strongly attracted to Fe3+ and A P than are NO,- and Cl-. When HNO, and HCl are dried onto goethite and evacuated, it appears that ligand exchange can occur (Parfitt and Russell, 1977). P d i t t and Russell (1977) examined the spectra of surface OH groups of goethite in the presence of a range of adsorbed anions. The experiments showed that sulfate and selenite could replace all A-type OH, suggesting that binuclear complexes were formed. This was confirmed by the observation that at half-
ANION ADSORPTION BY SOILS
15
coverage the remaining A-type OH hydrogen bonds were lengthened by an amount that depended on the anion oxygen-oxygen distance. This conclusion is consistent with (a) the strong adsorption observed for selenite and sulfate on iron surfaces (Aylmoreetal., 1967; Hingstonetaf., 1968b, 1971), ( 6)theobservation that selenite cannot be desorbed from goethite (Hingston et al., 1974), and (c) an adsorption maximum of about 200 pmol g-', giving one anion per two A-type FeeOH. Parfitt and Smart (1977, 1978) examined the S-0 stretching spectrum of sulfate adsorbed on iron oxides and confirmed that sulfate formed binuclear bridging complexes. A similar spectrum was obtained on a wet film of sulfate and goethite, suggesting that the complex is present not only on drying but also in suspension (R. L. Parfitt, unpublished results). However, Yates and Healy (1975) compared the rate and extent of sulfate adsorption with phosphate adsorption at pH 6.15 and suggested that sulfate was not involved in ligand exchange on goethite. Borate, molybdate, and silicate are adsorbed on goethite by ligand exchange (Hingston er al., 1972), and infrared spectra show that A-type OH are replaced during the reaction (Pditt and Russell, 1977). Silicic acid behaves as a weak monobasic acid (pK = 9.6); thus, only one goethite OH can be replaced by silicate. The surface complex may be in the form Fe.OSi(OH),. Boric acid is a Lewis acid that accepts OH- (pK = 9.0), and the reaction with goethite probably gives Fe.OH.B(OH),. Hingston et al. (1974) and Bowden et al. (1974) showed that fluoride is adsorbed on goethite by a ligand exchange reaction. More recently Parfitt et a f . (1976) found that only A-type OH were exchanged, and the B- and C-type OH were inert to fluoride. This is consistent with an isotherm maximum near 400 pmol g-' for fluoride adsorption on a high-surface-area goethite (Hingston et al., 1974). Clean preparations of goethite adsorb atmospheric carbon dioxide to give a surface carbonate species (Russell et al., 1975). However, most anions, except for NO;, are adsorbed more strongly on goethite and block the adsorption sites. The halogens Br- and I- show adsorption behavior similar to that of Cl- . On air drying HCl, HBr, or HI with goethite, some ligand exchange with A-type OH takes place, but some HCl, HBr, and HI is also adsorbed by hydrogen bonding (Parfitt and Russell, 1977). In work with organic ligands Watson et a f . (1973) suggested that 2,4-D was weakly adsorbed on goethite by ligand exchange. P d i t t et al. (1977a) showed that benzoate was also weakly adsorbed, with one carboxylate oxygen replacing one A-type OH and the other carboxylate oxygen keyed into the surface groove so that the aromatic ring is at a high angle to the (100) face. Appelt et al. (1975a) showed that p-OH benzoate, salicylate, and phthalate were more strongly adsorbed than was benzoate. Low levels of oxalate are strongly adsorbed on goethite in the binuclear form
16
R. L. PARFIlT
(Fe * 00CC00 * Fe). At higher surface coverage oxalate is more weakly adsorbed and is in the monodentate form (Parfitt et al., 1977a). The adsorption of fulvic acid and humic acid on iron oxides has been studied by a number of workers (Greenland, 1965). Recently it has been shown that both fulvic acid and humic acid are strongly adsorbed on goethite by ligand exchange. The carboxylate groups replace several A-type OH, giving multiple points of contact. Additional mechanisms of adsorption include hydrogen bonds and entropy effects. Van der Waals bonding did not appear to contribute significantly to the adsorption (Parfitt et al., 1977~).
B . HEMATITE ( o l - F ~ O 3 )
Although the oxygen within hematite crystals is present as oxide, the ideal surface is composed entirely of hydroxide ions in one and two coordination (Parfitt et al., 1975). However, Atkinson et al. (1967) suggested that the actual degree of order on the hematite surface depended on the method of preparation. Most studies have been carried out with hematite that is prepared by hydrolysis of ferric nitrate solutions under reflux conditions. Such preparations usually contain a small amount of goethite as a separate phase. Some goethite may also be present as a surface coating on hematite, but differences between the infrared spectra for phosphated goethite and hematite suggest that the hematite surface is different from the goethite surface. An infrared study of phosphate adsorbed by drying on hematite gave results that were consistent with an ordered hematite surface, (Parfitt et al., 1975), but Breeuwsma and Lyklema (1971), using potentiometric titration, DTG, and N2 adsorption data, concluded that the surface was porous to cations, N2,and water, but not to anions. It is likely that hematite suspensions have a coating of amorphous iron hydroxide, which becomes more crystalline on drying (Atkinson et al., 1967; Breeuwsma and Lyklema, 1971, 1973). Jurinak (1966) found that surface OH groups on hematite heated to 500°C were regenerated by water vapor adsorption and that each OH regenerated occupied 0.22 n d . Jurinak (1966) and Parfitt et al. (1975) found that one phosphate ion was adsorbed also on each 0.22 n d of surface. Jurinak (1966) also found that phosphated hematite had one surface OH per 0.125 n d , which suggested that two POH groups now occupied one FeOH site (that is, the phosphate was monodentate Fe - 0 .PO(OHX, with two OH groups exposed). However, Parftt et al. (1975), on the basis of infrared spectra, suggested that phosphate was adsorbed in the bridging form. Breeuwsma and Lyklema (1973) used a hematite which they considered was slightly porous to cations, and they concluded that phosphate replaced O&+ groups at low pH and OH at high pH. Kuo and Lotse (1974) investigated the
ANION ADSORPTION BY SOILS
17
kinetics of adsorption and desorption of phosphate and found that the adsorption process required a very low activation energy. Sulfate adsorption has been studied by Jurinak (1966), Aylmore ef al. (1967), and Breeuwsma and Lyklema (1973); the results were consistent with ligand exchange. Jurinak (1966) suggested that sulfate was adsorbed in the monodentate form, but infrared results indicated that the bridging complex was formed (Parfitt and Smart, 1978). Molybdate adsorption is more complex, and paramolybdate probably forms on the surface at high concentrations (Reyes and Jurinak, 1967). Microcalorimetry showed that the surface energy of hematite was lower after anion adsorption (Jurinak and Burau, 1967).
C. OTHER IRON OXIDES
The crystal structure of lepidocrocite (7-FeOOH) is given in diagrammatic form by Wells (1962). The ideal surface consists of rows of one-, two-, and three-coordinated OH ions. The crystal structure of a akaganeite @I-FeOOH)consists of multiple octahedral chains joined up along their lengths by sharing comers. It probably has rows of OH ions in one, two, and three coordination exposed at the surface (Gallagher, 1970; Parfitt ef al., 1975). Infrared spectroscopy has shown that both phosphate and sulfate are adsorbed as binuclear bridging complexes on lepidocrocite and akaganeite (Parfitt et al., 1975; Parfitt and Smart, 1978).
D. GIBBSITE AND OTHER ALUMINUM SURFACES
The mechanism of anion adsorption on aluminum hydroxides is less well understood than that on iron oxides because the surfaces are less well defined. However, synthetic gibbsite can be prepared in a very well crystallized form (Russell et al., 1974). The crystal structure of gibbsite is given by Bragg and Claringbull (1965). Synthetic gibbsite consists of well-ordered hexagonal crystals, and, although the (001) face predominates, the edge faces contain the reactive sites (Parfitt et al., 1977b). In the ideal structure each A13+ ion exposed on the edge faces is coordinated to one H,O and one OH ion, while each OH on the (001) face is coordinated to two A13+ ions below the surface layer. Russell et al. (1974) found that five different types of surface OH on the (001) face could be detected by infrared spectroscopy. None of these surface OH groups exchanged with phosphate or oxalate (Parfitt et al., 1977b). Alvarez et al. (1976), using laser Raman spectroscopy, observed four bands,
18
R. L. P A R F m
at frequenciesclose to the infrared frequencies of bulk OH. Although the gibbsite had a low surface area, it was suggested that they were surface OH, since the intensity varied with anion adsorption. Muljadi et al. (1966a,b,c) suggested that phosphate adsorption on gibbsite from dilute phosphate solutions took place on the edge faces, and entropy factors were a very significant driving force in the reaction. They indicated that ligand exchange occurred and that phosphate was adsorbed in a monodentate form. However, later isotopic exchange work by Kyle et al. (1975) showed that phosphate was probably adsorbed as a bridging complex. Parfitt et al. (1977b) studied the adsorption of phosphate, oxalate, and benzoate on a synthetic gibbsite that had about 60pmol g-' Al.(OH)(H,O) exposed on the edge faces. The adsorption isotherms showed that 25 pmol g-' phosphate and 21 pmol g-' oxalate were strongly adsorbed on this preparation; for phosphate the adsorption maximum occurred at 70 pmol 8'. Infrared data showed that up to 80 pmol g-' oxalate was adsorbed by ligand exchange with Al. This supports earlier conclusions that the edge Al*(OH)H,O groups react with phosphate and oxalate and suggests that the complexes probably are in a binuclear or bidentate form. No reaction with the (001) face Al*OH*AIgroups was observed, since the five infrared bands due to these surface OH were unchanged, as were the four bands due to OH within the crystal. Muljadi ef al. (1966a) and Kyle et al. (1975) showed that gibbsite adsorbed more than 200 pmol g-' phosphate at high solution concentrations, but their preparations may have included some less-crystalline but very reactive phase. At high concentrations at pH 5 , phosphate adsorption continued to increase, and some concomitant potassium adsorption occurred at the higher concentrations. Muljadi et al. (1966a) suggested that this adsorption occurred on the lesscrystalline phase. Pditt et al. (1977b) found the plateau was at 70 pmol g-' in a well-crystallized gibbsite, and thus the crystallinity of gibbsite is important in controlling the amount of phosphate adsorbed. Helyar et al. (1976a,b) studied the kinetics of adsorption and desorption of phosphate on a commercial synthetic gibbsite and found that the initial rapid adsorption was followed by a slow reaction. More phosphate is adsorbed in the presence of Ca ions than in the presence of Na, K, or Mg, and it was postulated that two phosphate ions on the (001) face formed a complex with one Ca ion, which allowed increased phosphate adsorption. However, this seems unlikely, since infrared results show that phosphate is not adsorbed on the (001) face, and Ca causes increased phosphate adsorption on other oxides and soils (Ryden and Syers, 1975b; R. L. Parfitt,unpublished results). It also seems likely that the commercial gibbsite contained a small amount of high-surface-area amorphous material, since the calculated surface area is much less than the measured surface area.
ANION ADSORPTION BY SOILS
19
At very high concentrations of phosphate, precipitation or growth of a new crystal phase probably takes place (Bache, 1964; Muljadi et al., 1966a). Hingston et al. (1972, 1974) determined adsorption envelopes for sulfate, selenite, molybdate, and silicate on gibbsite and concluded that the results were consistent with ligand exchange reactions. Boehmite (7-A100H) has a structure similar to that of lepidocrocite, and pseudoboehmite is a less-crystalline form of boehmite. Aylmore et al. (1967) showed that sulfate was strongly adsorbed on pseudoboehmite at pH 4.6, and little sulfate could be desorbed. When phosphate is adsorbed on mixed aluminum hydroxides, OH ions are released and surface positive charge is decreased (Rajan et al., 1974; Rajan, 1975a, 1976). Interpretation of these data is difficult, since both the phosphate and the aluminum hydroxide can accept or release protons. Rajan (1976) suggested that, at pH 4, H,POr was adsorbed initially on Al.OH,+, sites and with increasing phosphate coverage Al. OH sites reacted to form monodentate complexes, A1 * H,PO,". At high phosphate concentrations the phosphate reacted with surface A1 * OH * A1 groups. Chen et al. (1973a,b) studied phosphate adsorption on a-alumina and found that 15 pmol g-' was strongly adsorbed. The adsorption envelopes had a maximum at pH 4, and phosphate adsorption was decreased by the presence of chelating anions. The rapid phosphate adsorption reaction was followed by a slow reaction when hexagonal crystals were formed. Van Riemsdijk et al. (1975, 1977) also observed the growth of hexagonal crystals with aluminum hydroxide and x-ray data and suggested that they were crystals of sterrettite. In a study using y-A&O,, Huang (1975a) also found that maximum adsorption occurred near pH 4 with high levels of phosphate but shifted to higher pH with low levels of phosphate. The specific chemical energy of adsorption was estimated to be -16 kl mol-I. At high phosphate concentrations the reaction is complicated, since surfaces containing aluminum react to form new phases such as taranakite (Wada, 1959; Taylor et al . , 1965; Tamimi et al., 1968). It has been suggested that when A1 occupies cation exchange sites it is present both as A P and as a basic cation with an empirical formula Al,(OHX+ (Brown and Newmann, 1973). Muljadi et al. (1966b) have shown that aluminum in this form is able to react strongly with phosphate, and it is likely that exchangeable aluminum is a site for ligand exchange. Rich (1968) has reviewed the literature on the formation of hydroxyaluminum species. It appears that interlayer hydroxyaluminum does not take part in anion exchange reactions in the interlayer surface region of expansible layer silicates (Huang, 1975b). Colombera et al. (1971) showed that hydroxyaluminum species could form on illite surfaces; Greenland (1971) gave them the name outlayers and
20
R. L. PARlTlT
suggested that reactive aluminum in soils was in this form and probably is involved in ligand exchange reactions.
E. AMORPHOUS HYDROXIDES Amorphous iron and aluminum hydroxides have less well defined surfaces than the crystalline hydrous oxides. Femhydrite, amorphous ferric hydroxide, consists of spherical particles, 10-20 nm in diameter, with a large surface area (100-300 m2 g-') and a defective hematite structure where some 0 ions are replaced by water and some Fe positions are vacant (Schwertmann et al., 1974). Amorphous aluminum hydroxide is unstable and rapidly crystallizes to pseudoboehmite and bayerite. Reactions that have been studied include adsorption with silicic acid (Beckwith and Reeve, 1963; Hingston and Raupach, 1967), borate (McPhail et al., 1972; Sims and Bingham, 1968a), phosphate (De, 1961; Hsu and Rennie, 1962; Bache, 1964; Hasan and Pollard, 1966; Ryden and Syers, 1975a), iodide (Whitehead, 1974), arsenate (Anderson et al., 1976), and molybdate (Jones, 1957; Reisenauer et al., 1962), and the results are consistent with ligand exchange. Ryden and Syers (1975a) suggested equations for phosphate adsorption at different surface coverage, but pH was not controlled in these experiments. Parfitt et al. (1975) and Parfitt and Smart (1978) have shown that phosphate and sulfate are adsorbed on amorphous Fe(OH),, and both form a binuclear bridging complex. Allophane has been defined as a naturally occurring hydrous aluminosilicateof varying composition yet with short range order, Si-0-A1 bonds, and a distinct DTA trace (Wada and Harward, 1974). Allophane particles are probably spherical, with a diameter of about 5 nm and a structure consisting of a glass-like aluminosilicate core with an outer surface of aluminum surrounded by an octahedral arrangement of water and OH- ions. Some SiOH groups may also be exposed at the surface. F8+ ions are often found in association with allophanes, and reactive OH and H,O ligands are probably associated with the iron as well as the aluminum. The negative charge of an allophane measured by Na+ adsorption at (pH 7) was 135 meq g-' , and the pH of zero charge varied from pH 7 to lower values with different allophanic material (Wada and Harward, 1974). Ghosh and Battacharyya (1930) and Cloos et al. (1968) studied the adsorption of phosphate on synthetic silica aluminas and found that adsorption increased as the aluminum content increased. It was suggested that adsorption took place on positively charged sites and with OH groups on surface hydroxyaluminum cations.
ANION ADSORPTION BY SOILS
21
Rajan (1975a,b,c) and Rajan and Perrott (1975) showed that low concentrations of phosphate were adsorbed by ligand exchange on silica aluminas and soil allophanes. Some adsorbed sulfate, and silicate was displaced in addition to OH ions and H,O. At higher concentrations hydroxyaluminum polymers were disrupted, and structural silicate was displaced by phosphate but not by selenite (Rajan and Watkinson, 1976). Inoue and Wada (1968, 1971a,b) found that the adsorption of humic material on allophane, imogolite, and montmorillonite followed a Langmuir curve. Allophane adsorbed the largest amount, and it was concluded that adsorption was by ligand exchange of carboxylate groups with the coordination shells of A1 atoms and by van der Waals bonding. The structure of imogolite has been determined by Cradwick et al. (1972). The external surface of the tubes has a face similar to the gibbsite (001) face. Phosphate adsorption probably can occur on this face as well as on sites at the ends of the tubes where broken bonds occur. Other sites within the tubes contain Al-OH and Si. OH groups, and it is unlikely that anion adsorption occurs in this region (Parfitt et al., 1974). P d i t t et al. (1974, 1977b) suggested that phosphate and oxalate were adsorbed on imogolite by ligand exchange on the external surfaces of the tubes and at the end of tubes. However, the adsorption was weaker than on gibbsite or allophane.
F. CLAY MINERALS Some clay minerals have a pH-dependent charge which occurs at the edges of the crystals where Al.(OH)H,O groups are exposed. The Al*OHgroups are the sites that accept a proton at low pH to become AlaOH; (Schofield, 1949; Schofield and Samson, 1953; Bolland et al., 1976). Muljadiet al. (1966a) found that the positive charge on a high-surface-area kaolinite was 0.95 meq per 100 g at pH 3. At low solution concentrations phosphate is adsorbed on some clay mineral surfaces (Low and Black, 1950; Kuo and Lotse, 1972), but at high phosphate concentrations precipitation occurred, resulting in the formation of new crystalline phases such as taranakite (Kittrick and Jackson, 1954, 1955, 1956; Wada, 1959). The mechanism of adsorption is by exchange of phosphate with AI-OH groups on the edge sites of clay minerals (Kelley and Midgley, 1943; Low and Black, 1950; Mehlich, 1964; Pissarides et al., 1968). Muljadi et al. (1966a,b) showed that phosphate adsorption on K-kaolinite occurred on the edges of the crystals, and each phosphate ion occupied 0.28-
22
R. L. P A m
0.41 d . Each Al-(OH)H20exposed on the edges occupies 0.33 n d , suggesting that one phosphate ion is adsorbed on each A1 ion. Kafkafi et al. (1967) suggested that, since two adjacent Al*OHwere 0.296 nm apart, some phosphate cou€dbe fixed on kaolinite as a binuclear complex. However, the major part of the phosphate was exchangeable and was thought to occur in a monodentate form. Kuo and Lotse (1972) suggested that phosphate exchanged with Al*&O groups on kaolinite rather than AlmOH, since (a) adsorption increased with decrease in pH, and (6) no release of OH was observed. These effects can now be explained by considering surface OH; groups. When exchangeable A1 i s present in kaolinite, approximately half of the A1 is able to adsorb phosphate (Muljadi et al., 1966b). It is possible that only one of the two forms of A1 described by Brown and Newmann (1973) is reactive. This is also consistent with earlier observations (Coleman, 1944, 1945; Haseman et al., 1950; Russell and Low, 1954; Hemwall, 1957b; Coleman et al., 1960; HSU, 1965, 1968; Hall and Baker, 1971). Pissarides et al. (1968) showed that the exchangeable cations influenced the amount of phosphate that could be adsorbed on edge sites of clay minerals, and on Na-montmorillonite there was negative adsorption owing to diffuse doublelayer effects. Parfitt (1972) also found that negative adsorption occurred r - ~ Na-montmorillonite with uronic acid at pH 6, but positive adsorption took place at low pH or if Al was present on the exchange sites. When aluminum and iron hydroxyl species are present on mica surfaces, phosphate and sulfate adsorption are greatly increased (Rankin and Wilson, 1969; Langdon et al., 1973; Perrott et af., 1974a). It was suggested that phosphate reacted initially by adsorption on A1 and Fe sites, and then this was followed by a rearrangement reaction. Kodama and Singh (1972) and Kodama and Webber (1975) found that phosphate and sulfate could be incorporated into montmonllonite interlayers. However, the reaction involved a precipitation reaction with aluminum, giving AlPO4nHzO and A~(OHX.S(SO~)O.,S. Hudcova (1970) also studied the adsorption of phosphate on clay minerals and found that, although kaolinite adsorbed most phosphate, the adsorption energy was greatest on illite. Schell and Jordan (1959) showed that montmorillonite adsorbed more phosphate and sulfate than did halloysite or kaolinite. When silica was adsorbed on kaolinite, equal amounts of phosphate were desorbed, which suggested that adsorption occurred on similar sites (Kafkafi and Bar-Yosef, 1969). Maximum silica adsorption occurred near the pK value of the silicic acid (Bar-Yosef et af., 1969). The surface area, the surface aluminum and iron, the pH, and the exchangeable cations are the major factors influencing phosphate adsorption on clay minerals, and they explain the differences between clay minerals (Edzwald et al.,
ANION ADSOWION BY SOILS
23
1976). Results of Frost and Griffin (1977) suggest that these factors also control the extent of selenite and arsenate adsorption. Aylmore et al. (1967) found that sulfate was adsorbed by kaolinite, and they suggested that adsorption occurred by ligand exchange on edge sites. Initially sulfate was strongly adsorbed, but at higher supernatant concentration the adsorption was reversible with respect to concentration. At maximum adsorption one sulfate ion would be adsorbed on 0.67 n d and 1.33 n d for the two kaolinites studied, assuming an edge area of 4 d g-' (Muljadi et al., 1966a). This represents lower surface coverage than with phosphate. Bower and Hatcher (1967) studied the adsorption of fluoride on clay minerals and found that more fluoride was adsorbed on kaolinite and halloysite than on montmorillonite and vermiculite. In two studies of borate adsorption on clay minerals, Hingston (1964) and Sims and Bingham (1967) found that maximum adsorption occurred at pH values of 9-10. Borate retention was largely attributed to hydroxyiron and hydroxyaluminum inpurities in the clay minerals. Couch and Grim (1968) showed that borate adsorption on illite consisted in a rapid reaction and a slow adsorption process. They suggested that the former was due to B(OH)L adsorption on edge sites and the latter to diffusion of boron into tetrahedral sites. The results of Jasmund and Lindner (1973) support these conclusions. The mechanisms of adsorption of organic anions on clay minerals have been reviewed by Greenland (1965, 1971) and Mortland (1970).
G . CALCITE (CaC03)
Phosphate adsorption on calcite surfaces can be described by a Langmuir equation (Cole et al., 1953; Kuo and Lotse, 1972; Holford and Mattingly, 1975b). The reaction involves three steps: (a) chemisorption of phosphate accompanied by heterogeneous formation of nuclei of amorphous calcium phosphate; (b) a slow transformation of these nuclei into crystalline calcium phosphate; and ( c ) crystal growth of calcium phosphate (Stumm and Leckie, 1971; Griffin and Jurinak, 1973, 1974). The calcium phosphate species which is nucleated depends on the initial phosphate concentration. At low phosphate concentrations Griffin and Jurinak (1974) found that hydroxyapatite was formed, although Amer and Ramy (1971), using solubility measurement and isotopic exchange, suggested that octacalcium phosphate was formed from phosphate concentrations between 1.1 and 1.7pg ml-' . Lahav and Bolt (1963) showed that the calcite surface was modified by dissolved components from soil solution, suggesting that calcite in soil will have different surface properties from pure calcite. This has been discussed in more detail by Mattingly (1975).
R. L. PARFI’IT
24
V. Identification of Adsorption Sites in Soils
A. SURFACE IRON AND ALUMINUM
The reactive sites for anion adsorption in pure systems are the singly coordinated Al-OH and Fe-OH groups, which are exposed at surfaces. These groups are present at the edges of clay minerals as well as on the surfaces of hydrous oxides, and therefore they are present in most soils (Mott, 1970). The nature and distribution of the reactive iron and aluminum components in soils have been described elsewhere (Oades, 1963; Mitchell et al., 1964; Rich, 1968; Coulter, 1969; Greenland, 1971; Wada and Harward, 1974; Jones and Uehara, 1973). The OH sites in two-coordinated Al-(OH).Al do not usually take part in ligand exchange reactions in pure systems, although they can form hydrogen bonds. The Lewis acid sites Al*OHz and Fe.OHz also occur on the edges of minerals. Under certain conditions they can react with Lewis bases such as ammonia to give Fe-NH, and Al-NH,, although under soil conditions water is held more strongly than ammonia. At high pH these sites become negatively charged: AI.OH,
+ O K + A I . O K + H,O
The sites that adsorb protons at low pH are probably the one-coordinated A1 * OH and Fe * OH groups, which give A1 * O&+ and Fe * OH: . On some oxides and oxyhydroxides FeO groups may also be exposed at the surface, and they may become protonated (FeOH+) at low pH. Several workers have used Schofield’s (1949) method of chloride adsorption to measure the positive sites on soils (Deshpandeel al., 1964; Tweneboah et al., 1967; Moshi et al., 1974). Others have measured the charge characteristics of soils by potentiometric titration (van Raij and Peech, 1972; Espinoza et al., 1975; El-Swaify and Sayegh, 1975; Gallez et al., 1976). However, some sites will not be detected by these methods if they are blocked by other adsorbed anions (Deshpande et al., 1968). Fieldes and Perrott (1966) have used fluoride exchange as a test for allophane, and Brydon and Day (1970) showed that this reaction also occurs with other amorphous soil material. The test is probably not specific for identifying ligand exchange sites in soil, since Al-OH.A1 and Si-OH groups react with fluoride in the pH range 6-7 where this reaction is carried out (Perrott et al., 1976a). However, tests with fluoride do give an indication of the amount of poorly ordered material present in soils. If Fee OH and Ale OH sites already hold organic anions, fluoride must compete with these anions for the sites, and therefore tests with fluoride are not suitable for surface soils (Perrott et al., 1976a). Recent results suggest that fluoride exchange at pH 8 may be useful in estimating ligand exchange sites (Perrott et al., 1976b). Many workers have correlated extractable iron and aluminum in soils with
ANION ADSORF’TION BY SOILS
25
anion adsorption data. Tamm’s acid oxalate reagent has been widely used to extract iron and aluminum from soils (Tamm, 1922; Schwertmann, 1964; Schwertmann et al., 1968; Daly and Binnie, 1974; Juo et al., 1974). Landa and Gast (1973) have shown that amorphous Fe(0Hh was extracted by this reagent but goethite was not dissolved. When used on soil, Tamm’s reagent is thought to cause dissolution of amorphous iron and aluminum compounds. The reaction with iron is largely a photochemical reaction that requires ultraviolet light (Schwertmann, 1964), but aluminum ions are brought into solution as oxalate complexes. Exchangeable aluminum, AI*OB+ions, and A1 at edge sites are probably dissolved in addition to amorphous aluminum hydroxides and amorphous aluminosilicates. The exact nature of amorphous or disordered material is not well understood, but such material has been found in measurable amounts in volcanic ash soils, poorly drained soils, tropical soils, and podzols (Mitchell et af., 1964; Wada and Harward, 1974). Buffered dithionite solutions have been used to reduce free iron oxides, which can then be extracted in citrate or acid solutions. These reagents will dissolve crystalline iron oxides in addition to the amorphous material dissolved by Tamm’s acid oxalate. Thus, dithionite extracts give no indication of the surface Fe-OH, since the iron oxides are completely dissolved. Dithionite is not completely specific for extraction of iron from soil, since aluminum and silicon are also brought into solution (Habibullah et af., 1972; Juo et al., 1974). It is doubtful if iron and aluminum can be separated completely, since soil goethites contain some aluminum, which substitutes for ferric ions in the structure (Nomsh and Taylor, 1961). Therefore, both iron and alunimum are brought into solution during dissolution of soil goethite. Surface coatings of iron and aluminum hydroxides can be formed on the external surfaces of clay minerals (e.g., Greenland, 1971; Colombera et al., 1971), and these substances have been shown to adsorb anions (Sims and Bingham, 1968b; Langdon et al., 1973; Perrott et al., 1974a,b; Huang, 1975b). Sree Ramulu et al. (1967) and Huang (1975b) have shown that similar material in 2: I clay interlayers is not active in anion adsorption. Tweneboah et al. (1967) suggested that aluminum coatings could be extracted with 0.5 M CaCI, at pH 1.5; however, a small amount of iron and silicon is also extracted with this reagent. Dithionite citrate bicarbonate solution has also been used to remove coatings from clays, but silicon and aluminum as well as iron are dissolved (e.g., Roth et al., 1969), so the exact nature of the iron compounds is uncertain. B. ORGANOMETALLIC COMPLEXES
Phosphate can react with metal ions such as cobalt to form monodentate or bidentate complexes in the presence of other organic ligands-for example,
26
R. L. PARFTlT
Co(en)P04 and Co(N&kP04 (Lincoln and Stranks, 1968). Phosphato-iron-III and aluminum complexes form in solution only at low pH (Bohn and Peech, 1969), but humic acid containing ferric ions is able to hold P in solution at higher pH (Weir and Soper, 1963). Humic acid and fulvic acid form complexes with aluminum as well as ferric ions (Schnitzer and Skinner, 1964; Schnitzer, 1969). Aleksandrova (1954) reported that part of the aluminum that reacted with humic acid was exchangeable and could adsorb phosphate and other anions (Appelt et al., 1975b). Although iron is strongly complexed by humic acids, Fokin and Sinkha (1970) showed that phosphate can be adsorbed by the complex, part of the phosphate being isotopically exchangeable at pH 4. Phosphate was adsorbed more strongly by an ironfulvic acid complex. Iron and aluminum components of soil organic matter are also important in the adsorption of anionic surfactants (alkylate sulfonates) by soils (Krishna Murti et al., 1966).
C. EXTRACTION OF PHOSPHATE
Chang and Jackson (1957) developed an extraction procedure that they claimed was able to separate the phosphate held by aluminum, iron, and calcium. In their procedure 0.5 M NaF was used to dissolve aluminum from soils, thus releasing phosphate held by the aluminum. However, some phosphate held by iron and calcium is also released in the process, and some of the aluminumbound phosphate is readsorbed on other sites (Bromfield, 1967, 1970). Then 0.1 M NaOH was used to displace phosphate held by ferric ions, but some aluminosilicates will be dissolved by this treatment, and phosphate substituting for silicate will also be released. Calcium phosphates are dissolved in a final treatment with 0.5 M sulfuric acid. Because of the uncertainty of the method, results obtained with the Chang and Jackson procedure should be treated with caution. VI. Adsorption by Soils
A. PHOSPHATE
Phosphate adsorption by soils has been the subject of extensive reviews (Dean, 1949; Wild, 1950b; Hemwall, 1957a; Smith, 1965; Larsen, 1967; Ryden ef al., 1973). Most reviewers have dealt with the precipitation mechanism and the adsorption mechanism of phosphate “fixation. ” Some papers suggest that, in acid soils, phosphate is associated with hydrous oxides of iron and aluminum, and it is unlikely that discrete crystalline iron and aluminum phosphates persist in
ANION ADSORITION BY SOILS
27
soils (e.g., Bache, 1963, 1964). However, in the immediate vicinity of phosphate fertilizer particles, there are local conditions of low pH and high phosphate concentration, which may cause dissolution of clays and reprecipitation of phosphates. Later work suggests that basic aluminum phosphates may form in acid soils even at low phosphate concentrations (White and Taylor, 1977; van Riemsdijk et al., 1975, 1977). In neutral and calcareous soils calcium phosphates are formed, and, if calcite is present, hydroxyapatite and calcium phosphates are adsorbed on the CaCO, surface. 1 , Adsorption Sites
Phosphate adsorption by soils is usually determined by shaking the soil with phosphate solutions in an indifferent electrolyte for a given time. The phosphate remaining in solution is measured, and the phosphate adsorbed can be calculated. The adsorption isotherm is given by a plot of adsorbed phosphate against phosphate remaining in solution. This procedure has been used by many workers to measure the phosphate adsorption capacity of soils (e.g., Fox et al., 1969; Barrow, 1970; Bache and Williams, 1971; Rajan and Fox, 1972; Anderson et al., 1974; Gebhardt and Coleman, 1974~). Many workers, often using a single point on the isotherm, have correlated the phosphate adsorption capacity with other soil factors. Most results show that phosphate adsorption is better correlated with extractable aluminum than with iron (Williams et al., 1958; Saunders, 1965; Fassbender, 1969; Harter, 1969; Syers er al., 1971; John, 1972; Udo and Uzu, 1972; Leal and Velloso, 1973; Lopez-Hernandez and Burnham, 1974a; Evans and Smillie, 1976). However, some results show that either aluminum (Bromfield, 1965; Schwertmann and Knittel, 1973) or iron alone (Myszka and Janowska, 1973) is correlated with phosphate adsorption. Other workers have found a correlation with organic matter (Saunders, 1965; Myszka and Janowska, 1973; Leal and Velloso, 1973; Harter, 1969; Fassbender, 1969; John, 1972; Lopez-Hernandez and Burnham, 1974a), pH or clay content (Udo and Uzu, 1972; Schwertmann and Knittel, 1973), or calcium carbonate (Kacar, 1967). Phosphate adsorption can also be correlated with exchangeable aluminum in soils (Franklin and Reisenauer, 1960; Syers et al., 1971; Udo and Uzu, 1972), although Fitter and Sutton (1975) found this correlation only in soils with pH 5 the correlation was with exchangeable calcium. This agrees with the results on pure systems containing exchangeable aluminum, which show that phosphate does react with aluminum held on cation exchange sites. Wild (1950a, 1953) and Thorup and Mehlich (1961) showed that other exchangeable cations were important in affecting phosphate adsorption. The order of decreasing effect was A1 > Ca > Mg > K > Na = N h .
28
R. L. PARFITT
Many uncertainties are associated with this type of work. The phosphate adsorption capacity will vary with shaking time, the indifferent electrolyte, the initial phosphate concentration, and the solid-to-solution ratio for each soil (Rajan and Fox, 1972; Ryden and Syers, 1975b; Hope and Syers, 1976). However, Ryden and Syers (1975b) suggested that ionic strength and different cations affected only the rate at which equilibrium was attained. The phosphate adsorption will also increase with a decrease in pH if sodium or potassium phosphate is used (Obihara and Russel, 1972; Parfitt, 1977), since a lower pH will give the hydrous oxides more positive charge (Hingston et al., 1972; Bowden et al., 1974). Phosphate adsorption on synthetic hydrous oxides such as goethite, alumina, and gibbsite shows that most of the adsorption is complete within several hours. At concentrations of less than 0.005 pmol cm-3 (0.15 pg ~ m - ~ phosphate ), is adsorbed very strongly by surface OH groups to give binuclear or bidentate complexes. It seems likely that the same reaction will occur in soils wherever there are Al.(OH)&O or pairs of surface FeOH separated by about 0.3 nm (Parfitt, 1977; Ryden et al., 1977b). This reaction probably explains the correlation between rapid phosphate adsorption and extractable aluminum and iron in soils. Complete correlation is not possible, since some of the iron and aluminum that is extracted will also be inaccessible to phosphate ions, or in the forms FeOHFe, FeOHFe, and AlOHAl, which do not react with phosphate at low equilibrium concentrations. If crystalline iron oxides are not present, the best measure of the amounts of surface iron and aluminum are probably the methods based on Tamm’s acid oxalate extractant. For soils with large amounts of crystalline iron oxides, dithionite may be a more suitable extractant, although it will give an overestimate of surface aluminum and iron. Taylor and Schwertmann (1974) have shown that hematite and goethite are sinks for phosphate in fermginous soil concretions, and the ratio of P to Fe depends on weathering conditions and mode of genesis. In one Australian soil the sites for anion adsorption were shown to occur mainly on goethite particles (Fordham and Nomsh, 1974). Several workers have investigated phosphate adsorption on soils after extraction of iron and aluminum. The extraction procedure is important because reagents such as oxalate or citrate will block the remaining adsorption sites (Nagarajah et al., 1970). Interpretation of the results is difficult because removal of iron and aluminum sites can also cause dispersion of clay particles, thus giving an increased surface area for phosphate adsorption (Habibullah et al., 1972). Therefore it is not surprising that phosphate adsorption is increased after extraction with some soils and decreased in others (Coleman, 1944, 1945; Russell and Low, 1954; Bromfield, 1964, 1965; Hudcova and Kovarova, 1969; Fox et al., 1971; Galindo et al., 1971; Syerser al., 1971; Habibullahetal., 1972; Moshi et al., 1974). The significant correlations obtained between soil organic matter and phosphate adsorption suggest that some phosphate is adsorbed by the iron or
ANION ADSORPTION BY SOILS
29
aluminum ions, which are also chelated by large organic molecules in soils such as humic acid and fulvic acid (Fokin and Sinkha, 1970; Williams, 1960). Weir and Soper (1963) found that, although phosphate in this form is held against exchange by an anionic resin, part of the phosphate is isotopically exchangeable and available to plants. However, Fox and Kamprath (1971) showed that phosphate is weakly held on organic soils and can be removed by leaching. Thus, phosphate can be adsorbed at different energy levels by ferric or aluminum ions, which are held by organic matter but which are accessible to phosphate in solution. Soil organic matter can also block sites on iron and aluminum hydrous oxides and reduce phosphate adsorption by soil (Hashimoto and Takayama, 1971; Moshi et al., 1974). Attempts to remove organic matter with hydrogen peroxide results in the formation of oxalate, which blocks more sites and further reduces phosphate adsorption (Hudcova and Kavarova, 1969). 2 . Langmuir Isotherms Many workers have determined phosphate adsorption isotherms on soils and then have attempted to fit the results to a Langmuir or Freundlich equation (Fried and Shapiro, 1956; Olsen and Watanabe, 1957; Woodruff and Kamprath, 1965; du Plessis and Burger, 1966; Rennie and McKercher, 1959; Weir, 1972; Karim et al., 1973; Leal and Velloso, 1973; Schwertmann i d Knittel, 1973; Fitter and Sutton, 1975; Ballaux and Peaslee, 1975). Straight lines are obtained when the results from a limited concentration range are plotted according to the Langmuir equation. In some cases the data can best be fitted with two straight lines, and this has been taken to indicate two different phosphate adsorption sites (Shapiro and Fried, 1959; Syers et al., 1973; Karim et al., 1973; Juo and Maduakor, 1974; Rajan and Fox, 1975). Holford et al. (1974) used a Langmuir two-surface equation and obtained quite good agreement with experimental data. In calcareous soils the high-energy phosphate-adsorbing surface was correlated with free iron oxides and the low-energy surface was correlated with organic matter and the surface area of calcium carbonate (Holford and Mattingly, 1975a). Isotherms for the adsorption of phosphate by soils, over a large concentration range, may be described by a series of Langmuir equations. Ryden et al. (1977a,b) used five Langmuir equations, which then were reduced to three equations by successive approximations, thus dividing the isotherms into three regions. The conclusions were similar to those of Muljadi et al. (1966a), who also invoked three types of adsorption region. The nature of adsorption in Regions I and I1 is largely chemical, whereas more physical adsorption may occur in Region 111. It was suggested that OH,+exchanged with phosphate in Region I, and OH exchanged in Region II. However, the results of Rajan (1976) and Parfitt and Atkinson (1976) suggest that both OH 2' and OH participate in an exchange reaction with phosphate in Regions I and 11, with more OH,+being ex-
30
R. L. PARFITT
changed initially. If pH is not controlled, then more OH will be exchanged as the pH increases. Ryden et al. (1977a,b) have suggested that adsorption in Region I11 is more physical than chemical. However, this conclusion was based on Langmuir isotherms of adsorption, kinetics, and charge relationships in situations where pH was not controlled. Since adsorption at constant ionic strength in Region I11 was not completely reversible, this weak adsorption may be due to ligand exchange on a nearly saturated surface where the surface charge is much reduced. In other words, the mechanism of adsorption in all regions or for the whole isotherm may be essentially the same. Nevertheless, phosphate can be readily desorbed from “Region 111,” and this is obviously important for plant growth (Ryden and Syers, 1977). Results with goethite, which has only one site where phosphate adsorption occurs, show that the Langmuir equation does not apply throughout the whole concentration range because the sites are not isolated and the surface charge changes with surface coverage (Atkinson, 1969; Bowden et al., 1974). The equations developed by Bowden et al. (1973, 1977) give much better agreement with the experimental data, since the charges on the surface and on the anion are taken into account. Therefore conclusions, based on Langmuir equations, suggesting that there are two or three sites of phosphate adsorption must be treated with caution. Since the slope of an isotherm is an index of adsorption energy, it can be used to compare the performance of different soils with respect to phosphate adsorption. Bache and Williams (1971) used the Temkin equation, which takes into account the decrease in adsorption energy with surface coverage. The plot of phosphate adsorbed against log (equilibrium concentration) was linear over a larger concentration range as compared with the Langmuir plot. They also found that the slope of this curve at c = lW4 M was correlated with x , which was determined from a single point on the isotherm where 150 mg P was added per 100 g soil. They suggested that this single-point determination could be used as a simple method of describing the phosphate sorption isotherm. Gunary (1970) showed that inclusion of a square-root term in the Freundlich equation gave a better fit with the data, but there was no theoretical basis for this. Very few workers have measured the surface areas of soils used for phosphate adsorption experiments. Habibullah et al. (1972) and Ryden and Syers (1975b) used nitrogen adsorption to determine surface areas, although the area occupied by each phosphate ion was not calculated. Olsen and Watanabe (1957) used ethylene glycol surface areas and found that each phosphate ion occupied 22 n d for an acid soil and 5.2 n d for a calcareous soil. This is a less dense surface coverage compared with one phosphate per 0.66 n d on goethite, 0.22 nrnZ on hematite, 0.33 n d on lepidocrocite, and 0.45 nm2 on Fe(OHh gel for each phosphate ion ( P d i t t et al., 1975). Some tropical volcanic ash soils adsorb phosphate up to 200 pmol g-’ and
ANION ADSORPTION BY SOILS
31
oxisols can adsorb up to 50 pmol g-' (Rajan and Fox, 1972; Fox, 1974; Gebhardt and Coleman, 1974c; Parftt, 1977), which is similar to the amounts adsorbed by synthetic hydroxides. This suggests that soil allophane and soil iron oxides have large surface areas. Phosphate retention on allophane soils increases with the degree of weathering (Saunders, 1965; Fox, 1974), probably because of the increased aluminum content of weathered allophane (Wada and Harward, 1974). Blanchar and Hossner (1969) studied the adsorption of polyphosphates by some corn belt soils. They found that trimetaphosphate was not adsorbed, but tripolyphosphate and pyrophosphate adsorption was higher than orthophosphate adsorption.
3 . Ligand Exchange Reactions Kolthoff (1936) and Kelley and Midgley (1943) suggested that phosphate could exchange with OH- on an edge Al. OH; this type of reaction is referred to as a ligand exchange reaction. Recent evidence for ligand exchange in soils comes from studies of competitive adsorption of anions, adsorption isotherms, and change in adsorption at different pH values (Obihara and Russell, 1972; Rajan and Fox, 1975; Parfitt, 1977). Ligand exchange reactions in soils involve competition between different anions in the soil for adsorption sites (Rajan and Fox, 1975). The most abundant anions naturally present are probably the organic anions, bicarbonate, nitrate, silicate, sulfate, phosphate, and hydroxyl. Rajan and Fox (1975) found that phosphate displaced sulfate and silicate during ligand exchange, but at high phosphate concentrations structural silicate was displaced. Nagarajah et al. (1968, 1970) studied the competitive adsorption and desorption of organic acids and phosphate on gibbsite, goethite, and kaolinite. They found that phosphate adsorption was reduced considerably by polybasic acids, the effect being more marked with the aluminum surfaces. Polygalacturonic acid, a component of root exudates, was also quite effective in reducing phosphate adsorption. Fulvic acid and humic acid react strongly with hydrous oxides (Greenland, 1971; Parfitt ef al., 1977c), and they can reduce phosphate adsorption on soils (Leaver and Russell, 1957; Manojlovic, 1965; Bhat and Bouyer, 1968; Hashimoto and Takayama, 1971; Weir, 1972; Moshi et al., 1974). Hingston et al. (1971) found that selenite and arsenate partly reduced phosphate adsorption on goethite. These and other results lead to the conclusion that, although phosphate is very strongly adsorbed by most soils, it can be partly displaced by the anions Ass-, SeG- HCQ-, OH-, and some polybasic organic anions (Deb and Datta, 1967; LeFleur and Craddock, 1967; Barrow, 1970; Syers et al., 1971; Nagarajah et al., 1968, 1970; Kinjo and Pratt, 1971b; Gebhardt and Coleman, 1974c; Hingston et al., 1974; Lopez-Hernandez, 1974). When some soils are shaken with distilled water some phosphate is desorbed
32
R. L. PARFITT
into solution (White and Beckett, 1964), but when phosphate is added adsorption occurs. Ryden et al. (1972) found that adsorbed phosphate could be desorbed from an A horizon, but it was probably readsorbed in the B horizon. In soils developed on silica sands, phosphate is often lost by leaching, since the phosphate reacts largely with the soil organic matter (Mattingly, 1965; Fox and Kamprath, 1971; Humphreys and Pritchett, 1971; Ballard and Fiskell, 1974). Silicate has been applied to some tropical soils in an attempt both to make adsorbed phosphate more available and to reduce phosphate adsorption (Roy et al., 1971). Obihara and Russell (1972) and Russell (1973) have dealt with this in some detail. They concluded that silicate, which is adsorbed more strongly at high pH, may increase phosphate availability, particularly near pH 7. However, Kafkafi and Giskin (1970) showed that silica was desorbed by soils when phosphate fertilizer was added, which indicates that phosphate is more strongly adsorbed than silicate. Laboratory experiments have shown that sulfate is also desorbed when phosphate is adsorbed onto soils, suggesting that the same sites are active in adsorbing ligands other than phosphate (Rajan and Fox, 1975). Use is made of desorption reactions in chemical analysis of soils for available phosphate. The soils are shaken for short periods with a small volume of extracting solution. Many of the extractants contain an anion that, if present in the correct concentration, can desorb phosphate from soils. Such extractants include 0.5 M sodium bicarbonate, acid fluoride solutions, acetic acid, and citric acid. Evans and Syers (1971) showed that, although citrate and bicarbonate desorbed phosphate from the face of a soil crumb, subsequently some phosphate was redistributed within the crumb. Beckwith (1965) developed a method based on adsorption isotherms that predicted the amount of phosphate fertilizer a soil would require to give an adequate level of phosphate in solution. The phosphate required to give 0.2 pg cmF3 (ppm) in solution after adsorption was assumed to be the amount of phosphate that was required for good plant growth in some Australian soils. Ozanne and Shaw (1967, 1968) have used 0.3 pg cm-3 for pastures in Western Australia. Fox et al. (1974) has found that the equilibrium concentrations required to give 95% of maximum yield were 0.4 pg cm-3 for lettuce, 0.1 pg cm-3 for sweet potato, and 0.06 pg cm-3 for corn in Hawaiian soils. The amount of phosphate required to give these equilibrium concentrations in solution varied greatly, with weathered volcanic ash soils needing very large amounts of phosphate, and alluvial soils needing small amounts (Fox et al., 1968; Fox and Kamprath, 1970). Jones and Benson ( 1975) found that a value of 0.13 p g was required for sweet corn on a high-phosphorus-fixing soil. Singh and Jones (1977) have shown that phosphate desorption increases with temperature in the range 12"30°C, and lettuce required lower concentrations of phosphate in soil solution at higher temperatures.
ANION ADSORPTION BY SOILS
33
Phosphate adsorption decreases as pH increases if NaCl is used as the indifferent electrolyte (Hingston et al., 1972; Obihara and Russell, 1972; Parfitt, 1977). Adsorption can also be reduced by adding lime to acid soils to raise the pH (Woodruff and Kamprath, 1965; Lopez-Hernandez and Burnham, 1974b). However, Reeve and Sumner (1970) and Lucas and Blue (1972), using limed and unlimed soils from the field, showed that liming did not reduce phosphate retention in some tropical soils. Mokwunye (1975) found similar results using buffer solutions at different pH and suggested that hydroxyaluminum material was activated at pH above 5 and provided sites for phosphate adsorption. Recent results indicate that adsorption on goethite is less dependent on pH if CaCI, is used as the electrolyte instead of NaCl, although bridging phosphate complexes are formed in both systems (R. L. Parfitt, unpublished data). The work of White and Taylor (1977) suggests that systems containing aluminum and calcium behave in a more complicated manner. Several workers have noted that, after phosphate is adsorbed on soils, the cation exchange capacity is increased (Mehlich, 1961; Schalscha et al., 1972, 1974b; Mekaru and Uehara, 1972; Sawhney, 1974; Juo and Maduakor, 1974; Ryden and Syers, 1975a, 1976; El-Swaify and Sayegh, 1975). Parfitt and Atkinson (1976) indicated that this was because of phosphate blocking positively charged sites as well as the adsorbed phosphate itself carrying a negative charge at higher pH values. 4 . Slow Reactions of Phosphate in Soil
Evidence from model systems suggests that ligand exchange reactions should occur rapidly between exposed Al*OH and FeeOH groups and phosphate in solution, and indeed there is an initial rapid uptake of phosphate by soils (Low and Black, 1950; Haseman et al., 1950; Hsu, 1964; Evans and Syers, 1971; Vanderdeelen et al., 1973). However, this rapid adsorption is followed by a period when slow adsorption occurs (Haseman et al., 1950; Low and Black, 1950; Hsu, 1964; du Plessis and Burger, 1966). Russell (1973) and Kuo and Lotse (1974) indicated that the reaction was a diffusion-controlled process. Phosphate that is added to soil is initially exchangeable with 32P-labeledphosphate solutions, indicating that all the phosphate is exposed. With time the amount drops, until about 40% remains exchangeable (Talibudeen, 1958; Mattingly and Talibudeen, 1961; Johnston and Poulton, 1977), which suggests that some of the phosphate is no longer exposed and possibly is held within the precipitates. Larsen et al. (1965) showed that half the phosphate that was applied as fertilizer became nonlabile within one to three years on most British soils. Munns and Fox (1976) found that, after 50-200 days, 30-50% of added phosphate remained labile in tropical soils and thus should be available for plant growth.
34
R. L. PARFITT
This slow reaction may be due to the precipitation of phosphates (Chen et al., 1973b; van Riemsdijk et al., 1975, 1977), or to penetration of the surface by phosphate ions (Holford and Mattingly, 1976), or to diffusion into pores. It is unlikely that variscite (AlP04.2H20)and strengite (FePO4.2H2O) are present in soils (Ryden et al., 1973), but at high phosphate concentrations and at low pH, clays react to form taranakite, (NH4,K)3A15H,(P04)8* 18H20 (Kittrick and Jackson, 1954, 1955). Although these conditions do prevail close to phosphate fertilizer granules, taranakite has been found only in soils treated with saturated fertilizer solutions. Bell and Black (1970) have listed other crystalline phosphates that have been identified in soils under these conditions. However, Norrish (1968) and Adams et al. (1973) have identified the plumbogummite minerals, gorceixite [BaA13(P04),(OH)5H20] and crandallite [CaA13(P04)2(0H)5 H,O], in soils and have suggested that they can be formed as a soil develops. Sawhney (1973) found some silt-size grains in soils that contained phosphate together with Al, Fe, Si, and some Ca. Barrow (1973a, 1974a,b,c) and Barrow and Shaw (1974, 1975a,b) studied the slow reaction between phosphate and soils and found that the rate was temperature-dependent but was not greatly affected by differences between soils or by a range of water contents. This suggested that the reaction through solution was not rate-limiting or, possibly, was not involved at all. The proportion of phosphate that was immobilized by the slow reaction as measured by arsenate displacement, plant uptake, and isotopic exchange was independent of the level of application. Much of the phosphate blocked the initial adsorption sites but in a form not readily exchangeable with labeled phosphate. It was suggested that the slow reaction was due to phosphate changing from a monodentate form to a bridging form. This conclusion is not consistent with the observations that pure iron oxides rapidly form the bridging complex, but the postulated reaction may occur with some aluminum hydroxide surfaces (Kafkafi et al., 1967). Several authors have used Chang and Jackson’s (1957) method in an attempt to follow the reactions of phosphate with time, and results suggest that phosphate that is initially “Al-bound” (NaF-extractable P) migrates to the “Fe-bound” fraction (NaOH-extractable P) (e.g., Hubbard and Walmsley, 1974; Smith, 1965). For calcareous soils Probert and Larsen (1972) suggested that the slow reaction involved a crystallization or recrystallization reaction. B. ARSENATE
The arsenate ion and the phosphate ion are chemically alike; thus, the adsorption behavior of arsenate on hydrous oxides is similar to phosphate adsorption (Hingston et al., 1968a, 1971).
ANION ADSORPTION BY SOILS
35
Fordham and Nomsh (1974) showed that arsenate and phosphate were both retained in a Western Australian soil by goethite particles rather than by iron in a more dispersed form. Several workers using less direct methods have suggested that arsenate adsorption by soils is controlled largely by the hydrous oxides of iron and aluminum (Holobrady et al., 1969; Holobrady and Galba 1970; Jacobs et al., 1970). Galba (1972) showed that arsenate adsorption decreased with pH, and Woolson et al. (1973) found that arsenate could be desorbed from soils by leaching with phosphate, which indicates that arsenate, like phosphate, is adsorbed in soils by a ligand exchange mechanism.
C. MOLYBDATE
Molybdate is adsorbed on goethite and gibbsite by a ligand exchange reaction (Hingston et al., 1972), which suggests that molybdate will react with exposed FeOH and AlOH groups. In soils, adsorption increases from low values at pH to a maximum at pH 4 (Jones, 1957; Reisenauer et al., 1962; Catani et al., 1970), and adsorbed molybdate can be released after treatment with dithionite or oxalate (Jones, 1956, 1957; Reisenauer et al., 1962; Trobisch and Schilling, 1963; Smith and Leeper, 1969; Cheng and Ouellette, 1973), which is consistent with ligand exchange on iron and aluminum surfaces. Similar results have been obtained with volcanic ash soils (Theng, 1971; Gonzalez et al., 1974). Barrow (1970, 1972) determined adsorption isotherms for molybdate on soils and found that molybdate adsorption on different soils paralleled phosphate and sulfate adsorption on the same soils. The adsorption of molybdate by soils is similar to the adsorption of phosphate in many respects. Initially the molybdate is rapidly adsorbed from solution, but with time a slow reaction takes place independent of the water content of the soil. The product of this reaction cannot be displaced with hydroxide solutions (Smith and Leeper, 1969; Barrow, 1973b; Barrow and Shaw, 1974); thus, the slow reaction may be due to reorganization of molybdate on surfaces or to polymerization of molybdate, which normally occurs at low pH. Molybdate can be desorbed from soils by phosphate or sulfate (Stout et al., 1951; Barrow, 1973b). Desorption with phosphate increased with pH. Phosphate was more effective than hydroxide at pH 7, but at pH above 9 hydroxide alone was nearly as efficient as phosphate (Barrow, 1973b). Phosphate also reduced molybdate adsorption from solution, but sulfate had no effect (Gorlach et al., 1969). The effects with phosphate decreased with time, which shows that molybdate is more strongly adsorbed after initial adsorption.
36
R. L. PARFI’IT
D. SELENITE The selenite (Se03*-’ anion is more stable in soils than selenate (Se042-), which is rapidly leached or reduced to selenite. Selenite is strongly adsorbed by ligand exchange on goethite (Hingston et al., 1968b, 1971, 1974), where it probably forms a bridging binuclear complex (Parfitt and Russell, 1977; Parfitt and Smart, 1977). Cary et al. (1967) also found that selenium is immobilized by sesquioxides in acid soils and later showed that selenium concentration in soil solution is governed primarily by a femc oxide-selenite adsorption complex, which forms rapidly when selenite is added to soils (Geering et al., 1968; Cary and Allaway, 1969). However, Levesque (1974a,b) found that selenite was also associated with aluminum and organic matter in Canadian podzols, and Jones and Belling (1967) showed that selenite was retained by calcareous soils. Brown and Carter (1969) found that selenite leaching was increased by additions of sulfate, suggesting that both ions are adsorbed on the same sites. Selenite adsorption by New Zealand soils was related to the degree of weathering and the allophane content of these soils (John et al., 1976).
E. SULFATE
Reviews by Freney ef al. (1962) and Harward and Reisenauer (1966) have included sections on the adsorption of sulfate by soils. Many soils retain sulfate, particularly soils with large amounts of hydrous oxides of iron and aluminum. Thus, weathered tropical soils and volcanic ash soils retain sulfate strongly. Adsorption isotherms for sulfate adsorption on soils have been determined by Chao et al. (1962), Hasan et al. (1970), Barrow (1972), Haque and Walmsley (1973), and others. At low solution concentration, the isotherms have been described by the Langmuir or Freundlich equations (Harward and Reisenauer, 1966; Bornemisza and Llanos, 1967; Hasan et al., 1970). Hingston et al. (1972) showed that sulfate adsorption on goethite and gibbsite decreased with increase in pH up to 8, beyond which no adsorption occurred; similar results are found for soils (Harward and Reisenauer, 1966; Gebhardt and Coleman, 1974b; Scott, 1976). Haque and Walmsley (1974a) and Barrow (1967) have shown that sulfate adsorption is correlated with extractable aluminum rather than extractable iron, although both iron oxide and hydrous aluminum surfaces strongly adsorb sulfate (Aylmore et al., 1967). Scott (1976) suggested that sulfate adsorption at low levels depended on active iron rather than active aluminum in Scottish soils, but aluminum was more important near to the saturation level. Sanders and Tinker
ANION ADSORPTION BY SOILS
37
(1975) suggested that hematite was responsible for sulfate adsorption in an Oxford soil. Volcanic ash soils usually adsorb considerable amounts of sulfate, probably because of the presence of allophane and other hydrous oxides (Ayers and Hagihara, 1953; Fox et al., 1971; Mekaru, 1969; Haque and Walmsley, 1973, 1974b; Gebhardt and Coleman, 1974b). However, Hogg and Toxopeus (1966) and Fox (1974) have shown that younger allophane soils retain little sulfate against leaching, whereas the older allophane soils retain sulfate strongly. This suggests that either there are additional sites in older soils owing to the increased aluminum levels, or the sites are more accessible. Losses of sulfate by leaching have been reported by a number of investigations, which were reviewed by Harward and Reisenauer (1966). Leaching is more significant in soils that are low in hydrous oxides of iron and aluminum, particularly in A horizons. Swoboda and Thomas (1965) showed that sulfate is leached even in red-yellow podzolic soils, which have significant amounts of iron oxides, if large volumes of water are used. Leaching also can occur if positive sites are blocked by organic ligands (for example, in highly organic soils), since the sulfate has a lower binding constant than polycarboxylic acids (Haque and Walmsley, 1974b). Thus, Gillman (1974) found more phosphateextractable sulfate in lower horizons where the pH of zero charge was higher and where there were more positive sites. Harward and Reisenaur (1966) have reviewed the papers dealing with mechanism of adsorption. The most likely mechanism of ligand exchange is the replacement of M OH: or M * OH groups by sulfate ions, and Bornemesza and Llanos (1967) have shown that OH ions are released during sulfate adsorption. P d i t t and Russell (1977) and P d i t t and Smart (1977) showed that sulfate is adsorbed as the binuclear bridging complex Fe*OS(OO)O*Feon goethite, and recent work shows that the same complex is formed on all iron oxides (Parfitt and Smart, 1978). The data of Gallez et al. (1976), which showed that the pH of zero charge for soils shifts to higher pH when sulfate is adsorbed, are consistent with the formation of a binuclear bridging complex. The ligand exchange mechanism accounts for the rapid reaction of sulfate with soils. This may be followed by a slow reaction, which Chang and Thomas (1963) suggested was due to rupture of bridging A1 * OH A1 and A1 * 0 A1 groups followed by adsorption. Sulfate is adsorbed by soils less strongly than phosphate (Hasan et al., 1970; Haque and Walmsley, 1973), and phosphate solutions are used to extract sulfate in soil tests for sulfur (Chaoer al., 1962; Fox et al., 1964; Barrow, 1967; Peverill et al., 1975). It is likely that the same sites are involved in adsorption in soils (Scott, 1976) as has been shown for goethite ( P d itt and Russell, 1977). The order of adsorption for different anions is phosphate > molybdate > sulfate >
-
-
R. L. PARFITT
38
chloride > nitrate (Ayers and Hagihara, 1953; Fieldes and Schofield, 1960; Singh and Kanehiro, 1969; Kinjo and Pratt, 1971b; Barrow, 1972; Gebhardt and Coleman, 1974b).
F. BORIC ACID Boron is probably present in soil solution as boric acid, H3BG, a weak monobasic acid, which acts not as a proton donor but as a Lewis acid, which accepts OH. B(OH),
+ H20= B(OH);
t H+
pK
=
9.0
Boric acid has been shown to react with goethite, possibly by accepting OH from FeOH groups (Parfitt and Russell, 1977). At higher concentrations boric acid fmns polymers, and the acidity increases. 3B(OHh = B&(OH)L
+ H+ + 2 & 0
pK = 6.84
Illite adsorbs more boron than kaolinite or montmorillonite clays (Fleet, 1965; Hingston, 1964), and Couch and Grim (1968) suggested that B(0H); was held at the edges of illite crystals, while some boron slowly diffused into the tetrahedral sites. Maximum adsorption on soils and soil materials is observed at pH 9 close to the pK as predicted by Hingston ef af. (1972) for ligand exchange, which suggests that boron adsorption is similar to a ligand exchange reaction (Hingston, 1964; Sims and Bingham, 1967, 1968a,b; Okazaki and Chao, 1968; Bingham et af., 1971; Metwally ef af., 1974). This is consistent with the observation that hydrous oxides of iron and aluminum adsorb boric acid (Sims and Bingham, 1968a; McPhail et af., 1972; Metwally ef af., 1974; El-Damaty et af., 1974). Other workers have shown that boric acid adsorption is correlated with extractable aluminum (Harada and Tamai, 1968; Hatcher ef af., 1967) and organic matter (Gupta, 1968). Russell (1973) suggested that boric acid is also held by humic colloids in soils, since carboxylic acids can condense with boric acid. This may explain the correlation of adsorption with organic matter content. Volcanic ash soils adsorbed large amounts of boric acid; the adsorption was correlated with allophane but not with free iron oxides or organic matter (Schalscha ef al., 1973). Boron adsorption follows a Langmuir isotherm over a limited concentration range (Hatcher and Bower, 1958; Biggar and Fireman, 1960; Hingston, 1964; Singh, 1964; Okazaki and Chao, 1968; Bingham et al., 1971; El-Damaty et al., 1974); Hatcher and Bower (1958) and Singh (1964) suggested that multisite adsorption was occurring.
ANION ADSORPTION BY SOILS
39
In a study of the desorption of boron from soil, Griffin and Burau (1974) concluded that boron was held on magnesium sites as well as on hydroxyiron and hydroxyaluminum surfaces. Rhoades et al. (1970) showed that boron is adsorbed by hydroxymagnesium [Mg(OHk] clusters; they suggested that these clusters are important in controlling boron adsorption in arid soils. Leaching experiments suggest that some boron is dissolved from soils, but part of the boron remains even after prolonged leaching (Reeve et al., 1955; Rhoades et al., 1970). G . SILICIC ACID
Silicon is present in soil solution as silicic acid, Si(OH),. The solubility of silicic acid in pure systems is constant in the pH range 2-9, but in soils it decreases with increase in pH (Russell, 1973). Silicic acid is known to be adsorbed in certain soils by hydrous iron and aluminum oxides (Beckwith and Reeve, 1963, 1964; McKeague and Cline, 1963a,b; Jones and Handreck, 1965, 1967; Wada and Inoue, 1974). Adsorption increased with>pHup to 9, which equals the pK for silicic acid dissociation: Si(OH), = H3SiOc
+ H+
Thus, the amount of silicic acid in soil solution can be explained by adsorption on iron and aluminum surfaces (Miller, 1967;McKeague and Cline, 1963a;Beckwith and Reeve, 1964). Maximum adsorption in soils occurred at pH 9 (McKeague and Cline, 1963b; Obihara and Russell, 1972), which suggested that ligand exchange reactions were involved. Obihara and Russell (1972) found that silicate adsorption followed the Langmuir equation, while Wada and Inoue (1974), for volcanic ash soils, used a Freundlich equation at higher silicate concentrations. Silicate adsorption is decreased by the presence of phosphate (Ohihara and Russell, 1972; Kafkafi and Giskin, 1970) and by humus (Wada and Inoue, 1974), which suggests that the same sites are active in adsorption. H. FLUORIDE
The fluoride ion reacts with goethite by ligand exchange with the onecoordinated OH group (Hingston et al., 1972, 1974; Parfitt et al., 1976; Parfitt and Russell, 1977). With gibbsite a large number of OH groups are replaced, suggesting that A1 * OH * A1 as well as A1 * OH groups can exchange with fluoride. Bower and Hatcher (1967) showed that fluoride adsorption followed a Langmuir equation and was accompanied by release of OH. Gibbsite, [Al(OH)J, halloysite, and kaolinite adsorbed more fluoride than goethite, montmorillonite, and vermiculite.
40
R. L. P M l T
Fieldes and Perrott (1966) used the release of OH ions by fluoride as a field test for allophane, but Brydon and Day (1970) showed that soils containing aluminum soluble in Tamm’s acid oxalate solution also reacted in the same way. Perrott et al. (1976a) developed a laboratory method whereby OH released by fluoride could be used as an indicator of the amount of disordered material in subsoils.
I. HALIDES
Chloride adsorption has been used to measure the positive charge of soils (Schofield, 1949). Gebhardt and Coleman (1974a) found that andepts adsorbed up to 6 meq Cl- per 100 g at pH 6 and up to 32 meq per 100 g at pH 3.8. Adsorbed Cl- was exchangeable with NQ- and could be desorbed by washing with water. Smith and Davis (1974) found that bromide was adsorbed in only one of the soils studied; the other soils exhibited anion exclusion properties with bromide. Iodide is adsorbed by soils as well as by iron and aluminum hydroxides (Whitehead, 1973, 1974; Seleznev and Tyuryukanov, 1970). Adsorption increased with concentration until a plateau was reached in the isotherm. Maximum adsorption occurred at low pH. The halides can be adsorbed in small amounts on goethite by ligand exchange (Parfitt and Russell, 1977), but in soils they are probably adsorbed by electrostatic attraction to M*OHZ sites, which occur at low pH.
J . NITRATE
Nitrate is weakly adsorbed on goethite and gibbsite by electrostatic attraction (Hingston et al., 1972), but ligand exchange does occur in evacuated systems at low pH (Parfiitt and Russell, 1977). In most cases nitrate is leached quite readily from soils (e.g., Smith and Davis, 1974) and is held more weakly than chloride, sulfate, or phosphate (Kinjo and Pratt, 1971a,b; Singh and Kanehiro, 1969). However, soils that have positive sites are able to prwent rapid leaching of nitrate (van Raij and Camargo, 1974; Wild, 1972; Jones, 1975; B!ack and Waring, 1976). Volcanic ash soils retain nitrate more strongly than other soils presumably because of the presence of many positive sites on allophane (Singh and Kanehiro, 1969; Kinjo et af ., 1971; Schalscha et al., 1974a; Espinoza et af., 1975). Kinjo and Pratt (1971a) found that adsorption followed the Langmuir isotherm and increased with decreasing pH to pH 3.5.
ANION ADSORPTION BY SOILS
41
VII. Summary and Conclusions
The literature covering anion adsorption by soils and soil materials leads to the following conclusions: (1) Surface Al. OH and FeaOH groups are the important sites for the adsorption of anions. The amount of adsorption is controlled by the number of these sites that are exposed at surfaces, and therefore soils that contain large amounts of high-surface-area hydrous iron and aluminum oxides will be extremely efficient in adsorbing anions. (2) Anion adsorption involves an electrostatic interaction as well as some chemical interaction between the surface and the ion. The Langmuir model is incomplete in describing this adsorption, because it takes no account of charge. The adsorption of anions is dependent on pH, with maximum adsorption usually occumng for fully dissociated ions at low pH where the surface becomes positively charged, owing to protonation of surface M.OH groups. Maximum adsorption of anions of incompletely dissociated acids occurs at a pH value close to the pK of the acid. (3) The order of adsorption by a soil is probably phosphate > arsenate > selenite = molybdate > sulfate = fluoride > chloride > nitrate. The more strongly adsorbed ions will react with M * OH and M *OHgroups in a ligand exchange reaction where the anion becomes coordinated to the metal ion. For phosphate and sulfate it is likely that binuclear bridging complexes [Fe-OP(O0)O-Feand Fe*OS(OO)O.Fe] are formed on iron oxide surfaces. (4) Anion adsorption reactions in soil are complicated by competition for adsorption sites from other anions, including carboxylates, and also by the presence of cations such as calcium and aluminum. These reactions require further study under controlled conditions if anion adsorption in soils is to be more fully understood. It has become clear that there are many similarities between the adsorption reactions of different anions, and results obtained with one anion are an indication of what may happen with another anion. It is hoped that the ideas and conclusions that have emerged will lead to a better understanding of the factors controlling anion availability in soils.
ACKNOWLEDGMENTS I am grateful to Drs. R. J . Atkinson, G.J. Churchman, R. J . Furkert, and B . K. Theng and to Mr. A. J . Metson for helpful criticism and advice in the preparation of this manuscript.
42
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ADVANCES IN AGRONOMY, VOL. 30
COLD HARDINESS AND FREEZING INJURY OF AGRONOMIC CROPS Peter L. Steponkus Department of Agronomy, Cornell University, Ithaca, New York
I. Introduction ............ ........... .. . .. . .. . A. Winter B. Cold Hardiness ...................................................... 11. Characterization of the Freezing Process and Freezing Injury A. The Freezing Process.. . . . . . , . . . . . B. Factors Affecting the Freezing Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111. Environmental Factors Affecting Cold Hardiness
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Repercussions of Freezing on the D. Manifestations of Freezing Injury 111. Environmental Factors Affecting Cold C.
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IV. Effect of Developmental Stage on Cold ss . . . . . . . . . . . . . V. Physiological and Biochemical Aspects A. Biochemical Alterations from a D B. Biochemical Alterations from an Environmental Perspective . C. Biochemical Alterations from a Stress Avoidance Perspective D. Biochemical Alterations from a Stress Tolerance Perspective VI. Screening and Stress Procedure VII. A. Summary and Conclusions Acclimation Procedures. . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ..... . . . . . . ., . . . . . . . . B. Freezing Procedures.. . . . . . . . . . . . . . .
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I. Introduction
A. WINTER HARDINESS
Winter hardiness of cereal grains and perennial forage crops is of considerable concern to agronomists in cold northern temperate regions of the world. Reductions in grain yield are incurred not only as a direct result of winter damage to fall-seeded crops but also as a result of limiting the areas where such crops can be 'Department of Agronomy Series Paper No. 1225. 51 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN-0- 12-000730.4
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sown, as the alternative is to use spring-sown cultivars, which are generally less productive (Salmon, 1917a; Hill and Salmon, 1927; Warnes et al., 1971). In addition, winter hardiness is a prime factor limiting perennation of forage crops grown in northern regions. A stand reduction of 25% or more is considered to be sufficient deterioration to warrant replanting (Heinrichs, 1973). As a result, winter hardiness of agronomic crops has been the subject of considerable investigation, and development of varieties with improved winter hardiness for northward expansion is of primary concern (Warnes and Johnson, 1972b). In spite of these efforts, however, the introduction of new cultivars that possess increased winter hardiness has not always been so successful (Grafius, 1974). Although Grafius (1974) concludes that present methods are inadequate for increasing winter hardiness, it might be more appropriate to consider that the primary objective to increase winter hardiness per se is too broad an undertaking. Winter hardiness implies avoidance of or tolerance to all the cumulative effects of winter that a plant encounters-including freezing, heaving, smothering, desiccation, and diseases (Salmon, 1917b). The combined effects of climatic, soil, plant, and cultural factors interact to determine the degree of injury incurred by a crop following the rigors of winter. Soil factors, such as compaction or waterholding capacity, may accentuate or diminish the influence of several climatic factors, including temperature and precipitation. Conversely, climatic conditions, such as the amplitude of diurnal temperature fluctuations or snow cover, may mitigate soil factors. Cultural practices and previous climatic conditions may influence the plant’s resistance, which has considerable diversity within a species, to the stresses imposed on it by the combined climatic and soil factors. Plant resistance may take the form of certain physiological or morphological adaptations that allow the plant either to tolerate or to avoid the imposed stresses. Tolerance and avoidance mechanisms may reside at either the whole plant, tissue, or cellular level. Thus, the undertaking to increase winter hardiness is indeed a rather ambitious objective, and one that is probably insurmountable if approached in its entirety. In addition to the various factors that affect cold hardiness, numerous logistical difficulties are encountered in assessing winter hardiness. The usual approach is to subject the plants to the rigors of winter in a field situation. Such a situation lacks any control over the severity of the stresses imposed on the plants and also lacks any degree of consistency (Hill and Salmon, 1927). In this respect, Levitt (1956) has indicated that winter conditions that result in differential survival occur only once every ten years. Salmon (1933) indicates that between 1917 and 1933, in the location of the experiment, no winter was severe enough to distinguish between relatively hardy and less hardy varieties of wheat. Thus, it is often necessary to grow material for many years and in several locations in order to maximize the probability of achieving the desired degree of injury (Andrews, 1958). The need to delineate the various components of winter hardiness and to devise appropriate stress conditions for each is evident.
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B. COLD HARDINESS Of all the factors relating to the winter hardiness complex, cold hardiness, the ability to withstand low freezing temperatures, is of paramount importance. In most winter-wheat-producing areas, temperature is the single most important factor limiting plant survival (Johnson et al., 1970). In an evaluation of the survival of wheat varieties grown at nearly thirty experiment stations in the Great Plains for several decades, direct freezing injury appeared to be the principal cause of killing (Quisenberry, 1938). Heifer and Kline (1960) indicated that soil temperatures below -6°C at a 2.5-cm depth caused significant winter killing of winter oats. The ability of plants to withstand low temperatures is a latent trait, which exhibits an annual periodicity. It is only through the interaction of appropriate environmental cues and the genetic potential of a species that an increase in cold hardiness is manifested. Therefore, of prime concern are the environmental factors that affect the annual process of cold acclimation and, of equal significance, the annual process of deacclimation. Although the usual approach has been to question what alterations are occurring in the tissue in response to the environmental cues, attention should be directed first to understanding what conditions or stresses the plant is expected to avoid or tolerate during the freezing process. Thus, before an attempt is made to assess what biochemical or physiological alterations are causally related to the acclimation process, knowledge of what constitutes freezing damage is essential. It is well established that the order of cold hardiness is rye > wheat > barley > oats, but it is not known whether these differences are due to differences in the tolerance of freezing stresses or to differences in the freezing stresses that arise in the different species during freezing. Questions of why freezing to - 18°C allows for survival whereas freezing to -20°C results in death are not adequately answered. What changes in the cellular environment are effected by this slight decrease in temperature so that survival is now precluded? The answers to these questions are needed to assess accurately the significance of biochemical changes occumng during cold acclimation in order to determine which are causally related to the ability to withstand freezing stresses. Much of the research directed toward an understanding of the mechanism of cold acclimation has centered on the analysis of various cellular constituents in plants that have been subjected to acclimating conditions. This wealth of information cannot be fully appreciated, however, because of a lack of understanding of what constitutes freezing injury. Unfortunately, the basic premise of such correlative investigations is that an increase (or decrease) in a particular cellular constituent implies a causal relationship to cold hardiness. This rationale has even been extended to assuming that relative differences in cold hardiness between cultivars or even between species should be accompanied by corresponding increments in the particular constituent under investigation. However, this
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would hold true only if the investigator were fortunate enough to choose the one cellular constituent that was the rate-limiting step in the entire cold acclimation process. It is not too surprising that this approach frequently parallels current technology in laboratory methodology and instrumentation. Thus, in order to ultimately improve the winter hardiness of agronomic crops, we must first address the problem of cold hardiness. Fundamental to such an endeavor is the need to characterize the freezing stresses that are imposed on the plant tissue, to understand the repercussions of these stresses on the cellular environment and architecture, and to determine what constitutes freezing injury at the cellular and molecular level. Only when such informatiofi is available can the physiological and biochemical aspects of cold acclimation be fully elucidated. With such an approach, procedures for artificial acclimation, freezing, and assessment of subsequent viability will emerge for use in evaluation of germplasm in breeding programs and for formulation of appropriate cultural and management practices. Moreover, such information would provide direction for breeding programs and cultural practices rather than merely serving as a postmortem evaluation. It is within the above format that this review will be presented. II. Characterization of the Freezing Process and Freezing Injury
In recent years, several comprehensive reviews on the freezing process and resultant injury in plants have been written. These include the extremely comprehensive treatise by Levitt (1972); the reviews by Mazur (1969, 1970), with special emphasis on the physicochemical aspects of the freezing process; the review of Olien (1967a), which discusses freezing stresses that result from macro and micro redistribution patterns of water in relation to survival; the review of Heber and Santarius (1973), which places particular emphasis on the possible mechanisms of protection by various cellular constituents; and the review of Burke ef al. (1976) on freezing injury. For the purpose of this review, consideration of the physical and chemical events that occur during freezing, the ensuing changes in the cellular environment, and the repercussions on cellular structure and function are of prime concern. Current knowledge of the physicochemical aspects of the freezing process is quite extensive (Mazur, 1969, 1970; Olien, 1967a, 1977). In comparison, our knowledge of what constitutes freezing damage at the molecular level is rather deficient. Although numerous theories on the mechanism of freezing injury have been postulated, in 1969 Mazur considered them all to have deficiencies. Unfortunately, this still holds true. Contributing to this situation is the fact that the multiple stresses that arise during freezing are viewed simply as causing death, without any delineation of the numerous ways in which death can be effected. Even at the cellular level this deficiency prevails, and, instead of death, loss of
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semipermeability is the measure of damage. Such deficiencies in understanding what constitutes freezing damage have precluded an accurate assessment of the proposed mechanisms of injury and cold acclimation.
A.
THE FREEZING PROCESS
Freezing in plant tissues involves the redistribution of water with respect to both its physical state and its location. The freezing process and the resultant alterations in the plant can be studied at various levels of organization within the plant. However, regardless of the level of ultimate concern, the freezing process in an individual cell should be of immediate concern, and it is at the cellular level that an understanding of the freezing process must begin. Mazur (1969, 1970) has detailed the physicochemical events occurring during freezing. In summary, when intact plants, tissues, cells, or isolated organelles are subjected to temperatures that are decreasing below the freezing point of water, the water both in the cell and in the extracellular surroundingswill initially supercool. Because the extracellular solution has a lower solute concentration and more effective ice nucleators, initial ice formation will occur extracellularly. Generally at temperatures above - lOT, subsequent nucleation of the intracellular water is prevented by the plasma membrane. As the vapor pressure of the extracellular ice-water mixture is less than that of the intracellular water, a vapor pressure gradient results. Vapor pressure equilibrium can be achieved either by the efflux of water out of the cell to the extracellular ice, which results in cellular dehydration, or by intracellular ice formation. The manner in which equilibrium is achieved depends on the rate at which the cells are cooled in relation to the permeability of the plasma membrane and the surface-area-to-volumeratio. The temperature of the external ice will establish the lower value of the vapor pressure gradient, and hence will be responsible for water to be removed from the cell to achieve equilibrium. However, the actual amount that is removed will depend on the initial osmolality of the intracellular solution. Whether this amount of water will be removed depends on the membrane permeability and the surface area available for efflux. If the flux is not adequate, the cells will equilibrate by intracellular ice formation-which is generally considered to be lethal (Levitt, 1972). However, under the freezing rates normally encountered in nature, the cells will equilibrate by extracellular ice formation. It should be emphasized that, even though ice formation occurs at only a few degrees below O'C, liquid water still remains. In dilute solutions the entire system will eventually be composed of ice, and the solutes will precipitate (Mazur, 1969). This point is referred to as the eutectic point of the solution. Eutectic points are usually not observed in complex systems such as cells, and a small fraction of water will remain unfrozen even at low temperatures (Heber and Santarius, 1973). This point is very important in
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considering the ways in which freezing can affect cellular structure. If thermal equilibration is achieved and is followed by vapor pressure equilibrium, transitions are still occurring within the frozen tissue. Recrystallization-the conversion of small or nonspherical crystals to large spherical crystals-will occur because of the higher surface free energy of the former. Although the above events are associated with the freezing process, injury is usually apparent after thawing. Although this can, in part, be due to the necessity of a thawed condition for manifestation of injury that was incurred during freezing to be visible, there are numerous reports that events during thawing may be injurious (Levitt, 1972). During thawing, the vapor pressure of the external ice-water solution rises, and a vapor pressure gradient will again be established. Equilibration requires the movement of water back into the cell. It is within this setting of freezing and thawing that questions arise as to how these various cellular environmental changes result in injury and how plants in an acclimated state can survive the freezing process.
B. FACTORS AFFECTING THE FREEZING PROCESS
The preceding discussion provides a general description of the freezing process that is widely used to consider the various stresses that a cell will be subjected to during freezing. There is general agreement that intracellular ice formation subjects the plant cells to mechanical stresses of ice, and extracellular ice formation subjects the cells to dehydration, which results in several subsequent stresses. Although there is no question that, under certain conditions, the two distinct types of freezing can occur as described, additional stresses can occur in intact plants composed of several different tissue types. These additional stresses can occur after the initial extracellular ice nucleation event but before equilibration is achieved. In these situations, several factors, both internal and external, can affect the freezing process and alter the stresses that arise in the cellular environment. Olien (1961, 1964, 1965) has indirectly studied the patterns of extracellular water content during freezing by observing the electrophoretic behavior of various charged dyes. Several patterns of fluctuation in extracellular water content during freezing have been observed and have been interpreted as indicative of different types of freezing stress. Following the initial event of extracellular ice formation, two types of freezing are inferred: equilibrium and non-equilibrium freezing. Olien (1977) considers the distinction between the two as being a function of the intensity of the freezing process-with intensity referring to the amount of energy required for ice crystal growth that acts in a specific time interval and region of tissue (Olien, 1973). Low-intensity freezing induces
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equilibrium water transitions, and high-intensity freezing results in nonequilibrium transitions. Nonequilibrium freezing involves a sudden drop in the content of liquid between protoplasts, during which time the amount of water frozen is not a function of temperature. This displacement from equilibrium is large, especially as freezing begins (Olien, 1967a). Depending on conditions in the tissue and the type of tissue, nonequilibrium freezing, which originates in the extracellular region, can result in two forms of stress. The sudden formation of ice can cause nucleation of the intracellular water and result in intracellular freezing as described previously. Alternatively, the nonequilibrium freezing energy can be dissipated by explosive formation of large ice masses along the cell walls, resulting in separation and disruption of the tissues (Olien, 1967a). Although both processes are nonequilibrium freezing events, the former results in intracellular ice formation, whereas the latter results in extracellular ice formation. Equilibrium freezing involves a continuous exponential decrease in liquid between protoplasts as temperature decreases and results in cell contraction due to dehydration. Thus, equilibrium freezing would describe the process of extracellular ice formation as outlined in the previous section. However, dehydration of the cell can occur either by having the extracellular ice and unfrozen water in contact with the cell surface, or it can be achieved through a vapor phase with the ice spatially separated from the cell surface (Olien, 1977). The energies of freezing and frost desiccation have been discussed by Olien (1971a). Adhesion stresses can also result from equilibrium freezing. Several factors determine whether equilibrium or nonequilibrium freezing will occur, and significant differences exist in the patterns of water transitions, ice structures, killing temperature, and injury (Olien, 1967a). These factors include the amount of supercooling, moisture content of the tissue, heat transfer, and tissue type. Although some factors can quantitatively affect the degree of stress and result in the degree of injury being a function of temperature, other factors can qualitatively affect the freezing process and produce a different type of stress, which is not a function of temperature. It is clear that Olien’s work demonstrates the importance of how one views the freezing process. Consideration of whether a given factor is affecting the freezing process is usually not done directly, and alterations in the freezing process are usually inferred from evaluation of the damage or injury incurred. Factors that have been shown to affect the degree of injury include duration and intensity of the cold, rate of freezing and thawing, degree of supercooling, and multiple freezing cycles; these factors relate to the physical parameters of the test environment. Other factors that affect the degree of injury include stage of growth or development, anatomical differences, moisture content, and growth habit; such factors relate to parameters of the test subject. In determining how or why these
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factors affect the degree of injury, concern should be directed to whether they alter the freezing process and resultant stresses, or whether they affect the sensitivity of the plant to the stresses. Such a distinction would be helpful in considering the process of cold acclimation, to be discussed in a subsequent section (Section V). However, for now it may be introduced by the question of whether cold acclimation involves an alteration in the freezing process and resulting stresses, or whether the tolerance or sensitivity of the plant is altered. It is clear from Olien's work that the former is a valid and documented possibility. With reference to factors that affect the freezing process, it is possible that the wellestablished and substantial differences in hardiness that exist in different plant organs (leaves versus roots) may be due to differences in the freezing stresses imposed on the tissues rather than strictly due to differences in "hardiness"-if hardiness is interpreted to mean tolerance (see Levitt, 1972). Therefore, careful consideration of the factors that affect the freezing process is warranted, especially in assessing what factors are causally related to the acclimation process. 1 . Moisture Content
Numerous reports by some of the earliest workers (Chandler, 1913; Schaffnit, 1910; Sinz, 1914) demonstrated that winter injury was greater with high levels of tissue moisture. Salmon (1917b) considered moisture content of plant tissue to be among the most important of internal factors that influence winter hardiness. Subsequently, alterations in moisture content were shown to occur during both cold acclimation (Newton, 1924; Gruentuch, 1935; Andrews et al., 1974a) and deacclimation (Laude, 1937; Gusta and Fowler, 1976). In the above studies, there was a high correlation between the degree of injury incurred and water content; however, it was not established as to whether this was due to an alteration in the freezing process or to some influence on the plants' tolerance. In 1964, Olien reported that decreasing the moisture content of barley leaves from 3.5 to 1.5 g H,O per gram dry weight decreased the lethal temperature from 25" to O"F, and the freezing point decreased from 30" to 25°F. In this and subsequent papers, Olien and co-workers (1965, 1974a, 1975; Olien and Marchetti, 1976) concluded that the lower moisture content was altering the freezing process from an injurious nonequilibrium pattern to a less-injurious equilibrium pattern. The killing temperature of acclimated barley is lowest (- 10" to -20°C) at 65% tissue moisture, increasing to 0°C when the tissue moisture approaches 80% (Olien, 1974b). In wheat, high moisture content resulted in LQ, values between - 12" and - 16°C; under low moisture content the LQ, values were between -15" and -23°C. Although Olien considers moisture content to alter the freezing stress rather dramatically, reduced moisture levels can also result in an increased solute concentration, which would influence the amount of water removed from the intracellular solution.
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2 . Rate of Cooling The rate of freezing and thawing is a primary factor that influences freezing injury. As previously discussed, the rate at which cooling occurs will determine the type of freezing (Mazur, 1969, 1970) or the intensity and type of stresses involved (Olien, 1967a). As early as 1917, Salmon (1917a) suggested that slow freezing decreases injury by preventing ice formation within the cells, giving the tissue an opportunity to dry out and permitting the protoplasm to adjust to the new conditions. From observations of the freezing of various cell types, Mazur (1969) surmises that slow cooling rates promote long exposures to solution effects-that is, exposure to critical concentrations of solute or to critical levels of dehydrationand faster rates increase the probability of intracellular freezing. Such interpretations are based on a wide range of cooling and thawing rates associated with the freeze preservation of cells and tissues. Although it is frequently observed in intact tissues that fast freezing rates increase injury (Sprague, 1955), it has not generally been observed that extremely slow rates are necessarily more injurious than intermediate rates. However, in very few instances has a sufficiently wide range of freezing rates been investigated in intact plants. Furthermore, it is also important to ascertain whether the fast rates of cooling that increase injury are actually experienced under natural conditions. For example, Sprague (1955) demonstrated that cold-acclimated ladino clover stolons and alfalfa roots and crowns were injured less when cooled slowly than when cooled rapidly. However, most significant was the fact that measured midwinter plant temperatures on clear days and nights approached those found to be lethal. In addition to the rate of cooling, the rate of thawing may also influence the degree of injury. Gusta and Fowler (1977) demonstrated that wheat plants thawed slowly (0.5"-2"C/hr) had an LDs0 of -18"C, whereas those thawed rapidly (2"-4"C/min) had an LQ, of - 15°C. Although the incidence of such a rapid thawing rate under natural conditions is extremely unlikely, this observation must be recognized in artificial freezing tests.
3 . Duration of Freezing Once thermal equilibration of the tissue with the ambient temperature occurs, vapor pressure equilibration will follow. Once vapor pressure is achieved during extracellular freezing, cellular dehydration should be maximal and further increases in injury should be minimal. However, numerous reports have demonstrated that the length of time the tissue is frozen can influence the degree of injury that results (Hudson and Brustkern, 1965; Greenham, 1966; Rammelt, 1972). Similarly, Gusta and Fowler (1977) report that at a given freezing temperature the duration between 0 and 1 hour had no effect on injury, but after
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24 hours the injury increased. One might question whether this was a manifestation of a longer period of time in the frozen condition or whether temperature and vapor pressure equilibration was not achieved after 1 hour. Pomeroy et al. (1975) have reported that the influence of duration of freezing on injury depends on whether the freezing temperature is near the lethal temperature. For instance, in wheat, survival was 100% at -6"C, regardless of the length of time frozen (1-120 Fours); at - 12'C, survival decreased from 100% after 12 hours; and at- 16"C,'survival decreased from 80% after 1 hour to 10% after 48 hours. If vapor pressure equilibration occurred, other stresses, such as grain growth, are contributing to injury. 4 . Supercooling
Siminovitch and Scarth (1 938) have shown that supercooling in both tender and hardy cells often results in intracellular freezing. When extensive supercooling occurred before ice inoculation, the degree of intracellular freezing was much more extensive in tender tissues than in hardy tissues. Olien (1964) considers supercooling to be more injurious because it promotes nonequilibrium freezing. Whereas Gusta and Fowler (1977) found that supercooling followed by freezing resulted in death at higher temperatures than when freezing was initiated at just below O'C, Andrews et al. (1974a) contend that, under conditions that induce supercooling, the cold hardiness is increased considerably. If supercooling occurs without freezing, it will be a very effective mechanism for avoiding freezing damage (Levitt, 1972). However, if freezing follows supercooling, the rate of freezing incurred by the tissue is much greater than the cooling rate of the ambient environment. Thus, the detrimental effects associated with supercooling are related to the extremely rapid rates of freezing and the resultant stresses that occur under such conditions. Although Nath and Fisher (1971) have shown that alfalfa will supercool 8'-9'C in artificial freezing tests, few studies report either the amount of supercooling or the resultant freezing rate the tissues are subjected to. Until such information is available, the degree of supercooling that can be tolerated remains to be resolved.
C. REPERCUSSIONS OF FREEZING ON THE CELLULAR ENVIRONMENT
The repercussions of the freezing process on the cellular environment of the cell and its components are numerous. These changes include the obvious decrease in temperature, the presence of ice crystals, and dehydration of the cell. There are several consequences of dehydration, which include a reduction in cell volume and surface area, an increase in concentration of solutes, precipitation of
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some salts resulting in pH changes, and removal of water of hydration of macromolecules. Mazur (1969, 1970) refers to these as solution effects and notes that all these repercussions occur as a monotonic function of temperature. There is general agreement that, under conditions of extracellular ice formation, decreases in temperature or the presence of ice crystals per se are not responsible for injury (Heber and Santarius, 1973), and that the process of cellular dehydration is the most disruptive and injurious repercussion of the freezing process (Mazur, 1970). However, dehydration results in a multitude of effects, and it is within this array that there is a great divergence in hypotheses on the mechanism of freezing damage. All the repercussions, either singularly or in various combinations, have served as the basis for mechanisms of freezing damage (see Mazur, 1969; Heber and Santarius, 1973). As mentioned before (Mazur, 1970), none is entirely satisfactory. It would appear that the major shortcoming of each lies in the attempt to explain all the manifestations of freezing. Since freezing results in a multitude of stresses, it is reasonable to assume that the overall mechanism of freezing injury is a composite of many of the hypotheses put forth and that they should not be considered as mutually exclusive.
I . Direct Effects of Temperature In general, direct effects of low temperature are not considered to be responsible for the damage incurred during freezing (Heber and Santarius, 1973). However, such conclusions can be prejudiced, because damage may result from numerous stresses, and one factor may mask or preclude manifestation by another. Generally, temperature has been deemed a minor component in studies comparing the survival of tissues in a supercooled state with those that have been frozen. However, there have been examples of how temperature can affect protein denaturation (Brandts, 1967). 2 . Ice Crystal Formation Aspects of mechanical damage and the incidence of physical abrasion of the cells have been reviewed extensively by other authors (Levitt, 1972; Heber and Santarius, 1973). Such physical damage is generally acknowledged to result from intracellular ice formation and is presumably the reason why this type of freezing is always lethal. Although the usual conclusion is that extracellular ice formation does not cause injury through physical damage of the ice crystals, Olien (1964) indicates that under certain conditions nonequilibrium freezing can result in the rapid formation of ice masses and large perfect crystals that can split the tissues and destroy cells. The structure of crystals forming between the protoplasts is considered to be very important in affecting injury. Masses of small or imperfect crystals cause little damage, but they may become more perfect and
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destructive as the temperature drops. The type of ice crystal formation may vary considerably in winter cereals, and certain cellular components can influence ice crystal formation (Olien, 1965). As early as 1906, Wiegand reported that ice formation may physically separate plant tissues.
3 . Cellular Dehydration There is general concensus that cellular dehydration is the primary cause of freezing damage. Dehydration results in multiple effects, as delineated earlier. At a given temperature, the extent of dehydration will be a function of the initial osmolality of the cell, since the temperature of the extracellular ice will determine the vapor pressure that must be achieved by removal of water. In cells with a higher osmotic concentration, less water will have to be removed to achieve vapor pressure equilibrium at a given temperature. The concentration of cellular solutes required to achieve vapor pressure has been invoked by many as the primary cause of freezing injury (see Mazur, 1970). The dehydration and resultant concentration of solutes may result in injury in a number of ways: Some indicate that the actual concentration of various toxic compounds is responsible, whereas others indicate that only the osmotic removal of water is important (see Mazur, 1970). Removal of water causes a reduction in cell size, which has been considered to be important, as it could lead to deformation of the plasma membrane. As early as 1940, Scarth et al. indicated that injury to the plasma membrane was related to plasmolysis and deplasmolysis. Wiest and Steponkus (1978) have shown that protoplasts that have been contracted in relatively high osmolalities and subsequently induced to expand by dilution of the osmoticum lyse before they regain their original size. Furthermore, the amount of injury to frozen and thawed protoplasts could be quantitatively accounted for by injury that occurs when the plasma membrane is osmotically induced to contract and expand. Both the extent and kinetics of injury in protoplasts exposed to a freeze-thaw cycle and in those subjected to osmotic manipulation are similar. These facts strongly suggest that injury to protoplasts during a freeze-thaw cycle is due to the same stresses of contraction and expansion that result from osmotic manipulation in the absence of ice. C. R. Olien (personal communication) has indicated that such an agreement is to be expected, because when isolated protoplasts are frozen in an aqueous medium, the major stress is due to desiccation by the osmotic removal of water.
D. MANIFESTATIONS OF FREEZING INJURY
The visual manifestations of freezing injury-a darkened, water-soaked, flaccid appearance-are very apparent immediately following thawing. The gross disruption of cellular architecture is evidenced by extremely leaky cellular mem-
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branes. It is unfortunate that the indications of freezing injury are so vivid, so sudden, and so easily measured by techniques such as the release of electrolytes (Dexter et a l . , 1932), because inquiries into the manifestations of freezing injury at a more fundamental level have been stifled. Although there is a general concensus that freezing injury results in the loss of semipermeability, this is, at best, a very broad generalization, as there are many ways in which the loss of semipermeability can be achieved.
I . Injury at the Tissue Level In considering cold hardiness in relation to plant morphology, it is generally agreed that the crown is the most vulnerable (Kneen and Blish, 1941), as survival of both cereal grains (Pauli, 1960) and forage crops (Jung and Larson, 1972) is dependent on the survival of regenerative and conductive tissues. Within the crown, three distinct regions can be distinguished on the basis of major differences in the tissue: the upper region, containing the apical meristem; the basal region, where large vascular elements of the mesocotyl and roots enter and continue toward the central transitional region; and lateral regions, where much finer vascular elements branch out from the central region to leaf sheaths (Olien, 1974b). Crown survival depends on the extent of injury in these three regions, and the type of freezing process that can occur varies among the regions (Olien, 1964). Generally, equilibrium freezing will occur in the upper region, unless the moisture content is excessively high, and nonequilibrium freezing is usual in the basal region, unless the moisture content is exceptionally low. The different types of freezing processes can occur simultaneously in the different regions. Even though the crown is the most critical for survival, Jung and Larson (1972) state that cold tolerance is usually greatest for crowns, intermediate for roots, and least for leaves. This statement is based on the decreased incidence of freezing temperatures in addition to actual tolerance. With respect to individual tissues of alfalfa, the stelar tissue of roots is considered more tolerant than cortical tissue, as the latter develops little cold tolerance (Jung and Larson, 1972). 2 . Injury at the Cellular Level In recent years, it has been commonly inferred that the primary cause of freezing injury is damage incurred by cellular membranes, especially the plasma membrane. Although freeze-induced membrane damage has received widespread attention since the late 1960’s, it is not an entirely new revelation (Levitt and Dear, 1970). As early as 1912, Maximov concluded that freezing injury was due to damage to the plasma membrane. A resurgence in the view pointing to the plasma membrane as the site of freezing injury was provided by
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Levitt and Scarth (1936a,b) and Siminovitch and Scarth (1938). In the ensuing years, these observations gradually lost their significance, as attention was diverted to soluble cytoplasmic components as the site of freezing injury. Fortunately, in the late 1960’s there was a renewed emphasis on cellular membranes as the site of freezing injury (Heber, 1967, 1968; Siminovitch et al., 1968; Mazur, 1969, 1970; Olien, 1967a; Heber et al., 1973; Levitt and Dear, 1970; Sakai and Yoshida, 1968). However, in spite of this renewed interest, characterization of membrane damage did not progress much further than the acknowledgment that freezing results in the loss of semipermeability. This is, at best, a very broad generalization, but it has served as the basis for evaluating the various effects of freezing and speculating on mechanisms of injury. Thus, all too frequently, the multiple stresses arising during freezing have been interpreted with respect to a single general type of strain-the loss of semipermeability. In the early 1960’s, interest in membranes was stimulated by studies of alterations in chloroplast membranes in relation to freezing injury (Heber and Santarius, 1964). Numerous papers by Heber and co-workers (Heber, 1967, 1968, 1970; Heber and Emst, 1967; Heber and Santarius, 1964, 1967, 1973; Heber et al., 1971, 1973; Santarius, 1971, 1973a,b; Santarius and Heber, 1970, 1972)provided considerable insight into the effects of freezing on the function of chloroplast membranes. Subsequently, Garber and Steponkus (1976a) and Steponkus et al. (1977) extended this work, providing information on the repercussions of freezing on chloroplast thylakoid structure and function at the molecular level. Heber (1967) attempted to localize the site of freezing injury and concluded that uncoupling of photophosphorylation was a result of altered permeability of membranes. A similar conclusion was reached by Uribe and Jagendorf (1968). However, Garber and Steponkus (1976a) demonstrated that the situation was considerably more complex-ven though their attention was restricted to the effects of freezing on photophosphorylation as measured by light-induced proton uptake (see Jagendorf, 1975). Following a slow freeze-thaw cycle, there were three lesions in light-induced proton uptake: loss of plastocyanin, a protein in the electron transport chain; loss of chloroplast coupling factor, the protein responsible for coupling ATP synthesis to electron transport; and loss of osmotic responsiveness. Delineation of the freeze-induced damage to light-induced proton uptake into three separate lesions clearly demonstrated the complexity of freezing on biological systems. If this system can be considered as a simplification of the situation that exists in comparison with intact cells, the dangers of making inferences regarding mechanisms of freezing injury from observations of survival are evident. The question of what constitutes plasma membrane damage at the molecular level remains to be answered, although several recent reports have addressed the question (Palta et al., 1977a,b; Steponkus and Wiest, 1978; Wiest and Stepon-
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kus, 1978). In these reports, questions of what constitutes plasma membrane damage is approached by studying the plasma membrane in siru in either tissue slices or isolated protoplasts. Whereas the work of Palta et al. (1977a,b) is concerned with alterations in plasma membrane permeability following sublethal injury, the work of Wiest and Steponkus (1978) is concerned with the damage resulting in rupture of the plasma membrane. As early as 1940, Scarth et al. indicated that injury to the plasma membrane occurred during deplasmolysis. Wiest and Steponkus (1978) determined that, on thawing, the degree of expansion that can be tolerated before lysis occurs is dependent on the degree of contraction incurred during the prior freeze-induced contraction. It was apparent that some membrane alteration occurred when the protoplasts were contracted, which subsequently limited the protoplast size that could be achieved on dilution. The contraction-inducedalteration in itself did not result in lysis, but affected the resilience of the plasma membrane. Furthermore, there was a critical increment in surface area that could be tolerated, which was independent of the degree of contraction. The fact that injury was correlated with the surface area expansion of the plasma membrane immediately suggests that injury is related to the disruption of intermolecular forces occumng in the plane tangent to the membrane surface. As lysis was correlated with an absolute increase in surface area, this implied that disruption of the membrane requires the same amount of work regardless of the surface area. This fact suggests that the work required for disruption of the plasma membrane is equal to the magnitude of the weakest intermolecular forces joining the membrane together and is constant. The preceding work demonstrates the nature of a specific lesion in the plasma membrane that develops as a result of a specific stress that occurs during extracellular freezing-desiccation by the osmotic removal of water (Olien, 1967a; see Section 11,C,3). Such a lesion could be wholly responsible for injury incurred in tender tissues at relatively high subfreezing temperatures when the majority of water is removed from the cells. However, other lesions would be more likely to appear in tissues that are injured at lower temperatures, where only a small fraction of the initial water content is remaining and can be removed by lowering the temperature. Elucidation of these lesions requires that the stresses be imposed in a singular fashion, which is sometimes difficult. However, such information on the nature of both the stress and the resultant lesion that occurs at any given time during freezing is needed in order to determine the mechanism by which plants acclimate. 111. Environmental Factors Affecting Cold Hardiness
Temperate-zone crop plants exhibit an annual periodicity in their tolerance of freezing temperatures; in the winter they are able to withstand freezing temperatures of -30°C or lower, but in the spring or summer they are susceptible to
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freezing temperatures and are easily killed by temperatures of -3” to -5°C. The increase in cold hardiness during the fall is referred to as cold acclimation, and the loss of hardiness in the spring is termed deacclimation. The cold hardiness of a given species is dependent on two factors: (a) the inherent or genetic capacity of the species to acclimate in order to withstand freezing temperatures, and (b) the conditioning or expression of this heritable capacity. Plants lacking the genetic capacity are considered unhardy orfrost-sensitive species; those that possess the genetic capacity but have not experienced the proper cues for its expression are considered to be in an unhardy Condition; and those that possess the genetic capacity and have received the proper environmentalcues are considered to be in a hardy condition. Heslop-Harrison (1969), in discussing how development and differentiation determine yield of economic plants, introduced the subject with the following eloquent statement: “What is incontestable is that development and differentiation are manifestations of gene function, so that the fundamental problem can at least be defined: it is to understand how gene action is governed in ontogeny so as to give an orderly expression to the potentialities attained during the evolutionary history of a species, producing an organism that is harmoniously coordinated both within itself and with the environment. The process of cold acclimation and the development of cold hardiness of a species is a prime example of the interaction between a plant and its environment as outlined in this statement. The problems of cold hardiness that are now confronting agronomists concerned with world food production are due to the fact that man, out of necessity, is disturbing the “orderly expression” of, “the potentialities attained during the evolutionary history of a species,” so that at times the organism is no longer “harmoniously coordinated. . . with the environment. It is from this perspective that the problems of cold hardiness and the environmental cues that influence cold acclimation should be viewed. When one is considering how plants respond to the environmental cues, it must not be assumed that all plants should respond similarly to the same cues. Thus, what are considered to be “proper,” “optimum,” or “necessary” environmental cues for one species may vary considerably for different cultivars or ecotypes within that species. It is within this context that the great diversity of reports in the literature should be viewed. Furthermore, although the environmental cues serve to synchronize plant development with the environment, the plant’s responsiveness has taken centuries to evolve; and freezing injury in cultivated species can result from any factor that disrupts this synchrony. Some varieties may not be responsive to the surrounding environmental cues or may not respond rapidly enough; some may not develop a sufficient degree of hardiness; and some may deacclimate too rapidly. Each of these factors may arise because individual varieties are being introduced into areas that are vastly or even slightly different from their natural habitat, where centuries of selection pressures have evolved those individuals most closely and appropriately ”
”
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synchronized with the prevailing environment. Although it is the burden of the breeder to work within these confines, it is the responsibility of physiologists to provide understanding and direction for the new horizons in improving cold hardiness of a species. Cold hardiness may be influenced by radiation, temperature, photoperiod, precipitation, and stage of development of the plant, with different optimum conditions for different species and cultivars. Suneson and Peltier (1938) characterized the seasonal progression of cold hardiness of winter wheat in relation to environmental factors during a 6-year period. Initially, high daily temperature maxima in conjunction with high radiation and shortened photoperiods were conducive to increased hardiness. At this time, xeric conditions favored acclimation. Subsequently, exposure to sustained low temperatures resulted in maximum hardiness, but the actual level was dependent on the preceding stage. Finally, a decrease in hardiness was associated with the warmer temperatures of spring. In considering aspects of the environment that are conducive to acclimation, this interplay and progression must be kept in mind in regard to the effects of temperature, light, and moisture.
A. TEMPERATURE
Temperature is the key environmental parameter for synchronizing a plant's capacity to withstand freezing temperatures with the prevailing ambient temperatures (Olien, 1967a; Paulsen, 1968; Svec and Hodges, 1972a; Gusta and Fowler, 1976). Low, above-freezing temperatures are conducive to an increase in hardiness in the fall, and warm temperatures are responsible for the decrease in the spring. Generally, it is considered that most plants will acclimate as temperatures are gradually lowered below 10°C (Alden and Hermann, 1971). For instance, winter cereals are considered to be in a tender state if plants have been growing at a temperature above 1O"C, with optimal temperatures for acclimation near 3°C (Olien, 1967a). Maximum cold acclimation of alfalfa and other perennial legumes develops about the time the soil freezes (Hodgson, 1964). However, during acclimation the progressive decline in temperatures from the relatively high temperatures in early fall, followed by the low, above-freezing temperatures in late fall and early winter, followed by freezing temperatures in winter, is extremely important in the acclimation process. Each stage has a distinctive role in the overall process of acclimation with respect both to succeeding stages and to the influence of other environmental factors. This is an important point to remember in attempting to define precisely the role of temperature in the acclimation process. In cereal grains, noticeable increases in hardiness occur in a few days at 3°C with the maximum occumng in about 3 weeks (Olien, 1964). Longer periods at
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above-freezing temperatures result in a gradual loss of hardiness, even under apparently optimum conditions of nutrition and light (Andrews, 1958; Roberts and Grant, 1968), a factor alluded to by Molisch in 1897. However, if after the hardening period the plants are maintained at temperatures slightly below freezing, hardiness will be retained (Olien, 1967a). Similarly, Andrews er af. (1974b) found that diurnal freezing did not significantly increase maximum hardiness but reduced the rate of loss of hardiness after the maximum had been attained. This fact would probably account for the report of a specific freezing temperature requirement for hardening of winter wheat seedlings (Siminovitch er al., 1967). In this case, seedlings grown in the light at 2°C increased in hardiness after transfer to -3°C in the dark for 2 weeks. However, the comparison was made with seedlings maintained at 2°C for 8 weeks, during which time hardiness may have been declining. On this basis, one would have to question whether there are two separate phases of acclimation-one at above-freezing temperatures and one below, as was claimed (Siminovitch er af., 1967). The importance of the progression of temperatures can be supported by the fact that Pomeroy er af. (1975) found that high levels of hardiness can be rapidly induced in 4-6 days in wheat if the hardening temperatures are preceded by warm temperatures. Previous reports (Pomeroy and Fowler, 1973; Andrews et af., 1974b) showed that 6-8 weeks at 2"-4°C were required for maximum hardiness.
B . LIGHT
Many authors, from the very earliest workers to the most contemporary, have considered light to be a major factor influencing cold acclimation. In 1899, Toporkov (cited by Vasil'yev, 1961) concluded that intense light is one of the major factors promoting the development of resistance to low temperatures, and in 1974, Andrews er al. (1974b) indicated that levels of cold hardiness in winter wheat plants are directly associated with the length and intensity of daily light exposure. Although the involvement of light in the cold acclimation process has endured the test of time, reports in the literature appear, at times, to be conflicting. For instance, Dexter (1933a,b) is often cited to support the concept that winter annuals are incapable of cold acclimation in the absence of light, but Tysdal (1933) found that light intensity was important only when it reached a minimum so that it weakened the plant. Andrews (1958) states that, in germinating seeds, hardiness can be induced in the dark. In this instance, the apparent differences in reports regarding the requisite for light can be attributed to the stage of development the plants are in at the time of acclimation. Thus, in germinating seeds the endosperm is a source of energy, so that acclimation can occur in the dark, but in older seedlings the endosperm is no longer an effective source of reserves, and there is a light dependency. Even Dexter (1933a,b)
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concluded that the role of light was only to act as a source of photosynthate, and in plants such as alfalfa, which have large reserves, cold hardiness can increase in the dark. Rather conclusive evidence that light per se is not a requirement of the acclimation process is supported by the numerous reports that the light requirement can be replaced by incubation on sugar solutions (Tumanov and Trunova, 1963; Steponkus and Lanphear, 1967b). Clarification of the point that light functions to provide a source of photosynthates still leaves an additional area of conflict with respect to whether the photosynthates produced are merely a source of respiratory substrates enabling the acclimation process to proceed or whether in fact a portion of the photosynthates results directly in increased hardiness. This point will be addressed in a subsequent section (Section V). Steponkus and Lanphear (1968) interpret the low light saturation of the acclimation response to be due to the fact that the requirements of the cold acclimation process are only a small proportion of the total mount of the photosynthates produced. Similarly, Hiinsel (1972) indicates that the endosperm might, under certain conditions, exert a decisive influence on the degree of frost resistance and on seedling survival. In young seedlings, light-dependent processes, as well as mobilization of endosperm reserves, are important for optimal hardening, and light can compensate for the grain effect more effectively with respect to hardening than with respect to dry matter production. A second area of apparent conflict in the literature in regard to the role of light in the acclimation process relates to whether light functions in a photoperiodic role in addition to a photosynthetic role. It is commonly accepted that short photoperiods stimulate acclimation, partly because of the annual decline in photoperiod that is coincident with the time of natural acclimation, but also because of reports that some species fail to acclimate under long photoperiods (Hodgson and Bula, 1956). However, Trunova (1965) reports that hardiness of winter wheat increases more rapidly under long light exposures (16-24 hours) than under short light exposure (8-12 hours). Paulsen (1968) also reported that winter wheat hardened to the greatest extent under a long photoperiod and a decreasing temperature, and Rimpau (1958) observed a direct correlation between length of the critical photoperiod and freezing tolerance in wheat. Paulsen (1968) suggested that low light intensities under artificial conditions may be the reason for reports that long photoperiods favor acclimation, whereas under field conditions short photoperiods promote hardiness. Such an explanation is only partially correct; the distinction should be based on whether the species or cultivar in question is photoperiodically responsive in relation to growth cessation or induction of dormancy in those species with a true physiological rest period. Although Andrews (1960b) reported an apparent photoperiodic response of wheat, the results are difficult to interpret, owing to a temperature interaction resulting from the increased light duration. More frequently, the reports indicate that short photoperiods are not required for acclimation of cereal grains
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(Trunova, 1965; Paulsen, 1968; Rimpau, 1958), but short photoperiods are required for acclimation of certain perennials such as alfalfa (Bula ef al., 1956; Hodgson, 1964; Hodgson and Bula, 1956). This difference may be due to the selection for photoperiodic insensitivity in winter cereal grains, which may be of questionable value (Johnson et ul., 1970). According to Young and Feltner (1966), winter annuals have no permanent period of dormancy, and growth processes cease only on exposure to low temperatures. A photoperiodic stimulation of cold acclimation in photoperiodically responsive cultivars underlines the statement made by Heslop-Harrison (1969) in regard to the synchronization of the plant with its environment and the fact that it is dependent on the potentials attained during the evolutionary history of the species. Thus, Hodgson and Bula (1956) found that hardy but southern ecotypes of sweet clover (Melilotus spp.) failed to develop maximum hardiness in Alaska, even though they were exposed to normally acclimating temperatures. Similarly, Bula et al. (1956) found that northern ecotypes of alfalfa developed resistance earlier under a 12-hour photoperiod and at a faster rate than southern ecotypes. Medicagofalcata, a far-northern ecotype, developed cold resistance equally well under long, normal, or shortened photoperiods;M. sativa, cultivars Moapa and Caliverde, failed to develop any appreciable degree of hardiness under any photoperiod, whereas M . sativa cv. Ranger increased in hardiness in response to shortened photoperiods. Thus, in Alaska cold resistance must be developed by mid-October, and hardening must occur under photoperiods of 15 hours (midAugust) to 11 hours (early October). It would appear that both southern ecotypes and M. falcatu are photoperiodically insensitive, but the latter can become acclimated under any photoperiod, whereas in the southern ecotypes appreciable acclimation does not occur. Only in the intermediate temperature varieties such as Ranger was photoperiod instrumental in synchronizing the plant with the environment. Thus, in the case of perennial forage species, photoperiodic stimulation of acclimation influences the hardiness of plants with respect to the timing of the onset of acclimation, and the genetic potential for cold acclimation is achieved only after exposure to the photoperiod to which a cultivar is adapted (Hodgson, 1964). Thus, in these species photoperiod may be as important as temperature in influencing cold acclimation.
C . MOISTURE
Conflicting reports exist for the role of moisture in hardiness. In regard to soil moisture, Salmon (1933) and Tysdal(l933) indicated that soil moisture acts as a buffer against sudden temperature changes. However, Gruentuch (1935) specifically pointed to high soil moisture as a factor reducing the degree of plant hardening. Earlier, Newton (1924) found that a reduction in available water content within
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the plant was the most important quantitative change associated with hardiness. Not only is decreased moisture content associated with increased survival, but Gusta and Fowler (1976) concluded that in dehardening studies there was a high positive correlation between cold survival and water content of crowns. However, the greatest increase in water content in both artificially and naturally acclimated crowns occurred during the first 3 days of dehardening and then became constant or decreased slowly, whereas deacclimation proceeded almost linearly for 15 days. Metcalf et al. (1970) found that a slight change in moisture content of winter cereal crowns had a significant effect on the lowest temperature that plants could survive. Although other environmental parameters affect the hardiness of the plants through their interaction with the hardening process and the development of tolerance to the stresses of freezing, Olien (1964) and Suneson and Peltier (1934a,b) consider that changing moisture content greatly affects the freezing process. Thus, whereas light and temperature effects on acclimation are probably mediated through the development of resistance, moisture content directly affects the stresses that the plant must withstand. IV. Effect of Developmental Stage on Cold Hardiness
Given that several factors affect the freezing process and resultant stresses and that several environmental factors alter the plant’s resistance to these stresses, the plant must be viewed as the integrator of these opposing series of events. The effectiveness of the integration will determine the incidence of injury. The stage of development can influence both the stresses incurred and the resistance acquired. Plant age, or, more properly, the stage of development, appears to have a profound effect on the plant’s capacity for both acclimation and maintenance of hardiness. However, many studies that have attempted to elucidate these aspects have been confounded with other factors. Field studies in which the effect of sowing date is considered in relation either to hardiness at different times during the winter (Andrews ef al., 1960) or to overall winter survival (Roberts and Grant, 1968) confound stage of development with the duration of the acclimation period. Nevertheless, interesting observations have been made from such studies that cannot be explained by differences in the duration of the acclimation period. For instance, Roberts and Grant (1968) observed that relatively old plants, which had been growing 11 weeks or more before growth cessation, experienced more winter injury than did plants that were seeded at a later date. Similar results were obtained when cold hardiness, rather than winter hardiness, was determined (Andrews et a!., 1960). Injury to plants grown for 16 weeks in the fall before exposure to a freezing temperature was greater than injury to younger plants. No
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satisfactory explanation for this decreased hardiness of mature plants has been proposed. Many controlled experiments have been performed by sowing dry or recently imbibed seeds directly at acclimating temperatures and determining hardiness as a function of time (Andrews et al., 1974a). Prior to imbibition, a dry seed is capable of surviving exposure to -196°C. This hardiness decreases during 5 days at 2°C or 24 hours at 24°C (Andrews et al., 1974a). This early minimum in seedling hardiness may be due to an imbibition-induced alteration of the freezing process or to an effect of the initiation of growth processes in the seed, or to both. An influence of the former would be through increased water content. An influence of the latter appears to occur in the case of dormoats. Andrews and Burrows (1974) have observed that field survival of a dormoat crop, treated so as to induce secondary dormancy, survived the winter better than an untreated crop. Although the seeds of the former group had obviously become imbibed during the fall, as evidenced by the ability of the untreated seeds to germinate, the secondary dormancy prevented germination and thus allowed the crop to survive the rigors of winter. Following the imbibitiodgermination-induceddecline in hardiness is a rather steady increase in hardiness of cereals grown at low temperatures for about 3-6 weeks (Andrews et al., 1974b). The exact stage of development at which this maximum occurs, as well as its duration, is dependent on the specific variety (Roberts and Grant, 1968) and can be used to distinguish genetic differences between varieties (Andrews, 1958). Klages (1926a,b) demonstrated that young winter wheat seedlings exhibited the greatest cold acclimation potential before achieving the three- to four-leaf stage. Suneson and Peltier (1934b) and Peltier and Kiesselbach (1934) reported similar results in seedlings of oats, barley, and wheat. However, Roberts and Grant (1968) reported a maximum in winter hardiness of winter wheat at the four- to six-leaf stage of plants grown in the dark. Similarly, maximum hardiness was observed in the four- to six-leaf stage in rye (Andrews, 1960b) and barley (Dantuma and Andrews, 1960). When seedlings are grown in the dark, maximum hardiness is achieved when the coleoptiles reach 45-55 mm in length (Andrews, 1960a). In general, hardiness of various legumes is minimal when they are forming the first pair of permanent leaves (Steinbauer, 1926) and subsequently increases with age up to 60 days (Peltier and Tysdal, 1932). However, although this is true for alfalfa and red clover, Tysdal and Pieters (1934) found that hardiness of lespedeza decreases after the two-leaf stage. Thus, lespedeza cultivars were distinctly hardier than red clover or alfalfa in the unhardened cotyledonary stage, whereas in the hardened cotyledonary stage the reverse was true. In cereals, the second decline in hardiness has been attributed to the exhaustion of endosperm reserves (Suneson and Peltier, 1934a,b; Peltier and Kiesselbach, 1934). Suneson and Peltier (1934b) observed that the youngest seedlings exhib-
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ited the greatest cold acclimation potential, and seedlings in the transition stage between endosperm dependence and tillering exhibited the least potential. Others attribute the loss of hardiness to the completion of the vernalization process (Andrews, 1960a). However, Voblikova (1965) cited several reports indicating that vernalization was completed in 45-60 days, whereas cold hardiness was maintained for several months. However, hardiness determinations were made at 2-month intervals, and thus he was unable to determine precisely the time at which the decline in hardiness began. Vasil'yev (1961) conducted a series of enlightening studies to determine the effect of vernalization on tissue hardiness. Seeds are capable of completing vernalization when held at 0°C for 40-50 days, depending on the variety. Oneweek-old seedlings grown from vernalized seeds were found to be less injured by exposure to - 10" to -12°C than were seedlings of the same age grown from nonvernalized seeds. On the other hand, when the hardiness of plants in the tillering stage was compared, it was observed that plants grown from vernalized seeds were more susceptible to freezing temperatures than were plants grown from nonvernalized seeds. Vasil'yev (1961) concluded that the vernalization process per se does not affect tissue hardiness but, rather, that vernalization affects the growth rate of the plants. The development of plants grown from vernalized seeds appeared to be slower initially than that of nonvernalized plants. However, at about the four-leaf stage, the growth rate of vernalized plants was much higher than that of nonvernalized plants. Thus, the possibility exists that this increase in the growth rate is the primary cause for the decline in hardiness-perhaps by depleting the already diminishing endosperm reserves. Further evidence to support this view comes from observations that plants held continuously at -4°C for 22 weeks after exposure to 2"-5"C for 12 weeks retained a high degree of hardiness, even though vernalization had been completed (Voblikova, 1965). The possibility exists that vernalization predisposes plants to a more rapid exhaustion of endosperm reserves and in this way has an indirect effect on plant hardiness. However, a rigorous attempt to demonstrate conclusively the cause of this second decline in the cold hardiness of winter cereals has yet to be made, and some reports indicate that the decline in hardiness may not be associated with decreases in stored sugars (Tsenov, 1972). V. Physiological and Biochemical Aspects of Cold Acclimation
The majority of research concerned with cold hardiness of higher plants has dealt with the biochemical changes that occur during the period of cold acclimation. It would not be too great an exaggeration to state that almost all cellular constituents have, at one time or another, been analyzed with respect to a possible involvement in the cold acclimation process. In spite of the extensive amount
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of work and the overwhelming number of publications in this area, there is little agreement on the significance of the measured changes-a fact that has been cited by several review authors (Smith, 1968; Mazur, 1970; Alden and Hermann, 1971; Levitt, 1972; Heber and Santarius, 1973). Several factors have contributed to the dilemma that one of the most extensively studied areas of cold hardiness is the least understood. First, in much of the work there is a prevailing attitude that a single compound is responsible for cold hardiness and serves as a basis for the innumerable correlative studies. Associated with this attitude is the concept that, the greater the quantitative increase (or decrease) in a given compound, the more likely it is responsible for cold hardiness. In many instances, the correlation coefficient between hardiness and a cellular component in either a series of different cultivars or different species, or at different times of the year, has served as justification for the inclusion or exclusion of a particular component in the cold acclimation process. One must be very cautious of such reasoning, for only if the particular compound were the product of the rate-limiting step in the entire cold acclimation process would such a correlation exist. Second, there tends to be the assumption that freezing injury is the result of the same stress either in all plants or, more important, within the same plant at different stages of acclimation. Although the work of Olien (1967a) has shown evidence to the contrary, such an attitude is derived from the delineation of the various stresses that occur during freezing (see Levitt, 1972). Too often it is inferred that different types of freezing stresses are separate and distinct events that are mutually exclusive in a given plant. Rather, it would be more appropriate to view the stresses that arise during freezing as a sequential series of events. They may be envisioned as successive stress barriers, and survival depends on the successful and sequential avoidance or tolerance of each individual stress barrier. Hence, cold acclimation can be envisioned to involve a sequential series of alterations that would allow each stress barrier to be overcome, rather than one particular alteration being responsible for the total increase in hardiness. With such a view, it is easy to understand why numerous biochemical and physiological changes may be associated with the cold acclimation process, rather than one single biochemical event. Although little would be gained from yet another cataloguing of these reports, the significance of the various physiological and biochemical alterations can be assessed from various perspectives. Are the changes associated with other alterations in growth and development that coincide with cold acclimation and depend on some of the same environmental cues as cold acclimation? Are the changes merely occurring in response to the prevailing environment without any causal relationship to the cold acclimation process? Are the changes directly instrumental in either circumventing or tolerating the various stresses that occur during
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freezing, or are they only preliminary metabolic changes that precede the alterations that are directly involved? Rather than present a comprehensive listing of all changes, attention will be given to certain biochemical and physiological changes that serve to illustrate these points. Of ultimate concern is the elucidation of the changes that result in either preclusion, circumvention, or tolerance of the stress barriers that can arise during freezing.
A. BIOCHEMICAL ALTERATIONS FROM A DEVELOPMENTAL PERSPECTIVE
As was previously mentioned, cold acclimation is occumng during a period of very dynamic and dramatic fluctuations in the ambient environment. In addition, the cold acclimation process is only one of several developmental events that are occurring during this period. Cold acclimation is preceded by the slowdown or cessation of growth, the onset of dormancy, and the development of tillers or rhizomes, to mention only a few developmental processes. In addition, in cereal grains, the process of vernalization is occurring during the winter period. In such a developmental setting, is there any question that alterations in plant hormones, nucleic acid components, or protein complement might be detected? Thus, reports of alterations in RNA and DNA (Jung ef al., 1967; Shih ef al., 1967) are not unexpected. In fact, it would be very surprising if such changes did not take place. Similarly, the appearance of altered electrophoretic patterns of watersoluble proteins from acclimated alfalfa roots (Coleman er al., 1966; Gerloff et al., 1967) or from rhizomes of Bermuda grass (Davis and Gilbert, 1970) or from leaves of perennial ryegrass (Draper and Watson, 1971) is not unexpected. Although vernalization may influence the cold acclimation potential (Vasil'yev, 1961; Voblikova, 1965; Vincent, 1972), very few studies have considered the biochemical similarities or dissimilarities between the two processes. One exception is in the area of lipid alterations. Redshaw and Zalik (1968) demonstrated that, although noticeable differences in lipids were found after exposure to low temperatures, both spring and winter varieties exhibited similar trends in polar and neutral lipids and the constituent fatty acids. Subsequently, Thomson and Zalik (1973) concluded that changes in lipids that occur on exposure to low temperatures are not unique to vernalization. However, de Silva et al. (1975) studied lipid alterations in two near-isogenic lines of wheat (spring and winter) and observed that higher levels of phospholipids were found in the winter genotype than in the spring genotype. Also, in wheat grown at 2"C, the lipids of the winter type exhibited larger increases in linoleic and linolenic acids than did the lipids of the spring genotype grown at the same temperature. It was further suggested that these increases may be associated with the vernalization gene.
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Although the data are not conclusive, they serve to point out that some biochemical changes associated with the vernalization process may occur during the period of cold acclimation. B. BIOCHEMICAL ALTERATIONS FROM AN ENVIRONMENTAL PERSPECTIVE
Of prime concern is whether alterations in biochemical constituents are associated with the cold acclimation process, or whether they are merely manifestations of metabolic adjustments in response to low temperatures. Such metabolic adjustments in response to low temperatures is a common response in many organisms that do not undergo cold acclimation. An important and relevant example of such a low-temperature metabolic adjustment relates to qualitative changes in lipids and constituent fatty acids. Effects of temperature on the fatty acid composition of higher plants (Harris and James, 1969a,b; Hitchcock and Nichols, 1971). animals (Hilditch and Williams, 1964), and several microorganisms (Weete, 1974) have been well documented. In this diverse range of organisms, low temperature will generally stimulate the accumulation of unsaturated fatty acids-ven though the ability to survive freezing is not a universal phenomenon. However, since such a change coincides with the period of cold acclimation, it has frequently been inferred to be associated with the cold acclimation process. A positive correlation between lipid content and hardiness was reported by Sinnott in 1918; his rather comprehensive survey included more than 300 species of 100 genera over a 3-year period! Although many such early observations employed histological techniques that may have resulted in erroneous interpretations (Alden and Hermann, 1971), numerous studies using other techniques have since established this correlation (Levitt, 1972). Although the association of lipids and constitutive fatty acids with cold hardiness has had a long history, a renewed interest occurred in the early 1960’s and continues to this date. Such interest was stimulated by the initial reports of Lyons el al. (1964) that the degree of unsaturation of fatty acids of mitochondrial lipids was associated with chilling injury. One of the first investigations on agronomic crops subsequent to the report of Lyons er al. (1964) was with alfalfa (Gerloff et al., 1966). The fatty acid composition of root tissues of a nonhardy cultivar (Caliverde) and a hardy cultivar (Vernal) increased approximately twofold during the fall, and the increase was the result of a preferential increase in polyunsaturated fatty acids. Although the increases were similar in both cultivars, it was concluded that the large increase may play a role in acclimation. Using the same two cultivars, Kuiper (1970) demonstrated that at lower growth temperatures (15°C) changes in the
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lipid composition also occurred, as manifested by increases in the mono- and digalactosyl diglyceride, phosphatidyl choline, and phosphatidyl ethanolamine fractions. Furthermore, the hardier cultivar, Vernal, exhibited a higher percentage of these components than did the less hardy cultivar, Caliverde. Although the lowest temperature used (15°C) was considered to be conducive to hardening, no hardiness determinations were made, and it is extremely doubtful that cold acclimation was occumng. However, Grenier et af. (1972) demonstrated that total lipids in alfalfa roots increase during acclimation. Subsequently, it was shown that fatty acids increased in unsaturation, particularly in linoleic acid (18:2) (Grenier and Willemot, 1974). It was suggested that linoleic acid synthesis and hardening were related and that the mechanism suggested by Lyons et al. (1 964) may explain, in part, the varietal differences in hardiness of alfalfa. At this time, reports indicated that significant increases in the phospholipid content and a marked increase in linolenic acid (18:3) content occurred in wheat seedlings grown at 2"C, as compared with seedlings grown at 24°C (de la Roche et al., 1972). It should be noted that, in order to have material at a comparable stage of morphological development, the 2°C seedlings were 5 weeks old, whereas the 24°C seedlings were 72 hours old. In any case, the observations of increased lipid content and unsaturation were similar to those for alfalfa, although the increase in unsaturation was due to increased linolenic rather than linoleic acid. de la Roche et al. concluded that increased synthesis and unsaturation of fatty acids may contribute to increased freezing resistance. Membranes containing lipids with higher proportions of unsaturated fatty acids were viewed as being more fluid and less likely to be irreversibly damaged by freezing temperatures. They also considered that higher unsaturation increases membrane permeability as reported by Lyons and Asmundson (1965). In a subsequent paper, the increase in unsaturation was considered to be the result of altered fatty acid desaturase activity rather than a preferential synthesis of individual phospholipids (de la Roche et al., 1973). In 1974, Miller et al. considered the lipid composition of mitochondria of several hardy and unhardy cultivars of wheat with respect to total lipid content, fatty acid composition, respiratory activity, and electron spin resonance behavior. Although linolenic acid content increased after growth at 2"C, the increase in total unsaturation in all four cultivars, both hardy and unhardy, was quite similar. In addition, three temperature-dependent structural transitions, identified by electron spin resonance, occurred at lower temperatures in the seedlings grown at 2°C. Because of the lack of any significant differences in both of these parameters in either hardy or unhardy cultivars, it was concluded that the ability to withstand freezing at low temperatures could not be accounted for by lipid or functional changes in the mitochondria1 membrane. Although this might be interpreted to mean that lipid changes in membranes other than mitochondria may be related to cold acclimation, a subsequent paper (de la Roche el al., 1975)
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concludes that the increase in unsaturation is only a low-temperature response. In this study, changes in the lipid and fatty acid constituents were measured in the four different cultivars of varying hardiness in an attempt to dissociate changes due to low temperature from those related to cold acclimation. In all cultivars there was a uniform stimulation of linolenic acid synthesis, which resulted in a net increase in the unsaturation of membrane lipids. Since the acclimated plants of the different cultivars ranged in hardiness from -5°C to -18”C, it was concluded that the increase in linolenic acid per se was not a primary factor in cold acclimation between -5°C and -18°C. At this time, Willemot (1975) directed his efforts to wheat and concluded that increased phospholipid synthesis was not a prerequisite to hardening in winter wheat; however, in a subsequent paper (Willemot et al., 1977) he still maintained that varietal differences in frost hardiness may be related to changes in fatty acid content in alfalfa but not in wheat. This stance was based on additional work by Grenier et al. (1975), who studied the incorporation of I4C-acetateinto lipids of alfalfa roots. It is interesting to note, though, that Willemot (1977) later concluded that increased unsaturation of fatty acids is probably an important part of the mechanism of cold adaptation-even in winter wheat. Treatment of winter wheat plants with a derivative of pyridazinone (BASF 13-338) 36 hours before cold acclimation completely inhibited both the accumulation of linolenic acid in the roots and any increase in cold hardiness. The authors were satisfied that the failure to acclimate was not a secondary effect of phytotoxicity. The above reports are presented to illustrate the oscillations that exist in the interpretation of biochemical changes occurring during cold acclimation. Actually, “fluidity in interpretation’’would be a more aproposphrase! Usually such oscillations are the result of differingopinions of different investigatorsworking with different species. However, in this case both groups were working with the same species. Furthermore, while one laboratory (de la Roche) has apparently made a 180-degreechange from their initial stance that changes in unsaturation were related to cold acclimation, the other (Willemot) has also made an apparent 180-degree change in their stance-but from an initial point of concurrence with the conclusion that changes in unsaturation were not related to cold acclimation of wheat. Although this appears to be a somewhat bewildering dilemma, there are some subtleties in the terminology in the recent papers of the two groups that bear further discussion. For example: de la Roche et al. (1975) stated that “the increase in linolenic acid per se is not a primary factor in cold hardening, at least at temperaturesfrom -5” to -18°C . . .” Singh et al. (1977) stated: “. . . both unsaturation and bulk fluidity of the isolated lipids from these plants bear little relationship to their degree of freezing tolerance. ” Willemot et al. (1977) stated: “The lack of differences between fatty acid profiles of the two cultivars rules out the explanation of varietal diferences on the basis of major changes in fatty acid unsaturation.” And Willemot (1977) stated: “It is possible that
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linolenic acid accumulation is a prerequisite to hardening, but that all cultivars have acquired this characteristic and that less hardy cultivars have their frost resistance limited by otherfactors. ” Thus, if one delves deeply (emphasis added in the above statements) into the various reports, there appears to be some inkling of agreement. Although the strongest evidence that the increase in unsaturation is a lowtemperature response comes from the observations of de la Roche er al. (1975) where cultivars of contrasting hardiness were used, some caution must be exercised before summarily concluding that such a comparison could serve to distinguish biochemical alterations that are low-temperature responses from those that are causally related to cold acclimation. Although all the cultivars had similar killing points (-2°C) in the nonacclimated condition, they varied in their killing points (Marquis, -5°C; Cappelle-Desprez, -6°C; Rideau, - 13°C; Kharkov, - 18°C) after 5 weeks of acclimation. Thus, it was not as though acclimation was totally precluded in the less hardy varieties; only the final extent of hardiness varied. In other words, they all increased in hardiness from -2°C to at least -5°C. If freezing injury were viewed only as resulting from one stress, this might not have any significance; however, there is compelling evidence (Olien, 1967a) to indicate that freezing injury is the result of several stresses. Such evidence allows for the possibility that stresses are encountered in a sequential manner. Hence, it is entirely possible that the increase in unsaturation was associated with the increase in hardiness beyond -2°C but not beyond -5°C. Although de la Roche et al. (1975) alluded to such a possibility with the statement that “the increase in linolenic acid per se is not a primary factor in cold hardening, at least at temperatures from -5” to - 18°C. . . ,” they concluded that “stimulation of linolenic acid biosynthesis is merely a general response to the low temperature growth condition. ” And in a later paper (Singh et al., 1977), it is concluded that “both unsaturation and bulk fluidity. . . bear little relationship to their degree of freezing tolerance. It is important to note the expression “degree of freezing tolerance,” which may be interpreted to imply that the changes may be related to the initial stages of acclimation; however, this was not stated. Similarly, Willemot et al. (1977) demonstrated that both the hardy cultivar, Kharkov, and the unhardy cultivar, Champlein, increased in hardiness to the same extent (from -5°C to -12°C) after 1 week of acclimation. Furthermore, it was during this time that the largest increase in linolenic acid occurred in the roots-whether expressed as an absolute amount or as a percentage of the total fatty acids. Thus, the possibility remains that increases in unsaturation may be related to overcoming one of the initial stresses that is incurred in the extremely tender stages. If such were the case, then increases in fatty acid unsaturation might be related to the cold acclimation process but not be responsible for the extent of hardiness in hardier varieties and hence not responsible for varietal differences. If ”
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this were so, then changes in unsaturation of fatty acids might be one of the initial events occurring during cold acclimation, and any such changes should be detected after relatively short periods of time. Lending support to this possibility, Farkas et al. (1975) indicated that changes in the fatty acid composition of wheat and rye leaves were readily apparent after 2 days of exposure to cold. Furthermore, in the data of Willemot (1977) significant increases in the incorporation of 32Pinto lipids (expressed as percentage of radioactivity absorbed) occurred within 14 hours. Thus, the efforts of de la Roche and Willemot and their co-workers may have provided some valuable evidence to support the concept that cold acclimation involves a sequential series of events necessary to ensure survival after exposure to a series of freezing stresses. However, one problem remains. Specifically, in no instance, to the author’s knowledge, has it been experimentally demonstrated how an increase in fatty acid unsaturation would contribute to either the mitigation or the avoidance of any of the stresses currently known to occur during freezing. This includes any demonstrations that increases in unsaturation of fatty acids of plant membrane lipids could result in increased membrane water permeability in order to achieve the avoidance of intracellular ice formation. (This point will be addressed in the following section.) Furthermore, this is the same problem that existed when increases in fatty acid unsaturation observed in chilling resistant species (Lyons et al., 1964) were generally assumed to be beneficial in frost-hardy species. However, this is only an opinion formed from the lack of a complete understanding of the stresses arising during freezing and should not be construed to mean that increases in unsaturation cannot be beneficial-nly that sufficient information on the subject is lacking.
C. BIOCHEMICAL ALTERATIONS FROM A STRESS AVOIDANCE PERSPECTIVE
There are numerous factors that can influence the degree of injury incurred during freezing and that can be considered from the viewpoint of whether the stresses of the freezing process are avoided, mitigated, or tolerated. Levitt (1972) has delineated resistance into evasion, avoidance, and tolerance mechanisms, which is helpful in providing some uniform terminology in an area fraught with a multitude of interchangeable terms. Furthermore, Levitt has indicated that the problem of cold resistance should be analyzed and viewed in the context of stresses and resultant strains. Although Levitt has further distinguished strains as being either elastic or plastic, a strict and parochial usage of the terms evasion, avoidance, and tolerance or stresses and strains will be possible only in situations clearly defined with respect to time and location. For example, consider low-temperature tolerance: Tolerance of low tempera-
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tures can be achieved either by avoiding or by tolerating ice formation in the plant; if ice formation is tolerated, it can be either by avoidance or by tolerance of various mechanical stresses, depending on the location of ice formation in tissue; if intracellular ice formation is avoided and extracellular ice formation is tolerated, the latter can be through either the avoidance or the tolerance of the associated dehydration of the cell; if dehydration of the cell can be tolerated, then either the avoidance or the tolerance of the concentration of toxic solutes or other solution effects can be important. Thus, only each event and its subsequent repercussions can be considered as an avoidance or tolerance situation, and not the overall sequence of events. A similar picture can be considered for stresses and strains in that a particular stress can result in a strain that may, in turn, be considered as a stress. For example, extracellular ice formation is a stress that results in dehydration. It can be debated whether to consider the dehydration as a strain of the system (the cell) or as a resultant stress impinging on the system. Clearly, the semantics associated with cold resistance can be interpreted in several ways, and strict interpretations can diminish their usefulness as conceptual guides. As there is general agreement that membrane damage is a universal manifestation of freezing injury, it could be suggested that avoidance or mitigation of stresses would generally be a result of changes in the membrane environment, whereas tolerance mechanisms would be a result of changes in the membrane per se. There is little doubt that cold resistance is a composite of both possibilities. From a stress avoidance perspective, biochemical changes occurring during the period of cold acclimation should be first viewed in the context of the known physicochemical events and ensuing changes in the cellular environment (see Mazur, 1970) in order to determine whether these changes can result in an alteration of the various freezing stresses (Olien, 1967a). Such a sequence would include the physicochemical events that occur after the moment of extracellular ice formation. These events, which are amenable to avoidance or mitigation, include avoidance of intracellular ice formation, mitigation of the amount or type of extracellular ice formation, mitigation of cellular dehydration, and mitigation of toxic solute concentration and other solution effects.
I . Avoidance of Intracellular Ice Formation One of the initial potential stresses that can be encountered during freezing is the formation of intracellular ice. Siminovitch and Scarth (1938) observed that lethal intracellular ice formation occurred at a slower freezing rate in nonacclimated tissues than in acclimated tissues. Salcheva and Samygin (1963) reported that intracellular ice can form in wheat cooled at the relatively slow rates of 20"C/hr. Olien (1 967a) considers the ease with which ice can be induced to form within the protoplasts as an index of the transition from the tender (unhardy) to
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the hardy state. Several factors could be instrumental in preventing intracellular ice formation. Increases in cellular solute concentration, one of the most universal manifestations of cold acclimation (see Alden and Hermann, 1971; Levitt, 1972), would serve to depress the freezing point of the intracellular solution so that freezing would occur initially in the extracellular solution. Such an increase in solute concentration could be achieved either by an increase in solutes or by a decrease in the amount of solvent. Johansson (1970) and Johansson and Krull(l970) have suggested that cold acclimation of winter wheat may be due, in part, to an increase in solute concentrations. Thus, increases in sugars, amino acids, organic acids, and other osmotically active compounds could serve to achieve the necessary freezing-point depression. However, if viewed strictly from a colligative basis, such increases could only lower the freezing point to the extent of 1.86"Cl osmolal. To prevent intracellular ice formation at lower temperatures, there must be sufficient water efflux from the cell. As early as 1936, it was postulated that cold acclimation could result in an alteration in membrane water permeability in order to permit the rapid removal of water to extracellular sites of ice nucleation (Scarth, 1936; Levitt and Scarth, 1936b). This provided an explanation for the observation that intracellular ice formation occurred at slower freezing rates in nonacclimated tissues than in acclimated tissues. This concept has endured and has been cited by numerous individuals throughout the subsequent years (Levitt, 1972). However, later evidence by Stout et al. (1977) indicated that there may not be a direct cause-and-effect relationship between the two observations. calculations of the rate of ice formation during freezing at a relatively high rate, viewed in relation to membrane water permeability, as measured by a nuclear magnetic resonance technique, indicated that the amount of water efflux required for extracellular ice formation would not be limited by the water permeability of the plasma membrane in nonacclimated tissue. Hence, the decreased incidence of intracellularice formation in tissue may not be a result of increases in membrane water permeability. Sukumaran and Weiser ( 1972) also reported that no differences in water permeability were found between hardy and unhardy cultivars of potatoes. The data of Stout et al. (1977) indicate that the resistance to water efflux may be controlled by heat transfer mechanisms. If so, the magnitude of this resistance would decrease with an increasing freezing rate. Other factors that affect the rate of ice formation could contribute to the resistance. Olien (1967b) has described cell wall polymers that act as competitive inhibitors of ice formation; such inhibitors could explain the observation that acclimated cells survive faster cooling rates than do nonacclimated cells (Siminovitch and Scarth, 1938). The rate of ice formation can also be influenced by whether supercooling can occur (Burke ef al., 1976). The larger the amount of supercooling, the greater
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will be the freezing rate when ice nucleation does occur. However, for supercooling to be a factor in decreasing the freezing rate, less supercooling would have to be associated with acclimation, and this is the opposite of what is usually observed (Burke et a f . , 1976). Another possibility is that, since the plasma membrane serves as a significant barrier to nucleation of the cytoplasm (Levitt, 1962; Olien, 1971b; Mazur, 1970), an avoidance of intracellular ice formation may result from an alteration in the plasma membrane, which increases its effectiveness as a barrier to ice nucleation of the cytoplasm. 2 . Mitigation of Extracellular Ice Formation Considerable evidence has accumulated to indicate that an increase in cold resistance can be achieved by altering the stresses that occur during freezing. Whereas the previous section addressed alterations in the location of ice formation (intracellular versus extracellular), there is evidence to suggest that alterations in the patterns and types of extracellular ice formation can also occur. Thus, although Olien (1967a) considers the ease with which ice can be induced to form within the protoplasts (intracellular ice nucleation) as associated with the transition from an unhardy to a hardy state, resistance also involves modification of some stresses by production of substances that alter the water redistribution pattern (extracellular propagation). Both cases involve nonequilibrium freezing stresses. With respect to extracellular propagation, Olien (1964) has indicated that the structure of crystals forming between the protoplasts is the most important factor affecting initial injury, and the degree of hardiness depends on factors that modify this stress (Olien, 1965, 1968). In subsequent papers, however, it was acknowledged that inhibitors of freezing are not the sole factor in determining a cultivar’s degree of winter hardiness (Shearman er al., 1973) and that the relative degree of hardiness involves traits that affect both the stresses that develop as well as the resistance of the tissues to other stresses, which permits them to tolerate greater distortion (Olien, 1977). Olien (1964) indicates that masses of small or imperfect crystals cause little damage, but damage is increased if the masses are more perfect and solid. The presence of various cell wall mucilages can affect the structure of ice masses in the vicinity of cell walls (Olien, 1965). Also, smaller and more imperfect crystals were observed in hardier tissues (Olien, 1967b), and it was concluded that polymers (arabo-xylans) from winter cereals interfered with freezing by competing with water for positions in the ice lattice. Although the polymers did not prevent freezing, they greatly altered the structure of the ice. In artificial systems, extracts from barley were shown to interact weakly with the ice lattice, whereas polymers from rye interact strongly (Olien, 1967b). In subsequent work, the relationship between the average inhibitor rating of the polymers and the
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plant survival rating of various cultivars was not very good (Olien, 1970). Although activity of the kinetic inhibitors was commensurate with survival ability in some cultivars, other cultivars exhibited better kinetic inhibitor activity than was indicated by their survival rating (Shearman et al., 1973). However, such a demonstration would be expected only if the inhibitors were the sole factor involved in preventing freezing injury in the winter cereals studied, and this should not be assumed. In addition to modifying the type of ice crystals formed, the polymers may also influence the location of ice formation and prevent ice crystals from growing into critical regions (Olien, 1974a). The polymers are considered to give a plant control over both the location and the macro-strbcture of ice formation. It is envisioned that the polymers coat the ice crystal and tend to impede its growth. One point of concern relates to whether the polymers are specifically associated with the cold acclimation process or whether they are constitutive. Shearman ef al. (1973) demonstrated that the polymers were isolated from seeds rather than from plants exposed to acclimating conditions. The monosaccharide composition of the polymers isolated from seeds was similar to that of those isolated from crown tissue. Also, no compositional differences were evident between polymers showing activity and those that were inactive. However, Olien’s work indicates that one important aspect of the cold acclimation process involves alterations in the freezing stresses, and there is the possibility that differences in the hardiness of different tissues may be due to differences in the freezing stresses that occur.
3 . Mitigation of Dehydration There are numerous and varied hypotheses regarding the mechanism of freezing damage, but many of them share the basic belief that injury is a function of dehydration. Although there are several events associated with dehydration and individually they serve as the basis for the various hypotheses, any alteration that occurs during acclimation that reduces the amount of water removed from the cell during freezing could be considered beneficial. However, this statement must be qualified, and it is applicable only for the consideration of injury incurred during extracellularice formation. As previously mentioned, the most common change occurring during cold acclimation is an increase in the osmotic concentration, with either sugars, amino acids, or organic acids being responsible. Such an increase in osmotic concentration will influence the amount of water that must be removed from the cell in order to achieve vapor pressure equilibrium with the extracellular solution. Thus, Johansson (1970) and Johansson and Krull (1970) suggested that increases in hardiness of wheat plants resulted from increased solute concentration, which decreased the extent of cell dehydration at freezing temperatures. How-
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ever, Gustaet al. (1975) demonstrated that, in wheat, the amount of unfreezable water at a given temperature was not strongly dependent on the degree of cold acclimation. In contrast, the amount of water frozen that could be tolerated was significantly less in hardy plants. In other words, during cold acclimation resistance increased, but Johansson observed that injury was always associated with a constant degree of dehydration, whereas Gusta, Burke and Kapoor observed that injury occurred at different (increasing) degrees of dehydration. The former finding would imply that cold acclimation involves avoidance mechanisms, whereas the latter would imply tolerance mechanisms. Although Gusta et al. (1975) rationalized the differences, in either case a reduction in the amount of cellular dehydration would reduce the amount of injury. Therefore, regardless of whether the cell must avoid dehydration or whether it must tolerate dehydration, increases in osmotic concentration will be beneficial. 4. Mitigation of Toxic Solute Concentration Mitigation of toxic solute concentration is closely associated with mitigation of cellular dehydration. The distinction is that some hypotheses on the mechanism of injury indicate that the absolute degree of dehydration and solute concentration incurred are not by themselves injurious; rather, the concentration of specific and toxic compounds, generally considered to be salts, is responsible for injury. Thus, Heber and Santarius (1973) have considered that one component of cold acclimation involves the formation of protective compounds, which can result in protection through nonspecific colligative dilution of toxic compounds. On achieving vapor pressure equilibrium at a given freezing temperature, the protective compounds are concentrated along with the toxic compounds. Since the total concentration of the intracellular solution will be a function of the temperature, the toxic compounds will account for only a portion of this concentration. A further distinction from merely mitigation of dehydration, where the extent of protection will be a function of the initial concentration of solutes, is that protection on a colligative basis will also be a function of the ratio of protective compounds to the toxic compounds. Several compoundscan act as cryoprotectants, providing they are nontoxic over a wide range of concentrations and are osmotically active. Thus, most sugars and some organic and amino acids can function in this role (Heber et al., 1973). Frequently it is noted that on a molar basis some sugars are more effective than others (Tumanov and Trunova, 1963; Heber and Santarius, 1973), usually following the order trisaccharides > disaccharides > monosaccharides. Although this may be indicative of some specificity, differences in activities, especially in concentrated amounts, may account for the differential protection as observed in cryoprotection of chloroplast thylakoids (R. D. Lineberger and P. L. Steponkus, unpublished results).
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Heber (1968) has also indicated that mitigation of toxic substances may also be effected by cryoprotectiveproteins in a manner other than a colligative reduction of toxic compounds. Certain proteins isolated from hardy tissues of rye and barley can protect isolated chloroplast thylakoid membranes against freezing injury (Heber and Emst, 1967; Heber, 1968, 1970). The compounds are very effective at low concentrations and are more than twenty times as effective as sucrose. Whether the increases in total soluble proteins during cold acclimation of alfalfa (Bula et al., 1956; Wilding et af., 1960; Jung and Smith, 1961; Coleman ef al., 1966; Shih et al., 1967; Gerloff et al., 1967; Jung et al., 1967; Brown et af., 1970; Faw and Jung, 1972; Faw et af., 1976) or red and sweet clover (Bula and Smith, 1954; Hodgson and Bula, 1956) or wheat (Pauli and Mitchell, 1960; Zech and Pauli, 1960; Pauli et af., 1961; Pauli and Zech, 1964) contribute to cold resistance in a similar manner requires the demonstration of a cryoprotective influence of the water-soluble proteins.
D. BIOCHEMICAL ALTERATIONS FROM A STRESS TOLERANCE PERSPECTIVE
As cellular membranes are the primary site of freezing injury, it follows that cold acclimation must involve cellular alterations that allow the membranes to survive lower freezing temperatures. Such alterations may be in the cellular environment, as was just discussed, so that either the freezing stresses are altered or there is a direct protection of the membranes. However, cold acclimation may also involve changes in the membrane itself, so that its susceptibility to the freezing stresses is decreased. In 1937, Scarth and Levitt indicated that cold acclimation may render the plasma membrane more tolerant to freezing stresses. Evidence that cold acclimation results in changes in the tolerance of the plasma membrane has been provided by Scarth et al. (1940), and later Siminovitch and Levitt (1941) indicated that the plasma membrane of hardy protoplasts is more resistant to dehydration and is less easily ruptured by deplasmolysis or tension. Efforts by Siminovitch and co-workers (1967a,b, 1968) have been concerned with the localization of injury and resistance in cellular membranes. They concluded that resistance is an intimate property of the components of the plasma membrane, rather than some property arising from the purely colligative action of solutes. Resistance was considered to be associated with the observed increases in membrane structures, which was termed augmentation. Pomeroy and Siminovitch (1 97 1) provided electron microscopic evidence that the process of augmentation was manifested by marked invaginations in the plasma membrane. Wiest and Steponkus (1978) have shown that freezing injury in isolated protoplasts is the result of a membrane alteration that occurs when the protoplast is frozen and contracted, which subsequently limits the size that can be achieved on
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dilution and expansion. The contraction-inducedalteration does not result in lysis but affects the resilience of the protoplast so that there is a critical increment in surface area that can be tolerated. Augmentation of the plasma membrane could conceivably have an important protective effect in increasing the tolerance of the membrane to such an expansion-induced rupture. The concept of augmentation is specifically concerned with quantitative increments in the plasma membrane, rather than major qualitative changes (Siminovitch et al., 1975; Singh ef al., 1975). Other studies indicate that functional alterations in plasma membrane may occur following cold acclimation (Wiest and Steponkus, 1977); however, there is relatively little information regarding qualitative changes in the plasma membrane per se. This fact is attributable to the difficulties encountered in isolating plasma membranes in sufficiently pure form and in a state that can be proved to be identical to their state in vivo. Alternatively, other cellular membranes have been investigated with respect to alterations that occur during freezing (Steponkus ef al., 1977). There is some concern that changes in organelle membranes may not be related to freezing injury, since the plasma membrane is most often considered as the primary site of injury. However, if the plasma membrane is the site of freezing injury and if during cold acclimation protection of this membrane is achieved, it is reasonable to assume that other membranes should also acclimate, lest they become the primary site of freezing injury by default. Several reports of alterations in mitochondria structure and function have been presented, which may indicate that a link between cold acclimation and cold hardiness involves alterations in membrane structure (Miller et al., 1974). However, the membrane changes involve alterations of swelling and contraction characteristics that are influenced by membrane fluidity and may only be manifestations of a low-temperature stimulation of fatty acid unsaturation (Pomeroy, 1976, 1977). Garber and Steponkus (1976b) and Steponkus et al. (1977) have presented biochemical and electron microscopic evidence that chloroplast thylakoid membranes are altered during cold acclimation. Specifically, it was shown that lower concentrations of sucrose afford greater protection of proton uptake in thylakoids isolated from acclimated tissue than in those isolated from nonacclimated tissue. In addition, electron microscopy of acclimated and nonacclimated thylakoids revealed that there was a decreased protein particle concentration on the innerfracture face of acclimated thylakoids. This observation has been recently conf m e d to occur in the plasma membrane of acclimated cells grown in tissue culture (Sugawara and Sakai, 1978). It is interesting to note that the altered particle density occurs at a site-the inner-fracture face of the thylakoidsthat also exhibits alterations when a specific freeze-induced lesion, release of chloroplast coupling factor (CF,), is incurred. This would indicate that there is some structural dependence between CF,, which is released by freezing,
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and the inner-fracture face, where manifestations of cold acclimation can be observed. Such observations would support the hypotheses of several investigators (Tumanov, 1967; Sakai and Yoshida, 1968; Steponkus, 1971) that cold acclimation involves a change both in the cellular environment and in the membrane itself; in the final analysis they justify consideration of alterations in the cellular membranes as being partly responsible for an increased tolerance to freezing. However, future progress will depend on characterization of the specific membrane lesions in order to assess the significance of the various changes that occur during freezing. Although some problems are associated with isolation of the plasma membrane, recent studies with protoplasts (de la Roche et al., 1977; Steponkus and Wiest, 1978; Wiest and Steponkus, 1978) may contribute significantly to this area. Even with the information presently available, however, it is clear that cold acclimation can involve numerous events that alter both the stresses in the cellular environment and the tolerance of the membranes to these stresses. VI. Screening and Stress Proceduresfor Determining Cold Hardiness
Methods used for screening plant cultivars on the basis of cold hardiness have been varied and numerous. Field tests afford a direct measure of winter hardiness, but they are extremely variable from year to year, owing to differences in weather conditions during the fall (which may affect the extent of acclimation of plants entering the winter) as well as the weather conditions during the winter. Because of these yearly variations, trials must be run for a number of years; they are, therefore, extremely time-consuming. Furthermore, winter hardiness is a composite of cold hardiness, desiccation resistance, ability to tolerate or resist frost heaving, disease resistance, and probably several other factors as well. Advances in the genetic manipulation of plants are made much easier if the improvement of only one factor at a time is attempted. Although breeding for increased cold hardiness alone may result in increased susceptibility to other winter factors, the location of germplasm with high degrees of cold hardiness and possibly the chromosomal mapping of the genes conducive to increased cold hardiness may be found by such studies. With such information, attempts to breed for increased winter hardiness would be significantly enhanced. The development of rapid techniques for the screening of plants with high acclimation potential has led to the development of indirect screening methods for determining potential cold hardiness. These methods have arisen largely as a result of empirical correlations between some physiological or anatomical characteristic of seeds or seedlings of a few varieties and the cold hardiness of these varieties. However, as early as 1927 Hill and Salmon stated: “Many
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attempts have been made to correlate cold resistance with some easily observed morphological or physiological character. The relation of winter hardiness to the size of cells, the habit of growth, osmotic pressure of the cell sap, the water and sugar content of the tissue, the hydrophilic colloids of the protoplasm, and other characters have been studied. No results of great value have been secured, so far as finding a practical means of detecting winter-hardy varieties or strains is concerned. Nevertheless, research is still directed toward finding a foolproof indirect method of determining cold hardiness. For instance, Heinrichs (1959) observed that nonhardy cultivars of alfalfa germinate more rapidly and completely in the presence of 6-atm salt or sugar solutions than do hardy cultivars, but many exceptions were noted. Dovrat and Waldman (1967) observed a better inverse correlation between varietal hardiness and germination rate in 6-atm mannitol solutions than was found with the germination percentage after 5 days, although their method did not distinguish between the hardy cultivar Rambler and the intermediate Laduk. Present knowledge concerning the acclimation process, the freezing process, and the mechanisms of freeze-thaw injury to plants casts doubt that any single, indirect method of screening for cold hardiness will be found. Levitt (1972) has pointed out that direct freezing, followed by tests of viability, is the only fully reliable method of assessing cold hardiness. However, several methodological problems exist with this direct test of cold hardiness. The age of the plant, the acclimation procedure, the type of freezing test imposed, the type of tissue used, and the analysis of viability are factors that must be considered and optimized before the potential for acclimation can be determined. ”
A . ACCLIMATION PROCEDURES
The age of the seedling entering the acclimation process has a profound effect on its acclimation rate (Suneson and Peltier, 1934a), the killing point of the plant in the nonacclimated state (Kinbacher, 1962), and probably the acclimation potential of the plant. Seeds are commonly sown at the “typical” time in the fall and brought indoors for controlled freezing tests at various times throughout the winter (Weibel and Quisenberry, 1941). However, cultivars may be produced that possess the greatest acclimation potential when, for instance, a large number of tillers are present rather than at the three- or four-leaf stage. The cold hardiness of these varieties could be quite useful to the fanner, who would have to sow the seed earlier than normal, but the acclimation potential of these varieties would not be detected by the breeder. A comparison of plant age versus acclimation potential is rarely if ever done during screening, and the possibility therefore exists that a number of such varieties have been made but overlooked. Because of this it is suggested that plants of several ages be used during screening.
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An optimum acclimation procedure must be used before the plants are tested for hardiness. Seeds can be sown directly in the field where the natural acclimation process can occur. However, this procedure induces yearly variability and limits the researcher to conducting one experiment per year. Development of artificial acclimation procedures would eliminate both of these problems. From studies of environmental factors affecting the acclimation process, we know that low temperatures and light (especially in the case of plants whose endosperm reserves have been exhausted) are required. Since the farmer has no control over these environmentalfactors, the optimal artificial acclimation procedure conducted by the breeder should involve a simulation of “typical” weather conditions in that geographical area during the fall and early winter before the probability of a serious freeze becomes great.
B. FREEZING PROCEDURES
Two types of freezing tests are commonly practiced, each with its advantages and disadvantages. In many cases a large number of plants are exposed to a single freezing temperature, and varieties are ranked according to the percentage of plants surviving this single temperature (Hill and Salmon, 1927). The primary advantage of this technique is that only a single freezer is required, and information can be obtained concerning the relative ranking of varieties. However, it has been noted that more than one freezing temperature is required if varieties with a wide range of hardiness are to be compared (Warnes and Johnson, 1972a). Fowler et al. (1973) have concluded that freezing tests using one temperature are statistically valid only when differentiating varieties with large differences in hardiness. A great deal more valuable information can be obtained by exposing plants to a variety of freezing temperatures. The minimum temperature at which 50% (or any economically significant percentage) of the test population is killed can be immediately obtained from such a study (Pomeroy and Fowler, 1973). The temperature-versus-survival profile contains extremely valuable information in itself. As an example of this little-recognized importance, consider two genotypes, A and B. For the sake of hypothesis let us say that 50% of the populations of both A and B survive exposure to - 10°C. That is, both genotypes have an LD5,,of - 10°C. However, after exposure to, for instance, - 15”C, only 10% of population A survives, whereas 40% of population B survives. The L&, value implies that both genotypes are of equal cold hardiness, whereas the temperature-versus-survival profile demonstrates that B is clearly a superior genotype in terms of its cold hardiness. Although exposing the plants to a number of freezing temperatures requires more work than exposure to a single temperature, the additional information that can be obtained renders the procedure well worth the effort.
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If freezing is carried out in large rooms, entire plants can be exposed to the freezing process. This is an ideal situation. Unfortunately, the freezers available to most researchers are rather limited in size; thus, selection of a portion of the plant to test cold hardiness would afford maximum utilization of space. Kneen and Blish (1941) observed that, although the cold hardiness of neither the roots nor the leaves corresponds to that of the whole plant, only survival of the crown tissue appears to be related to plant survival. Many workers are currently conducting freezing tests on the crown and making inferences about the cold hardiness of the entire plant (Warnes er af., 1971; Warnes and Johnson, 1972a,b). Since the crown region is the growth center of winter cereals, and is capable of regenerating leaves and roots if they are excised, the assumption that survival of the crown is related to plant survival appears to be a valid one. Although such techniques are commonly used, they may not provide an accurate simulation of the freezing environment that the plants encounter under field conditions. From the extensive work of Olien, it should be evident that injury is not a function of temperature alone. As discussed, several factors can influence the freezing stresses incurred at a particular temperature and require standardization. Furthermore, since these factors may arise in certain regions of cultivation and not in others, or during certain years and not during others; a comprehensive analysis of stress resistance, based on current knowledge (Olien, 1977), should be attempted if any broad extrapolations are to be made and universally applied.
C. VIABILITY ASSAYS
After the freeze-thaw stress is imposed, a test of viability is required. The most direct method, but also the most time-consuming, is to determine the ability for regrowth. Numerous investigators have pointed out that viability determinations are accurate only after 3 weeks or more have elapsed from the time of the freezing test (Anderson and Kiesselbach, 1934; Warnes et al., 1971). The problem with this direct method of assessing viability is the time requirement. It can take a month or longer before the results of a freezing test are known. For this reason artificial tests capable of predicting tissue viability have been developed. These tests are based on the principle that freezing injury is a result of membrane disruption. Dexter et al. (1930, 1932) have developed a technique that measures the release of cellular electrolytes after freezing, which has been used quite extensively. Siminovitch et al. (1962, 1964) have used an analogous test of viability based on the release of amino acids from injured tissues. Metabolic competence of tissue after a freeze-thaw cycle can also be determined by the tissue’s ability to reduce triphenyltetrazolium chloride ( n C ) (Steponkus and Lanphear, 1967a; Ahnng and Irving, 1969). Although all these techniques have been used extensively for measuring tissue
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survival, several problems associated with their use should be acknowledged. First, since they are being used to “predict” plant survival, a calibration curve to determine the extent of leakage of compounds or TTC reduction that corresponds to death should be made for each species under investigation. In many cases this is not done. Second, although the techniques are extremely useful for determining injury in relatively homogeneous masses of tissue, such as roots or leaves, there is still the problem that survival of such tissues is not indicative of plant survival. However, if crown tissues are used to circumvent this problem, none of the indirect tests of viability will be reliable because of the inherent heterogeneity of the crown tissue. Injury to minute, but specific and highly critical, regions of the crown will not be reflected when efflux of compounds from (or reduction of TTC in) the total tissue mass is measured. Very specific techniques for these specialized regions need to be developed. Alternatively, the critical regions can be isolated, but this would eliminate any expediency factor. Thus, because of the above problems and the fact that crown survival most accurately predicts plant survival, direct regrowth measurements remain the best measure of freezing injury. However, there is one serious deficiency in this approach with respect to agronomic crops and especially winter cereals. Generally, researchers will choose an arbitrary survival percentage for comparison and evaluation of cultivars. Although this provides a relative evaluation of the freezing resistance of the tissue, it cannot be inferred that subsequent yield of the different cultivars with the same LD5, will be the equal. Some studies have considered the recovery of cereals from winter injury (Olien and Marchetti, 1976), but few, if any, have equated percentage survival ratings to yield. Considerable genetic variability may exist in this area, which may be overlooked in tests based solely on percentage survival. VII. Summary and Conclusions
Cold hardiness, one of the key components of the winter hardiness complex, is a significant problem confronting agronomists concerned with crop production in cold northern regions of the world. Ever since the earliest reports of the agricultural implications of low temperatures were recorded in 1127 (see Vasil’yev, 1961), considerable attention has been devoted to the problem. However, as is characteristic of most biological phenomena, that which initially appears to be a seemingly simple problem becomes an increasingly complex situation composed of many paradoxical facts. For example, although low temperature is responsible for the potentially lethal stresses that are imposed on a plant, it is also the primary environmental cue responsible for eliciting the plant’s potential to survive freezing temperatures. Although an understanding of freezing injury and cold acclimation has steadily
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evolved, the final answers have not yet been realized. A significant amount of information exists in regards to the physicochemical events associated with the freezing process, but the manner in which injury is effected is not fully understood. The importance of several environmental cues in the cold acclimation process is well established; however, the manner in which these cues are translated into increased resistance remains to be resolved. Although numerous biochemical changes occur during cold acclimation, the significance of most can only be speculated. Insufficient information on what constitutes freezing injury at the molecular level precludes the final integration of the many, already known facts. In addition, the lack of an appropriate conceptualization of the freezing process and cold acclimation can hinder our usage and interpretation of the known facts. Too often, individual aspects of both processes have been considered as mutually exclusive. Rather than searching for any one specific event to explain freezing injury, we might better view the freezing process as a sequential series of potentially lethal stress barriers. In turn, the cold acclimation process can be envisioned as a sequential series of events that enable the plant to avoid, mitigate, or tolerate the stress barriers as they arise. In such a conceptualization, any one single freezing stress would only become the limiting factor at a particular moment in time, depending on the immediate conditions and the successful swnounting of prior stress barriers. Similarly, whether any one particular resistance mechanism would become the primary factor depends on the immediate conditions. Thus, differences in hardiness between species may be due to distinctly different stress barriers that arise during freezing, whereas differences in hardiness between cultivars within a species may be due to differences in the extent of resistance to a given stress barrier. The improvement of agronomic crops with respect to cold hardiness will require considerable input and coordination of numerous disciplines and individuals. And, although an extreme range of diversity in the ability to survive freezing temperatures exists in the plant kingdom-between 0" and - 196°Conly relatively small increases (5°C) in the hardiness of a particular agronomic crop need be achieved in order to have a significant impact on world food production. REFERENCES Ahring, R. M., and Irving, R. M. 1969. Crop Sci. 9, 615-618. Alden, J . , and Hemann, R. K . 1971. But. Rev. 37, 37-142. Anderson, A , , and Kiesselbach, T. A. 1934. J . Am. Soc. Agron. 26, 44-50. Andrews, C. J . , and Burrows, V. D. 1974. Can. J . Plant Sci. 54, 565-571. Andrews, C. J . , Pomeroy, M . K . , and de la Roche, I. A. 1974a. Can. J . Plant. Sci. 54, 9-15. Andrews, C. J . , Pomeroy, M. K . , and de la Roche, I. A. 1974b. Can. J . Bof. 52, 2539-2546. Andrews. J . E. 1958. Can. J . Plant Sci. 38, 1-7.
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Scarth, G. W., and Levitt, J. 1937. Plant Physiol. 12, 51-78. Scarth, G. W., Levitt, J., and Siminovitch, D. 1940. Cold Spring Harbor Symp. Quant. Biol. 8, 102-109. Schaffnit, E. 1910. Mitt. Kaiser- Wilhelm-Inst. Landw. Bromberg 3, 93-1 15 (cited by Chandler, 1913). S h e m a n , L. L., Olien, C. R., Marchetti, B. L., and Everson, E. H. 1973. Crop Sci. 13, 514-519. Shih, S. C., Jung, G. A., and Shelton, D. C. 1967. Crop Sci. 7, 385-389. Siminovitch, D., and Levitt, J. 1941. Can. J. Res. Sect. C 19, 9-20. Siminovitch, D., and Scarth, G. W. 1938. Can. J . Res. 16, 467481. Siminovitch, D., Thenien, H., Wilner, J., and Gfeller, F. 1962. Can. J . Bor. 40, 1267-1269. Siminovitch, D., Thenien, H., Gfeller, F., and Rheaume, B. 1964. Can. J. Bor. 42, 637-649. Siminovitch, D., Rheaume, B., and Sacher, R. 1967a. In “Molecular Mechanisms of Temperature Adaptation” (C. L. Prosser, ed.). Publ. No. 84, pp. 3 4 0 . Am. Assoc. Adv. Sci., Washington, D.C. Siminovitch, D., Gfeller, G., and Rheaume, B. 1967b. In “Cellular Injury and Resistance in Living Organisms” (E. Asashina, ed.), pp. 93-117. Inst. Low Temp. Sci., Sapporo, Japan. Siminovitch, D., Rheaume, B., Pomeroy, K., and Lepage, M. 1968. Cryobiology 5, 202-225. Siminovitch, D., Singh, J., and de la Roche, 1. A. 1975. Cryobiology 12, 144-153. Singh, J., de la Roche, I. A., and Siminovitch, D. 1975. Nature (London) 257, 669-670. Singh, J., de la Roche, I. A., and Siminovitch, D. 1977. Cryobiology 14, 620-624. Sinnott, E. W. 1918. Bor. Gaz. (Chicago) 66, 162-175. Sinz, E. 1914. J. Landwirtsch. 62, 302-312. Smith, D. 1968. Cryobiology 5 , 148-159. Sprague, M. A. 1955. Plant Physiol. 30, 447451. Steinbauer, G. 1926. Plant Physiol. 1, 281-286. Steponkus. P. L. 1971. Plant Physiol. 47, 175-180. Steponkus, P. L., and Lanphear, F. 0. 1967a. Plant Physiol. 42, 1423-1426. Steponkus, P. L., and Lanphear, F. 0. 1967b. Plant Physiol. 42, 1673-1677. Steponkus, P. L., and Lanphear, F. 0. 1968. Physiol. Plant. 21, 777-791. Steponkus, P. L., and Wiest, S. C. 1978. In ”Plant Cold Hardiness and Freezing Stress” (P. H. Li and A. Sakai, eds.), pp. 75-91. Academic Press, New York. Steponkus, P. L., Garber, M. P., Myers, S. P., and Lineberger, R. D. 1977. Cryobiology 14, 303-321. Stout, D. G., Steponkus, P. L.,and Cotts, R. M. 1977. Plant Physiol. 60, 374-378. Sugawara, Y.,and Sakai, A. 1978. In “Plant Cold Hardiness and Freezing Stress” (P. H. Li ano A. Sakai, eds.), pp. 197-210. Academic Press, New York. Sukumaran, N. P.,and Weiser, C. J. 1972. Plant Physiol. 50, 564-567. Suneson, C. A., and Peltier, G. L. 1934a. J. A m . SOC.Agron. 26, 50-58. Suneson, C. A,, and Peltier, G. L. 1934b. J. Am. SOC. Agron. 26, 687-692. Suneson, C. A , , and Peltier, G. L. 1938. J. Am. SOC.Agron. 30, 769-778. Svec, L. V.,and Hodges, H. F. 1972a. Can. J. Plant Sci. 52, 165-175. Svec, L. V., and Hodges, H. F. 1972b. Can. J. Plant Sci. 52, 955-963. Thomson, L. W., and Zalik, S. 1973. Plant Physiol52, 268-273. Trunova, T. 1. 1965. Sov. Plant Physiol. (Engl. Transl.) 12, 70-77. Tsenov, A . 1972. In “The Winter Hardiness of Cereals” (S. Rajki, ed.), pp. 61-70. Agric. Res. Inst. Hung. Acad. Sci., Martonvasar. Tumanov, I. 1. 1967. Sov. Planr Physiol. (Engl. Transl.) 14, 440453. Tumanov, I. I., and Trunova, T. I. 1963. Sov. Plant Physiol. (Engl. Transl.) 10, 140-149. Tysdal, H. M. 1933. J. Agric. Res. 46, 483-515. Tysdal, H. M., and Pieters, A. J. 1934. J. Am. SOC. Agron. 26, 923-928.
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Uribe, E. G.,and Jagendorf. A. T. 1968. Arch. Biochem. Biophys. 128, 351-359. Vasil’yev, I. M. 1961. “Wintering of Plants.” Am. Inst. Biol. Sci., Washington, D.C. Vincent, A. 1972. In “The Winter Hardiness of Cereals” (S. Rajki, ed.), pp. 31-70. Agric. Res. Inst. Acad. Sci., Martonvasar. Voblikova, T. V. 1965. Sov. Plant Physiol. (Engl. Transl.) 12, 63-69. Wames. D. D., and Johnson, V. A. 1972a. Agron. J. 64, 285-288. Wames. D. D., and Johnson, V. A. 1972b. Crop Sci. 12, 403-405. Wames, D. D., Schmidt, J. W., and Johnson, V. A. 1971. Barley Gene:. 2 , Proc. Inr. Symp.. 2nd. 1969 pp. 364-377. Weete, J. D. 1974. Monogr. Lipid Res. 1, 1-393. Weibel, R. O., and Quisenberry, K. S. 1941. J. Am. Soc. Agron. 33, 336-341. Wiegand, K. M. 1906. Planr World 9, 31-32. Wiest, S. C., and Steponkus, P. L. 1977. J. Am. SOC.Hor:. Sci. 102, 119-123. Wiest, S. C., and Steponkus, P. L. 1978. Planr Physiol. 62 (in press). Willemot, C. 1975. Plant Physiol. 55, 356-359. Willemot, C. 1977. Planr Physiol. 60, 1-4. Willemot, C., Hope, H.J., Williams, R. J., and Michaud, R. 1977. Cryobiology 14, 87-93. Young, A. L., and Feltner, K. C. 1966. Crop Sci. 6, 547-551. Zech, A. C., and Pauli, A. W. 1960. Agron. J. 52, 334-337.
ADVANCES IN AGRONOMY, VOL. 30
THE ROLE OF ROOTING CHARACTERISTICS IN THE SUPPLY OF WATER TO PLANTS' H. M. Taylor and Betty Klepper USDA, Science and Education Administration, Federal Research, Iowa State University, Ames, Iowa, and Columbia Plateau Conservation Research Center, Pendleton, Oregon 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 . . . . . . . . . . . . . . loo 11. A Model of Water Upt t System . . . . . . . . . . . . . . . . 105 111. Diurnal Water Potentials in IV . Axial Resistances . . . . .
Resistances in the Abso Rooting Volume. . . . . The Real World . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors That Man Can Control ............................................. 1x. A Final Thought . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................................ References
V. VI . VII. VIII.
120 122 125 125
I. Introduction
Nearly every terrestrial plant undergoes a reduction in water potential each day. This reduction develops because water vapor diffuses out of the open stomata, whereas CO, diffuses into the substomatal cavity. Loss of water causes tissue to dehydrate and lowers the total chemical potential of the leaf water. Water then moves from the adjacent plant tissue along a path of decreasing potential. During morning hours, the radiant energy load on the leaves steadily increases, and the water potential decreases. The energy load decreases in the afternoon, there is less evaporation from the leaves and the tissues rehydrate until they become turgid during the night if sufficient water is supplied by the roots. Three major factors control how much plant water potential decreases during the daily stress period. These factors are ( a )the input of energy to the leaf and the resultant evaporation of water; (b)the difficulty with which water can move from internal evaporating surfaces through the stomata to the ambient air; and (c) the supply of water from the soil to substomatal evaporating surfaces within the leaves. We shall stress the third factor, the water-supplying capability of root systems in field situations, in this article. We shall not attempt to include all pertinent literature but shall present an account of the factors that affect water flow from the soil to the xylem of the plant at ground level, quoting extensively 'Journal Paper J-8906 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 1941, and Technical Paper 4673 of the Oregon State University Agricultural Experiment Station, Pendleton, Oregon. 99 Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISBN 0-12-ooO730-4
100
H. M. TAYLOR AND BETTY KLEPPER
from our research. This article will not cover physiological effects of a continuing water deficit on top growth and yield but will, from time to time, mention responses of plant tops to water stress because of a feedback response on root functions. Excellent reviews have been published recently on plant response to water stress (Hsiao, 1973), water stress and growth (Hsiao ef al., 1976), photosynthesis in water-stressed plants (Boyer, 1976), and plant hormone interactions with water stress (Vaadia, 1976). In addition, Jarvis (1975) has reviewed water transfer in plants, Tinker (1976) has reviewed transport of water to plant roots in soil, and Newman (1976) has reviewed water movement through root systems. This article will cover some material specifically covered by the last three authors, but will stress behavior of root systems under field conditions more than they did. II. A Model of Water Uptake by Roots
Many conceptual models have been developed to describe the uptake of water from soil by a root system. These models assume, either explicitly or implicitly, that uptake rate is a function of (a) transpiration rate, (b)plant root length (either total or active roots), (c) water uptake rate per unit of root length (either measured or calculated), and (d) a water potential difference between some point in the soil and some point in the plant or atmosphere. The models often differ only in assumptions incorporated to simplify calculations. Availability of largestorage-capacity computers has made several of these simplifications unnecessary. We therefore can use a comparatively complex method for combining root length, conductivity, and water potential effects on root system uptake patterns. This model provides a framework that allows us to pinpoint and discuss major gaps in our knowledge about patterns of plant water uptake. This partly tested model starts with assumptions that accurate rates of plant transpiration and root water uptake from each soil layer can be obtained. Evapotranspiration (Ritchie et al., 1972), evaporation of water from the land surface (Hillel, 1975), and water fluxes between soil layers (Klute, 1973) can be calculated, but direct measurements are preferable. Transpired water is withdrawn from leaf tissues, which in turn are continually resupplied through the vascular system by water taken up by roots. The source of transpiration water can be expressed by the equation Ta = A B p
+ Ua
where Ta = transpiration rate (cm3 H,0/cm2 land surface/min)
A& = rate of decrease of water stored in plants occupying the specified land surface (cm3 H,O/cmZ land surface/min)
101
ROLE OF ROOTING CHARACTERISTICS
V , = water uptake rate from soil by roots of plants occupying the specified land surface (cm3 H,0/cm2 land surface/min) Many models ignore A& and equate transpiration to root water uptake. This procedure usually provides satisfactory answers when only daily values are required and plants rehydrate to the same early morning water potentials on successive days. Under these specified conditions, the error due to ignoring a daily growth component of A&, usually will be less than 5% of transpiration-1-2% for soybeans [Gfycine max (L.) Merr.] in Iowa. If instantaneous transpiration rates and root water uptake rates are required, however, A&, can be appreciable. For example, Kramer (1937) found that the difference between transpiration and absorption (A&,) sometimes was greater than root absorption for sunflowers (Helianthusannuus L.) (Fig. 1). The AO,, values will be especially large, relative to transpiration, when succulent plants are first watered after undergoing severe water stress, at night when transpiration rates are low, or when major changes in transpiration rates occur rapidly. Under field conditions, roots absorb water from soils at different depths, with different water contents and with different physical properties. Therefore, it is convenient to partition water uptake among several soil volumes, such as the horizontal layers shown in Fig. 2. Then uptake from a layer can be determined as a function of (a) root length in the layer, (6) total water potential difference between bulk soil in the layer and plant xylem at the land surface, and (c)
0 '
oeoo
I
I
loo0
1200
I
I
1400 1600
I
I
I
1800 Po0 2200
I
2400
Time (hours)
FIG. I . Rates of transpiration and absorption of sunflowers (Heliutirhus utinuus L.) for 2-hour periods. Redrawn from Kramer (1937).
102
H. M. TAYLOR AND BETTY KLEPPER
FIG.2. A schematic definition of several components of Eqs. (2) and (3) superimposed on the root distribution (after Bohm, 1977) of 85day soybeans growr. in 100-cm-wide rows on loess soil in western Iowa. Each dot represents one root projecting from a prepared trench wall.
resistances to flow between bulk soil and xylem at the land surface. Water uptake of an isolated plant can then be calculated as 11
u = ix= l u i
(2)
where Uiis positive water uptake from a layer of soil (cm3/min). Water extraction by a densely planted crop can be partitioned equally among individual plants, and results can be stated on a land area basis, using the relationship U,,= (U)(planting density). For the sake of simplicity, however, consider the comparatively isolated plant in Fig. 2. Water uptake (U,) from soil volume, Vi, can be calculated by using the equation
ROLE OF ROOTING CHARACTERISTICS
ui
= (Vi)(Li)(qi)($si-
103
(3)
$ri)
where Vi = a rooted volume of soil (cm3) with uniform properties including uniform root density and water potential hi = root length density in Vi (cm/cm3) qi = average root water uptake rate in Vi (cm3 H,O/cm root/bar/min) t,bSi = total soil water potential in Vi (bars) $fi = total water potential in root xylem at midpoint of V, (bars) This equation implies that water uptake in the ith layer is equal to the product of ; rate of water three terms: total root length in the layer-that is, ( V i ) ( L U i )the uptake per unit length of root per bar; and the potential difference from bulk soil into the root xylem at the midpoint of Vi (bar). Field measurements of root xylem water potential deep in the soil profile are not available. Generally, however, accurate values can be obtained for h, the total water potential in plant xylem at the ground surface. An estimate of t,hfi can be made from JlP by subtracting from $ p the loss in potential due to elevation (a generally negligible loss) and the loss in potential due to friction developed between moving water columns and xylem vessel walls. Water potential loss due to elevation can be calculated from $ = - (0.001 bar/cm)(cm) (4) where z is distance (cm) from the land surface (negative). To calculate frictional losses, we must assign resistance values to the main roots carrying water upward in the profile. Figure 3 shows a taprooted system. The diagram shows the location of midpoints in successive layers. All water uptake from a layer is assumed to be channeled through this midpoint. In addition, all water from lower (or more distal) layers is assumed to be channeled through this same midpoint. Beginning at the bottom (nth) layer, Eq. (3) provides the rate of delivery of water to the midpoint, m,,:
un = (vn)(&m)(qn)($sn
(5)
- $rn)
A resistance to flow, R , , is located between the point m and men+,. Thus the potential decrease due to frictional forces ($Y,bars) from m, to qn-j, is, by an Ohm's law analogue, $fn = ($r
+
$.)n
- ($r
+
-
=
$z)n--I
= UnRn
(6)
Similarly, for the top layer, I1
IClf.1
=
$ri
+
$21
$p
2 Ui(RJ i=l
(7)
In general, for flux from the ith layer to the (i - 1) th layer, +fi
= ($r
+
- ($r + + z ) i - l =
i:
ut(Ri)
i=l
(8)
104
H . M. TAYLOR AND BETTY KLEPPER
FIG. 3. A schematic representation of a taprooted plant as visualized for the present model. Symbols m , to m, indicate midpoints of soil volumes V , to V , . R , to R,, indicate axial resistances between midpoints, and qP is xylem water potential at the land surface.
Thus, the xylem potential, t,bP, increases to $,., at the midpoint of the ith layer, where $A
= $p
- $zi -
Wi
(9) Since frictional loss between two points is not constant but depends on flow rate, values must be summed for individual layers for each situation. For example, if upper layers of soil are wet so that fluxes are high into adsorbing roots in the upper part of the profile, then resistance values will be high in xylem elements, and low midday values of JlP will not be transmitted to root xylem vessels in deep soil layers. The equation for uptake from a soil layer can now be written by substituting Eq. (9) into Eq. (3):
ui = ( v t ) ( L i ) ( q i ) ( $ s i
-
+ $zi + x$fi)
(10) The relationship of some of the components of this equation to a plant-soil system in the field can be seen in Fig. 2. We shall now discuss experimental data $p
ROLE OF ROOTING CHARACTERISTICS
105
showing patterns of diurnal changes in water Fotential values. We shall then review information available about axi, ’ 0- Actional resistances, which may cause $r to be higher than JlP, and shall discuss the patterns of rooting to be expected in field situations and how those rooting patterns can influence water extraction. 111. Diurnal Water Potentials in the Soil-Plant System
Figures 4, 5, and 6 show generalized patterns of water potential for three points in a soil-plant system. Data for such field crops as cotton (Gossypium hirsutum L.) and soybeans have been used to estimate $p and $s at some specified depth for a sunny (Fig. 4) and a cloudy day (Fig. 5) (wet soil) and for a sunny day (dry soil) (Fig. 6). Values for +,., root xylem water potential, have been plotted on the assumption that, in wet soil, most resistance to flow occurs during absorption, not axially (longitudinally) in xylem. The way in which $,. varies during the day depends on how much axial resistance occurs in the species under the conditions being studied. If axial resistances are very great, $,. will not vary nearly as much diurnally as is shown in Figs. 5 and 6. At a low flow rate, $T will nearly equal $ p . Total water potential in leaf xylem, $ I , of cotton usually is - 1 to -2 bars before dawn if most of the roots are in wet soil (Browning et al., 1975). For illustrative purposes, we shall assume that total xylem water potential at the land surface is about equal to because within-canopy xylem resistances do not appear to be large in cotton (Klepper et al., 1973). Leaf water potential, and thus JlP, decreases to -10 to -12 bars by 1400 hours during a sunny day, then increases to - 1 to -2 bars by 2000 hours (Fig. 4). During a cloudy day, $ p at 1400 hours usually is -8 to - 10 bars (Fig. 5). It may decrease to -20 to -25 bars at 1400 hours if most of the roots are in dry soil (& drier than -1 to -5 bars) (Fig. 6).
m
L.-.-.-I-.-.-.-.
E
!2
r
.-.L.2.-.L.L -.-.-.-!
4-
._ c
2 -8-
: L
s
P
-12-16‘
FIG. 4. A generalized diurnal pattern of soil (&), root xylem &), and plant &) water potentials on a “sunny” day when cotton and soybean roots are located in “wet” soil.
O L b -
.
.-.
.-L.
.-l-.L.-.L-.-l-.4.-.
-.
.-.
4-
1-8-
a
u:
5 -12-16 -
s
IV. Axial Resistances
We shall now consider the resistance to flow axially within the xylem elements that conduct water from layer to layer in the root profile. These resistances arise because of frictional forces between the moving water column and the vessel walls. Osmotic potentials (Fiscus, 1977), temperature differentials (Taylor and Cary, 1961), and electrical force fields (Briggs, 1967) may also be important. We do not yet have sufficient information to deal with these latter components of total water potential during axial flow. Water moves upward only slowly when transpiration is low, but as rapidly as 1 m d s e c when transpiration is high (Nobel, 1970). Frictional forces along unit length of xylem are low when water movement is slow, but it has been postulated that these frictional forces are high, at least in some species, when transpiration rates are high (Hellkvist et al., 1974). Richter (1973) discussed the difficulties Time (Hours) Midnight 0 2
Noon
4
6
8
10 ,
12 I
14 1
16
18
20
22
Midnight 24