Plant Physiol. (1986) 81, 792-797 0032-0889/86/81 /0792/06/$0 1.00/0 Effects of NaCl and CaCl2 on Ion Activities in Complex Nutrient Solutions and Root Growth of Cotton' Received for publication December 5, 1985 and in revised form March 18, 1986

GRANT R. CRAMER, ANDRfE LAUCHLI*, AND EMANUEL EPSTEIN Department ofLand, Air and Resources, University ofCalifornia, Davis, California 95616

ABSTRACT MATERIALS AND METHODS Sodium displaces Ca2 from membranes (GR Cramer, A Liiuchli, VS Analysis of Ion Activities in Nutrient Solutions. Ion activities Polito Physiol 1985 79: 207-211) and this can be related to the in the nutrient solutions were calculated using the computer (Ca2`)/(Na')2 activity ratio in the external solution (GR Cramer, A program, GEOCHEM (25). The rationale for the use of this Lauchli 1986 J Exp Bot 37: 321-330). Supplemental Ca2" is known to program has been described previously (7). Briefly, the program mitigate the adverse effects of salinity on plant growth. In this report we calculates ion activities by a method of successive approxima- investigated the effects of NaCl (0-250 millimolar) and Ca2" (0.4 and 10 tions, taking into consideration ion-pair formation and precipi- millimolar) on the ion activities in solution and on root growth of cotton tation. (Gossypium hirsutum L.). Ion activities were analyzed using the computer Plant Growth Conditions. Cotton seeds (Gossypium hirsutum program, GEOCHEM. Most ion activities in a 0.1 modified Hoagland L. cv Acala SJ-2) were planted in germination paper 'sandwiches' solution were significantly reduced by both NaCl and supplemental Ca2". (16). The sandwiches were inserted into slots of plastic racks Ion-pair formation and precipitation were significant for some ions, which were placed in aerated treatment solutions in 3.7 L plastic especially phosphate. Root growth of 6-day-old seedlings was stimulated containers for continual wetting of the germination paper. The by low NaCl concentrations (25 millimolar). At higher NaCl concentra- tops ofthe containers were covered with BM and T multipurpose tions, root growth was inhibited; the concentration at which this occurred plastic wrap to minimize evaporation from the germination depended on the Ca2" concentration and the growth index used. Supple- paper and salt accumulation. The control solution consisted of mental Ca2" mitigated the inhibition of root growth caused by NaCl. a 0.1 modified Hoagland solution (16) containing 0.4 mM Ca", There was a curvilinear relationship between root growth and the (Ca2")/ with a pH of 6.5. Treatments of NaCl (0-250 mm final concen- (Na+)2 ratio in the nutrient solution. The mechanisms by which Na+ and tration), NaCl and supplemental Ca2" (9.6 mM CaC12 to make a Ca2 may affect root growth are discussed. 10 mm final Ca2+ concentration), or KCI (50 mM final concen- tration) were added to the control solution. The temperature of the nutrient solution was maintained at 27°C using a Lauda K- 2/RD constant-temperature circulator. When the cotyledons emerged, the were irradiated with incandescent lighting (400 ,umol m-2 s-'; 15:9 h day:night cycle). Plants were har- vested 6 d after the start of seed imbibition. Root growth was measured as length, fresh weight, and dry weight at harvest time.

Salt stress may reduce plant growth by water deficits, ion RESULTS toxicity, ion imbalance, or a combination ofany ofthese factors. Cotton is one ofthe most salt-tolerant crops species (19). Growth Ion Activities in Relation to NaCI and Ca2 Concentration. of cotton is most salt-sensitive in the seedling stage (1, 3). In Additions ofNaCI or CaCl2 significantly affected the activities of previous studies, we have shown that Na+ displaces Ca2" from most ions in solution. Here, we report only those ions (Mg2", membranes and alters K+ and Ca2" transport in cotton roots (8; Ca2+, Na+, P04 species, and SO42-) whose concentrations were GR Cramer, J Lynch, A Lauchli, E Epstein, unpublished results). reduced by ion-pair formation and precipitation by 10% or more Supplemental Ca2` partly restores perturbations of selective ion of their original concentration. Sodium activity in a complete transport (GR Cramer, J Lynch, A Lauchli, E Epstein, unpub- nutrient solution rose almost in direct proportion to increasing lished results), ionic balance (5, 16), and growth (5, 10, 16) concentrations of NaCl; supplemental Ca2" had no significant incurred by salt stress in cotton. effect (Fig. la). Magnesium (Fig. lb) andS42- (Fig. lc) activities Salinity can also affect the ion activities in solution by changes declined with increasing NaCl concentrations. Supplemental in ionic strength (which affects the activity coefficient), ion-pair Ca2" reduced activity of these ions further, particularly at low formation, and precipitation (7). Previously, we showed that NaCl concentrations. One should note that the original Mg2' consideration of the ion activities in solution was important in and S042- concentrations in solution were 100 ,uM. describing the Na+-Ca2+ interactions at the plasmalemma; spe- activity was also markedly reduced by increasing NaCl concen- cifically, that the Ca2" activity at the plasmalemma surface was trations (Fig. 2). Activity in the low Ca2" treatment (Fig. 2a) did related to the (Ca2+)/(Na+)2 ratio (parentheses denote activities) not decrease in a smooth manner as did that of the previously in the external solution (7). In that study, we considered the ionic discussed divalent ions. This is the result of changes in ion-pair interactions only in simple salt solutions. In this report, we formation and precipitation with phosphate (data not shown). consider the ionic interactions in complete nutrient solutions Figure 3 demonstrates the complex ion interactions that can and describe their relationship to root growth. occur in a complete nutrient solution when the composition and ionic strength are altered. Phosphate is particularly susceptible 'Supported by National Science Foundation grant DMB84-04442. to precipitation and ion-pair formation. As percentages of all 792 EFFECTS OF NaCl AND CaC12 ON ION ACTIVITIES 793

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._ U .S- 3.5 - 4 N 3.0 F 2.5 F 20 2I^n L..I 80. 0 50 100 I50 200 250 NoCI (mM) FIG. 2. Effect of added NaCl on Ca2" activity in a 0.1 modified 60 Hoagland solution (pH 6.5). a, 0.4 mm Ca; b, 10 mm Ca.

s - Effects of NaCI and Supplemental Ca2" on Root Growth. Having examined the effects of NaCI on the activities of various 0 40 ._ ions in a complete nutrient solution, we attempted to relate these 0 findings to the growth of cotton roots in solutions characterized 4 by their ion activities. Root growth was measured by several 20 indices: tap root length (lateral root development was minimal at this age), fresh weight, and dry weight. At 0.4 mM Ca2", tap root length was significantly reduced at 50 mM NaCl and above

(Fig. 5). At 10 mm Ca2 , tap root length was increased at low 0 0 50 100 150 200 250 NaCl concentrations, reaching its maximum at 100 mM NaCl, NaCI (mM) and was only reduced to values significantly below its control at 250 mm NaCI. At low Ca2" and high NaCl concentrations, the FIG. 1. Effect of added NaCl on ion activities in a 0.1 modified roots turned brown, particularly at their tips, whereas with the Hoagland solution (pH 6.5). a, Na+; b, 100 AM Mg; c, 100 AM SO42. (/- high Ca2+ treatment, the roots appeared healthy and white, even -A), 0.4 mM Ca; (LI---U), 10 mm Ca. at 250 mm NaCl. Similar trends were evident for the fresh weight of roots (Fig. 6a). There was an increase at low and a reduction phosphate present, the phosphate species bound with hydrogen at high NaCl concentrations. The dry weight of roots was not (H3PO4, H2P04-, and HP042-) increased with NaCl concentra- significantly increased at low, but was significantly reduced at tions (up to 50 mM) for the low Ca2+ treatment (Fig. 3a), while high NaCl concentrations (Fig. 6b). phosphate bound or precipitated with Ca decreased (95% of the The NaCl concentration at which there was a decline in growth phosphate is as a precipitate at 0 mm NaCI). As NaCl concentra- varied with the index measured. Of the three indices measured, tions increased, Na+ formed ion-pairs with phosphate, reducing tap root length was increased to the largest extent, and reduced phosphate activity even more. At 10 mm Ca>, phosphate activity the least, for the high Ca2+ treatment. In contrast, tap root length was completely dominated by precipitation with Ca2 (Fig. 3b). appeared to be the most NaCl-sensitive parameter at the low The activity of H2P04- was affected differently by increasing Ca2" concentration. The fresh weight:dry weight ratio was re- NaCl concentrations at low and high external Ca>2 concentra- duced to below its control value at NaCl concentrations of 150 tions (Fig. 4). At the low Ca>2 concentration, H2PO4- activity mm and beyond (Fig. 6c). The high and low Ca2+ treatments had varied between the range of 61 and 95 AM, increasing at low, but no significant effect on this ratio. declining at high NaCl concentrations (Fig. 4a). At high Ca>2 Root Growth in Relation to Ion Activities in Solution. Since concentration, H2PO4- activity was very low and varied between supplemental Ca2+ stimulated root growth, tap root length at the range of 0.3 and 0.9 uM, increasing steadily with increasing varied NaCl concentrations was plotted against the Ca2' activity NaCl concentrations. The original concentration of phosphate in the external solution for both Ca2' treatments (Fig. 7). Al- was 200 uM. though root growth within each Ca2+ treatment appeared to be 794 CRAMER ET Al. Plant Physiol. Vol. 81, 1986 100 a 80S

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20 _ 0.3 0 50 100 150 200 250 0 -1 NoCI (mM) a1r I I i I 50 100 150 200 2250 FIG. 4. Effect of added NaCl on H2PO4- activity in a 0.1 modified NoCI (mM) Hoagland solution (pH 6.5). The phosphate concentration was 200 uM. a, 0.4 mM Ca; b, 10 mm Ca. FIG. 3. Effect of added NaCI on phosphate species (% of total phos- phate concentration). a, 0.4 mM Ca; b, 10 mM Ca. (A-A), P04 bound 20 with H; (@---4), P04 with bound and precipitated Ca; (O --O), P04 bound with Na. E related to the reduction in Ca2" activity, the latter could not '5 account for the differences in root growth between Ca2" activity 0 of 2.34 mM (250 mM NaCl and 10 mM Ca2") and 0.2 mm Ca2" z w (25 mM NaCl and 0.4 mM Ca2"). Previously, we showed that the -J Ca2" activity at the plasmalemma surface was related to the I0 0 (Ca2+)/(NaNY ratio in the external solution (7). Since growth 0 might also be related to this ratio, root length was plotted against W. ratios were it (Fig. 8). The (Ca2+)/(Na+)2 log transformed since 4 5 they differed by 10 orders of magnitude between the low and I.- high salt treatments. The log transformation did not affect the conclusions derived from this plot. There was a distinct relation- ship between root growth and ion activity ratios (Fig. 8). Root 0 0 50 100 ISO 200 250 growth was maximal at a (Ca2+)/(Na+)2 ratio ofapproximately 1 (log 1 = 0), declining sharply at values below this ratio. At higher NoCI (mM) ratio values root growth declined, but to a lesser extent. Similar FIG. 5. Effect of added NaCI on cotton tap root length. The error relationships were found for fresh and dry weights of roots (data bars represent 95% confidence intervals for their respective curves. Each not shown). value is the mean of3 replications, with at least 17 subsamples (seedlings) Ion-Specific Effect on Root Growth. To test whether the inhi- per replication (A-A), 0.4 mm Ca; (---O), 10 mM Ca. bition by salt of root growth was cation-specific, tap root length in the absence of salt was compared (Table I) with treatments of interactions in a complex solution are much more significant 50 mM NaCl or KCI in a modified Hoagland solution (0.4 mM and complex than those found in a simple salt solution (7). These Ca2+). NaCl inhibited tap root length to a significantly greater interactions cannot be descibed by the simple assumption that extent than did KCI. ion activities are determined principally by their activity coeffi- cient. Some ions, such as K+, NO3-, and Cl- may fall into this DISCUSSION category, but others (particularly phosphate) do not, and require one to consider the formation ofion pairs and precipitates. Salt Effects on Ion Activities in Solution. The calculated results Phosphate precipitation is not always readily visible in a nu- of ion activities in a complete nutrient solution show that ion trient solution, but may occur nonetheless, resulting in a sus- EFFECTS OF NaCl AND CaCl2 ON ION ACTIVITIES 795

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0 0 1 2 3 4 5 7 Co Activity (mM) FIG. 7. Relationship between tap root length and Ca2+ activity in 6 solution (NaCl concentrations varied from 0 to 250 mM). (A-A), 0.4 m Ca; (0-0), 10 mm Ca. I 5 0 0~~~~~~~~~~-- 0 20 4 0 E - 3 o O

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A I I I I I I I. 0 ¢ 20 -2 0 2 4 6 8 2+ +2 1 a:>. log (Coa) /(No ) (mM) FIG. 8. Relationship between tap root length and the (Ca2`)/Na')2 in 0.4 mm 10 mM Ca. The parentheses denote X ratio solution. (A), Ca; (0), 15 activities. NaCl concentrations varied from 0 to 250 mM. U. Table I. Cation-specific Effects on Tap Root Length The control was a 0.1 modified Hoagland solution (pH 6.5). 10 Ion Activity Ratioa 0 50 1OO 150 200 250 Treatment (Ca2+) Tap Root Length NOCI (mM) (Total Cations) on and FIG. 6. Effect of added NaCl (a) fresh weight, (b) dry weight, mM cm % ofcontrol (c) fresh weight:dry weight ratio of cotton roots. The error bars represent Control 2.05 x 10-' 11.9 ± 0.6b 100 95% confidence intervals for their respective curves. (A-A), 0.4 mM 50 NaCl 3.87 x 10-3 7.0 ± 0.9 58 Ca; (0---0), 10 mm Ca. 50 KCI 3.75 x 10-3 8.6 ± 0.6 73 pended, invisible precipitate. Once precipitated, calcium phos- ' Parentheses denote activities. b Mean ± 95% confidence interval. phate redissolves very slowly. The pH ofthe solution can have a There were 3 replications per mean value with at least 13 subsamples substantial effect on these interactions. For example, using GEO- (seedlings) per replicate. CHEM, we have calculated that a 0.1 modified Hoagland solu- tion at pH 6.5 has approximately 50% of the phosphate precip- when one wants to study the effect of a specific ion in solution, itated as calcium phosphate, but at pH 5.5 no precipitation because ofthe usual implicit assumption that the activities ofthe occurs. This effect may be significant for many nutrient solutions other ions do not change. For the studies of such ion effects, it since plants often alter the pH of the solution in which they is therefore important to develop a system such as that ofBloom grow. and Chapin (4), in which the activities in the nutrient solution These findings indicate that addition of any salt not only can be closely monitored or have a computer program (such as changes the concentration and activity of the ions added, but GEOCHEM) available for calculation of ion activities. can substantially alter the concentrations and activities of the Effects of Na' and Ca2" on Root Growth. It is clear that NaCl other ions present in solution. This is particularly important at high concentrations inhibited root growth and that this re- 796 CRAMER ET AL Plant Physiol. Vol. 81, 1986 sponse was mitigated by supplemental Ca2", regardless of the selectivity (8, 16; GR Cramer, J Lynch, A Lauchli, E Epstein, index ofroot growth examined. This effect was most pronounced unpublished results). Potassium is important for normal protein when tap root length was investigated. function and is an important solute for the maintenance of cell Cotton root growth appears to be very sensitive to Ca2" con- turgor (18). A reduction in K+ tissue concentration could impair centrations (3, 10, 15, 16, 22, 23). Supplemental Ca" increased these processes, essential for expansive cell growth. However, the Ca2 influx (GR Cramer, J Lynch, A Lauchli, and E Epstein, loss of K+ did not correlate well with the inhibition of root unpublished results) and Ca2 tissue concentrations (16) for salt- growth by NaCl at 0.4 mM Ca". Root growth was inhibited by stressed cotton roots, supporting this hypothesis. However, the 50 mM NaCl (Fig. 5), but K+ efflux only increased at 150 mM Ca>2 activities in the nutrient solutions did not correlate well NaCl and beyond (8). Furthermore, 50 mM KCl inhibited root with tap root length (Fig. 7). Root growth was better correlated growth (Table I), although this treatment did not displace mem- with the (Ca2+)/(Na+)2 ratio in the solution (Fig. 8). brane-associated Ca> (8). The leakage of K+ may indeed lead to Adams and co-workers (2, 14) suggested that it is the (Ca")/ impaired growth of salt-stressed cotton roots, but only at high (total cation) ratio (where parentheses denote activities) in the NaCl concentrations. nutrient solution that influence growth and Ca> sufficiency. Supplemental Ca> reduced the phosphate activity in the nu- They found that cotton root length began to be inhibited at ratios trient solution. Phosphate concentrations similar to those used between 0.1 and 0.15, and markedly declined at ratios below this in this study were thought to be toxic in salt-stressed soybeans value. Their hypothesis implies that ion-specific effects are not (1 1). Cotton may respond similarly to these unrealistically high important; they did not, however, investigate the effects ofNaCl. phosphate concentrations under salt-stress. The nutrient solution In our study, root length was not inhibited by 25 mM NaCl at with the low Ca2"/salt treatments had 100 times the H2PO4 the low Ca> concentration, which has a (Ca2")/(total cation) activity than did the high Ca2+/salt treatment (Fig. 4). However, ratio that is less than 0.008. In addition, this ratio is slightly less the changes in phosphate bound with H (Fig. 3) or H2P04- with KCI (3.75 x 1-0) than with NaCl (3.87 x 10-3) at 50 mM, activity (Fig. 4) did not correlate well with root growth (Fig. 5). yet root growth was inhibited by NaCl to a greater extent than Root growth was stimulated at low concentrations of NaCl by KCI (Table I). Thus, the hypothesis of Adams and co-workers (Figs. 5, 6, a and b). This may be due to increased turgor as a (2, 14) does not seem to take into account differences in growth result of a lower osmotic potential in the cell generated by the due to ion-specific effects, particularly in relation to salt stress. uptake of NaCl. However, the high Ca>2 treatment stimulated In light of the present findings, their hypothesis requires modi- growth more than did the low Ca>2 concentration, yet Na+ influx fication to improve its general applicability. Our data were was significantly less in the high Ca>2 treatment (GR Cramer, J plotted using their ion ratio; it did not fully account for the Lynch, A Liuchli, E Epstein, unpublished results). Calcium may differences observed between the two Ca>2 treatments (data not affect growth by effects on cell wall extensibility (i.e. macromol- shown). Our growth data were best described by the (Ca>2)/ ecule secretion). At higher NaCl concentrations other factors (Na+)2 ratio, for which there is a theoretical basis (7). This implies may come into play, such as the disruption ofnormal membrane that it is the interaction between the Ca> and Na+ activities in function, which could then lead to further physiological dysfunc- solution that is important for determining root growth of cotton tions (8; GR Cramer, J Lynch, A Lauchli, E Epstein, unpublished in saline solutions. results). Thus, there appear to be complex interactions between Calcium is important for normal plant function. It is involved Na+, Ca2+, and growth. in cell wall extensibility and membrane stability and function, In summary, NaCl at increasing concentration significantly and acts as a second messenger for many biochemical processes affected the activity of most ions in the nutrient solution. This within the cytosol (13). Cleland and Rayle (6) concluded that complicated the analysis of the growth data. Root growth was Ca2> did not have any direct physical role in cell wall extensibil- stimulated by low NaCl concentrations, but was inhibited at ity. Nakajima et al. (21), however, showed that cell wall extension higher concentrations. Supplemental Ca>2 improved root growth was inhibited by 50 mm CaCl2 and that this could be reversed under saline conditions. KCI (at 50 mM) inhibited root growth by replacement with 50 mM MgCl2. Furthermore, Soll and to a lesser extent than did NaCl, indicating that this inhibition Bottger (24) found that concentrations of NaCl or KCI between was partially ion-specific. Root growth was best related to the 100 and 1000 mm induced extension of coleoptile epidermal (Ca2+)/(Na+)2 ratio in the external solution. strips. These authors concluded that Ca> was involved in cell wall stiffening and that the exchange of Ca> from the cell wall LITERATURE CITED caused cell wall loosening. 1. ABUI.-NAAS AA, MS OMRAN 1974 Salt tolerance of seventeen cotton cultivars Sodium Ca> from the cell wall and during germination and early seedling development. Z Ack Pflanzenbau probably displaces (24) 140: 229-236 should allow for rapid expansion, yet under saline conditions at 2. BENNETT AC, F ADAMS 1970 Calcium deficiency and ammonia toxicity as a low Ca> concentration (Fig. 5), cotton root growth was not separate causal factors of (NH4)2HP04- injury to seedlings. Soil Sci Soc Am stimulated but inhibited. Extension or growth of the cell wall is Proc 34: 255-259 partly a result of processes Plant cell wall 3. BERNSTEIN L, HE HAYWARD 1958 Physiology of salt tolerance. Annu Rev synthetic (17). poly- Plant Physiol 9: 25-46 saccharides and proteins are secreted to the apoplast by exocy- 4. BLOOM AJ, FS CHAPIN III 1981 Differences in steady-state net ammonium tosis ( 13). Griffing and Ray ( 12) suggested that cell wall secretion and nitrate influx by cold- and warm-adapted barley varieties. Plant Physiol may be regulated by intracellular Ca". Meindl (20) stated that 68: 1064-1067 Ca> seemed to be a general mediator for recognition and fusion 5. CALAHAN JS JR 1977 Some physiological effects ofhigh sodium, calcium, and chloride concentrations on cotton. PhD thesis. Texas A&M University, processes of different types of vesicles at the plasmalemma. College Station According to Hanson (13) various ionic perturbations adversely 6. CLELAND RE, DL RAYLE 1977 Reevaluation of the effect of calcium ion on affect normal secretion, indicating that these ionic perturbations auxin-induced elongation. Plant Physiol 60: 709-712 also alter membrane and Ca> fluxes. He 7. CRAMER GR, A LAUCHLI 1986 Ion activities in solution in relation to Na+- may permeability Ca2" interactions at the plasmalemma. J Exp Bot 37: 321-330 further stressed that the regulatory system for macromolecule 8. CRAMER GR, A LAUCHLI, VS POLITO 1985 Displacement ofCa2" by Na+ from secretion requires external Ca". the plasmalemma of root cells. A primary response to salt stress? Plant Calcium is also important for membrane stability and ion- Physiol 79: 207-21 1 9. EPSTEIN E 1972 Mineral Nutrition of Plants. Principles and Perspectives. Wiley selective transport (9, 13). Sodium displaced Ca> from the and Sons, New York plasmalemma of cotton root hairs (8). Associated with this was 10. GERARD CJ 1971 Influence ofosmotic potential, temperature, and calcium on a loss of K+ from the root tissue and an alteration of K+/Na+ growth of plant roots. Agron J 63: 555-558 EFFECTS OF NaCl AND CaC12 ON ION ACTIVITIES 797

11. GRATTAN SR, EV MAAS 1984 Interactive effects of salinity and substrate 19. MAAS EV, GJ HOFFMAN 1977 Crop salt tolerance-current assessment. ASCE phosphate on soybean. Agron J 76: 668-676 J Irr Drain Div 103: 115-134 12. GRIFFING LR, PM RAY 1979 Dependence of cell wall secretion on calcium. 20. MEINDL U 1982 Patterned distribution ofmembrane associated calcium during Plant Physiol 63: S-283 pore formation. Protoplasma 112: 138-141 13. HANSON JB 1984 The function of calcium in . In PB Tinker, A 21. NAKAJIMA N, H MORIKAWA S, IGARASHI, M SENDA 1981 Differential effect of ULuchli, eds, Advances in Plant Nutrition, Vol 1. Praeger, New York, pp calcium and magnesium on mechanical properties of pea stem walls. Plant 149-208 Cell Physiol 22: 1305-1315 14. HOWARD DD, F ADAMS 1965 Calcium requirement for penetration ofsubsoils 22. NELSON LE 1971 The effects of root temperature and Ca supply on the growth and transpiration of cotton seedlings (Gossypium hirsutum, L). Plant Soil by primary cotton roots. Soil Sci Soc Am Proc 29: 558-562 72 15. JOHANSON L, HE JOHAM 1971 The influence of calcium absorption and 34: 1-729 23. PRESLEY JT, OA LEONARD 1948 The effect of calcium and other ions on the accumulation on the growth of excised cotton roots. Plant Soil 34: 331-339 early development of the radicle of cotton seedlings. Plant Physiol 23: 516- 16. KENT LM, A LXUCHLI 1985 Germination and seedling growth of cotton: 525 salinity-calcium interactions. Plant Cell Environ 8: 155-159 24. SOLL H, M BOTTGER 1982 The mechanism of proton-induced increase of cell 17. LABAVITCH JM 1981 Cell wall turnover in plant development. Annu Rev Plant wall extensibility. Plant Sci Lett 24: 163-171 Physiol 32: 385-406 25. SPOSITO G, SV MATTIGOD 1980 GEOCHEM: a computer program for the 18. LEIGH RA, RG WYN JONES 1984 A hypothesis relating critical potassium calculation of chemical equilibria in soil solutions and other natural water concentrations for growth to the distribution and functions ofthis ion in the systems. The Kearney Foundation of Soil Science, University of California, plant cell. New Phytol 97: 1-13 Riverside