Cation Exchange Properties of the Cell Walls of ...

Journal ofExperimental Botany, Vol. 33, No. 132, pp. 125-139, February 1982

Cation Exchange Properties of the Cell Walls of Enteromorpha intestinalis (L.) Link. (Ulvales, Chlorophyta RAYMOND J. RITCHIE AND A. W. D. LARKUM

Received 8 July 1981 (revised)

ABSTRACT The cell wall of Enteromorpha intestinalis (a marine alga) has been found to behave as a weakly cross-linked cation exchanger in NaCl solutions from 0-1-1020 mMolal (0-1-1000 mMolar). Anion adsorption could be described by Freundlich isotherms over this concentration range. The large anion, inulin carboxylate, was found to be a tracer of the anion free space of plant tissues only in salt solutions above 10 mMolal. The cell wall of Enteromorpha has a cation exchange capacity of about 2500 fimo\ g~' dry wt. (Na + form). The cell wall volume is a complex function of pH and the NaCl concentration. As a result, the cation exchange capacity is only predictable on a dry weight basis. The fixed negative charges of the cell wall have a pKs of 2 in situ and 1 -75 in vitro, and seem to be a mixture of sulphate and carboxyl sugar esters. The applicability of the Donnan equation to plant cell walls is discussed. Interpretation of the cell wall as a single thermodynamic phase is shown to be inappropriate. A large proportion of the cell wall solution is unaffected by the fixed anions. INTRODUCTION

Consideration of the extracellular or free space ion compartments is an important aspect of the study of the ion relations of plant cells (Briggs, Hope, and Robertson, 1961). Cytoplasmic and free space compartments of tissues can be easily confused (Spanswick and Williams, 1965; Briggs et al., 1961; Walker and Pitman, 1976). Dainty and Hope (1959, 1961) and Dainty, Hope, and Denby (1960) identified the free space of Chara australis as primarily a cell wall phenomenon and showed that the cell wall behaved liked a cation-exchange resin. Demarty, Ayadi, Monnier, Morvan, and Thellier (1977) conducted similar, but more rigorously theoretical, studies on the cell walls ofLemna minor. Cation-exchangers have the following properties: (a) they adsorb more cations than anions from their environment due to the presence of fixed anions, (b) they partially exclude mobile anions, and (c) the fixed anions can be identified by their pKa (Helfferich, 1962, Ch. 4). Cation-exchangers also show complex swelling effects which are influenced by ionic strength. Previous studies on cell walls have involved freshwater plants in which the effects of ionic strength were negligible. However, for marine plants ionic strength is an important consideration and euryhaline species are exposed to a wide range of ionic strengths.

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The School of Biological Sciences, The University of Sydney, Australia

126

Ritchie and Larkum-Cation Exchange Properties of Enteromorpha Cell Walls

The ion exchange properties of the cell walls of living Viva plants have been studied (Cummins, Strand, and Vaughn, 1966, 1969), but not isolated cell walls. In this study the cation-exchange properties of the isolated cell walls of the euryhaline marine alga Enteromorpha intestinalis were investigated over a wide range of ionic strengths.

Anion-adsorption capacity Enteromorpha cell walls in the Na + form, from seawater plants and ACBSW plants, were equilibrated to NaCl solutions from 0-1 to 1020 mMolal. Samples were then equilibrated to 36 Cl~-labelled solutions of the appropriate concentration, shaken for 2 h, then replicate aliquots of


MATERIALS AND METHODS General Laboratory cultures of Enteromorpha intestinalis (L.) Link were grown from a single plant collected in an intertidal brackish soak at Cape Banks, La Perouse, Sydney, Australia in January 1977. Swarmers from this plant were used to set up a single cell line. The culture line had a very wide salinity tolerance and grew in salinities from 5% seawater (25 mM Cl~) to over twice normal seawater (> 1100 mM Cl"). Plants were grown in enriched seawater (f/2 as described by Stein, 1973) or an artificial K+-enriched brackish water medium, designated ACBSW, of the following composition; KC1 0-5 mM, NaCl 17-94 mM, CaCl 2 740 //M, MgSO4 211 fiM, Na 2 SO 4 920 fiM, NaHCO 3 400 //M, tricine or EPPS 2mM; pH 7-85; trace elements, phosphate, nitrate, and vitamins as for medium f/2. ACBSW closely resembled the brackish water medium in which the plant was found except that the K + level was raised from 140 fiM to 0-5 mM by substituting some of the Na + with K + . In 100 ml cultures the alga tended to suffer K + deficiency if not provided with extra K + in ACBSW because the plant depleted the medium of K + . Plants were used in experiments when about 2 months old. Cultures were kept under shaded conditions at about 20 °C and 100 fiE m2 s~' natural light. Cell wall material was prepared in two forms; (1) A cell wall powder form, prepared essentially as described by Demarty et al., (1977) except that methanol was found to be a better extractant than acetone. (2) A morphologically intact form (i.e. not pulverized) which had approximately the same morphology as the intact living plant. Filaments were cut into pieces 0-5-1-0 cm long, then soaked and washed alternately in 0-5% Triton X-100 and methanol over 3 d until all pigmentation was removed. The cell wall yield was 564 ± 38 mg dry wt. (Na + form) g~' dry wt. tissue (n = 6, ± 95% confidence limit). As in previous work (Demarty et al., 1977; Dainty and Hope, 1959) a simple cell wall—monovalent salt system was used to demonstrate the cation exchange behaviour of the cell walls. The crude cell wall preparations were converted to the Na + form by soaking in frequent changes of 1 -0 Molar NaCl, then equilibrating to the NaCl concentration of interest. The Na + form was taken as the standard state rather than the H + form, so dry weights were corrected for the mass of NaCl adsorbed by the cell walls. All experiments were conducted at 20 ±1 °C and at pH 8-0 ± 0-2 unless otherwise stated. The water contents and dry weights of cell wall preparations were measured by blotting samples dry with Miracloth, weighing, then drying at 80 °C to constant weight. Thus anion and cation contents of cell walls could be calculated as /imol g~' dry wt. (Na + form) and as a mean concentration (mMolal). The isotopes used were obtained from The Radiochemical Centre, Amersham. 36C1" was counted using a Nuclear—Chicago gas flow counter and [MC]inulin carboxylate and [14C|mannitol were counted using a Packard 3375 scintillation counter. The systematics of the genus Enteromorpha Link are in such a confused state that the only way to be sure one is working on one species is to set up a laboratory cell line. Our cultured plants appeared to be E. intestinalis (L.) Link (Bliding, 1963); however, no Australian Enteromorpha specimens have ever been checked against the European types specimens (H. B. S. Womersley personal communication). Live cultures of the cell line used in this paper are deposited with the 'Culture Collection of Algae and Protozoa', Cambridge and the 'Culture Collection of Algae", University of Texas, Austin.

Ritchie and Larkum—Cation Exchange Properties of Enteromorpha Cell Walls 127 the equilibration medium taken for measuring the specific activity. The dry weight, water content, and radioactivity of the cell walls were measured. Each experimental treatment was done in duplicate. Adsorption of anions by cation exchangers can be described by adsorption laws such as the Freundlich isotherm (Helfferich, 1962, Sec. 5-2), which is of the form:

y = axb where y is the adsorbed anion content in the cation exchanger, x is the anion concentration in the bulk electrolyte (mMolal), and a and b are empirical constants derived using an appropriate curve fitting method. Plotted on a log-log scale a Freundlich isotherm is a linear function. The exponent b of Freundlich isotherms of ion exchanger co-ions is always greater than unity because a Donnan-type equilibrium is involved (Helfferich, 1962, Sec. 5-2).

Adsorption of a high molecular weight anion Inulin carboxylate is commonly used as a tracer for extracellular or free space anion content of plant tissues because it is not readily taken up by plant cells. The inherent assumption that the inulin carboxylate behaves as a tracer for all mobile anions in the cell wall phase is often not tested (Bisson and Gutkneckt, 1975; Bisson and Kirst, 1979). [14C]Inulin carboxylate has a molecular weight of about 5000 (Radiochemical Centre data sheet), compared to 35-5 for chloride so the pressure terms of the electrochemical equation for the cell wall phase might be significant, because the effects of pressure on the distribution coefficient increases as the solute partial molal volume increases (Helfferich, 1962, Sec. 5-3). Cell walls (Na + form) from plants grown in seawater and ACBSW were equilibrated to NaCl solutions from 0-1 to 1020 mMolal. Samples were then transferred to 5 ml NaCl solution labelled with a trace amount of [l4C]inulin carboxylate. The total mobile anion concentration was increased by 5 //Molar by adding the inulin carboxylate: this was allowed for in calculations. The experiment was conducted in the same way as the 36C1~ experiments above. Calculations were made assuming inulin carboxylate acted as a perfect tracer for all mobile anions in the system. Freundlich isotherms were fitted to the data by regression analysis and compared to those obtained using 36C1~ to test if inulin carboxylate acted as a chloride tracer. A dsorption of a non-electrolyte An ideal non-electrolyte would distribute itself between a cation exchanger and the bulk electrolyte phases governed by the swelling pressure within the cation exchanger which in turn is governed by the ionic strength of the bulk electrolyte. The swelling pressure within the cell wall is due to the osmotic effects of the fixed negative charges and tends to squeeze non-electrolytes from


Total cation capacity and cation-exchange capacity Cell wall powder (Na + form) from Enteromorpha plants grown in seawater was equilibrated to a range of NaCl solutions from 0-1 to 1020 mMolal. After dry weight and water content had been measured, each sample was digested in hot HNO 3 (1-0 Molar) + NH 4 NO 3 (0-5 Molar). Chloride content was assayed using a chloride titrator and sodium content by flame photometry. The cation exchange capacity was taken as the difference between the total Na and Cl assays. Six independent samples per treatment were used to calculate means ±95% confidence limits. Cation exchange capacity and total cation capacity v. NaCl concentration were statistically analysed using separate one-way ANOVARs (Zar, 1974). The variances were found to be uniform within each ANOVAR and a Tukey test interval was calculated to compare any treatment mean with any other at the P < 0 05 level (Steel and Torrie, 1960). The plot of cation exchange capacity (mMolal units) v. NaCl concentration suggested that there was a significant swelling of the cell wall in dilute salt compared to strong salt solutions. Cell wall water content v. NaCl is shown in Fig. 4. The treatment variances were heterogeneous and so parametric statistics were inappropriate. The non-parametric Kruskal-Wallis test (analysis of variance by ranks) and the non-parametric multiple comparisons test were carried out to identify significantly different swelling volumes at the P < 0-05 level (Zar, 1974).

128 Ritchie and Larkum-Cation Exchange Properties of Enteromorpha Cell Walls the cell wall. The swelling pressure can be calculated from the ideal gas laws and would be negative (Helfferich, 1962, Sec. 5-3): RT . "= — — In m/m n

where 77 is the pressure, 7? is the gas constant, T is the absolute temperature, Vn is the partial molal volume of the solute, and m/m is the distribution coefficient of the solute. Samples of cell walls (Na + form) from seawater plants were equilibrated to NaCl solutions from 0-1 to 1020 mMolal. Each NaCl treatment was then labelled with a trace of [MC]mannitol. The total amount of mannitol was less than 100 //Molar. After labelling for about 30 min, the cell walls were collected, and aliquots of the loading solution taken for counting. The cells were dried to determine the water content then counted. The distribution coefficient m/m was the ratio of counts ml" 1 H 2 O in the two phases where m denotes the cell wall phase.

p7Ca = pH + log10 [Na+] - log10 (x/2) where pH is the ^-titration point, [Na + ] is the Na + concentration in the bulk electrolyte, and x is the mean concentration of fixed anionogenic groups in the cell wall. The Henderson-Hasselbalch equation for weak acids cannot be used for in situ determinations of the pATa of cation exchangers because the pH inside the cation exchanger cannot be easily measured. The pKa of the fixed anions can also be measured in vitro after first hydrolysing them out of the cell wall using hot HC1, then titrating the extracted free acid. Cell walls (Na + form) from seawater-grown plants were digested in 0-1 Molar HC1 overnight at 80 °C, the residue removed by centrifugation, and the supernatant dried to remove the HC1. The extract was redissolved in deionized water and titrated with NaOH. The starting pH was about 1-4. RESULTS

Adsorption of anions by cell walls Figure 1 shows log-log plots of adsorbed chloride expressed in /umo\ g"1 dry wt. (Na + form) and as a mean concentration (mMolal) plotted against the NaCl concentration (mMolal) for cell wall powders from ACBSW plants (solid line, dots) and seawater plants (dotted line, squares). The fitted regression lines show very high regression coefficients (P < 0001) showing that Freundlich isotherms describe the chloride adsorption by cell walls very well. Using a linear regression program on the log-log transformed data, the correlation coefficients, constant log a, and the exponent b of the fitted Freundlich isotherms could be calculated and compared for the two cell wall types (Table 1). Error bars were calculated for log a and b using the procedures of Zar (1974). Then the null hypothesis, that the chloride adsorption behaviour of the two cell wall types could be tested at the P < 0-05 level.


Determination ofpKe of the fixed negative charges Attempts to titrate cell walls with HC1 were unsatisfactory because it took hours for the pH of the bulk electrolyte to stabilize after each addition of HC1 (cf. Morvan, Demarty, and Thellier, 1979). A better approach was to equilibrate cell walls (Na + form) from seawater-grown plants to 10 mMolar NaCl with the pH values from 3 to 9 and assay the Na + content. Weak acid buffers (1 mMolar) were used to maintain the desired pH. The buffered NaCl was frequently changed to equilibrate the cell walls to the desired pH (usually took 24 h). Nearly all Na + was balanced by the fixed negative charges (see Figs 2 and 3) and so the cell wall Na + content would represent the number of fixed negative charges not balanced by H + . Following water content and dry weight determination, Na + was extracted and assayed as described above for cell walls in equilibrium with each pH regime. Six replicates per pH treatment were made. The pKa was calculated from the point at which one half of the fixed negative charges (as //mol g"1 dry wt. (Na + form)) were titrated by H+, as described by Helfferich (1962, Sec. 4-4):

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200 500 10-' 0.3 1 3 10 30 100 300 1000 Concentration of NaCl / mMolal FIG. 1. Log-log plots of adsorbed chloride in the cell walls of plants grown in ACBSW v. concentration of NaCl (mMolal) using 36C1~. The left ordinate is ftmo\ g"1 dry wt. (Na + form) and black circle data points are chloride adsorption in fimol g"1 dry wt. (Na + form) at each NaCl concentration. The right ordinate is mean adsorbed chloride concentration (mMolal) and the black square data points are mean chloride concentration (mMolal) in the cell walls at each NaCl concentration. The two regression lines were fitted by linear regression on the log-log transformed data (r = 0-999). The statistical data for the regressions are shown in Table 1).

1. Freundlich isotherms of chloride adsorption v. NaCl concentration using 3 6 C/A: Cell wall powder Dry weight basis (//mol g~' dry wt. (Na+ form)). Error bars are ±95% confidence limits. TABLE

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Comparison of the Freundlich isotherms obtained using 36C1~ and ['"Cjinulin carboxylate tracers for cell wall chloride (Tables 1 and 2) show that inulin carboxylate acts as a chloride tracer in the cell walls of seawater plants but has a slightly different adsorption isotherm to that of chloride in ACBSW cell walls. In Fig. 5 the 36C1~ and [14C]inulin carboxylate data for ACBSW cell walls are compared. The two fitted Freundlich isotherms are also shown. At very low NaCl concentrations inulin carboxylate actually overestimates the cell wall adsorbed chloride. At concentrations above 10 mMolal NaCl, inulin carboxylate acts as an efficient tracer for chloride in ACBSW cell walls. Adsorption of a non-electrolyte A one factor ANOVAR on [14C] mannitol adsorption by cell wall powder (from seawater plants) equilibrated to 0-1, 1-0, 10, 100, and 1020 mMolal NaCl showed no significant effect of ionic strength on the distribution coefficient m/m. Bartlett's test for homogeneity of variances showed that treatment variances were homogeneous at the P < 0-05 level. The / value of the ANOVAR was 1-23 with 4/25 degrees of freedom, this is not significant at the P < 0-05 level. A mean distribution coefficient could be calculated for all NaCl concentrations tested (1-03 ± 0-03, n = 29, mean ±29% confidence limit). The swelling pressure must be relatively low to allow mannitol molecules to have distribution coefficients of unity or marginally greater than unity. Polar solutes often have distribution coefficients greater than unity but mannitol is not polar (Helfferich, 1962, Sec. 5-3).


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3 5 7 8 9 pH of bathing electrolyte (10 mM NaCl) FIG. 6. Titration curve of seawater type cell walls showing the number of fixed anions in the ionized form (jimo\ g~' dry wt. (Na + form)) v. pH. The cell walls were equilibrated to a range of 10 mMolal NaCl solutions buffered to pH values from 3 to 9. The apparent half-titration point was at pH 3-25. Error bars are ± 95% confidence limits based on 6 replicates.


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pKa of the fixed negative charges of isolated cell walls Figure 6 shows the effect of pH on the total fixed anions balanced by Na + (in ^mol g"1 dry wt. (Na + form)) of seawater-type cell wall powder in equilibrium with buffered 10 mMolal NaCl. This shows that the fixed negative charges are titratable with H + . It can be estimated that one half of the fixed anions were titrated by H + at pH 3-25. The mean concentration of Na + in the cell wall was 170 mMolal at this pH. Using the equation of Helfferich (1962, Sec. 4-4), the apparent pA"a would be 2-0. Thus, in the physiological pH range (pH 7-9), the fixed anions would be all in the ionized state. The cell wall water volume was also found to be affected by the pH. Figure 7 shows a plot of the cell wall volume in ml H2O g"1 dry wt. (Na + form) v. pH of the bathing electrolyte. Using the Tukey test (Steel and Torrie, 1960) to separate means, the cell wall volume is maximal at pH 7, with significant shrinkage in both acid and alkaline conditions. Titration runs on the solubilized fixed anions of the cell walls showed no obvious inflection point which would be expected from a typical weak acid-strong base titrat|on. The titration curves showed asymptotic behaviour typical of strong acid-strong base titrations. Ionogenic groups such as pectins (pATa ~ 3-41; Kertesz, 1951) would have been easily detected in the titrations and so some of the fixed anions are strong acid

136 Ritchie and Larkum-Cation Exchange Properties of Enteromorpha Cell Walls residues with a pKa of 2 or less. The approximate isoelectric point (pi) was at pH 1 -78 ± 0-07 (« = 4, ± 95% confidence limit).


DISCUSSION As in the freshwater plants Chara australis (Dainty and Hope, 1959, 1961; and Dainty et al., 1960) and Lemna minor (Demarty et al., 1977; Morvan et al, 1979), we have found the the cell wall of Enteromorpha intestinalis behaves as a cation exchanger; it adsorbs an excess of cations over anions and partially excludes anions (Donnan exclusion) (Helfferich, 1962; Boyd and Bunzl, 1967). Dainty and Hope (1959) and Demarty et al., (1977) found that in dilute electrolytes the degree of anion adsorption by cell walls was directly proportional to the bulk electrolyte concentration. Since Enteromorpha is a euryhaline plant, we studied cation and anion adsorption over a much wider range of salt concentration than previously. This led to the important conclusions that; (1) anion adsorption obeys Freundlich isotherms typical of cation exchangers with values of exponent b greater than unity i.e. Donnan exclusion is not a constant and (2) the cell wall volume is a complex function of pH and the ionic strength of the bathing electrolyte. The value of exponent b of a Freundlich isotherm provides information about the type of cation exchanger involved. Highly cross-linked cation exchangers have Freundlich isotherms for anion adsorption with values of exponent b greater than unity and approaching the value of two which would occur in an ideal Donnan system (Helfferich, 1962; Sec. 5-3; Goldring, 1966). The values of exponent b found in this study for chloride and inulin carboxylate adsorption were only slightly greater than unity (see Tables 1 and 2); it can be concluded that the anion adsorption properties of the cell wall of Enteromorpha are analogous to a weakly cross-linked cation-exchanger (Helfferich, 1962, Sec. 5-3). A low degree of cross-linking is also associated with an increased deviation from an ideal Donnan system, thus the cell wall of Enteromorpha cannot be expected to quantitatively obey the Donnan equation. Some comparisons can be made of the Donnan systems of the cell walls of Chara and Lemna to that of Enteromorpha. Using 131 I-, Dainty and Hope (1959) found a distribution coefficient of 0-48 for anions in the cell walls of Chara, e.g. in 1-0 mMolar KI the cell wall anion concentration would be 0-48 mM. A similar distribution coefficient of 0-34 was found in Lemna cell walls (Demarty et al., 1977). Enteromorpha cell walls in equilibrium with 1 mMolal NaCl have a distribution coefficient for chloride of 0 1 0 ± 0-016 (n = 4, ±95% confidence limits). Thus Donnan exclusion of anions is much more efficient in Enteromorpha cell walls than in Chara or Lemna. Nevertheless the cell wall cannot be an ideal Donnan phase; in 1 0 mMolal NaCl the mean concentration of mobile cations in the cell wall is about 400 mMolal (Fig. 3) and so the cell wall anion concentration would be 2-5 //Molal if the cell wall of Enteromorpha constituted a single ideal Donnan phase, i.e. 2-5% of that observed. Dainty and Hope (1959) came to the same conclusion based on a similar calculation on anion adsorption by Chara cell walls. Synthetic cation exchangers also adsorb more anions in dilute electrolyte than predicted by the Donnan equation (Helfferich, 1962, Sec. 5-3). This was thought

Ritchie and Larkum—Cation Exchange Properties of Enteromorpha Cell Walls 13 7


to have been due to the activity coefficients of adsorbed cations approaching zero in dilute salt, but Boyd and Bunzl (1967) showed that the activity coefficients of counter-ions approach unity in dilute salt. Thus excessive anion adsorption must be accounted for in some other way. Dainty and Hope (1959, 1961) proposed that only a part of the cell wall volume acted as a Donnan phase (the Donnan free space, DFS), whilst the bulk of the cell wall solution was non-adsorbed electrolyte (water free space, WFS). Provided activity coefficients can be ignored, the ion contents and volumes of the DFS and WFS can be calculated from cell wall analyses by an iterative procedure. Activity coefficients cannot be ignored above about 10 mMolal NaCl, but if they are included, the system of equations cannot be solved explicitly. Thus, the DFS/WFS model is inappropriate for cell walls of plants in equilibrium with seawater or any other strong salt solution unless the activity coefficients of ions in the cell wall phase can be estimated. The activity coefficients of polyelectrolyte gels of similar chemistry to that of the fixed anions of the cell wall can be measured and could be used as an approximation (see Tomasula, Swanson, and Ander, 1978). Demarty et al., (1977) used an alternative approach in which all the electrochemical equilibrium terms were taken into account, in particular the pressure terms normally neglected, to yield the Donnan equation. This model implies that there is a very large swelling pressure in the cell walls of plants sufficient to cause large deviations from the Donnan equation. We have shown that the pressure difference (AP) between the cell wall and bulk electrolyte phases is not large enough to cause a significant exclusion of [14C]mannitol, even in very dilute electrolyte. Pressure effects are greater on large solutes than small ones, yet [14C]inulin carboxylate (molecular weight about 5000) is preferentially adsorbed over chloride ions in ACBSW cell walls (Table 2 and Fig. 5). Seawater cell walls do not discriminate between inulin carboxylate and chloride. These results support the conclusion from the [14C]mannitol work that the AP of cell walls is not large enough to affect distribution coefficients of anions and to attribute deviation from the Donnan equation to swelling pressure is incorrect. Figures 2 and 3 demonstrate the presence of fixed negative charges in Enteromorpha cell walls and show that the cell walls have similar cation adsorption properties to those of Chara and Lemna (i.e. a cation exchange capacity of about 2500//mol g"1 dry wt. (Na + form) and mean concentration of fixed anions of 400 mMolal in dilute salt). The total cation exchange capacity can be predicted by the sum of the cation exchange capacity and anion adsorption isotherm, on a dry weight basis by K = 2500 + 0-728 x 1 1 8 . Prediction of the total cation concentration would be much more useful because thermodynamic equations such as the Donnan equation use concentration terms. Unfortunately, Fig. 3 shows that the mean concentration of fixed anions cannot be taken as a constant and so a simple equation such as the above cannot be used to predict the total mean cell wall cation concentration. Thus the concentration of cell wall anions is predictable by a simple adsorption law but the mean concentration of cation is not. This complex effect is due to swelling and also occurs in synthetic cation exchangers (Helfferich, 1962, Sec. 5-2).

138

Ritchie and Larkum-Cation Exchange Properties of Enteromorpha Cell Walls

ACKNOWLEDGEMENTS

This work was partially supported by a research grant from the University of Sydney. One of the authors (R.J.R) would like to thank the Department of Veterans Affairs via the Soldiers Children Education Scheme for financial assistance. The authors would like to thank Professor M. G. Pitman for critically reading the manuscript. LITERATURE CITED BISSON, M. A., and GUTKNECKT, J., 1975. Osmotic regulation in the marine alga, Codium decorticatum. I. Regulation of turgor pressure by control of ionic composition. J. Memb. Biol. 24,183-200. and KIRST, G. O., 1979. Osmotic adaptation in the marine alga Griffithsia months (Rhodophyceae): The role of ions and organic compounds. Aust. J. PI. Physiol. 6, 523-38 BLIDING, C , 1963. A critical survey of European taxa of Ulvales. I. Caprosiphon, Percursaria, Blidingia, Enteromorpha. Opera Botanica, 8 (3), 1-160. BOYD, G. E., and BUNZL, K., 1967. The Donnan equilibrium in cross-linked polystyrene cation and anion exchangers. J. Am. chem. Soc. 89,1776-80. BRIGGS, G. E., HOPE, A. B., and ROBERTSON, R. N., 1961. Electrolytes and plant cells. Blackwell

Scientific Publ., Oxford. CUMMINS, J. T., STRAND, J. A., and VAUGHN, B. E., 1966. Sodium transport in Ulva. Biochim.

Biophys.Acta, 126, 330-7.

1969. The movement of H + and other ions at the onset of photosynthesis in Ulva. Ibid. 173, 198-205. DArNTY, J., and HOPE, A. B., 1959. Ionic relations of cells of Chara australis. I. Ion exchange in the cell wall. Aust. J. biol. Sci. 12,395-411. 1961. The electric double layer and the Donnan equilibrium in relation to plant cell walls. Ibid. 14,541-51.


The pKa value for the fixed anion of the cell wall of Enteromorpha is two or less and this is inconsistent with the fixed anions being entirely pectins, as assumed a priori by Morvan et al., (1979). A general characteristic of the cell walls of marine algae is the presence of sulphate esters of sugars (Percival and McDowell, 1967). The polysaccharide anionic group of the cell walls of Enteromorpha compressa is repeating units of sulphated aldbiouronic acid i.e. the fixed negative charges are a population of carboxyl and sulphate ester residues. Sulphate esters are strongly acidic (pKa ~ 1-0; Helfferich, 1962, Sec. 4-4) and would account for the low apparent pKa value of the fixed anions of the cell wall of Enteromorpha; Dainty and Hope (1959) did not test for sulphate esters in the cell wall of Chara, however, their pKa value of 2-2 suggests their presence. Taking the cell wall sulphate ester content of Enteromorpha compressa as typical of Enteromorpha species (16-8% of the cell dry weight; McKinnell and Percival, 1962), the sulphate ester content of the cell wall accounts for about 39% of the fixed anions. Finally, Figs 4 and 7 show that the cell water volume is a complex function of both the ionic strength and the pH of the bathing electrolyte. The effect of ionic strength is typical of a weakly cross-linked cation exchanger (Helfferich, 1962, Sec. 5-2), but neither the neutral pH swelling maximum, nor the shrinkage in alkaline salt (Fig. 7), is predicted by the Katchalsky model of cation exchanger swelling. It is clear that the cell wall water volumes of plants must be determined under carefully defined conditions.

Ritchie and Larkum-Cation Exchange Properties of Enteromorpha Cell Walls 139 and DENBY, CHRISTINE, 1960. Ionic relations of cells of Chara australis. II. The indiffusible anions of the cell wall. Ibid. 13,267-76. DEMARTY, M.,

AYADI, A.,

MONNIER,

A.,

MORVAN, CLAUDINE, and

THELLIER, M.,

1977.

Electrochemical properties of isolated cell walls of Lemna minor. In Transmembrane ionic exchanges in plants. Eds M. Thellier et al., CNRS et Universite de Rouen, Paris. Pp. 61-73. HELFFERICH, F., 1962. Ion exchange. McGraw-Hill Publ. New York. GOLDRING, L. S., 1966. Heterogeneity and the physical chemical properties of ion-exchange resins. In Ion exchange; a series of advances. Ed. J. A. Marinsky. Edward Arnold Publ. and Dekker Publ., New York. Pp. 205-25. KERTESZ, Z. I., 1951. TRepectic substances. Interscience Publ., New York. MCKINNELL, J. P., and PERCIVAL, E., 1962. The acid polysaccharide from the green seaweed,

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