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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.
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.
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.
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).
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).
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.
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.
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).
130 Ritchie and Larkum—Cation Exchange Properties of Enteromorpha Cell Walls Table 1 (part A) shows that the constant log a and the exponent b, whether calculated on a dry weight or a mMolal basis, are not significantly different for the two cell wall preparations. The exponents b were all greater than unity, as would be expected from a Donnan-type system. Freundlich isotherms obtained using cell wall powders have some limitations to their usefulness because a powder has a very different surface area/volume ratio and geometry to that of an intact cell wall. Table 1 (part B) shows that chloride adsorption by intact isolated cell walls from seawater plants also obeys a Freundlich isotherm but its log a and exponent b are different to those of the powder form.
F I G 2. Cation absorption behaviour of cell walls from seawater plants. The black squares show total absorpted Na + in //mol g"1 dry wt. (Na + form) v. NaCl concentration (mMolal), and the Na + balanced by the fixed negative charges (the cation exchange capacity) v. NaCl concentration is shown by the open squares. Error bars about means are + 95% confidence limits. The two separate Tukey test intervals can be used to detect significantly different means of total cell wall Na + and cation exchange capacity respectively, at the P < 0-05 level.
Concentration of NaCl /mMolal FIG. 3. Cation adsorption behaviour of cell walls from seawater plants expressed on a mean concentration basis. The black squares show mean concentration of cell wall Na + (mMolal) v. NaCl concentration (mMolal). Error bars are ± 9 5 % confidence limits. The two separate Tukey test intervals can be used to detect significantly different means at the P < 0-05 level as described for Fig. 2.
which shows that the water volume of cell walls is a function of the ionic strength of the bathing electrolyte. Figure 4 shows the cell wall water volume g"1 dry wt. (Na + form) v. the NaCl concentration of the bathing electrolyte. The non-parametric Kruskal-Wallis test gave a x1 value of 45-7 which was significant at the P < 0-001 level (Zar, 1974). The results of the non-parametric multiple comparisons test is included in the legend of Fig. 4. The overall statistical conclusion was that the cell wall was in a uniform swollen state in dilute NaCl but as the NaCl concentration was increased above 10 mMolal the cell wall volume progressively decreased.
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).
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.
4 5 6 7 8 9 pH of equilibration medium FIG. 7. Swelling behaviour of the cell walls of Enteromorpha plants grown in seawater v. pH of the bathing electrolyte (10 mM NaCl). The cell wall volume shows a clear maxima at neutral pH. The Tukey test interval can be used to compare any two treatment means (n = 6) at the P < 0-05 level.
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).
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).
Scientific Publ., Oxford. CUMMINS, J. T., STRAND, J. A., and VAUGHN, B. E., 1966. Sodium transport in Ulva. Biochim.
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.
sulphonated polysaccharides. In 'Carbohydrate Sulphates'. A.C.S. Symposium Series No. 77. American Chemical Society, Washington, D.C. Publ. Pp. 245-281. WALKER, N. A., and PITMAN, M. G., 1976. Measurement of Fluxes Across Membranes. In Encyclopedia of plant physiology, New Series, Vol. 2, Part A: Cells. Eds U. Liittge and M. G. Pitman. Springer-Verlag Publ., Heidelberg. Pp. 93-126. ZAR, J. H., 1974. Biostatistical analysis. Prentice-Hall Publ., Englewood Cliffs, N. J., U.S.A.
Lemna minor L. PL Physiol. 63,1117-22. PERCIVAL, E., and MCDOWELL, R. H., 1967. Chemistry and Enzymology of Marine Algal Polysaccharides. Academic Press, London. SPANSWICK, R. M., and WILLIAMS, E. J., 1965. Calcium fluxes and membrane potentials in Nitella translucens. J. exp Bot. 16,463-73. STEEL, R. G. D., and TORRIE, J. H., 1960. Principles and procedures of statistics. McGraw-Hill Publ., New York. STEIN, JANET R. (Ed.), 1973. Handbook of phycological methods: culture methods and growth measurements. Cambridge Univ. Press, Cambridge.
Report "Ion Exchange Fluxes of the Cell Walls of Enteromorpha intestinalis (L. ) Link (Ulvales, Chlorophyta)"

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