An Ecophysiological Study on the Response to Salt of Four Halophytic and Glycophytic Juncus Species

Flora, Bd. 165, S. 197-209 (1976)

An Ecophysiological Study on the Response to Salt of Four Halophytic and Glycophytic Juncus Species J ELTE ROZEMA Department of Ecology, Biological Laboratory, Free University, Amsterdam, Netherlands

Summary With respect to the degree of growth reduction, Juncu8 gerardii appeared to be more salt tolerant than J. alpino-articulatu8 ssp. atricapillu'i and the glycophytic species J. bufoniu8 ssp. bufoniu8. The slowly growing J. maritimu8 is hardly affected by an increase in salinity. Higher salt concentrations do not enhance the degree of succulence. Chlorophyll content is less affected by salt in the relative salt tolerant species. Analysis of pressed plant sap revealed the important contribution of potassium ions to the total osmotic potential. This phenomenon, together with the independence of the K + concentrations of the increasing N a + content as salinity increases, could form the basis of salt tolerance in J. maritimu8 and J. gerardii. Soluble sugars, P0 43-, Mg2+ and Ca2 + form a small contribution in osmotic adjustment. In acid extract of dry material Ca2 + is present in relatively large amounts. Concentrations of malate, citrate, oxalate and succinate were estimated by means of gas-liquid gaschromatography. No increase of oxalate with increasing salinity was observed. The highest amounts of malate and citrate were observed under fresh water conditions. Reduction of organic acid levels is greater in the relatively salt sensitive species.

Introduction JunCU8 gerardii, J. maritimu8 and J. alpino-articulatu8 ssp. atricapillu8 occupy different zones along a height gradient at the island of Schiermonnikoog. The latter species is dominant in the upper part of the salt marsh, which is relatively less saline and less often inundated by seawater (ROZEMA 1975a and b). Abiotic factors influencing plant zonation in salt marshes were investigated by ADAMS (1963). Salinity and inundation vary with elevation above tide level or distance to seawater, and plant zonation is explained by a difference degree of salt tolerance. But at which stage in the cycle of a plant is zonation established? In an earlier study (ROZEMA 1975a), it was studied whether a differential germination response to increasing salinity will account for plant zonation. This appeared to be so only to a certain extent. In this study the role of growth is investigated. Salinity puts various problems to plants, at the population level (e.g. breeding system, ASTON and BRADSHAW (1966)), at the organismal level (e.g. POLLAK and WAISEL (1970)) and at the physiological and molecular level (enzym tolerance e.g. KYLIN and GEE (1970)); or ion uptake mechanisms e.g. EpSTEIN et al. (1963); ion toxicity e.g. GREENWAY (1962). Plants may find many different solutions to the problems of the first two levels, depending on their life form, breeding system, geographical occurrence and climatical conditions. However, it seems likely to assume, that given a high salt concentration in the protoplasm, mechanisms which enable endurance of salt are of a more uniform character among vascular plant species.

198 The aims of this study are firstly, to describe the response to salt of some ecologically significant growth parameters (viz. fresh and dry mass, vegetative propagation); secondly, to investigate ways of adaptation to salinity (viz. osmotic adaptation (BERNSTEIN 1963) and varying degree of succulence); thirdly, to explain this osmotic adaptation by means of mineral analysis and finally to study a possibility in which toxicity of excess salt could be rendered harmless i.e. regulation of the content of organic acids (OSMOND 1967). J unCU8 bufoniu8 ssp. bufoniu8 has been involved in this comparative study to test whether the response to salt of the other Juncus species may be regarded as a typical halophytic feature or not.

Materials and Methods Four Juncu8 species were studied, viz. J. maritimu8 LAMK. Juncu8 gerardii LOISL., J. alpinoarticulatu8 ssp. atricapillu8 (DREF. ex LANGE) and J. bufoniu8 L. ssp. bufonius. The first three species are native to a salt marsh at the North Sea Island Schiermonnikoog, latitude 53° 30', longitude 60 15'. Seed of J. bufoniu8 ssp. bufoniu8 was collected from an extensive sandy area near Amsterdam, with water-logging conditions. Plants were grown from seed in a naturally illuminated greenhouse, 200C ± 10C, 70% R. H. Four-weeks old seedlings were placed on a circulating nutrient solution; using a reservoir of solution of 700 I no change of it during the experiment was necessary. Compositionofthenutrient solution was 3.5MmCa(N0 3 )2; 5mMKN0 3 ; 1 mMKH2P04; 1 mMMgS04 ' 7H 20; 0.2 mM FeS04' 7 H 20 as a Na 2 ' EDTA-complex; 4.10- 3 mM CuS04 ' 5 H 20; 5.10-2 mM H 3 B0 3 ; 5' 10-2 ZnS04 . 7 H 20; 10-3 (NH 4)6 • M0 7 • 24 ' 4 H 2 0; 3 . 10-2 mM MnS04 . 4 H 20' pH was 6.8 ± 0.3 during the whole experiment, oxygen content was about 8.9 mg 2 /1. NaCI was applied stepwise (intervals of four days), in order to avoid an osmotic shock. Salt concentrations in the experiment were 0 mM NaCI, 60 mM NaCl, 150 mM NaCI and 300 roM NaC!. 40 plants of each species were grown at all four salt levels. After two months of growth, plants were harvested and shoots and roots were washed extensively in demineralised water. Leaf area was measured by means of an optical leaf area planimeter. Plants were frozen or dried at 90°C. Data on osmotic potentials of undiluted pressed plant sap were obtained with the use of a temperature osmometer (KNAUER, Berlin). For other analyses fifty times diluted plant sap was used. Cations were determined by atomicabsorptionspectrofotometry, Na+, K+ (emission), Ca2+, Mg2+ (absorption), Cl- was titrated electrometrically by a chloro-counter (MARIUS, Utrecht). Phosphate was determined colorimetrically after CHEN et a!. (1956). Sugar content was determined using the Antron-method (MORRIS 1948). The acid soluble fraction was prepared by destruction of dried plant material of which first the water soluble fraction (extraction with aqua dest., 90°C, 60 minutes) was removed, with a 7: 1 mixture of HN0 3 (65 %) and HCl0 4 (70 %). Chlorophyll was extracted from fresh plants with aceton after BRUINSMA(1963). Organic acids were identified and measured, after having prepared methylesters of the acids by addition of methanol to undiluted plant sap, on a HEWLETT PACKARD 5750 gas chromatograph, column packing 12.5% Diethylene Glycol Succinate/Chromosorb, W 45-60 mesh (ZAURA and METCOFF 1969). For malate and citrate column temperature was set at 175°C, for oxalate and succinate at 135 °C (HOLDEMAN and MOORE 1972). Results were analysed statistically by analysis of variance. Procedures for calculating and testing F-values and L.S.D. values are mentioned by SOKAL and ROHLF

°

(1969), significance level used was


°

= 0.05.

Results Growth It is shown in Fig. 1 that Juncu8 species studied, all have their growth optimum under fresh water conditions. This is true for all growth parameters. As ecological relevant differences between the species can be mentioned that at 150 mM NaCl

199

An Ecophysiological Study on the Responsc to Salt

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Fig. 1. The growth response to salt of four Juncu/J species. L. S. D. = Least Significant Difference; n. s. = not significant at IX = 0.05 level; * = significant at IX = 0.05 level; ** = signifikant at IX = 0.01 level; *** = significant at IX = 0.001 level. X 10 = values magnified 10 times to obtain figures which can be plotted. Juncu8 bufoniu8 ssp. bufoniu8 is annual and does not form a rhizome.

J. alpino-articulatu8 ssp. atricapillu8 shows hardly any growth with respect to fresh and dry weight production and only some three new shoots have been formed. On the other hand J. gerardii shows even at 300 mM NaCI some biomass production, and up to ten new shoots are formed, whereas 300 mM NaCI showed to be lethal in the cases of J. alpino-articulatu8 and J. bufoniu8 ssp. bufoniu8. This response to salt of the latter species confirms the characterization as glycophyt from the literature (REICHELT 1964). J. maritimu8 is less comparable on account of its slow growth under the experimental conditions even under fresh water conditions. However J. maritimu8 should yet be regarded as a salt tolerant species, because there is no pronounced negative influence of increasing salinity. It is worthwhile to notice that the annual species J. bufoniu8 ssp. bufoniu8 exhibits some kind of vegetative propagation, particularly under fresh water conditions i.e. one individual produces a bush of some 40 new shoots. Taking the relative degree of growth reduction as a criterion for salt tolerance, tolerance to salt diminishes in the order from J. gerardii, J. maritimu8, J. alpinoarticulatu8 ssp. atricapillu8 to J. bufoniu8 ssp. bufoniu8, which is in agreement with zonation sequence in the natural habitat. The vigorous growth of J. bufoniu8 ssp. bufoniu8 at 0 mM and 60 mM explains its sudden occurrence on- temporarily-desalinized dutch reclamations as described by REICHELT (1964).

.J.

200

ROZEMA

Osmotic adaptation Salinity appeared to reduce values of several components of plant growth. Since most halophytes are unable to exclude selectively sodium chloride during ion uptake, or to desalinate seawater - except in the case of some mangroves with ultrafiltration as described by SCHOLANDER (1968) - sodium chloride enters by accompanying water in uptake. Osmotically active accumulated ions may fulfil a useful task by adjusting the osmotic potential of plant tissue to enable water uptake in a saline medium. Fig. 2 shows the osmotic adjustment of the four Juncu8 species investigated to increasing salinity. J. alpino-articulatu8 ssp. atricapillu8 and J. butoniu8 ssp. butoniu8 are not capable to adjust at 300 mM NaCl. All plants died off, or, as in the latter species, just some shoots remain green and show overadjustment (W AISEL 1972), whereas roots already expire. Only J. maritimu8 and J. gerardii adapt over the whole salinity range, tolerating sodium and chloride concentrations, which are lethal to J. butoniu8 ssp. butoniu8. Looking at the mineral composition of plant sap of shoot and root tissue and the contribution of the components to the osmotic potential, the relatively small contribution of potassium ions in the relatively salt sensitive species J. blljoniu8 ssp. butoniu8 and J. alpino-articulatu8 ssp. atricapillu8 is remarkable. The latter species seems to be somewhat overadjusted at 150 mM, which might explain its natural occurrence in a zone which is approximately the mean upper level of inundation in winter. J. maritimu8 and J. gerardii differ in their osmotic adaptation, in

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An Ecophysiological Study on the Response to Salt

201

that the former maintains more or less the same osmotic potentials at all salt levels, whereas the latter follows stepwise the salt content of the medium, according to field observations of WALTER (1968). Further it is shown that relatively high osmotic potentials can be achieved in these species by large amounts of potassium, particularly at 0 mM NaCl. The increasing contribution of CI- and Na+ to the osmotic potential with increasing salinity is obvious in all species. The decrease of the partial osmotic potential of K+ is far less significant. This indicates a K+ uptake mechanism which is independent of a Na+ uptake mechanism. In most cases osmotically active ions of Na+, CI- and K+ may account for about 75-80 % of the osmotic potential of the plant sap. Calculations on the total contribution by Mg2+, Ca 2+, P0 43-, and sugars (Table 1) do not even add up to 1 or at most 2 atmospheres. In none of these data on ion contents a significantly negative or positive influence on account of salinity could be demonstrated. Apart from the somewhat lower osmotic potentials of root pressed sap the mineral composition of pressed root sap does not differ"essentially from the data obtained from the shoots. Succulence Halophytes may develop xeromorphic structures under saline conditions, which is regarded as a direct effect of the uptake of sodium (WAISEL 1972), and is believed to be one of the major factors in salt tolerance by improving a plant's water relations. However no significant increase of the degree of succulence was found in the leaves of the four JunCU8 investigated, as is shown in Table 2. Thus, these JunCU8 species are not to be considered as succulent-halophytes as described by WAISEL (1972). Chlorophyll content Reduction of growth can be explained by a lower content of chlorophyll in leaf tissues, which might be in agreement with the frequent appearance of chlorosis in shoots under saline conditions. Table 3 shows that in leaves of J. maritimu8 and J. gerardii no significant effect of salinity is found, whereas in J. alpino-articulatu8 ssp. atricapillu8 a slight decrease, and in J. bufoniu8 ssp. bufoniu8 an increase is obtained. These results confirm the salt tolerance of the first two mentioned species, whereas the response of the other species cannot be understood easily. Salt damage may lead to higher chlorophyll content (STROGONOV 1964, cited by WAISEL 1972), but also the reverse effect has been noticed. Acid soluble minerals Plant press sap contains water soluble ions. Toxicity of excess sodium and chloride ions !night be reduced by accumulation of these ions into cell wall or cuticula material or into supporting structures; besides this, sodium and chloride ions may be precipitated as unsoluble crystals. LARCHER (1973) and WAISEL (1972) state that the salt resistent J. maritimu8 and J. gerardii remove large amounts of excess salt by abscis-

202

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Table 1. Additional composition of plant press sap in relation to salinity of the nutrient solution OmM

Shoot

Root

60mM

M g 2+

Ca2+

POl- sugars

M g 2+ Ca2+

P0 4 3- sugars

J. maritimus J. gerardii J. alp.·art. ssp. atricapillus J. buf. ssp. bufoniu8

0.23 0.05 0.04

0.108 0.04 0.04

2.64 1.88 1.7l

1.37 1.09 1.62

0.06 0.04 0.07

0.101 0.040 0.050

2.65 1.77 1.59

1.55 1.10 0.87

0.060

0.045

0.93

0.86

0.045 0.040

1.02

0.80

J. maritimu8 J. gerardii J. alp.-art. ssp. atricapillus J. buf. ssp. bufonius

0.190 0.01 0.024

0.080 0.20 0.04

2.47 1.12 1.75

1.03 1.75 1.10

0.023 0.091 0.02 0.015 0.067 0.040

2.70 1.09 2.64

1.17 0.90 0.14

0.06

0.030

1.15

0.59

0.04

1.03

0.63

0.018

sugars = total content of soluble sugars expressed as g glucose/liter plant sap, Mg++, Ca++, P0 4 3-

Table 2. Effect of salinity (mM NaCI) on the degree of succulence, calculated as water content (g)/leaf surface (cm2) of the shoot of four Juncu8 species (averages of four replications) species

Juncu8 maritimus Juncu8 gerardii Juncu8 alpino-articulatus ssp. atricapillu8 Juncu8 bufonius ssp. bufoniu8

*

salinity

OmM

60mM

150mM

300mM

0.05026 0.02630 0.03954

0.04796 0.02890 0.04038

0.04676 0.02696 0.04371

0.03586 0.02133

0.03445

0.03380

0.03113

statistical significance L.S.D. value (IX = 0.05)

*

0.0085 0.0029 0.0112

*

0.0108

plants died off at this salt level.

sion of salt saturated leaves. Therefore acid soluble minerals were analysed. The results are given in Fig. 3 and 4. Whereas almost no Ca 2+ and Mg2+ ions could be demonstrated in pressed plant sap, acid extract of the dry material contains larger amounts of these ions, relatively, as compared to Na +. It may be calculated that from the total content of Na+ of a Juncus plant, only a small fraction (1-5 promille of the total contE'ut) is found in the acid soluble fraction. It can be seen from Fig. 3 and 4, that an increased salinity leads to increased N a + contents. Again, the K + content hardly decreases as salinity increases and also this diminution in the relatively tolerant species is lower than in the sensitive species. A decrease of Ca 2+ when salinity increases, can also be demonstrated. A similar relation for Mg2+ is not valid.

203

An Ecophysiological Study on the Response to Salt (mM NaCl). The values are the means of five replications Statistical significance L.S.D. (
300mM

150mM

Mg2+ Ca2+

POl- sugars

1.49 0.92 0.83

0.05 0.0l0 0.11 0.07 no growth

2.69 2.32

0.95

1.41

no growth

0.150 0.02 0.07l

0.17l 1.79 0.030 1.23 0.08 1.78

1.33 0.77 1.05

0.07 0.10 0.02 0.075 no growth

0.05

0.041 1.36

1.49

no growth

P0 43- sugars

Mg2+

Ca 2+

0.06 0.03 0.08

0.022 1.24 0.06 1.20 1.83 0.13

0.059

0.30

1.79 1.59

1.81 2.00

1.23 0.97

Mg2+

Ca2+

P0 43- sugars

0.090 0.090 0.040

0.073 0.003 0.060

0.67 0.45 0.95 0.83 0.650 0.700

0.032

0.020

0.40

0.87 0.019 0.040

0.095 0.091 0.05

0.95 0.57 1.65 0.43 2.620 2.800

0.040

0.032

0.85

0.40

0.94

expressed in gjliter plant sap. Table 3. The effect of NaCI concentration of the nutrient solution (mM NaCI) on the content of chlorophyll (mg chlorophylljg freshweight of four JuncuB species species

chlorophyll

60

0

150

300

F-

L.S.D. value

value

(
J. maritimuB

a b a+b

1.405 .611 2.043

.991 .050 1.041

1.415 .352 1.768

1.267 .330 1.597

n. s n. s n. s

1.580 0.356 2.561

J. gerardii

a b a+b

1.232 .365 1.597

1.242 .340 1.581

1.310 .369 1.679

1.172 .320 1.492

n. s n. s n. s

.410 .126 .528

J. alp. ·art. ssp. atricapilluB

a b a+b

1.227 .232 1.459

1.018 .324 1.327

.892 .266 1.158

**

0.103 0.081 0.121

J. bufoniu8 ssp. bufoniuB

a b a+b

.493 .146 .639

.613 .212 .824

1.058 .298 1.356

n. s

* * *

*

0.135 0.098 0.167

Organic acid meta bolism Organic acids might play an important role in the ionic balance of plant metabolism (OSMOND 1963). In many halophytes oxalate is supposed to function as a counter ion for excess cations. Presence of calciumoxalate crystals in vacuoles is a well-known phenomenon in piant life. Also it has been demonstrated that organic acids can reduce toxic influence of large amounts of cations by forming complexes as is discussed for zinc tolerance in plants by MATHYS (1975). A first attempt to establish the total amount of acids in plant sap (by means of a titration with 0.005-0.010 N NaOH) did not show a significant effect of salinity

204

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Fig. 4. Mineral composition of an acid extract of dry material of two relatively salt-sensitive JuncUBspecies. At 300 mM NaCI no growth was observed of J. alpino.articulatu8 ssp. atricapillu8 and strongly reduced growth of J. bufoniu8 ssp. bufonius.

205

An Ecophysiological Study on the Response to Salt

on total acid amount, neither did potentiometric titration till pH 8.40 (pH pressed sap, 20 X diluted: 5.75 ± 0.2), nor did titration of HN0 3 produced after precipitation of acids with a solution of Ca 2 +(N0 3 )2 -. Titration curves did not show sharp changes. These results suggest no important contribution of organic acids to the osmotic potential. Total titrated acid amount was far less as compared with a mixture of 0.01 M malate, 0.01 M citrate, 0.008 M oxalate, 0.003 M succinate. This mixture showed an osmotic potential of - 0.04 atm. These amounts will probably not affect the ionic balance. In further experiments, undiluted pressed sap samples of shoot tissue were examined by means of gas-liquid chromatography. Four main peaks could be distinguished in all chromatograms. Identification revealed malate and citrate to be the most important organic acids (Table 4). So, in contrast with many other halophytic species (e.g. Atriplex species) the Juncus species do not contain large amounts of water soluble oxalate. Table 4 shows the negative effect of salinity on the content of most acids in all species, although citrate amounts are less influenced as compared with malate. The oxalate amount does not increase at increased salinity. Totalling the amounts of organic acids in pressed plant sap, the total contribution to the osmotic potential does not exceed - 1.0 or at most - 2.0 atm. Table 4. Effect of salinity (mM NaCI) on the content of malate, citrate, oxalate and succinate (mM, of the press sap of 3 Juncu8 species* (means of 4 replications) species organic acid

salinity mM NaCI

Juncu8 ge'f'ardii

Juncu8 alp.-art. ssp. atricapillu8

Juncu8 bufoniu8 ssp. bufoniu8

malate

0 60 150

50.800 37.620 30.980

25.600 6.600 2.600

16.800 9.600 0.146

citrate

0 60 150

19.240 16.820 16.060

10.000 9.350 8.580

22.800 14.300 6.000

oxalate

0 60 150

0.471 0.282 0.050

0.478 0.094 0.013

1.884 1.906 1.879

succinate

0 60 150

1.958 4.742 3.000

3.844 4.540 4.260

2.844 1.122 0.732

* J. ma'f'itimu8 yielded insufficient press sap, so that no values are available, the same applies for the absence of data at 300 mM. Discussion Growth reduction caused by increasing salinity is a well-known phenomenon (e.g. PHLEGER 1971), although growth of a few strict halophytes may even be stimulated at 250 mM NaCI (e.g. Salicornia europaea L., AusTENFELD 1974). At this salt

206

J.

ROZEMA

concentration the Juncus species investigated can only tolerate salinity. Using the degree of growth reduction as a measure of salt tolerance, these Juncus species may be characterised as euhalophytes, resisting salt to different degrees (WAISEL 1972). J. gerardii shows stronger growth at 300 mM NaCI, as compared with J. maritimus, which is however less affected by salt in general. J. alpino-articulatus ssp. atricapillus is already damaged by salt at 150 mM, which could account for its concentrated presence in a sn:.all fringe in natural zonation, a little above the upper level of inundation. J. bufonius ssp. bufonius, which is considered to be a glycophyt can not tolerate 300 mM NaCl. Although leaves of Juncus species are tough, at least in the field, plants grown on the culture solutions remained somewhat weak. This may be due to the lack of Silicium (ERNST, personal communication). Leaf or stem succulence generally occurs in halophytes (W AISEL 1972). Succulence degree as defined in STEUBING (1965), did not Iaise at increasing salinity. KAPPEN and MAIER (1973) also established no increase in the degree of succulence as a regulating mechanism for lowering the chloride content in Halimione portulacoides (L.) AELLEN. This species was regarded as a succulent halophyt by STEINER (1939), which is confirmed by the results of BAUMEISTER and KLOOS (1974). Chlorophyll content of leaves of J. gerardii and J. maritimus is not significantly influenced by increase of salinity, which agrees with their relatively high salt tolerance. On the other hand, the lower content in J. alpino-articulatus ssp. atricapillus and the increment of chlorophyll amounts in J. bufonius ssp. bufonius make an unequivocal conclusion questionable. So we cannot simply subscribe the theory of STROGONOV (1964), which suggests, that a decreasing chlorophyll content is caused by destruction of chlorophyll which has lost its bond with chloroplasts at high salt concentrations. Osmotic adaptation reveals over-adjustment of J. alpino-articulatus ssp. atricapillus and J. bufonius ssp. bufonius at 150 mM NaCI and 300 mM NaCl, although the other Juncus species adapt by limiting the concentration of sodium chloride in root and shoot tissue. At 0 mM NaCI J. maritimus and J. gerardii maintain osmotic potentials of - 18.8 atm. and - 9.40 atm respectively. In both cases absorption of potassium ions forms the major contribution to the osmotic potential, and not the sugar content as could be expected from studies of glycophytes, where sugar content may contribute more than 50 % of the total osmotic potential (STEINER 1939). Whereas Na+ and CI- concentration of the pressed sap increases regularly with increasing salinity, the antagonistic feature for K+ can not be established. Potassium concentrations seem to be constant over the whole range of salinity, although uptake of K+ at high salt concentrations may become difficult because uptake sites at the mem brane are occupied by N a + ions (EpSTEIN 1972; W AISEL et al. 1966). The rate of potassium uptake is independent of sodium uptake rates. This may form a decisive contlibution to the relative salt tolerance of J. maritimus and J. gerardii. RAINS and EpSTEIN (1967) drew a similar conclusion with regard to salt tolerance in leaves of

A vicennia marina.

An Ecophysiological Study on the Response to Salt

207

In a field study ALBERT and KINZEL (1973) tried to classify many halophytes of the Neusiedlersee. They distinguish the Gramineae, Juncaceae and Cyperaceae as represfmting a physiotype with preferential uptake of K+, which is in agreement with our results. Using the K+/Na+ ratio as a criterion for the same purpose seems to be contestable, because of the dependency of this ratio on the salinity of the substrate. Our study demonstrates a changing K+/Na+ ratio from 15.8 (0 mM), via 3.1 (70 mM) and 1.3 (150 mM) to 1.3 (300 mM). An analysis of the mineral composition of the acid soluble fraction of dry plant material shows rather high amounts particularly of Ca 2 + and to a lesser extent Mg2+ as compared to plant pressed sap., which is in agreement with AUSTENFELD (1974). In this extract certain antagonistic relations in the contents of Na+ and K+ and of N a + and Mg+ exist. Only a small fraction (1- 5 pro mille ) of the total amount of Na + and K+ is found in the acid soluble extract. The relatively high content of Ca2+ in this extract agrees with its general occurrence in the middle lamella of cell walls (EpSTEIN 1972). Although Ca 2+ concentrations in pressed plant sap appeared to be low, no large amounts of oxalate could be demonstrated. Increasing salinity does not enhance the oxalate content of pressed sap, such as is the case in Salicornia europaea L. (AuSTENFELD 1974). A stimulation of oxalate production by CI- and Na+ as described by WILLIAMS (1960) seems to be absent in the Juncus species investigated. Organic acids may reduce the toxic effects of excess salts as was demonstrated for zinc, which forms complexes with malate (MATHYS 1975). However, in the Juncus species, increasing salinity does not have a positive influence on the content of malate, citrate, oxalate and succinate. Studying frost resistance in Halimione portulacoides (L.) AELLEN. KAPPEN and MAIER (1973) found enhanced citrate concentrations and to a lesser extent higher malate contents during a period with increased CI+ concentration in leaf tissue, indicating a protective function of organic acids to salt stress. Looking at the Juncus species, one may notice, that the relatively salt sentitive species show a stronger reduction in the content of malate as compared with J. gerardii. Especially citrate amounts are hardly reduced at 150 mM NaCl. So, at least some organic acids i.e. citrate may playa role in the metabolism of salt tolerance of these Juncus species, but less clearly as in some Chenopodiaceae with regard to oxalate (OSMOND 1963). Besides a possible function in the ion balance, organic acids may playa role in osmotic adaptation (BERNSTEIN 1961). However, the amounts of organic acids lead to a contribution of the osmotic potential of only - 2 atm. at the most. So, the role of organic acids in the salt tolerance seems to be rather small as yet.

Acknowledgements The author is indebted to Prof. Dr. v\T. H. O. ERNST for his continuous commenting and critical advice. Thanks are due to Mrs. T. F. LUGTENBORG and to Mr. H. J. M. NELISSEN for technical assistance, to Mr. G. W. H. VAN DEN BERG for drawing the figures and to Drs. F. W. VAN DER VEGTE for correcting the English text.

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Received July 1, 1975. Author's address: Drs. J. ROZEMA, Department of Ecology, Biological Laboratory, De Boelelaan 1087, Amsterdam-Bv., Netherlands.

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