V.5

Biochemical and Cytochemical Stud ies of Suaeda maritima τ. J. Flowers and J . L. Hall School of Biological Sciences, University of Sussex England

I. Introduction II. Materials and Methods

III. Results . . . . A. G r o w t h . . . . B. Enzyme Activity C. Protein Synthesis . D. Cytochemical Observations

IV. Discussion . . . . References . . . .

357 358 358 358 361 362 364 366 368

I. I n t r o d u c t i o n

In general plants are not very tolerant of high ionic concentrations and the growth of most crop plants is inhibited by sodium chloride levels above 0· 1 Μ (Nieman, 1962). There are, however, a number of plant species, including many members of the Chenopodiaceae, that are exceptionally tolerant of saline conditions and whose growth may even be stimulated by the high levels of sodium (048 N ) and chloride (0-56 N ) found in sea water. Growth under these conditions must, however, be associated with an adjustment of the cellular water potential at least to less than - 3 0 bars, the osmotic potential of sea water, if water is to be maintained within the symplast. This could be achieved either by the uptake of ions from the environment or, if ion exclusion occurs, by the internal production of an osmotically active solute. In the former case the ions may be either sequestered within the vacuoles or distributed throughout the protoplast. This second alter­native is exhibited by halophilic bacteria and the high cytoplasmic ion content is associated with the evolution of enzyme systems which require high salt concentrations for optimal activity (Baxter and Gibbons, 1956; Larsen, 1967).

357

358 Τ. J . FLOWERS AND J . L HALL

The question thus arises of whether the plant enzymes are tolerant of high sodium chloride levels, as found in halophilic bacteria, or whether they are protected from these high ionic levels by the sequestering of ions within the vacuoles. The determination of the effect of sodium chloride on enzyme systems from halophytes in vitro should therefore indicate whether the enzymes are adapted to these saline conditions and also give an indication of the levels of salt to be found in the cytoplasm.

This report describes a biochemical and cytochemical study of Suaeda maritima grown under saline and non-saline conditions. The effect of increasing salt concentration was studied in relation to a number of soluble enzymes and to an amino acid-incorporating microsomal fraction. The sites of ATPase activity and salt accumulation were investigated by cyto­chemical techniques. The results are discussed in relation to the mode of salt accumulation and salt tolerance by halophytes.

I I . M a t e r i a l s a n d M e t h o d s

Suaeda maritima (L . ) Dum. was grown much as previously described (Flowers, 1972). Briefly, seeds were germinated in sand and transferred to solution culture after about 3 weeks. For plants grown under saline conditions, sodium chloride was added at not more than 2 g l ~ ^ day~^ to give a final concentration of 0-34 Μ sodium chloride and the plants were used approxi­mately five weeks after transfer to the solution culture. Peas (Pisum sativum L. cv. Alaska) were grown either in sand irrigated with culture solution or on grids over water.

Microsomal fractions were prepared and amino acid incorporation measured as described elsewhere (Hall and Flowers, 1973). Chlorophyll con­centration was estimated by the method of Arnon (1949). Otherwise the analytical methods and enzyme preparations were as described by Flowers (1972).

For the cytochemical studies, ATPase activity was localized by the method described by Hall (1971). Material for electron microscopy was processed by standard procedures and embedded in an Epikote-Araldite mixture. Sections were post-stained with uranyl acetate and lead citrate unless stated otherwise.

I I I . R e s u l t s

A. G r o w t h

Suaeda maritima is a characteristic plant of the lower levels of salt marshes and thus tolerates high levels of salinity in the field. Grown in culture solution, both fresh and dry weight production were maximal with the

v.5 STUDIES OF SUAEDA MARITIMA 359

addition of 0-2-0·35 Μ sodium chloride (Fig. 1). Even at 0-68 Μ sodium chloride, grow^th measured in these terms was 1-5 times as great as in the absence of sodium chloride. The protein content per plant was only about 1-3 times as great under saline conditions (0-34 M ) and when expressed on a dry weight basis was half that of plants grown in the absence of salt (Table I). In contrast, peas were very sensitive to sodium chloride (Bernstein and Hayward, 1958; Nieman, 1962) and both fresh and dry weight were much reduced by 01 Μ sodium chloride.

The increase in weight of the Suaeda plants grown in the presence of salt was associated with an increase in leaf size. Sections of leaves showed that this was largely associated with an increase in the size of the cells surrounding the vascular tissue (Fig. 2). N o distinct morphological differences could be detected between sections of stems and roots of plants grown in the presence and absence of salt.

LSDp=005

_1_ - i -0 0 1 0 2 0 3 0 4 0 5 0 6 0 7

1% 2 % 4 % Molarity of NcCL in culture solution

FIG. 1. The effect of sodium chloride added to the culture solution on dry weight (open symbols) and fresh weight (black symbols) of Suaeda maritima (circles) and Pisum sativum (triangles). Reproduced with permission from Flowers (1972).

As well as the marked increase in fresh and dry weight of Suaeda produced by 0-34 Μ sodium chloride, there was a pronounced difference in the colour of the plants. Those grown in the presence of sodium chloride were lighter in colour than those grown in its absence and had a specific chlorophyll content lower by some 3-5 times (Table I) . Salt also reduced the dark respira­tion rate of the leaves (Table I) .

Salinization of the culture solution causes a considerable increase in the sodium and chloride content of the plant tops. Sodium and chloride levels

360 Τ. J . FLOWERS AND J . L HALL

FIG. 2. Paraffin-embedded sections of leaves of Suaeda maritima grown in the presence (a) and absence (b) of salt, χ 125.

were some thirty times higher in plants grown in the presence of 0-34 Μ sodium chloride, with concentrations reaching about 0-6 Ν when based on the overall water content. Potassium levels, on the other hand, were some 5-5 times lower in the salt-grown plants. The pea plants, whose growth was so noticeably reduced by salt showed a similar pattern of ion uptake although

v.5 STUDIES OF SUAEDA MARITIMA 361

Added sodium chloride (M) 0 0-34

Protein (mg/g dry weight) 60 30 Chlorophyll (mg/g fresh weight) 1-34 0-38

(mg/g dry weight) 103 3-6 Respiration (^1 0 2 / g fresh weight/h) 349 117

Ion contents Sodium (mequiv/g dry weight) 0-21 66

(mequiv/g water) 002 0-65 Chloride (mequiv/g dry weight) 020 61

(mequiv/g water) 0-02 059 Potassium (mequiv/g dry weight) 1.77 0-32

(mequiv/g water) 019 003

of much lower magnitude. The sodium, potassium, and chloride levels recorded after growth in 0-2 Μ sodium chloride were 0-2, 0-1 and 0-3 Ν respectively.

The major difference, therefore, between the response of the two species to saline conditions was the stimulation of growth in Suaeda associated with a high ion content and the inhibition of growth in Pisum associated with a low ion content.

B. Enzyme A c t i v i t y

T o study the effect of salinization on enzyme systems of halophytes and salt-sensitive plants, the effect of sodium chloride was determined on the activity of four soluble enzymes isolated from the tops of Suaeda and Pisum. The enzymes investigated were malic dehydrogenase, glucose-6-phosphate dehydrogenase, peroxidase and an acid phosphatase. The effect of 0-17 Μ and 0-33 Μ sodium chloride was determined at the optimal pH and at pH values above and below this. For the dehydrogenases, the effect of added salt was independent of the pH and in both cases the enzymes prepared from Suaeda, whether grown in salt or not, were just as sensitive to salt as the enzymes prepared from Pisum (Table I I ) .

Unlike the dehydrogenases, the effect of sodium chloride on peroxidase was very much a function of the pH, the greatest inhibition occurring at pH values (4-9) on the acid side of the optimum (pH 5-5) with no significant effect being observed at pH 70. However, as with the dehydrogenases, there was no difference in the response to added salt between the enzymes prepared from Pisum and Suaeda (Table I I ) .

Acid phosphatase was assayed using A T P as substrate and the activity from both species was maximal in the pH range 4·5-5·5. This acid phos­phatase was relatively insensitive to added sodium chloride, the only

TABLE I . Some characteristics of Suaeda grown in the presence and absence of sodium chloride

362 Τ. J . FLOWERS AND J . L HALL

Enzyme Molarity of added sodium chloride

0 0167 0-33 LSD = 0-05 Malic dehydrogenase Pisum 54 3-3 (61) 2-2 (41) 0-5 (ZlOD/min/mg protein at Suaeda-(f 3-7 2-2 (59) 1-2 (32) 02

pH 7-1) Suaeda-0-34 41 2-5 (64) 1-6 (39) 0-35

Glucose-6-phosphate dehydrogenase Pisum 13-9 103 (74) 60 (43) 1-7

(zlOD X 10^/min/mg protein) Suaeda-O 300 192 (64) 13-5 (45) 4.4

(pH 7-1 for Pisum and 7-8 for Suaeda) Suaeda^'34 25-2 18-8 (75) 14-1 (56) 2-2

Peroxidase (zlOD/min/mg protein at Pisum 90 5.3 (59) 3-2 (36) 1-3

pH 49) Suaeda-ϋ 6-6 3-5 (53) 2-8 (42) 0-5 pH 49) Suaeda-0-34 4-3 1-9 (44) 1-3 (30) 04

Acid phosphatase Pisum 106 116 1 (109) 113 1 (107) 28-3 (nmol Pi/min/mg protein at Suaeda-0 30-3 28-8 (95) 27-2 (90) 1-5

pH 5) Suaeda-0'34 944 919 (97) 83-1 (88) 4.5

" Suaeda grown in the absence of sah. The figures in parentheses are the rates relative to those in the absence of added salt.

significant effect observed on the activity from Pisum being a 34 % inhibition by 0-33 Μ salt at pH 7-0. The enzyme activity from Suaeda was sensitive to 0-33 Μ sodium chloride at all three pH values employed (40, 5-5, and 6-6) with a mean inhibition of 19 %. It may be inferred from these results that the enzymes of Suaeda are not similar to those of the halophihc bacteria that are adapted to high sodium chloride concentrations but are in some way pro­tected from the high salt levels that build up in the aerial parts.

C. Pro te in Syn thes is

A microsomal fraction has been prepared from Suaeda shoots grown under both saline and non-saline conditions which incorporated ^"^C-leucine into protein at a rate of 468 χ 10"^^ and 796 χ 10"^^mol/mg protein/ 15 min respectively. The effect of increasing sodium chloride concentration on the rate of amino acid incorporation in the presence of 50 mM potassium is shown in Fig. 3. Incorporation by microsomal fractions from both salt-and non-salt-grown plants was inhibited by sodium chloride to a similar extent and this was marked even at the lowest concentration (25 mM) employed. The inhibition was independent of the time of incubation in the assay medium (from 15 to 40 min) and of both magnesium and potassium

TABLE Π. The effect of sodium chloride, added in vitro, on the activities of some enzymes of Pisum and Suaeda

v .5 STUDIES OF SUAEDA MARITIMA 363

5 0

0

100

50

No NaCl

0 34m NaCL

25 5 0 100 2 0 0 NaCL concentration (mM)

FIG. 3. The effect of sodium chloride on the relative incorporation rate of ^'^C-leucine into the microsomal fraction of Suaeda grown under saline and non-saline conditions. In addition to the sodium added as chloride, all the assays contained 15 mw sodium as the salts of phosphocreatine and A T P .

TABLE I I I . The effect of sodium and potassium concentrations on the rate of incorporation of leucine into the microsomal fraction (from Hall and

Flowers, 1973)

Potassium chloride (mM)

Sodium chloride" (mM)

Leucine incorporated (10" mol/mg protein/

15 min)

50 0 447 100 0 253 200 0 85

50 100 91 100 100 106 200 100 59

100 50 159 100 200 93

LSD. p = 0 0 5 158 " See also Fig. 3.

100 I

364 Τ. J . FLOWERS AND J . L HALL

concentrations. In fact at concentrations greater than 50 mM potassium was as inhibitory as sodium (Table I I I ) . Similar effects of monovalent cations have been reported for rat-liver microsomal preparations (Samson, personal communication).

D. C y t o c h e m i c a l Observa t ions

The general fine structure of the root cells (Figs 4 and 6) showed no distinct differences from the structure of non-halophytes and no marked changes in fine structure were observed between Suaeda roots grown under non-salt and saline conditions. Roots incubated in the ATPase staining medium showed heavy activity at the plasmalemma (Fig. 4) and at the tonoplast

FIG. 4. Electron micrograph of root cell of Suaeda maritima grown in the presence of salt and incubated for ATPase activity showing heavy staining in the plasmalemma and plasmodesmata. χ 38,000.

(Fig. 5). Frequently cells showing high tonoplast activity did not show staining at the plasmalemma. The fine precipitate visible in the vacuoles of the salt-grown plants (e.g. Fig. 6) was also present in Suaeda grown in the absence of salt. Such precipitates are not normally visible in the roots of glycophytic species such as peas and maize.

V.5 STUDIES OF SUAEDA MARITIMA 365

FIG. 5. Electron micrograph of vacuolated root cell grown and treated as in Fig. 4. Showing ATPase activity at the tonoplast. χ 42,000.

FIG. 6. General appearance of root cells of Suaeda maritima grown in the presence of salt and showing the fine precipitates in the vacuoles, χ 14,000.

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Τ. J . FLOWERS AND J . L HALL

FIGS 7 and 8. Electron micrographs of root cells of Suaeda maritima grown in the presence of salt and showing membrane vesiculations from the plasmalemma (Fig. 7) and tono­plast (Fig. 8). Fig. 7. X 34,500. Fig. 8. χ 27,500.

In the highly vacuolated cortical cells behind the meristem and in the leaf cells many large vacuoles filled with a fine precipitate were observed within the main vacuole close to the cell surface. These vesicles appeared to arise both from plasmalemma (Fig. 7) and tonoplast (Fig. 8) and frequently exhibited ATPase activity.

I V . D i s c u s s i o n

The increase in growth induced in Suaeda by sodium chloride is typical of the more tolerant halophytes such as the Salicornia spp. (van Eijk, 1939; Baumeister and Schmidt, 1962; Webb, 1966). In both, fresh and dry weights are promoted particularly by 0·2-0-3 Μ sodium chloride. Growth in these saline solutions is accompanied by a massive increase in sodium and chloride in the tissues indicating an ionic adjustment of the water potential. Depression of potassium in the plant in the presence of high sodium chloride levels appears to be a fairly common response (Black, 1956; Ashby and Beadle,

v.5 STUDIES OF SUAEDA MARITIMA 367

1957; Williams, 1960; Greenway, 1962, 1968; Norkrans and Kylin, 1969). It must be realized however, that these data give no indication of the distribu­tion of sodium and potassium throughout the protoplast.

The four soluble enzymes prepared from Suaeda and from Pisum showed no significant difference in their sensitivity to high sodium chloride concen­trations added to the incubation medium. All, except acid phosphatase, showed appreciable inhibition. These results support the observations of Greenway and Osmond (1972) who reported no differences in salt sensitivity when comparing a variety of enzymes extracted from both salt-tolerant and sensitive species and from commercial sources. Thus unlike halophilic bacteria (Larsen, 1967; Hochstein and Dalton, 1968; Aitken et ai, 1970), in higher plants there is no evidence of adaptation to the high ionic levels found in the cells of salt-tolerant species. This lack of adaptation is also illustrated by studies of the eff'ect of sodium chloride on amino acid incor­poration into protein by a microsomal fraction isolated from Suaeda. Although the rate of incorporation was generally higher in the preparations isolated from the plants grown without salt, the addition of sodium chloride had a similar inhibitory eíTect in both systems. It is interesting that incor­poration was even inhibited by low sodium chloride concentrations (40 and 65 m M ) since these levels often stimulate isolated enzymes (Evans and Sorger, 1966). Salt has previously been shown to inhibit both amino acid uptake and incorporation into acid precipitates by excised pea root tips and there was some evidence that growth under saline conditions caused damage to incorporation sites (PoljakoiT-Mayber and Meiri, 1969). These results contrast with those obtained with halophilic bacteria where high potassium levels (4 M ) are necessary for ribosomal stability (Bayley and Kushner, 1964).

This lack of adaptation of various enzymic and protein-synthesizing systems to growth at high salt levels strongly suggests that these systems are not subjected to high ionic concentrations in the living cell. The apparent enigma in Suaeda of sodium-inhibited protein synthesis and yet sodium-stimulated growth may be reconciled if the cytoplasm is low in sodium which is sequestered in large amounts in the vacuoles. The consequential osmotic movement of water into the vacuole and increase in turgor is seen as the factor promoting growth.

The vesiculation apparent in both roots and leaves of Suaeda may be important with regard to the sequestering of salt within the vacuoles. The possibility cannot, however, be ruled out at this time that the vesicles are artefacts of fixation. Staining for ATPase activity showed a high reaction at the plasmalemma and it is interesting that Jennings (1968) has proposed that a sodium activated ATPase at the plasmalemma could bring about A T P synthesis in the presence of high levels of sodium in the external medium.

368 Τ. J . FLOWERS AND J . L HALL

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