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Cropping on saline land is restricted by the low tolerance of crops to salinity and waterlogging. Prospects for improving salt tolerance in wheat and barley include the use of: (i) intra-specific variation, (ii) variation for salt tolerance in the progenitors of these cereals, (iii) wide-hybridisation with halophytic ?wild' relatives (an option for wheat, but not barley), and (iv) transgenic techniques. In this review, key traits contributing to salt tolerance, and sources of variation for these within the Triticeae, are identified and recommendations for use of these traits in screening for salt tolerance are summarised. The potential of the approaches to deliver substantial improvements in salt tolerance is discussed, and the importance of adverse interactions between waterlogging and salinity are emphasised. The potential to develop new crops from the diverse halophytic flora is also considered.

Rice (Oryza sativa) is sensitive to salinity, which affects one-fifth of irrigated land worldwide. Reducing sodium and chloride uptake into rice while maintaining potassium uptake are characteristics that would aid growth under saline conditions. We describe genetic determinants of the net quantity of ions transported to the shoot, clearly distinguishing between quantitative trait loci (QTL) for the quantity of ions in a shoot and for those that affect the concentration of an ion in the shoot. The latter coincide with QTL for vegetative growth (vigor) and their interpretation is therefore ambiguous. We distinguished those QTL that are independent of vigor and thus directly indicate quantitative variation in the underlying mechanisms of ion uptake. These QTL independently govern sodium uptake, potassium uptake, and sodium:potassium selectivity. The QTL for sodium and potassium uptake are on different linkage groups (chromosomes). This is consistent with the independent inheritance of sodium and potassium uptake in the mapping population and with the mechanistically different uptake pathways for sodium and potassium in rice under saline conditions (apoplastic leakage and membrane transport, respectively). We report the chromosomal location of ion transport and selectivity traits that are compatible with agronomic needs and we indicate markers to assist selection in a breeding program. Based upon knowledge of the underlying mechanisms of ion uptake in rice, we argue that QTL for sodium transport are likely to act through the control of root development, whereas QTL for potassium uptake are likely to act through the structure or regulation of membrane-sited transport components.

Osmotic potentials and individual epidermal cell turgor pressures were measured in the leaves of seedlings of Suaeda maritima growing over a range of salinities. Leaf osmotic potentials were lower (more negative) the higher the salt concentration of the solution and were lowest in the youngest leaves and stem apices, producing a gradient of osmotic potential towards the apex of the plant. Epidermal cell turgor pressures were of the order of 0.25 to 0.3 MPa in the youngest leaves measured, decreasing to under 0.05 MPa for the oldest leaves. This pattern of turgor pressure was largely unaffected by external salinity. Calculation of leaf water potential indicated that the gradient between young leaves and the external medium was not altered by salinity, but with older leaves, however, this gradient diminished from being the same as that for young leaves in the absence of NaCl, to under 30% of this value at 400 mM NaCl. These results are discussed in relation to the growth response of S. maritima.

Summary Leaf material from the halophyteSuaeda maritima L. Dum. grown under both saline and non-saline conditions was fractionated under non-aqueous conditions in order to determine the ion content of various subcellular compartments. Fractions containing cell walls, nuclei and chloroplasts were successfully prepared and contents of DNA, chlorophyll, protein and Na+, K+, and Cl- determined. The cell wall fraction was not apparently heavily contaminated by the other fractions and had a low ion content although the nuclear fraction was contaminated by other organelles. The ion contents of chloroplasts were determined and the results discussed in relation to earlier microscopical data.

Comparisons were made of niche breadths and niche overlaps of halophytes growing in two emergent saltmarsh communities differing in the number of their constituent species and in diversity. Niche parameters were calculated along the major niche dimension of tidal inundation which was assessed using three related measurements, namely height on the marsh, number of submergences per year, and longest period of continuous exposure. This reduces the danger of measuring niches on one resource dimension along which species exhibit non-linear responses. Results obtained using these three measurements were very similar. Mean niche overlaps were significantly smaller, and most species had lower niche breadths, on the marsh with more diverse vegetation. These results agree with Pianka's (1974) niche overlap hypothesis; they indicate that competitive interactions between species are acting with different intensity on the two marshes, and that such interactions are important in determining the distribution limits of both low-and high-marsh species in the emergent salt-marsh community.

Abstract.: In a previous study on the effects of N-supply on leaf cell elongation, the spatial distribution of relative cell elongation rates (RCER), epidermal cell turgor, osmotic pressure (OP) and water potential (Ψ) along the elongation zone of the third leaf of barley was determined (W. Fricke et al. 1997, Planta 202: 522–530). The results suggested that in plants receiving N at fixed relative addition rates (N-supply limitation of growth), cell elongation was rate-limited by the rate of solute provision, whereas in plants growing on complete nutrient solution containing excessive amounts of N (N-demand limitation), cell elongation was rate-limited by the rate of water supply or wall yielding. In the present paper, these suggestions were tested further. The generation rates of cell OP, turgor and Ψ along the elongation zone were calculated by applying the continuity equation of fluid dynamics to the previous data. To allow a more conclusive interpretation of results, anatomical data were collected and bulk solute concentrations determined. The rate of OP generation generally exceeded the rate of turgor generation. As a result, negative values of cell Ψ were created, particularly in demand-limited plants. These plants showed highest RCER along the elongation zone and a Ψ gradient of at least −0.15 MPa between water source (xylem) and expanding epidermal cells. The latter was similar to a theoretically predicted value (−0.18 MPa). Highest rates of OP generation were observed in demand-limited plants, with a maximum rate of 0.112 MPa · h−1 at 16–20 mm from the leaf base. This was almost twice the rate in N-supply-limited plants and implied that the cells in the leaf elongation zone were capable of importing (or synthesising) every minute almost 1 mM of osmolytes. Potassium, Cl− and NO3− were the main inorganic osmolytes (only determined for demand-limited plants). Their concentrations suggest that, unlike the situation in fully expanded epidermal cells, sugars are used to generate OP and turgor. Anatomical data revealed that the zone of lateral cell expansion extended distally beyond the zone of cell elongation. It is concluded that leaf cell expansion in barley relies on high rates of water and solute supply, rates that may not be sustainable during periods of sufficient N-supply (limitation by water supply: Ψ gradients) or limiting N-supply (limitation by solute provision: reduced OP-generation rates). To minimise the possibility of growth limitation by water and osmolyte provision, longitudinal and lateral cell expansion peak at different locations along the growth zone.

A multiple cross was constructed with the aim of combining component traits for the complex salinity resistance character. The aim was to combine donors for physiological traits with the agronomically desirable semidwarf/intermediate plant type and with the overall salinity resistance of the traditional tall land races. We report a study of selection strategies in the resulting breeding population. The effects of early selection for agronomic traits and early selection for low sodium transport were compared with a control population in which minimal selection was practised. Conventional selection for agronomic characters at early generations selected against low sodium-transporting (and thus potentially salt-tolerant) genotypes. In contrast, mild early selection for low sodium transport enriched the population in potentially salt-resistant genotypes but did not select against agronomic (semi-dwarf/intermediate) genotypes. It is concluded that selection for agronomic traits should be made after selection for salt resistance and, ideally, should be delayed until the population has reached near-homozygosity.

Halophytes are plants adapted to live in a saline environment, be it seawater, a salt-water marsh, or a salt-desert; nearly all are angiosperms. The growth of dicotyledonous halophytes is frequently stimulated by salt, although that of most monocotyledonous halophytes is not. Survival requires osmotic adjustment, which is commonly achieved by using the ions present in the environment and which is associated with succulence in dicotyledons but generally not in monocotyledons. At the cellular level, survival depends on the ability of the plant to compartmentalize, in vacuoles, the ions used for osmotic adjustment, particularly sodium and chloride. A vital feature of the physiology of these plants is their ability to accumulate sufficient ions to maintain growth while avoiding either a water deficit or an excess of ions, either of which could prove toxic. The means by which relatively constant ion concentrations in the shoot are maintained are considered. The conclusion is drawn that ion uptake by the roots is not controlled by the shoots; rather, shoot growth is regulated by the supply of ions from the roots. The agricultural importance of halophytes in a world where most of the water is salty at any given moment is emphasized.

The ion content of compartments within cortical cells of mature roots of the halophyte Suaeda maritima grown at 200 mol·m-3 NaCl has been studied by X-ray microanalysis of freeze-substituted thin sections. Sodium and Cl were found in the vacuoles at about four-times the concentration in the cytoplasm or cell walls, whereas K was more concentrated in the cell walls and cytoplasm than in vacuoles. The vacuolar Na concentration was 12- to 13-times higher than that of K. The Na concentration of cell walls of cortical cells was about 95 mol·m-3 of analysed volume. The cytoplasmic K concentration within the mature cortical cells was estimated to be 55 mol·m-3 of analysed volume.

Grown under saline conditions, Suaeda maritima accumulates Na+ and Cl- into its leaves, where individual mesophyll cells behave differently in their compartmentation of these ions. Measurements of ion concentrations within selected subcellular compartments show that freeze-substitution with dry sectioning is a valuable preparative technique for analytical electron microscopy of highly vacuolate plant material. Using this approach, absolute estimates were made of Na+, K+ and Cl- concentrations in the cytoplasm, cell walls, chloroplasts and vacuoles of leaf mesophyll cells.

An attempt has been made to localize glycinebetaine in shoots of Suaeda maritima L. Dum. using a technique based on the formation of an iodoplatinate precipitate. Deposits were largely restricted to the cytoplasm of salt-grown plants and were analysed by transmission analytical electron microscopy. The results are considered to support the hypothesis that glycinebetaine acts as a cytoplasmic osmoticum to balance high vacuolar salt levels in certain halophytes.

An amino acid-incorporating microsomal fraction has been isolated from the leaves of the halophyte Suaeda maritima and the characteristics of the incorporation described. There were no differences in the properties of the microsomes isolated from plants grown in saline and non-saline conditions. The incorporation was severely inhibited by high concentrations of sodium or potassium ions. The results are discussed in relation to the mechanism of salt tolerance in halophytes and the localization of salt in the cells.

Summary Phenotypic resistance of salinity is expressed as the ability to survive and grow in a salinised medium. Some subjective measure of overall performance has normally been used in plant breeding programmes aimed at increasing salinity resistance, not only to evaluate progeny, but to select parents. Salinity resistance has, at least implicitly, been treated as a single trait. Physiological studies of rice suggest that a range of characteristics (such as low shoot sodium concentration, compartmentation of salt in older rather than younger leaves, tolerance to salt within leaves and plant vigour) would increase the ability of the plant to cope with salinity. We describe the screening of a large number of rice genotypes for overall performance (using an objective measure based on survival) and for the aforementioned physiological traits. There was wide variation in all the characters studied, but only vigour was strongly correlated with survival. Shoot sodium concentration, which a priori is expected to be important, accounted for only a small proportion of the variability in the survival of salinity. Tissue tolerance (the cellular component of resistance reflecting the ability to compartmentalise salt within leaves) revealed a fivefold range between genotypes in the tolerance of their leaves to salt, but this was not correlated positively with survival. On the basis of such (lack of) correlation, these traits would be rejected in normal plant breeding practice, but we discuss the fallacies involved in attempting correlation between individual traits and the overall performance of a salt-sensitive species in saline conditions. We conclude that whilst overall performance (survival) can be used to evaluate the salt resistance of a genotype, it is not the basis on which parents should be selected to construct a complex character through breeding. It was the norm for varieties which had one good characteristic affecting salt resistance to be unexceptional or poor in the others. This constitutes experimental evidence that the potential for salt resistance present in the rice genome has not been realised in genotypes currently extant. The results are discussed in relation to the use of physiological traits in plant breeding, with particular reference to environmental stresses that do not affect a significant part of a species' ecological range.

The amount of salt-affected agricultural land is increasing globally, so new crop varieties are needed that can grow in salt-affected soils. Despite concerted effort to develop salt-tolerant cereal crops, few commercially viable salt-tolerant crops have been released. This is puzzling, given the number of naturally salt-tolerant grass species. To better understand why salt-tolerance occurs naturally but is difficult to breed into crop species, we take a novel, biodiversity-based approach to its study, examining the evolutionary lability of salt-tolerance across the grass family. We analyse the phylogenetic distribution of naturally salt-tolerant species on a phylogeny of 2684 grasses, and find that salt-tolerance has evolved over 70 times, in a wide range of grass lineages. These results are confirmed by repeating the analysis at genus level on a phylogeny of over 800 grass genera. While salt-tolerance evolves surprisingly often, we find that its evolution does not often give rise to a large clade of salt-tolerant species. These results suggest that salt-tolerance is an evolutionarily labile trait in grasses.

The ion relations of the halophytc Suaeda maritima are described. When plants grew in 340 mol m−3 sodium chloride (—1⋅76 MPa) leaf solute potentials decreased, and were sustained around —2⋅5 MPa Inorganic ion concentration (mostly of sodium chloride) accounted for this. Comparable shoot ion concentrations of potassium, nitrate and sulphate occurred when plants grew on different salinity types characterized by these ions. Net sodium transport and shoot sodium concentration increased dramatically with increases in external sodium chloride concentration up to 85 mol m−3; thereafter, further increases in external sodium chloride concentration had relatively little effect upon either shoot sodium concentration or upon net transport of sodium to the shoot. The net transport of sodium plus potassium only doubled when the external concentration of sodium plus potassium increased from 24 to 687 mol m−3 Shoot ion concentrations were remarkably constant with time, external concentration and salinity type. The membrane flux rates and symplasmic ion concentrations needed to sustain the observed net transport of sodium (of some 10 mmol g−1 dry wt. of roots d−1) are calculated from anatomical and stereological data for the root system of this species. The minimum net sodium chloride flux to load the symplasm would be 260 nmol m−2s−1 if the whole cortical and epidermal plasmalemmal surface area were used uniformly, but the flux rate required would be 3000 nmol m−2s−1 if uptake took place only at the root surface. A flux rate of at least 1000 nmol m−2s−1 is needed between symplasm and xylem. The symplasmic concentration of NaCl would be at least 80 mol m−3. It is argued (1), that both symplasmic and xylem loading are likely to be passive processes mediated by ion channels rather than active carriers, (2), that net ion transport at 340 mol m−3 sodium chloride is close to the maximum which is physiologically sustainable and (3), that growth of this halophyte is limited by NaCl supply from the root.

An apoplastic pathway, the so-called bypass-flow, across rice roots to the xylem has been investigated and approximately quantified using the apoplastic tracer dye 8-hydroxy-l,3,6-pyrenetrisulphonic acid (PTS); former nomenclature 3-hydroxy-5,8,10-pyrenetrisulphonic acid. It was confirmed that PTS was confined to a compartment no greater than the apparent free space in living rice roots. Experimental handling did not contribute to bypass-flow. Rice roots recovered rapidly from severe damage: following root pruning, sodium and calcium uptake returned to steady values in about 6 h. Apoplastic flow declined after damage as a first-order kinetic with a half time of 75 min. Analysis of the pattern of elution of PTS from preloaded roots (intact, excised and heat-killed), and from cellulose, was followed to compare PTS movement in the extracellular compartment with that of water and small hydrated ions. Consideration is given to the factor by which the bypass-flow estimated with the dye would need to be corrected to reflect the proportion of the transpiration stream flowing in the apoplastic pathway. The data suggest that this factor would be at least 10 for transpiring rice plants. There was large individual variation in the transport both of sodium and of the apoplastic tracer PTS to the shoot. Plants with high sodium transport also had high PTS transport and it is concluded that some proportion of the sodium reaching the xylem in rice does so by a pathway which is also available to PTS, presumably direct apoplastic contact across the endodermis. A median value for the bypass-flow of water (corrected from PTS) would be 0.5 to 1.0 percent of the transpirational volume flow, but individuals with the highest sodium transport would have bypass-flow values of several percent. Evidence is discussed which suggests that apoplastic transport may increase in stress conditions, and it is argued that bypass-flow is a major contribution to sodium uptake in rice in saline conditions.

Yeo, A. R., Caporn, S. J. M.and Flowers, T. J. 1985. The effect of salinity upon photosynthesis in rice (Oryza sativa L.): Gas exchange by individual leaves in relation to their salt content.—J. exp. Bot. 36: 1240–1248. The effect of salinity upon net photosynthesis and transpiration by individual leaves of rice has been investigated by gas exchange measurements in seedlings at the five to six leaf stage. Salinity did not, initially, reduce net photosynthesis in the whole plant but only in the older leaves in which sodium accumulated. Analysis of the course of events in leaf four following salinization of the medium showed that net photosynthesis was inversely correlated with the sodium concentration in the leaf tissue. There was no evidence of a threshold effect; net photosynthesis declined linearly with increasing leaf sodium concentration and was reduced by 50% at only 05 mmol sodium per gram dry weight. The relationship between transpiration rate and leaf sodium concentration closely paralleled that for photosynthesis; there was no effect of leaf sodium concentration on the carbon dioxide concentration in the intercellular spaces, showing that sodium accumulation in the leaf affected stomatal aperture and carbon dioxide fixation simultaneously. Photosynthesis was reduced by half at a sodium concentration in the leaf which did not reduce the concentration of chlorophyll. The nature of the effect of salinity upon leaf gas exchange is discussed.

Yeo, A. R., Yeo, M. E., Caporn, S. J. M., Lachno, D. R. and Flowers, T. J. 1985. The use of 14C-ethane diol as a quantitative tracer for the transpirational volume flow of water and an investigation of the effects of salinity upon transpiration, net sodium accumulation and endogenous ABA in individual leaves of Oryza sativa L.—J. exp. Bot. 36: 1099–1109. Oryza sativa L. (rice) seedlings growing in saline conditions exhibit pronounced gradients in leaf sodium concentration which is always higher in the older leaves than the younger ones. Individual leaf transpiration rates have been investigated to discover whether movement of sodium in the transpiration stream is able to explain these profiles from leaf to leaf. The use of 14C labelled ethane diol to estimate transpiration was evaluated by direct comparison with values obtained by gas exchange measurements. Ethane diol uptake was linearly related to the transpirational volume flow and accurately predicted leaf to leaf gradients in transpiration rate in saline and non-saline conditions. 14C-ethane diol and 22NaCl were used to compare the fluxes of water and sodium into different leaves. The youngest leaf showed the highest transpiration rate but the lowest Na accumulation in saline conditions; conversely, the older leaves showed the lower transpiration rates but the greater accumulation of Na. The apparent concentration of Na in the xylem stream was 44 times lower into the younger leaf 4 than into the older leaf 1. Exposure to NaCl (50 mol m−3) for 24 h elicited an increase in endogenous ABA in the oldest leaf only, but no significant changes occurred in the younger leaves.

A stereological analysis of cells from the mesophyll, vascular bundles and central parenchyma in leaves of the halophyte Suaeda maritima (L.) Dum is described. The volume fraction of vacuole was greater in plants grown under saline conditions when compared with those under non-saline conditions, and there was a concurrent increase in the surface density of the tonoplast. The volume fraction of chloroplasts and cell wall fell under saline conditions, while that of the intercellular spaces increased. Salinization of the growth medium was accompanied by a large increase in the fraction of the cell volume occupied by the mitochondria: the increase in surface density of mitochondrial membranes was some 72% when averaged over all the cell types analysed.

Hajibagheri, M. A., Flowers, T. J., Collins, J. C. and Yeo, A. R. 1988. A comparison of the methods of X-ray microanalysis, compartmental analysis and longitudinal ion profiles to estimate cytoplasnuc ion concentrations in two maize varieties.—J. exp. Bot. 39: 279-290. The ion content of compartments within plant root cells has been studied by three different methods; flux analysis using radioactive isotopes, longitudinal ion profiles and X-ray microanalysis. The data provide estimates of the concentrations of K+ and CI− in the cytoplasm of roots of culture solution and salt grown maize by three independent methods. In the cultivar LG11 grown in 50 mol m−3 NaCl X-ray microanalysis, compartmental analysis and longitudinal profiles yielded approximately agreeing values for cytoplasmic K+ of 90, 70 and 62 mol m−33 of tissue volume respectively. However, the methods disagreed on cytoplasmic Cl− where the value obtained by compartmental analysis was about four times that from X-ray microanalysis and longitudinal profiles. Possible reasons for this are discussed.

Hajibagheri, M. A., Gilmour, D. J., Collins, J. C. and Flowers, T. J. 1986. X-ray microanalysis and ultrastructural studies of cell compartments of Dunaliella parva. -J. exp. Bot. 37: 1725–1732. Ultrastructural studies of the unicellular green alga Dunaliella parva showed the presence of cytoplasmic vacuoles. X-ray microanalysis was performed on sections of cells which had been freeze substituted in acetone. It was found that the concentrations of both Na and Cl were much higher in the vacuoles than in the cytoplasm. When cells were grown in 0·4 kmol m−3 NaCl the Na and Cl concentrations in the vacuoles were 349 and 283 mol m −3 respectively, whilst cytoplasmic Na and Cl concentrations were 37 and 26 mol m−3. Corresponding values for cells grown in 1·5 kmol m−3 NaCl were 392 mol m−3 Na and 325 mol m−3 Cl in the vacuoles and 36 mol m−3 Na and 30 mol m−3 Cl in the cytoplasm. Immediately after exposure to an increase in external salinity Na and Q concentrations increased in both vacuoles and cytoplasm. The results are discussed with reference to compartmental models for the ionic relations of Dunaiiella.

The effects of the concentrations and species of ions in the external medium upon the growth of Suaeda maritima, a coastal halophyte, have been investigated. Plants grown in highly saline conditions (340 mol m−3 NaCl) had larger fresh and dry weights than plants grown in a standard culture medium (13 mol m−3). However, differing contributions of water and inorganic ions meant that these increases in fresh and dry weights greatly exaggerated the increase in growth, i.e. organic dry matter production. Enhanced growth in high salt concentrations was evident only after prolonged exposure and was attributable to increased cell size. Growth increased smoothly with increasing mineral concentration in the external medium and was maximal at 170 mol m−3 added NaCl, although 90% maximal growth was obtained at only one tenth of this concentration. Ion-specific growth enhancement was not apparent at low concentrations of NaCl. Growth at high concentrations (340 mol m−3) was maximal in NaCl and the effects of other salts ranged between this and toxicity in KC1. It is suggested (1) that the description of growth stimulation in halophytes by salt is somewhat arbitrary, (2) that increased organic growth with increasing salt concentration results from the relationship between turgor pressure and extension growth, and (3) that tolerance to extreme salinities is a separate phenomenon from the growth response.

Single channel properties, whole vacuole currents and proton pumping capacity were investigated in the intact vacuoles and membrane patches of leaf tonoplast from the halophyte Suaeda maritima. ATP-dependent proton pumping capacity was similar to non-halophytes whether the plants were or were not grown with added sodium chloride (200 mM). The most abundant ion channel was inward rectifying and had a single channel conductance of 58 pS in symmetrical KCl solutions (100 mM) to 170 pS using physiological conditions (50/150 mM KCl/NaCl cytoplasmic side, 50/450 mM KCl/NaCl vacuolar side). The channel showed all the characteristics of the SV type channel described in many other species. In the open state these channels caused tonoplast conductances in excess of 0.5 nS m2– but conductances were much lower using physiological ion concentrations and membrane potentials. In spite of the poor selectivity and the potentially large tonoplast conductance it is calculated that compartmentation of NaCl in leaf vacuoles can be sustained by about 30% of ATP-dependent proton pumping capacity. The results do not indicate any special adaptation of the tonoplast ion channels in the halophyte.

Addition of 50 mM NaCl to Oryza sativa L. had little effect upon the time of leaf initiation, but leaf mortality prior to the normal phase of senescence was increased and the onset of senescence was advanced. There was no significant effect upon the day-to-day pattern of growth, nor upon the ultimate length, of leaves that were developing at the time of, or shortly after, salinization with 50 mM NaCI. Leaves that developed after prolonged exposure of the plants to salinity were shorter. Addition of NaCl, KC1 or mannitol to the root medium brought about a cessation of leaf elongation within one minute. Growth at a reduced rate restarted abruptly after a lag period that depended upon the external concentration. Elongation rate recovered its original value within 24 h after exposure to 50 mM NaCl, though not at higher concentrations. Addition of NaCl at concentrations up to 100 mM elicited no short-term effects upon photosynthetic gas exchange. Na uptake contributed to osmotic adjustment of the growing zone. When plants were rapidly exposed to 50 mM NaCl, no change in turgor pressure was detectable in the growing zone with the resolution of the miniature pressure probe used (about 70 kPa). It is concluded that the initial growth reduction in rice caused by salinization is due to a limitation of water supply. A clear distinction is made between the initial effects of low salinity which are recoverable and the long-term effects which result from the accumulation of salt within expanded leaves.

Mitochondria were isolated from green leaves and stems of the glycophyte Pisum sativum and the halophyte Suaeda maritima. The preparations oxidized malate, succinate, and 2-oxoglutarate as well as externally added NADH. Acceptor control ratios were about 2.8 for mitochondria from Pisum and 1.8 for mitochondria from Suaeda oxidizing malate+pyruvate in 125 mM sodium chloride. The mitochondrial fraction was contaminated with chloroplast fragments which resulted in relatively low rates of oxygen uptake when these were expressed on a protein basis. The addition of sodium chloride at concentrations greater than 200 mM considerably reduced the rates of oxygen uptake by both species in the presence and absence of phosphate acceptor (ADP). Acceptor control ratios were reduced and there was a marked decline in the ADP/O ratio. Sucrose at equivalent molar concentrations had a much less drastic effect on the mitochondria. There was no significant difference in the effects of these two solutes on mitochondria from the two species and the similarity of response is discussed in relation to the cytoplasmic ion content of the halophyte.

Endogenous abscisic acid contents were measured by gas-liquid chromatography in shoots of Suaeda maritima growing both in the steady state over a range of salinities and over a time-course following an increase in the culture solution salinity of between approximately 100 and 400 mol m−3 NaCl. In steady-state plants, the ABA content was maximal in the absence of salt at 41 ng g−1 fr. wt., declining to a minimum at 200 mol m−3 NaCl of 24 ng g−1 fr. wt. Increase of culture solution salinity resulted in a marked increase in shoot ABA which was maximal after 6 h or 24 h in plants previously growing at 200 mol m−3 NaCl and in the absence of salt, respectively. Additionally, culture solution water potentials were lowered by 1.0 MPa (equivalent to raising the salt concentration by around 200 mol m−3); this resulted in a similar increase in endogenous ABA content to that brought about by an iso-osmotic salt increase. Results are discussed in relation to the possible role of ABA in halophyte salt tolerance mechanisms.

Vacuoles of high purity were isolated from the leaves of the halophyte Suaeda maritima (L.) Dum. The relative compositions of phospholipids, phytosterols, and fatty acids in the tonoplast membrane were determined and membrane fluidity was assessed by electron spin resonance. The characteristics of the tonoplast were consistent with minimizing passive permeability to NaCI. The phospholipid: protein ratio (1.1: 1.0) was higher than that recorded in other membrane preparations, including vacuoles from beetroot storage material, commensurate with the low density (1.05 g cm−3) of the S. maritima tonoplast. The tonoplast fatty acids were highly saturated and dominated by n-hexadecanoic acid and n-octadecanoic acid. Phytosterols identified by gas chromatography were cholesterol, campesterol, stigmasterol, and β-sitosterol. Cholesterol was a trace percentage in protoplasts, but comprised 30% of the tonoplast sterols. Semi-quantitative analysis by chromatography on silica gel revealed an enrichment in the tonoplast of glycolipid which was not accounted for as chloroplast contamination. The fluidity of the tonoplast, determined by electron spin resonance, was lower than the protoplasts, consistent with the high degree of saturation of the fatty acid chains. The relevance of the lipid composition of the tonoplast to its central role in ion compartmentation within the halophyte cell is discussed.

The tonoplast plays a crucial role in ion compartmentation, which is a central feature of the salt tolerance of halophytes, but we do not know the properties of the membrane that confer this ability. A method was, therefore, developed for the isolation of vacuoles from Suaeda maritima (L.) Dum. of sufficiently high purity to enable biochemical characterization of their lipid and protein composition. Tonoplast fractions produced by density gradient centrifugation, as well as vacuoles isolated by a variety of methods (including DEAE dextran lysis, digitonin lysis, and mechanical shear forces) were unacceptably contaminated. A highly pure vacuole preparation was obtained when protoplasts were lysed by a mild hypotonic shock in alkaline buffer, in the presence of the compatible cytosolute glycine-betaine, followed by shear force during ultracentrifugation; cytoplasmic contamination was prevented by the addition of the zwitterionic detergent 3-([3-cholamidopropyl]dimethylammonio)-l-propanesulphonate (CHAPS). Light microscopy of this preparation revealed no intact protoplasts and no contamination by chlorophyll could be detected. Electron microscopy showed the vacuoles to be single-membrane-bound structures, and was the only criterion upon which vacuoles could be separated reliably from vacuoplasts, in which the plasmalemma is collapsed on to the tonoplast. Analysis by SDS-PAGE showed that a total of 15 polypeptides were enriched in the tonoplast and 27 in the soluble fraction from vacuole preparations, with a pattern similar to that reported for glycophytic species. The pure tonoplast exhibited both vanadate-insensitive ATPase and pyrophosphatase activities, but the properties of these enzymes were broadly similar to those of glycophytes. Analysis of membrane fatty acids showed that the degree of saturation of the putative tonoplast preparation increased as the assessment of the purity of the preparation (made by microscopy) increased. The ATPase could be substantially purified by ion-exchange FPLC. The results are discussed in relation to the degree of purity needed in membrane preparations in order to be suitable for biochemical analysis.

The fine structure and certain cytochemical properties of the shoots from the halophyte Suaeda maritima have been examined. No major fine structural differences were observed between plants grown in the presence or absence of salt. Considerable evidence of vesiculation was observed, particularly in cells of the stelar tissue from the stems. The specificity of the phosphotungstic acid-chromic acid stain for plasma membranes was examined and some doubt raised as to its widespread applicability as a plasma membrane marker in plant cells. The suitability of other membrane markers such as ruthenium red and ATPase activity were investigated.

Membrane fractions from the shoots of Suaeda maritima have been separated by sucrose gradient centrifugation. Certain staining and enzymatic activities were studied with the aim of identifying specific membrane types, particularly the plasma membrane. Chloroplast and mitochondrial membranes were largely distributed in the denser regions of the gradient (ρ 1.16). A plasma membrane fraction was not easily identifiable and the problems of separating these membranes from plant cells are discussed.

The effect of sodium, chloride on the growth of a halophyte, Suaeda maritima (L.) Dum., was compared with its effect on Pisum sativum L. cv. Alaska under controlled environmental conditions. The salt stimulated the growth of Suaeda maximally at concentrations of 170 to 340 mM while the growth of Pisum was inhibited even by 100 mM. Both species accumulated ions in the tops and the maximum concentrations of Na+ and Cl− rose in Suaeda to 860 mM (based on the water content) and 730 mM and in Pisum to 170 mM and 300 mM respectively. Respiration in both species was inhibited as the NaCl level in the culture solution was raised. Four supernatant enzymes (malic dehydrogenase, glucose-6-phosphate dehydrogenase, peroxidase, and acid phosphatase) prepared from Pisum and from Suaeda (grown either in the absence of added NaCl or in the presence of 340 mM NaCl) were assayed in various levels of sodium chloride. The dehydrogenases were markedly inhibited by increasing salt concentrations while there was a smaller effect on the peroxidase and acid phosphatase. There was no difference in the effect of salt on the enzymes prepared from the two species although one is halophilic and the other halophobic.

The effects of osmotically induced water deficits on the metabolism of aged beetroot and potato discs were investigated. In this paper the tissue-water relationships in two osmotica, mannitol, and Carbowax 1540, are described. Tissues in equilibrium with Carbowax solutions had lower fresh weights than those in isotonic mannitol solutions, particularly at water potentials below −0.7 J g−1 with potato and −2.0J g− with beetroot. Potato discs killed by freezing and thawing lost water to Carbowax but not to mannitol solutions. The extra effectiveness of Carbowax solutions in lowering fresh weight was attributed to an osmotic effect across the cell wall. Carbowax was found to penetrate plasmolysed potato tissue, however, at a rate of about 3.0 mg (g fresh weight)−1 h−. The extent to which water uptake occured on retransfer to water was unaffected by the nature of the soulte used, but depend on the degree of dehydration. The following phases were clearly defined: (1) recovery to the fresh weight at full turgor, when the water potential of potato tissue was not reduced below −0.5 J g− and of beetroot below −1.2 J g−1; (2) a declining degree of recovery with decreasing water potential over the ranges −0.5 to −1.0 J g−1 and −1.2 to −3.0 J g−1 for potato and beetroot, respectively, and (3)in potato, the absence of recovery of fresh weight following reduction of the water potential below −1.0 J g−1.

The effects of osmotically induced water deficits on the respiration, permeability, and ultra-structure of storage tissue are described. As the water potential was lowered respiration in both aged beetroot and potato discs was retarded with a concomitant rise in respiratory quotient. On return to water, the degree of recovery of the oxygen uptake appeared to be a function of the number of cells plasmolysed. The permeability data corroborated other evidence that membrane damage resulted when cells were plasmolysed. Deplasmolysis resulted in further and more extensive injury. Ultra-structural studies confirmed these findings and suggested that plasmolysis also affected mitochondrial structure.

Evidence of some effects of water deficit on respiratory metabolism is presented. Oxygen uptake by aged potato tuber discs became increasingly resistant to the effect of 0.05 M malonate with increasing water deficit, while there was an increasing retardation of oxygen uptake by dinitrophenol at 10-4 M. The same range of water deficit suppressed the evolution of 14CO2 from labelled acetate, succinate, and glucose, indicating a progressive disorganization of the Krebs cycle.