Sodium chloride toxicity and the cellular basis of salt tolerance in halophytes

Background Halophytes are the flora of saline soils. They adjust osmotically to soil salinity by accumulating ions and sequestering the vast majority of these (generally Na⁺ and Cl⁻) in vacuoles, while in the cytoplasm organic solutes are accumulated to prevent adverse effects on metabolism. At high salinities, however, growth is inhibited. Possible causes are: toxicity to metabolism of Na⁺ and/or Cl⁻ in the cytoplasm; insufficient osmotic adjustment resulting in reduced net photosynthesis because of stomatal closure; reduced turgor for expansion growth; adverse cellular water relations if ions build up in the apoplast (cell walls) of leaves; diversion of energy needed to maintain solute homeostasis; sub-optimal levels of K⁺ (or other mineral nutrients) required for maintaining enzyme activities; possible damage from reactive oxygen species; or changes in hormonal concentrations.Scope This review discusses the evidence for Na⁺ and Cl⁻ toxicity and the concept of tissue tolerance in relation to halophytes.Conclusions The data reviewed here suggest that halophytes tolerate cytoplasmic Na⁺ and Cl⁻ concentrations of 100–200 mm, but whether these ions ever reach toxic concentrations that inhibit metabolism in the cytoplasm or cause death is unknown. Measurements of ion concentrations in the cytosol of various cell types for contrasting species and growth conditions are needed. Future work should also focus on the properties of the tonoplast that enable ion accumulation and prevent ion leakage, such as the special properties of ion transporters and of the lipids that determine membrane permeability.     

Ultrastructure of the perfused rat epididymis: Effect of luminal sodium ion concentration

Summary The appearance of the rat epididymal epithelium changed when it was perfused in vivo through the lumen with unphysiologically high sodium ion concentrations; dilatation of intercellular spaces (ICS) at threshold concentrations of 30mM-Na⁺ in the cauda and about 55mM-Na⁺ in the corpus was associated with absorption of water from the lumen. Despite the distended ICS, junctional complexes appeared intact, and their integrity was confirmed by the exclusion of luminal horseradish peroxidase (HRP) from the ICS, and by demonstrating that circulating [³H]inulin did not enter the lumen. Smooth ER and lipid droplets in the principal cells of the corpus epididymidis were well maintained, and the preservation of granular ER in principal cells of the cauda epididymidis lent morphological support to the continued secretion of protein in this segment. However, occasional distension or involution of inner Golgi cisternae was evident in principal cells after 3–6 h perfusion. In contrast to multivesicular bodies of principal cells, the apical and basal vacuoles characteristic of clear cells changed in size with different perfusing solutions. When low Na⁺ concentrations were perfused large translucent vacuoles were frequently found in the apical cytoplasm of clear cells in the corpus and cauda epididymidis, and filled vacuoles became larger and showed a decrease in content density in the cauda epididymidis. These large vacuoles were absent from tissue perfused with high Na⁺ concentrations. Normal pinocytotic activity of both cell types was demonstrated by perfusing HRP which was taken up by the normal route in principal cells, with some transfer to the Golgi cisternae. By far the most HRP was accumulated in clear cell vacuoles irrespective of the composition of the perfusing solution.     

Quantitative ion localization within Suaeda maritima leaf mesophyll cells

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.Abbreviation TAEM transmission analytical electron microscopy     

In-situ determination of intracellular concentrations of K+ in barley (Hordeum vulgare L. cv. Kara) using the K+-binding fluorescent dye benzofuran isophthalate

The tetra[acetoxymethyl] ester of the K⁺-binding fluorescent dye benzofuran isophthalate (PBFI-AM) was used to determine changes in intracellular potassium (K⁺) concentrations and to measure net transport of K⁺ in barley (Hordeum vulgare L. cv. Kara) root and leaf protoplasts. When this dye binds to free K⁺ inside the cytoplasm, the fluorescence intensity ratio 340/380 nm increases in direct relation to the K⁺ concentration. Because of a delay in the uptake of dye into the vacuoles, it is possible to determine K⁺ concentrations in the vacuoles and transport of K⁺ from the cytoplasm into the vacuole. The uptake of PBFI-AM in root and leaf protoplasts of barley differed in the absence or presence of external K⁺ and was faster at pH 5.5 than at pH 7.0. The fluorescence intensity of the dye was stable for at least 20 h when the protoplasts were kept at 4°C. In the presence of nigericin, the fluorescence intensity of both cells and protoplasts was linearly related to the external concentration of K⁺ (up to 100 mM).     

The effects of salinity on ion concentrations within the root cells of Zea mays L.

Zea mays is a salt-sensitive crop species which in saline (100 mol m^-3 NaCl) conditions suffers considerable growth reduction correlated with elevated Na⁺ and Cl^- concentration within the leaves. To increase understanding of the regulation of ion uptake and transport by the roots in saline conditions, ion concentrations within individual root cortical cells were determined by X-ray microanalysis. There was variation in Na⁺, K⁺ and Cl^- distributions among individual cells, which could not be correlated with their spatial position in the roots. Generally, however, in response to saline growth conditions (100 mol m³ NaCl) Na⁺ and Cl^- were mostly localized in the vacuoles, although their concentrations were also sometimes increased in the cytoplasm and cell walls. The concentration of K⁺ in the cytoplasm was usually maintained at a level (mean 79 mol m^-3) compatible with the biochemical functions ascribed to this ion.Abbreviation (T)AEM (Transmission) analytical electron microscopy     

Participation of vacuoles in regulation of levels of K+, Mg2+ and orthophosphate ions in cytoplasm of the yeast Saccharomyces carlsbergensis

Intracellular distributions of K⁺, Mg²⁺ and orthophosphate under various conditions of cultivation or incubation of the yeast Saccharomyces carlsbergensis were studied by differential extraction of ion pools. The decisive role of vacuolar compartmentation of ions in regulation of K⁺, Mg²⁺ and orthophosphate levels in the yeast cytoplasm was shown. The content of intracellular K⁺ and Mg²⁺ in yeast increased or decreased primarily depending on the increase or decrease in the vacuolar ion pool. The levels of K⁺ and Mg²⁺ in the cytoplasm were practically unchanged. Vacuoles were involved in regulation of Mn²⁺ concentration in the cytoplasm of the yeast S. carlsbergensis accumulating this ion in the presence of glucose. Alongside the vacuolar compartmentation, the chemical compartmentation, i. e. formation of bound Mg²⁺, Mn²⁺ and K⁺ was, evidently, also involved in the control of ion levels in the cytoplasm. The orthophosphate level in the yeast cytoplasm was regulated by its accumulation in vacuoles and biosynthesis of inorganic polyphosphates in these organelles. The biosynthesis of low-molecular weight polyphosphates occurred parallel to the accumulation of Mg²⁺ or Mn²⁺ in vacuoles, thus confirming the availability of the other mechanism for the transport of these ions through the tonoplast differing from the transport mechanism through the plasmalemma.     

Effect of sodium on potassium fluxes at the cell membrane and vacuole membrane of red beet

Slices of red beet (Beta vulgaris) washed for 5 to 6 days are known to accumulate Na⁺ in preference to K⁺ from solutions containing both ions. The present work, using ion concentrations of 1.0 mm or less, with Ca²⁺ added in some cases, shows that Na⁺ strongly inhibits K⁺ influx at the cell membrane (plasmalemma) while K⁺ efflux is increased to a lesser extent. This result from compartmental analysis is confirmed by short (15-minute) influx experiments, which indicate an immediate inhibitory effect of Na⁺ on K⁺ influx at the cell membrane. It is concluded that cation selectivity, even when Na⁺ is favored for uptake, is primarily determined at the cell membrane. Nevertheless, a high level of K⁺ in the cytoplasm is maintained during Na⁺ influx, by an inhibition of K⁺ transfer to the vacuole.     

Physiology of halophytes

Summary The cellular basis of salt tolerance in halophytes depends upon the compartmentation of ions necessary for osmoregulation in vacuoles and upon osmotic adjustment of the cytoplasm by compatible solutes. The central role played by Na⁺ and Cl^– in osmotic adjustment suggests that the transport of these ions and its regulation must be of primary importance in the physiology of the plant as a whole. There have been few investigations into the regulation of leaf ion concentrations, but such data as are in the literature suggest that limiting xylem Na⁺ (and Cl^–) concentrations, together with continued leaf expansion, are particularly important. The role of phloem in retranslocation is uncertain due to lack of data. Decreases in transpiration rate per unit area of leaf help to lower the ion input into leaves. Any linked reductions in photosynthesis appear to be due to decreases in stomatal frequency.     

Some Observations on the Effects of Salinity on Ion Distributions and Cell Ultrastructure in Wheat Leaf Mesophyll Cells

The distribution of ions within wheat leaf mesophyll cells fromplants grown in the presence of 100 mol m^–3 NaCl werestudied by X-ray micro-analysis, and the volume fractions occupiedby the analysed compartments were determined stereologically.Generally, the cells did not accumulate high ion concentrationsin their vacuoles, and the much higher Na⁺ and Cl^– concentrationsin the cell walls would produce an imbalance of cell water relations.The Na⁺, K⁺ and Cl^– concentrations in the cytoplasm wereoften too high to be compatible with biochemical activity. Inresponse to saline growth conditions, the volume fractions occupiedby the cytoplasm and chloroplasts increased. Correspondence to: Institut für Botanik, Fachbereich Biologie,Technische Hochschule Darmstadt, 3–5 Schnittspahnstr.F.R.G. Key words: Salinity sensitivity, X-ray micro-analysis, stereology     

Substitution of Na+ by K+ in Tissues and Root Vacuoles of Barley (Hordeum vulgare L. cv. Aramir)

The aim of this paper was to investigate whether Na⁺ accumulated in the vacuoles of barley roots can be replaced by K⁺, or else remain irreversibly trapped.Barley seedlings (Hordeum vulgare L. cv. Aramir) were grown for 2 days on nutrient solutions and then salinized over 24 h by a linear increase in NaCl concentration (50 mol · m⁻³). NaCl was replaced by an equimolar concentration of KCl and 1 day later the KCl concentration was gradually decreased over 23 hours. After this the plants were grown for 2 further days on basic nutrient solution. Plant material was harvested at each experimental step in order to measure plant growth and K⁺ and Na⁺ concentrations in vacuoles and tissues.The results from quantitated, energy dispersive X-ray analyses on frozen, hydrated vacuoles of the rhizodermls and 3 subsequent cortical cell rows, point to a proportional replacement of K⁺ by Na⁺ after NaCl stress. A replacement of Na⁺ by K⁺ was clearly found in the rhizodermal vacuoles after KCl treatment. Flame emission spectrometric data from corresponding root segments support these results.     

Adaptation of plants to salt stress: the role of the ion transporters

Adaptation to high salinity is achieved by cellular ion homeostasis which involves regulation of toxic sodium ion (Na⁺) and Chloride ion (Cl⁻) uptake, preventing the transport of these ions to the aerial parts of the plants and vacuolar sequestration of these toxic ions. Ion transporters have long been known to play roles in maintaining ion homeostasis. Na⁺ enters the cell through various voltage dependent selective and non-selective ion channels. High Na⁺ concentration in the plasma membrane is balanced either by uptake of potassium ion (K⁺) by various potassium importing channels, by salt exclusion mechanism or by sequestration of Na⁺ in the vacuoles. Therefore, the role of high-affinity potassium transporter, the salt overly sensitive pathway, the most well-defined Na⁺ exclusion pathway that exports Na⁺ from cell into xylem and tonoplast localized cation transporters that compartmentalizes Na⁺ in vacuoles need to be studied in detail and applied to make the plant adaptable to saline soil. Knowledge on the regulation of expression of these transporters by the hormones, microRNAs and other non-coding RNAs can be utilized to manipulate the ion transport. Here, we reviewed paradigm of the ion transporters in salt stress signalling pathways from the recent and past studies aiding transformation of basic knowledge into biotechnological applications to generate engineered salt stress tolerant crops.

     

An attempt to use isolated vacuoles to determine the distribution of sodium and potassium in cells of storage roots of red beet (Beta vulgaris L.)

Vacuoles isolated from red beet (Beta vulgaris L.) storage roots contain Na⁺ and K⁺ but their analysis does not give reliable information about the size of vacuolar pools of these ions in vivo. Analyses of isolated vacuoles indicated that between 53% and 90% of the Na⁺ was located in the vacuole and that the vacuolar concentrations of Na⁺ ranged between 4 and 45 mol m^-3. Calculated concentrations of K⁺ in the vacuoles varied between 32 and 72 mol m^-3 but, in contrast to Na⁺, only about 50% of the K⁺ was located in the vacuole. Considerations of the likely cytoplasmic concentrations of Na⁺ and K⁺ suggest that if these results indicate conditions in vivo a large proportion of these ions must be located in the extracellular space, where they would exert considerable osmotic pressure. To test this, the effect of washing on cell turgor (measured directly with a pressure probe) and on loss of Na⁺ and K⁺ was determined. Washing caused an increase in turgor of 5 bar but losses of Na⁺ and K⁺ were less than predicted by the experiments with isolated vacuoles. It is concluded that beet vacuoles leak Na⁺ and K⁺ when isolated resulting in an underestimation of the size of vacuolar pools of these cations in vivo. Nonetheless, the turgor measurements provide evidence for the presence of osmotically active solute in the extracellular space. The possible contribution of extracellular Na⁺ and K⁺ to the observed turgor reduction is calculated and the physiological importance of the accumulation of extracellular solutes is discussed.     

Effect of d-myo-Inositol 1,4,5-Trisphosphate on the Electrical Properties of the Red Beet Vacuole Membrane

The effect of channel opening in the tonoplast by d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] has been examined on red beet (Beta vulgaris) vacuoles. Patch-clamp measurements of the vacuolar potential and current were performed on vacuoles isolated in 0.1 micromolar free Ca²⁺ medium. With vacuoles clamped at +30 millivolts, the Ins(1,4,5)P₃ induced changes in current were depending on the Ca²⁺ buffer strength in the external medium. The spontaneous depolarization of vacuoles in which H⁺-pumps were activated by 5 millimolar MgATP was increased from +6 to +18 millivolts by 1 micromolar Ins(1,4,5)P₃. We have interpreted our data by assuming that even with 2 millimolar EGTA to buffer Ca²⁺ at 0.1 micromolar in the external medium, Ins(1,4,5)P₃ released enough Ca²⁺ from the vacuole to produce an accumulation of this ion near the tonoplast. Apart from their dependency with free Ca²⁺ in the cytoplasm, the electrical properties of the tonoplast could be depending on the Ins(1,4,5)P₃ and Ca²⁺ buffer values in the cytoplasm.     

Cell ultrastructure and fatty acid composition of lipids in vegetative organs of <Emphasis Type="Italic">Chenopodium album</Emphasis> L. under salt stress conditions

White goosefoot plants (Chenopodium album L. of the family Chenopodiaceae) grown at various NaCl concentrations (3–350 mM) in the nutrient solution were used to study the cell ultrastructure as well as the qualitative and quantitative composition of fatty acids in the lipids of vegetative organs. In addition, the biomass of Ch. album vegetative organs, the water content, and the concentrations of K⁺, Na⁺, and Cl^– were determined. The growth rates of plants raised at NaCl concentrations up to 200–250 mM were the same as for the control plants grown at 3 mM NaCl; the growth parameters remained rather high even at NaCl concentrations of 300–350 mM. The water content in Ch. album organs remained high at all NaCl concentrations tested. Analysis of the ionic status of Ch. album revealed a comparatively high K⁺ content in plant organs. At low NaCl concentrations in the nutrient solution, K⁺ ions were the dominant contributors to the osmolarity (the total concentration of osmotically active substances) and, consequently, to the lowered cell water potential in leaves and roots. As the concentration of NaCl was increased, the plant organs accumulated larger amounts of Na⁺ and Cl^–, and the contribution of these ion species to osmolarity became increasingly noticeable. At 300–350 mM NaCl the contribution of Na⁺ and Cl^– to osmolarity was comparable to that of K⁺. An electron microscopy study of Ch. album cells revealed that, apart from the usual response to salinity manifested in typical ultrastructural changes of chloroplasts, mitochondria, and the cytosol, the salinity response comprised the enhanced formation of endocytic structures and exosomes and stimulation of autophagy. It is supposed that activation of these processes is related to the removal from the cytoplasm of toxic substances and the cell structures impaired by salt stress conditions. The qualitative and quantitative composition of fatty acids in the lipids of Ch. album organs was hardly affected by NaCl level. These findings are consistent with the high salt tolerance of Ch. album, manifested specifically in retention of growth functions under wide-range variations of NaCl concentration in the nutrient solution and in maintenance of K⁺, Na⁺, and Cl^– content in organs at a constant level characteristic of untreated plants.     

Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system

The anion contents of young barley leaves and of mesophyll protoplasts from the leaves was compared. Anion loss from the protoplasts during isolation was small. Although only about 60% of the leaf cells were mesophyll cells, phosphate and sulfate contents of the mesophyll cells accounted for almost 90% of the leaf contents. Chloride accumulated in the leaf epidermis. The rapid isolation of vacuoles from mesophyll protoplasts permitted the determination of vacuolar ion concentrations. Sodium and nitrate levels were very low in the cytoplasm, and much higher in the vacuole. When barley plants were grown in the presence of low NaCl levels, chloride concentrations were comparable in cytoplasm and vacuole, and similar observations were made with sulfate. Cytoplasmic phosphate concentrations were close to 30 millimolar and potassium concentrations 100 millimolar. During a 30 minute incubation period at room temperature, anion contents of isolated vacuoles decreased considerably. Efflux of NO₃⁻ was faster than that of Cl⁻. Phosphate and sulfate crossed the tonoplast only slowly. 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid partially inhibited the efflux of nitrate and, to a lesser extent, that of chloride. Decreased efflux was also observed in the presence of MgATP. In remarkable contrast, p-chloromercuribenzene sulfonate and HgCl₂ stimulated the efflux of nitrate and chloride, but not of phosphate. Labeled chloride was taken up by isolated vacuoles. The apparent K_m for chloride uptake at low chloride concentrations was 2.3 millimolar. At elevated chloride concentrations, chloride did not display saturation characteristics but, rather, characteristics of a diffusional process. Uptake was stimulated by ATP.     

SIMS Investigation of the Distribution of K+, Na+, Mg2+ and Ca2+ Cations in Birch Pollen

Two microanalytical techniques were used to investigate the inorganic cation content and distributions in birch (Betula verrucosa Ehrh.) pollen. With intact pollen grains. X-ray microanalysis (EDX) could only give a mean ionic composition. Secondary Ion Microscopy and Spectrometry (SIMS) appeared to be a more suitable technique to image ion distributions in the different pollen structures. This was carried out with samples prepared using a new vapour phase technique designed to improve ion retention. Transmission electron microscopy (TEM)showed good structural preservation of the samples. Monovalent ion (K⁺, Na⁺) distribution showed features different from those of the divalent cations (Ca²⁺, Mg²⁺). In the vegetative cell, the alkaline cations were mainly distributed in the most internal part of the cytoplasm and they were probably associated with starch grains or concentrated in dry vacuoles. Calcium distribution correlated well with the areas in the cytoplasm of the vegetative cell containing a dense network of mitochondria and endoplasmic reticulum. Within the pollen grain, the sperm cell appeared to contain the most calcium. Calcium was also abundant in the sporoderm. These results reveal the potential of SIMS for pollen studies that include germination, the monitoring of air pollutants and the allergens-ion interactions.     

Identification and mitotic partitioning strategies of vacuoles in the unicellular red alga <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis>

Cyanidioschyzon merolae is considered as a suitable model system for studies of organelle differentiation, proliferation and partitioning. Here, we have identified and characterized vacuoles in this organism and examined the partitioning of vacuoles using fluorescence and electron microscopy. Vacuoles were stained with the fluorescent aminopeptidase substrate 7-amino-4-chloromethylcoumarin l-arginine amide, acidotrophic dyes quinacrine and LysoTracker, and 4′,6-diamidino-2-phenyl indole, which, at a high concentration, stains polyphosphate. Vacuoles have been shown to be approximately 500 nm in diameter with a mean of around five per interphase cell. The vacuolar H⁺-ATPase inhibitor concanamycin A blocked the accumulation of quinacrine in the vacuoles, suggesting the presence of the enzyme on these membranes. Electron microscopy revealed that the vacuoles were single membrane-bound organelles with an electron-dense substance, often containing a thick layer surrounding the membrane. Immunoelectron microscopy using an anti-vacuolar-H⁺-pyrophosphatase antibody revealed the presence of the enzyme on these membranes. In interphase cells, vacuoles were distributed in the cytoplasm, while in mitotic cells they were localized adjacent to the mitochondria. Filamentous structures were observed between vacuoles and mitochondria. Vacuoles were distributed almost evenly to daughter cells and redistributed in the cytoplasm after cytokinesis. The change in localization of vacuoles also happened in microtubule-disrupted cells. Since no actin protein or filaments have been detected in C. merolae, this result suggests an intrinsic mechanism for the movement of vacuoles that differs from commonly known mechanisms mediated by microtubules and actin filaments.     

Amine Accumulation in Acidic Vacuoles Protects the Halotolerant Alga Dunaliella salina Against Alkaline Stress

Amines at alkaline pH induce in cells of the halotolerant alga Dunaliella a transient stress that is manifested by a drop in ATP and an increase of cytoplasmic pH. As much as 300 millimolar NH₄⁺ are taken up by the cells at pH 9. The uptake is not associated with gross changes in volume and is accompanied by K⁺ efflux. Most of the amine is not metabolized, and can be released by external acidification. Recovery of the cells from the amine-induced stress occurs within 30 to 60 minutes and is accompanied by massive swelling of vacuoles and by release of the fluorescent dye atebrin from these vacuoles, suggesting that amines are compartmentalized into acidic vacuoles. The time course of ammonia uptake into Dunaliella cells is biphasic—a rapid influx, associated with cytoplasmic alkalinization, followed by a temperature-dependent slow uptake phase, which is correlated with recovery of cellular ATP and cytoplasmic pH. The dependence of amine uptake on external pH indicates that it diffuses into the cells in the free amine form. Studies with lysed cell preparations, in which vacuoles become exposed but retain their capacity to accumulate amines, indicate that the permeability of the vacuolar membrane to amines is much higher than that of the plasma membrane. The results can be retionalized by assuming that the initial amine accumulation, which leads to rapid vacuolar alkalinization, activates metabolic reactions that further increase the capacity of the vacuoles to sequester most of the amine from the cytoplasm. The results indicate that acidic vacuoles in Dunaliella serve as a high-capacity buffering system for amines, and as a safeguard against cytoplasmic alkalinization and uncoupling of photosynthesis.     

Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue

Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner G.J. and Siegelman, H.W. (1975) Science 190, (1298–1299). The ATPase activity of fresh vacuole suspensions was found to be 2–3 times that of protoplasts from the same tissue. 70–80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6 μg/10⁶ vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N′-dicyclohexylcarbodiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits.Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tulipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K⁺, Na⁺, Mg²⁺, Cl^?, and Ca²⁺ respectively, which are about the same as those in protoplasts.