N. A. Myasoedov

Significance of Na+ and K+ for Sustained Hydration of Organ Tissues in Ecologically Distinct Halophytes of the Family Chenopodiaceae

The contents of Na+, K+, water, and dry matter were measured in leaves and roots of euhalophytes Salicornia europaea L. and Climacoptera lanata (Pall.) Botsch featuring succulent and xeromorphic cell structures, respectively, as well as in saltbush Atriplex micrantha C.A. Mey, a halophyte having bladder-like salt glands on their leaves. All three species were able to accumulate Na+ in their tissues. The Na+ content in organs increased with elevation of NaCl concentration in the substrate, the concentrations of Na+ being higher in leaves than in roots. When these halophytes were grown on a NaCl-free substrate, a trend toward K+ accumulation was observed and was better pronounced in leaves than in roots. Particularly high K+ concentrations were accumulated in Salicornia leaves. There were no principal differences in the partitioning of Na+ and K+ between organs of three halophyte species representing different ecological groups. At all substrate concentrations of NaCl, the total content of Na+ and K+ in leaves was higher than in roots. This distribution pattern persisted in Atriplex possessing salt glands, as well as in euhalophytes Salicornia and Climacoptera. The physiological significance of such universal pattern of ion accumulation and distribution among organs in halophytes is related to the necessity of water absorption by roots, its transport to shoots, and maintenance of sufficient cell water content in all organs under high soil salinity.

Involvement of Long-Distance Na+ Transport in Maintaining Water Potential Gradient in the Medium-Root-Leaf System of a Halophyte Suaeda altissima

The aim of this study was to determine the range of NaCl concentrations in the nutrient solution that allow Suaeda altissima (L.) Pall., a salt-accumulating halophyte, to maintain the upward gradient of water potential in the “medium-root-leaf” system. We evaluated the contribution of Na+ ions in the formation of water potential gradient and demonstrated that Na+ loading into the xylem is involved in this process. Plants were grown in water culture at NaCl concentrations ranging from zero to 1 M. The water potential of leaf and root cells was measured with the method of isopiestic thermocouple psychrometry. When NaCl concentration in the growth medium was raised in the range of 0–500 mM (the medium water potential was lowered accordingly), the root and leaf cells of S. altissima decreased their water potential, thus promoting the maintenance of the upward water potential gradient in the medium-root-leaf system. Growing S. altissima at NaCl concentrations f 750 mM and 1 M disordered water homeostasis and abolished the upward gradient of water potential between roots and leaves. At NaCl concentrations of 0–250 mM, the detached roots of S. altissima were capable of producing the xylem exudate. The concentration of Na+ in the exudate was 1.3 to 1.6 times higher than in the nutrient medium; the exudate pH was acidic and was lowered from 5.5 to 4.5 with the rise in the salt concentration. The results indicate that the long-distance Na+ transport and, especially, the mechanism of Na+ loading into the xylem play a substantial role in the formation of water potential gradient in S. altissima. The accumulation of Na+ in the xylem and acidic pH values of the xylem sap suggest that Na+ loading into the xylem is carried out by the Na+/H+ antiporter of the plasma membrane in parenchymal cells of the root stele.

Structural and Functional State of Thylakoids in a Halophyte Suaeda altissima before and after Disturbance of Salt–Water Balance by Extremely High Concentrations of NaCl

Halophyte plants Suaeda altissima L. were grown in water culture at different concentrations of NaCl in the medium, and their leaves were sampled to examine the ultrastructure of chloroplasts. In parallel tests, the functional state of chloroplasts was assessed from parameters of chlorophyll fluorescence. In addition the effects of NaCl on plant growth and on the contents of Na+, K+, and water in organs of S. altissima were investigated. At a wide range of external salt concentrations (0–750 mM NaCl), S. altissima plants retained the chloroplast ultrastructure and photosynthetic function in an intact condition. The impairment of thylakoid ultrastructure and the accompanying increase in nonphotochemical quenching of excited states of chlorophyll was only observed at an extremely high concentration of NaCl in the medium (1 M) that led to disruption of ionic homeostasis and lowered water content in tissues.

Pinocytosis in the root cells of a salt-accumulating halophyte Suaeda altissima and its possible involvement in chloride transport

Ultrastructure of root cells in salt-accumulating halophyte Suaeda altissima (L.) Pall. was examined with transmission electron microscopy. Plants were grown hydroponically on nutrient media containing 3, 50, 250, and 500 mM NaCl. Some plants were exposed to hypersomotic salt shock by an abrupt increase in NaCl concentration from 50 to 400 mM. Growing S. altissima plants at high NaCl concentrations induced the formation of type 1 pinocytotic structures in root cells. Type 1 structures appeared as pinocytotic invaginations of two membranes, the plasmalemma and tonoplast. These invaginations into vacuoles gave rise to freely ‘floating’ multivesicular bodies (MVB) enclosed by a double membrane layer. The pinocytotic invaginations and MVB contained the plasmalemma-derived vesicles and membranes of endosome origin. The hyperosmotic salt shock led to formation of type 2 and type 3 pinocytotic structures. The type 2 structures were formed as pinocytotic invaginations of the tonoplast and gave rise to MVB in vacuoles. Unlike type 1 MVB, the type 2 MVB had only one enclosing membrane, the tonoplast. The type 3 structures appeared as the plasmalemma-derived vesicles located in the periplasmic space. The cytochemical electron-microscopy method was applied to determine the intracellular Cl− localization. This method, based on sedimentation of electron-dense AgCl granules in tissues treated with silver nitrate, showed that the pinocytotic structures of all types contain Cl− ions. The presence of Cl− in pinocytotic structures implies the involvement of these structures in Cl− transport between the apoplast, cytoplasm, and the vacuole.

Functional Identification of H+-ATPase and Na+/H+ Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte Suaeda altissima

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H+-ATPase in the PM of the root cells of S. altissima. The addition of Na+ and Li+ ions to the outer medium resulted in dissipation of ΔpH preformed by the H+-ATPase, which indicates the presence in PM of the functionally active Na+/H+ antiporter. The results are discussed with regard to involvement of the Na+/H+ antiporter and the PM H+-ATPase in loading Na+ ions into the xylem of S. altissima roots.

Contributions of inorganic ions, soluble carbohydrates, and multiatomic alcohols to water homeostasis in Artemisia lerchiana and A. pauciflora

Two morphological forms of wormwood Artemisia lerchiana (f. erecta and f. nutans) and A. pauciflora Web. (morphological form erecta) were grown on sand culture at a range of NaCl concentrations in the nutrient medium and then assayed for Na+, K+, and Cl− content in various organs. In addition, the content of mono-, di-, and trisaccharides and multiatomic alcohols (mannitol and glycerol); water content; and organ biomass were determined. All plants examined showed high NaCl tolerance, comparable to that of halophytes. They were able to maintain high tissue hydration under conditions of salinity-induced growth suppression. The intracellular osmotic pressure in wormwood organs was mainly determined by the presence of Na+, K+, and Cl−, as well as by mono-, di-, and trisaccharides, mannitol, and glycerol. The high content of Na+ and Cl− in wormwood organs was also observed in the absence of salinity, which implies the ability of these organs to absorb ions from diluted NaCl solutions and accumulate ions in cells of their tissues. With the increase in salinity, the content of Na+ and Cl− in roots and leaves increased to even higher levels. It is concluded that the ability of wormwood plants to absorb and accumulate inorganic ions provides for sustainable high intracellular osmotic pressure and, accordingly, low water potential under drought and salinity conditions. Growing plants under high salinity lowered the content of monosaccharides in parallel with accumulation of the trisaccharide raffinose. It is supposed that soluble carbohydrates and multiatomic alcohols are not only significant for osmoregulation but also perform a protective function in wormwood plants. The lower osmotic pressure in root cells compared to that in leaf cells of all plants examined was mainly due to the gradient distribution of K+ and Cl− between roots and leaves. The two Artemisia species and two morphological forms of A. lerchiana did not differ appreciably in the ways of water balance regulation. It is found that different morphologies of two A. lerchiana forms are unrelated to variations in intracellular osmotic and turgor pressures.

Increased content of very-long-chain fatty acids in the lipids of halophyte vegetative organs.

Qualitative and quantitative compositions of esterified fatty acids (FAs) in the total lipids from the leaves, shoots, and roots of halophile plants, such as suaeda (Suaeda altissima), samphire (Salicornia europaea), and wormwood (Artemisia lerchiana), collected in their natural environments were estimated by GLC techniques. It was shown that the vegetative organs of these halophytes contained 24 FA species, and 16 of them were tentatively identified as the very-long-chain FAs (VLCFAs). There were four VLCFA groups, viz. C20, C21, C22, and C23, each including saturated, mono-, and diunsaturated components; C24 and C25 FAs were also present. The concentration of VLCFAs in the total FAs comprised 4–64%. In vegetative organs of higher plants not subjected to genetic transformation, such a high VLCFA content was found for the first time. Saturated and even-numbered components predominated among the VLCFAs, and the roots exceeded severalfold the above-ground organs in the total VLCFA content. Possible pathways of VLCFA biosynthesis in plants, VLCFA content in the vegetative tissues, and the physiological role of membrane lipid FA composition in the plant salt metabolism are discussed.

Cell ultrastructure and fatty acid composition of lipids in vegetative organs of Chenopodium album 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.

Evidence for the functioning of a Cl−/H+ antiporter in the membranes isolated from root cells of the halophyte Suaeda altissima and enriched with Golgi membranes

Cl−/H+ exchange activity in the membranes isolated from the root cells of the halophyte Suaeda altissima (L.) Pall. was originally revealed and characterized. The membrane vesicles were isolated by centrifugation of microsomes in a continuous iodixanol density gradient. The highest activity of latent inosine phosphatase, a marker of Golgi membranes, was localized in the upper part of the gradient, indicating its enrichment with Golgi membranes. The same part of the gradient was characterized by the highest Cl−/H+ exchange rate. The Cl−/H+ exchange activity was detected as electrogenic ΔpCl-dependent H+ transport monitored as changes in differential absorbance of a ΔpH-probe acridine orange, or as changes in fluorescence excitation spectrum of a pH-probe pyranine loaded into the vesicles. Generation of transmembrane electric potential (Δψ) during the Cl−/H+ exchange was assayed as changes in differential absorbance of a Δψ-probe safranin O. Establishing the transmembrane ΔpCl inward vesicles resulted in H+ efflux sensitive to DIDS (4,4′-diisothiocyano-2,2′-stylbene-disulfonic acid), an inhibitor of chloride transporters and channels, and generation of Δψ negative inside. To maintain the ΔpCl-dependent H+ efflux from the vesicles, either the presence of a penetrating cation tetraphenylphosphonium neutralizing negative charges inside the vesicles or null K+ diffusion potential across the membranes was required. The results demonstrate the activity of an electrogenic Cl−/H+ antiporter in the fraction enriched with Golgi membranes. We hypothesize that the Cl−/H+ antiporter is involved into the regulation of cytoplasmic Cl− concentrations by vesicular trafficking of Cl− from the cytoplasm to the vacuole by endosomes, derivatives of Golgi membranes.

Fatty acid composition of lipids in vegetative organs of the halophyte Suaeda altissima under different levels of salinity

Qualitative and quantitative composition of fatty acids (FA) in the lipids of vegetative organs of the halophyte Suaeda altissima (L.) Pall. grown at different NaCl concentrations in nutrient solution was studied. Along with this, the biomass of these organs, the content of water and Na+, Cl−, and K+ ions in them, and the ultrastructure of root and leaf cells were determined. At both low (1 mM) and high (750 mM) NaCl concentrations in nutrient solution, plants could maintain growth and water content in organs, demonstrating a noticeable increase in the dry weight and a slight increase in the water content at 250 mM NaCl. At all NaCl concentrations in nutrient solution, S. altissima tissues contained a relatively high K+ amount. Under salinity, Na+ and Cl− ions contributed substantially into the increase in the cell osmotic pressure, i.e., a decrease in their water potential; in the absence of salinity, K+ fulfilled this function. In the cells of both roots and leaves, NaCl stimulated endo- and exocytosis, supposedly involved in the vesicular compound transport. 750 mM NaCl induced plasmolysis and changes in the membrane structure, which can be interpreted as degradation processes. Under optimal NaCl concentration in medium (250 mM), the content of lipids in plant aboveground organs per fresh weight was more than 2.5-fold higher than under 1 or 750 mM NaCl, whereas in the roots opposite patten was observed. When plants were grown under non-optimal conditions, substantial changes occurred in the qualitative and quantitative FA composition in lipids of both aboveground organs and roots. Observed changes are discussed in relation to processes underlying S. altissima salt tolerance and those of disintegration occurring at the high external NaCl concentration (750 mM).