Yu. V. Balnokin

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.

Physiological Aspects of Adaptation of the Marine Microalga Tetraselmis (Platymonas) viridis to Various Medium Salinity

We studied the capability of the marine microalga Tetraselmis (Platymonas) viridis to adapt to low and high medium salinity. The normal NaCl concentration for growth of this alga is 0.5 M. It was shown that T. viridis cells could actively grow and maintain osmoregulation and cytoplasmic ion homeostasis in the wide range of external salt concentrations, from 0.01 to 1.2 M NaCl. Using the plasma membrane vesicles isolated from T. viridis cells grown at various NaCl concentrations (0.01, 0.05, 0.5, 0.9, and 1.2 M), we studied the formation of the phosphorylated intermediate of Na+-ATPase, the enzyme responsible for Na+ export from the cells with a mol wt of ca. 100 kD. Na+-ATPase was shown to function in the plasma membrane even in the cells growing at an extremely low NaCl concentration (0.01 M). When alga was grown in high-salt media, the synthesis of several proteins with molecular weights close to 100 kD was induced. The data obtained argue for the hypothesis, which was put forward earlier, that a novel Na+-ATPase isoform is induced by T. viridis growing at high NaCl concentrations.

Further evidence for an ATP-driven sodium pump in the marine alga Tetraselmis (Platymonas) viridis

Summary The ATP-dependent 22 Na + accumulation by inside/out plasma membrane vesicles isolated from the marine green microalga Tetraselmis (Platymonas) viridis Rouch. has been studied. The 22 Na + uptake was observed only within a narrow region of weakly alkaline pH with a maxium at pH 7.8–8.0. When permeant anions such as NO 3 − , were absent from the reaction medium, ATP-dependent 22 Na + uptake was low but could be stimulated by 6 μmol/L ClCCP. In the presence of NO 3 − , however, the rate of ATP-dependent 22 Na + uptake was much higher than in the absence of nitrate and not affected by the uncoupler ClCCP. The pH optimum of ATP-driven 22 Na + uptake differed from that of ATP-dependent ΔpH formation across the vesicle membranes (optimum pH 6.0–7.0). These data indicate that a ΔμH + -dependent Na + /H + antiporter does not contribute to the observed uptake under the experimental conditions; 100 μmol/L orthovanadate completely inhibited ATP-dependent 22 Na + uptake, whereas the uptake was not affected by 50 μmol/L amiloride and only slightly reduced by 200 μmol/L of this inhibitor. It is concluded that the ATP-supported 22 Na + uptake by Tetraselmis (Platymonas) viridis plasma membrane vesicles is carried out by a mechanism independent of the proton-motive force. For example it could be catalysed by a primary Na + -pump. It is thought that this pump is an orthovanadate-sensitive electrogenic Na + -ATPase.

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.

Some Methods for Human Liquid and Solid Waste Utilization in Bioregenerative Life-Support Systems

Bioregenerative life-support systems (BLSS) are studied for developing the technology for a future biological life-support system for long-term manned space missions. Ways to utilize human liquid and solid wastes to increase the closure degree of BLSS were investigated. First, urine and faeces underwent oxidation by Kudenko’s physicochemical method. The products were then used for root nutrition of wheat grown by the soil-like substrate culture method. Two means of eliminating sodium chloride, introduced into the irrigation solution together with the products of urine oxidation, were investigated. The first was based on routine electrodialysis of irrigation water at the end of wheat vegetation. Dialysis eliminated about 50% of Na from the solution. This desalinization was performed for nine vegetations. The second method was new: after wheat cultivation, the irrigation solution and the solution obtained by washing the substrate containing mineral elements not absorbed by the plants were used to grow salt-tolerant Salicornia europaea L. plants (saltwort). The above-ground biomass of this plant can be used as a food, and roots can be added to the soil-like substrate. Four consecutive wheat and Salicornia vegetations were cultivated. As a result of this wheat and Salicornia cultivation process, the soil-like substrate salinization by NaCl were considerably decreased.

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.

Ion Specificity of Na+-Transporting Systems in the Plasma Membrane of the Halotolerant Alga Tetraselmis (Platymonas) viridis

Vesicular preparations of plasma membranes (PM) from the microalga Tetraselmis (Platymonas) viridisRouch were used to investigate the ion specificity of the Na+/H+antiporter and Na+-translocating ATPase, two Na+-transporting systems previously identified functionally by our studies of T. viridisPM. The Na+/H+antiporter and Na+-ATPase were shown to translocate, with similar efficiencies, Na+and Li+across the membrane, whereas other cations, such as K+, Rb+, and Cs+, were not transported by these systems. Transport of the latter cations across PM of T. viridisoccurred through the ion channels of PM, which were apparently selective for K+.

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.