L. G. Popova

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.

Functional identification of electrogenic Na+-translocating ATPase in the plasma membrane of the halotolerant microalga Dunaliella maritima.

The hypothesis that the primary Na+‐pump, Na+‐ATPase, functions in the plasma membrane (PM) of halotolerant microalga Dunaliella maritima was tested using membrane preparations from this organism enriched with the PM vesicles. The pH profile of ATP hydrolysis catalyzed by the PM fractions exhibited a broad optimum between pH 6 and 9. Hydrolysis in the alkaline range was specifically stimulated by Na+ ions. Maximal sodium dependent ATP hydrolysis was observed at pH 7.5–8.0. On the assumption that the ATP‐hydrolysis at alkaline pH values is related to a Na+‐ATPase activity, we investigated two ATP‐dependent processes, sodium uptake by the PM vesicles and generation of electric potential difference (Δψ) across the vesicle membrane. PM vesicles from D. maritima were found to be able to accumulate 22Na+ upon ATP addition, with an optimum at pH 7.5–8.0. The ATP‐dependent Na+ accumulation was stimulated by the permeant NO 3 ‐ anion and the protonophore CCCP, and inhibited by orthovanadate. The sodium accumulation was accompanied by pronounced Δψ generation across the vesicle membrane. The data obtained indicate that a primary Na+ pump, an electrogenic Na+‐ATPase of the P‐type, functions in the PM of marine microalga D. maritima.

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+.

The Na+-translocating ATPase in the plasma membrane of the marine microalga Tetraselmis viridis catalyzes Na+/H+ exchange

Our previous investigations have established that Na+ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na+-pump, Na+-ATPase, is accompanied by H+ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na+-ATPase of T. viridis operates as an Na+/H+ exchanger is tested in the present work. The study of Na+ and H+ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO3− caused (i) an increase in ATP-driven Na+ uptake by the vesicles, (ii) an increase in (Na++ATP)-dependent vesicle lumen alkalization resulting from H+ efflux out of the vesicles and (iii) dissipation of electrical potential, Δψ, generated across the vesicle membrane by the Na+-ATPase. The (Na++ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K+ abolished Δψ at the vesicle membrane. The fact that the Na+-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane Δψ is consistent with a primary role of the Na+-ATPase in driving H+ outside the vesicles. The findings allowed us to conclude that the Na+-ATPase of T. viridis directly performs an exchange of Na+ for H+. Since the Na+-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa+/nH+, where m>n.

Electrogenicity of the Na+-ATPase from the marine microalga Tetraselmis (Platymonas) viridis and associated H+ countertransport.

Sodium accumulation by the Na+‐ATPase in the plasma membrane (PM) vesicles isolated from the marine alga Tetraselmis (Platymonas) viridis was shown to be accompanied by ΔΨ generation across the vesicle membrane (positive inside) and H+ efflux from the vesicle lumen. Na+ accumulation was assayed with 22Na+; ΔΨ generation was detected by recording absorption changes of oxonol VI; H+ efflux was monitored as an increase in fluorescence intensity of the pH indicator pyranine loaded into the vesicles. Both ATP‐dependent Na+ uptake and H+ ejection were increased by the H+ ionophore carbonyl cyanide m‐chlorophenylhydrazone (ClCCP) while ΔΨ was collapsed. The lipophilic anion tetraphenylboron ion (TPB−) inhibited H+ ejection from the vesicles and abolished ΔΨ. Based on the effects of ClCCP and TPB− on H+ ejection and ΔΨ generation, the conclusion was drawn that H+ countertransport observed during Na+‐ATPase operation is a secondary event energized by the electric potential which is generated in the course of Na+ translocation across the vesicle membrane. Increasing Na+ concentrations stimulated H+ efflux and caused the decrease in the ΔΨ observed, thus indicating that Na+ is likely a factor controlling H+ permeability of the vesicle membrane.

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na+-dependent H+ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na+ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with 22Na+ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na+-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent 22Na+ uptake and abolished the electric potential generated by the Na+-ATPase at the vesicle membrane. A permeant anion NO−3 accelerated ATP-dependent 22Na+ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO−3 inhibited the ATP-and Na+-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na+-dependent H+ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na+-ATPase. Hence, the Na+-transporting ATPase of D. maritima carries only one ion species, i.e., Na+. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.

Responses of photosynthetic apparatus of the halotolerant microalga Dunalliella maritima to hyperosmotic salt shock

Chlorophyll fluorescence induction curves were used as a means to assess the functional condition of the photosynthetic apparatus in cells of the halotolerant green microalga Dunaliella maritima (Massjuk) (division Chlorophyta) exposed to hyperosmotic salt shock of various intensities. The shock was caused by the transfer of algal cells grown in the medium with 0.5 M NaCl to the media with elevated NaCl concentrations (1.0, 1.5, and 2.0 M). Parameters of chlorophyll fluorescence (F0, Fm, F0′, Ft′) were measured by means of a specialized pulse-amplitude-modulation fluorometer PAM 2100. In addition, the rate of photosynthetic oxygen evolution as well as the intracellular Na+ and glycerol content (the main osmolyte in this microalga) were determined. The hyperosmotic salt shock was found to elevate the intracellular Na+ content and reduce the functional activity of PSII in D. maritima. The suppression of PSII activity was evident from the decrease in the maximal quantum yield of photochemical energy conversion in PSII, the decreased rate of linear electron transport, the increased reduction of the primary acceptor QA, and the suppression of photosynthetic O2 evolution. The functional activity of PSII recovered gradually along with restoration of osmotic and ionic balance in algal cells. It is proposed that PSI ensures energy supply during cell responses of D. maritima to hyperosmotic salt shock.

Monophasic kinetics of electron donation from stromal reductants in unicellular halophytic alga Tetraselmis viridis: Relation of the function to characteristic structure of chloroplast membrane system

Redox conversions of P700, the primary donor of photosystem I (PSI), were investigated in cells of a halophytic alga Tetraselmis viridis Rouch. under irradiation with white light pulses that excite both photosystems of the chloroplast and with far-red light initiating photochemical reactions in PSI only. The P700+ dark reduction after irradiation with 50-ms pulse of white light comprised three kinetic components. The half-decay times and relative contributions of the fast, middle, and slow components were 38 ms (49%), 295 ms (26%), and 1690 ms (23%), respectively. The treatment with diuron, known to block electron transport between the photosystems, eliminated the middle exponential term having the half-decay time of 295 ms. After irradiation with far-red light, the kinetics of P700+ dark reduction comprised only two components with half-deacy times of 980 ms (72%) and 78 ms (31%). The component with a decay halftime of about 100 ms was fully inhibited after treating the cells with antimycin A, a specific inhibitor of ferredoxin-dependent cyclic electron flow around PSI. In addition, this kinetic component was strongly suppressed by methyl viologen known to inhibit this alternative pathway of electron transport. Both aforementioned reagents had no effect on the slow component of P700+ reduction; this component remained monophasic. Unlike higher plant chloroplasts, the chloroplasts of Tetraselmis viridis contained no stacked grana. Based on inhibitor analysis and electron microscopy data, it was concluded that the slow component of P700+ reduction in the cells of halophytic microalga reflects the electron donation to PSI from reductants localized in the chloroplast stroma. The monophasic kinetics of this process in the halophytic microalga, compared to the biphasic kinetic pattern in higher plants, is related to the lack of stacked grana in Tetraselmis viridis cells.

Possible changes of Na(+)-ATPase kinetic characteristics in Tetraselmis viridis microalga upon adaptation to various concentrations of NaCl.

The maintenance of ionic homeostasis in the cytoplasm is a crucial physiological process inherent to all live cells, plant cells in particular. Under salinity, plant cells experience salt stress, which disturbs intracellular ionic balance, leads to accumulation of Na + in the cytoplasm, and causes toxic effects. To cope with toxic action of Na + ions, plant cells exploit the following mechanisms: (1) restriction of Na + entry into the cell, (2) sequestration of Na + in the vacuole, (3) extrusion of Na + from the cell to the outer medium through the plasma membrane (PM) [1]. In poorly vacuolated unicellular algae, e.g., representatives of the genus Tetraselmis , the first and the third mechanisms are particularly important. Even under a slight salt stress, the transmembrane electrochemical gradient of Na + at PM is directed from the medium into the cell. Therefore, the third protective mechanism can play the major role in maintaining ionic homeostasis in the cytoplasm of salttolerant plants.

In silico Analyses of Transcriptomes of the Marine Green Microalga Dunaliella tertiolecta: Identification of Sequences Encoding P-type ATPases

De novo assembled transcriptomes of the marine microalga Dunaliella tertiolecta (Chlorophyta) were analyzed. Transcriptome assemblies were performed using short-read RNA-seq data deposited in the SRA database (DNA and RNA Sequence Read Archive, NCBI). A merged transcriptome was assembled using a pooled RNA-seq data set. The goal of the study was in silico identification of nucleotide sequences encoding P-type ATPases in D. tertiolecta transcriptomes. P-type ATPases play a considerable role in the adaptation of an organism to a variable environment, and this problem is particularly significant for microalgae inhabiting an environment with an unstable ionic composition. Particular emphasis was given to searching for a sequence coding Na+-ATPase. This enzyme is expected to function in the plasma membrane of D. tertiolecta like in some marine algae, in particular, in the closely related alga Dunaliella maritima. An ensemble of 12 P-type ATPases consisting of members belonging to the five main subfamilies of the P-type ATPase family was revealed in the assembled transcriptomes. The genes of the following P-type ATPases were found: (1) heavy metal ATPases (subfamily PIB); (2) Ca2+-ATPases of SERCA type (subfamily P2A); (3) H+-ATPases (subfamily P3); (4) phospholipid-transporting ATPases (flippases) (subfamily P4); (5) cation- transporting ATPases of uncertain specificities (subfamily P5). The presence of functional Na+-ATPases in marine algae is presently undoubted. However, contrary to expectations, we failed to find a nucleotide sequence encoding a protein that could unequivocally be considered a Na+-ATPase. Further study is necessary to elucidate the roles of in silico revealed D. tertiolecta ATPases in Na+ transport.