In the study, the partial sequences of several nitrate transporter/channel genes of the NPF, NRT2, NAR2 and SLAC/SLAH families from the halophyte Suaeda altissima (L.) Pall. were identified, and their relative transcript abundance in S. altissima plants grown in nutrient media with different concentrations of $${\text{NO}}_{3}^{ - }$$ and Cl– was examined by qRT-PCR. Identification of the partial S. altissima nitrate transporter sequences were carried out assuming similarity of putative S. altissima genes with homologous genes from the halophytes S. fruticosa and S. glauca, which are closely related to S. altissima. Nucleotide sequences of S. fruticosa and S. glauca homologous genes were obtained by in silico analysis of the de novo assembled transcriptomes of these halophytes. The short-read RNA arrays for the transcriptome assemblies were taken from the BioProject database ( https://www.ncbi.nlm.nih.gov/bioproject/ ), NCBI, Acc. no. #PRJNA279962 and #PRJNA295637. Based on the identified contigs in the assembled S. fruticosa and S. glauca transcriptomes that were characterised as putative nitrate transporters or channels, primer sets were designed for the amplification of sequences encoding S. altissima nitrate transporters: SaNPF6.3 (MK 580125.1), SaNRT2.1 (MK 580128.1), SaNRT2.5 (MK 580129.1), SaNAR2.1 (MK 580130.1), SaSLAH1.1 (MK 580131.1) and SaSLAH1.2 (MK 580132.1) (GenBank Acc. no. are given, https://www.ncbi.nlm.nih.gov/genbank/ ). It was shown that the expression of S. altissima genes under consideration is organ-specific and changed in response to changes in NO3ˉ and NaCl concentrations in the growth medium.

The effects of N,N′-dicyclohexylcarbodiimide (DCCD), non-specific inhibitor of various transport systems functioning in biological membranes, on Na+-transporting P-type ATPase of the green halotolerant microalga Dunaliella maritima were studied in the experiments with vesicular plasma membranes isolated from the alga cells. The effects of DCCD on electrogenic/ion transport function of the enzyme and its ATP hydrolase activity were investigated. Electrogenic/ion transport function of the enzyme was recorded as a Na+-dependent generation of electric potential on the vesicle membranes with the help of the potential-sensitive probe oxonol VI. It was found that unlike many other ion-transporting ATPases, the Na+-ATPase of D. maritima is insensitive to DCCD. This agent did not inhibit either ATP hydrolysis catalyzed by this enzyme or its transport activity. At the same time DCCD affected the ability of the vesicle membranes to maintain electric potential generated by the D. maritima Na+-ATPase. The observed effects can be explained based on the assumption that DCCD interacts with the Na+/H+ antiporter in the plasma membrane of D. maritima.

Partial sequences of P-type ATPases were cloned from the marine microalgae Dunaliella maritima: two presumably H+-ATPases (DmHA1 and DmHA2) and two putative Ca2+-ATPases (DmCA1 and DmCA2). The functions of cloned proteins were estimated on the bases of their primary structure similarity with the proteins whose functions have been already characterized. The transcriptional response of cloned ATPase genes to a sharp increase in the NaCl concentration in the culture medium (from 100 to 500 mM) was investigated by quantitative RT-PCR. Hyperosmotic salt shock led to a significant increase in DmHA2 expression and to a slight increase in DmCA2 expression, while the expression of two other ATPases, DmHA1 and DmCA1, was repressed. The obtained data indicate that DmHA2 is involved in maintenance of ion homeostasis in D. maritima cells under hyperosmotic salt shock.

Министерство науки и высшего образования РФ Российская академия наук Общество физиологов растений России Правительство Республики Татарстан Федеральный исследовательский центр «Казанский научный центр Российской академии наук» Казанский институт биохимии и биофизики ФИЦ КазНЦ РАН Институт физиологии растений им.К.А

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.

2 Федеральное государственное бюджетное образовательное учреждение высшего образования «Санкт-Петербургский государственный университет», Центр геномной биоинформатики им

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe2+-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn2+. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl– ions.

The involvement of endocytosis in the Na+ ion uptake from the external medium by the cells of suspension culture derived from A. thaliana (Col-0) leaves was investigated. Na+ ion uptake by endocytic structures occurred following the addition of NaCl at the final concentration of 100 mM to the incubation medium. The presence of Na+ in membranous structures was recorded using fluorescence microscopy by colocalization of FM4-64, a marker of endocytosis structures, and Asante NaTRIUM Green-2 TMA+ salt (ANG-2 TMA), a membrane impermeable probe for sodium ions, that enabled the detection of Na+ absorbed by the cells via endocytosis but not through ion channels or transporters of the plasma membrane. Following a 1.5-h incubation of the cells in the presence of NaCl, FM4-64 and ANG-2 TMA, fluorescence of the probes was colocalized in structures with sizes ranging from 800 to 3000 nm. It was shown by electron microscopy that NaCl added to the cell incubation medium stimulated vesiculation and vacuolization of the cytoplasm, formation of plasma membrane invaginations, as well as fusion of microvacuoles with each other. The size of the structures, in which the colocalization of the two probes was detected by fluorescent microscopy, matched the size of the microvacuoles revealed by the electron microscopy. The obtained results indicate the capture of sodium ions contained in the apoplast by endocytosis invaginations, their subsequent internalization by the cells, and transfer into microvacuoles.

ВОЗМОЖНАЯ РОЛЬ БЕЛКА МЕМБРАННЫХ МИКРОДОМЕНОВ ARABIDOPSIS THALIANA Flot1 В РЕГУЛЯЦИИ ЭКСПРЕССИИ ГЕНАNа +

Аннотация.Измерено содержание K + , Na + и Cl -в корне и листе, а также исследовано распределение элемента K + по тканям у галофита Suaeda altissima (L.) Pall.в условиях засоления.NaCl приводил к увеличению содержания ионов в корне.В листе содержание Na + и Cl -при этом возрастало, а K + снижалось.Продемонстрировано дифференциальное распределение K в тканях органов и влияние засоления на

2 Федеральное государственное бюджетное образовательное учреждение высшего образования "Санкт-Петербургский государственный университет", Центр геномной биоинформатики им

The total content of quaternary ammonium compounds (QAC) and the choline content in roots, leaves, and xylem exudates of the halophyte Suaeda altissima (L.) Pall. were determined after growing plants at various NaCl concentrations in the nutrient solution (1, 50, 100, and 250 mM). Based on the results obtained, the content of glycine betaine in organs and xylem exudates of S. altissima was estimated as the difference between the total content of QAC and the choline content. In roots choline accounted for the largest portion of QAC (from 69 to 96% at various NaCl concentrations in nutrient media), whereas in leaves it contributed only 12–23%. The contribution of choline to QAC content in the xylem exudates was 84–90%. It is concluded that choline in S. altissima is mainly synthesized in roots and is delivered with the ascending water flow to leaves where it is utilized as a substrate for glycine betaine synthesis. The content of glycine betaine in leaves increased with elevation of NaCl concentration in the nutrient solution, thus contributing appreciably to the maintenance of osmotic balance in the cytoplasm of S. altissima at high salinity.

Na+ homeostasis in the cytoplasm is a common property of all organisms, irrespective of their taxonomic position. Low Na+ concentrations in the cytoplasm of living cells are maintained by specialized Na+-transporting molecular machines operating in the cell membranes. In eukaryotic cells, Na+-transporting ATPases of P-type play the important role in keeping the Na+ homeostasis. This review summarizes the authors’ investigations demonstrating the operation of the Na+-transporting P-type ATPases in the plasma membrane of green marine microalgae. Experiments described here provided the first evidence for the existence of the primary Na+-pump in plasma membranes of organisms attributed to the plant kingdom. The significance of the Na+-ATPases in halotolerant microalgae Dunaliella maritima and Tetraselmis viridis inhabiting saline environments is discussed.

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 (F 0, F m, F 0′, F t′) 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.