Dmitrii E. Khramov

Soil salinization is a widespread abiotic stress factor that significantly limits the growth, development, and productivity of plants. One of the effects of salt stress on plants is a disruption in mineral nutrient uptake, particularly due to the competition between nitrate and chloride anions for the root ion transport systems. In this study, we identified key genes in the salt-tolerant barley Hordeum marinum that regulate nitrate uptake from the soil solution and encode membrane proteins of the NPF, NRT2, and NAR2 families. When the expression profiles of nitrate transporter genes in H. marinum and H. vulgare were compared, differences were found in the response of NPF family genes to the addition of NaCl to the medium: in H. marinum, transcription of the HmNPF6.3B1 gene was significantly induced, whereas the expression level of orthologous genes in H. vulgare was almost independent of the NaCl concentration in the medium. Changes in the expression of nitrate transporter genes are consistent with data on the accumulation of nitrate and chloride anions in the organs of H. marinum and H. vulgare and indicate a greater ability of H. marinum, compared to H. vulgare, to maintain nitrate influx and limit chloride entry under salt stress conditions. Using the bimolecular fluorescence complementation (BiFC) method, the interaction of NRT2 family proteins of H. marinum with the HmNAR2.3 protein was detected, and using a heterologous expression system based on the yeast Ogataea parapolymorpha, the nitrate transport activity of the HmNPF6.3B1 protein was demonstrated.

NAR2 family proteins are involved in the uptake of nitrate by plant roots as partners of high-affinity nitrate transporters of NRT2 family. In this study, we cloned the coding sequence of a gene, which encodes a protein consisting of 196 amino acids with a calculated molecular weight of 21.7 kDa, from the euhalophyte Suaeda altissima. The protein was characterized in terms of structure and phylogeny. On the phylogenetic tree of NAR2 family proteins, the protein from S. altissima is located in a clade with proteins from the NAR2.2 subfamily found in species within the Amaranthaceae. Therefore, on the basis of phylogenetic relationships, we named it SaNAR2.2. According to a prediction of cellular localization and protein topology, SaNAR2.2 is a likely resident of the plasma membrane. We detected the interaction between SaNAR2.2 and putative partner proteins SaNRT2.1 and SaNRT2.5, which are members of the high-affinity nitrate transporter family NRT2 in S. altissima, using the bimolecular fluorescence complementation (BiFC) assay in Nicotiana benthamiana leaves and the yeast mating-based split-ubiquitin system (mbSUS). SaNAR2.2 was expressed predominantly in roots of S. altissima. The level of SaNAR2.2 transcripts significantly increases at low nitrate concentrations in the medium at the background of high NaCl concentrations (up to 0.5 M). These findings point to the important role of SaNAR2.2 as a participant in the high-affinity nitrate transport system under saline conditions.

Water Use Efficiency (WUE) regulation plays a key role in the plant response to global climate changes such as warming (New et al. in Philos Trans R Soc A Math Phys Eng Sci 369:6–19, 2011). Nowadays enhanced plant drought tolerance has become a priority trait in current crop improvement efforts. To gain insight into water requirement regulation of the drought-resistant halophyte Sueda altissima we determined WUE_i under various light conditions. RBL combination (50% red, 50% blue) contributed to twice stomatal conductance/transpiration decreases and a more pronounced response to green/far-red supplementation compared to glycophytic species. The most rapid WUE_i improvement was observed under far-red light conditions (0% R₆₆₀ + 100% FR). The critical role in halophyte growth and development played optimization of red-blue-green light balance in the spectrum. Besides, we also investigated moderate or strong (r > 0.5, r > 0.8) correlations between stomatal conductance (g _s ), transpiration (E), WUE_i and consequently SaSLAH3 expression. Moreover, this report identified 14- and 20-fold higher than control expression increases in responce to R/FR used together. These results suggested that SLAH3 channels have to be involved in phytochrome-controlled pathways of stomatal aperture regulation.

Plasma membrane-associated proteins of NAR2 family are involved in the uptake of nitrate by plant roots as partners of NRT2 family proteins, which carry out high- affinity nitrate transport across membranes of plant cells. In this study, the coding sequence of NAR2 family gene, SaNAR2.2, was cloned from the euhalophyte Suaeda altissima . This species is able to withstand high concentrations of NaCl, up to 750 mM, in the nutrient medium. The protein SaNAR2.2, of 196 amino acids in size, with a calculated molecular weight of 21.7 kDa, has been characterized in terms of structure and phylogeny. According to the prediction of cellular localization and protein topology, SaNAR2.2 is a resident of plasma membrane. It has one transmembrane segment at the C-terminal part and a small C-end located in cytoplasm. The large N-terminal part of SaNAR2.2 protein is located in the extracellular space. On the phylogenetic tree of NAR2 family proteins, the protein from S. altissima lies in a clade with the proteins of NAR2.2 subfamily from other plants of Amaranthaceae family. We detected the interaction between SaNAR2.2 and putative partner proteins SaNRT2.1 and SaNRT2.5, which are the members of high-affinity nitrate transporter family NRT2 from S. altissima , using the bimolecular fluorescence complementation (BiFC) assay in Nicotiana benthamiana leaves. The expression of SaNAR2.2 gene in organs of S. altissima plants grown at low (0.5 mM) or high (15 mM) nitrate concentrations in the nutrient medium, without NaCl or in the presence of increasing salt concentrations, was analyzed using RT-qPCR method. It has been found that SaNAR2.2 is expressed nearly exclusively in roots of S. altissima . As the concentrations of NaCl in the nutrient medium increased (up to 500 mM), the expression of SaNAR2.2 decreased at high external nitrate concentrations and significantly increased, up to ten-fold, at low nitrate concentrations; this points to the significant role of SaNAR2.2 protein under salinity conditions as a participant of high-affinity nitrate transport system. SaNAR2.2 is the first components of a high affinity nitrate transport system characterized in a terrestrial halophytic plant.

Plant nitrate transporters in the NPF (NRT1) family are characterized by multifunctionality and their involvement in a number of physiological processes. The proteins in this family have been identified in many monocotyledonous and dicotyledonous species: a bioinformatic analysis predicts from 20 to 139 members in the plant genomes sequenced so far, including mosses. Plant NPFs are phylogenetically related to proton-coupled oligopeptide transporters, which are evolutionally conserved in all kingdoms of life apart from Archaea. The phylogenetic analysis of the plant NPF family is based on the amino acid sequences present in databases; an analysis identified a separate NPF6 clade (subfamily) with the first plant nitrate transporters studied at the molecular level. The available information proves that proteins of the NPF6 clade play key roles not only in the supply of nitrate and its allocation within different parts of plants but also in the transport of chloride, amino acids, ammonium, and plant hormones such as auxins and ABA. Moreover, members of the NPF6 family participate in the perception of nitrate and ammonium, signaling, plant responses to different abiotic stresses, and the development of tolerance to these stresses and contribute to the structure of the root–soil microbiome composition. The available information allows us to conclude that NPF6 genes are among the promising targets for engineering/editing to increase the productivity of crops and their tolerance to stresses. The present review summarizes the available published data and our own results on members of the NPF6 clade of nitrate transporters, especially under salinity; we outline their molecular, structural, and functional characteristics and suggest potential lines for future research.

Two genes of nitrate transporters SaNRT2.1 and SaNRT2.5, putative orthologs of high-affinity nitrate transporter genes AtNRT2.1 and AtNRT2.5 from Arabidopsis thaliana, were cloned from the euhalophyte Suaeda altissima. Phylogenetic bioinformatic analysis demonstrated that the proteins SaNRT2.1 and SaNRT2.5 exhibited higher levels of homology to the corresponding proteins from the plants of family Amaranthaceae; the similarity of amino acid sequences between proteins SaNRT2.1 and SaNRT2.5 was lower (54%). Both SaNRT2.1 and SaNRT2.5 are integral membrane proteins forming 12 transmembrane helices as predicted by topological modeling. An attempt to demonstrate nitrate transporting activity of SaNRT2.1 or SaNRT2.5 by heterologous expression of the genes in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the only yeast nitrate transporter was not successful. The expression patterns of SaNRT2.1 and SaNRT2.5 were studied in S. altissima plants that were grown in hydroponics under either low (0.5 mM) or high (15 mM) nitrate and salinity from 0 to 750 mM NaCl. The growth of the plants was strongly inhibited by low nitrogen supply while stimulated by NaCl; it peaked at 250 mM NaCl for high nitrate and at 500 mM NaCl for low nitrate. Under low nitrate supply, nitrate contents in S. altissima roots, leaves and stems were reduced but increased in leaves and stems as salinity in the medium increased. Potassium contents remained stable under salinity treatment from 250 to 750 mM NaCl. Quantitative real-time PCR demonstrated that without salinity, SaNRT2.1 was expressed in all organs, its expression was not influenced by nitrate supply, while SaNRT2.5 was expressed exclusively in roots—its expression rose about 10-fold under low nitrate. Salinity increased expression of both SaNRT2.1 and SaNRT2.5 under low nitrate. SaNRT2.1 peaked in roots at 500 mM NaCl with 15-fold increase; SaNRT2.5 peaked in roots at 500 mM NaCl with 150-fold increase. It is suggested that SaNRT2.5 ensures effective nitrate uptake by roots and functions as an essential high-affinity nitrate transporter to support growth of adult S. altissima plants under nitrogen deficiency.

The coding sequence of EhNPF6.3, a homolog of the Arabidopsis thaliana dual-affinity nitrate transporter gene AtNPF6.3 (NRT1.1/CHL1), was cloned from the halophyte Eutrema halophilum (C.A.Mey.) O.E.Schulz / Thellungiella halophila. Expression of EhNPF6.3 in cells of a yeast Ogataea (Hansenula) polymorpha mutant strain for YNT1, the gene of the only nitrate transporter in this organism, restored nitrate uptake by the mutant cells and their ability to growth in selective media containing nitrate as the sole nitrogen source. Examination of nitrate ion uptake from the culture media by cells of O. polymorpha (Morais & M.H. Maia) Y. Yamada, K. Maeda and Mikata strains of wild-type, the Δynt1 knockout mutant, and the knockout mutant expressing EhNPF6.3, using a nitrate-selective electrode, showed that the rate of nitrate uptake by the halophyte transporter, EhNPF6.3, was comparable to that by the endogenous yeast nitrate transporter, YNT1. The results obtained indicate that the protein cloned from the halophyte E. halophilum, EhNPF6.3, is an ortholog of the dual-affinity nitrate transporter and, most likely, plays an important role in nitrate uptake and nitrate distribution among organs and tissues in E. halophilum plants.

The SaNPF6.3 gene, a putative ortholog of the dual-affinity nitrate (NO₃⁻) transporter gene AtNPF6.3/AtNRT1.1 from Arabidopsis thaliana, was cloned from the euhalophyte Suaeda altissima. The nitrate transporting activity of SaNPF6.3 was studied by heterologous expression of the gene in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the original nitrate transporter. Expression of SaNPF6.3 in Δynt1 cells rescued their ability to grow on the selective medium in the presence of nitrate and absorb nitrate from this medium. Confocal laser microscopy of the yeast cells expressing the fused protein GFP-SaNPF6.3 revealed GFP (green fluorescent protein) fluorescence localized predominantly in the cytoplasm and/or vacuoles. Apparently, in the heterologous expression system used, only a relatively small fraction of the GFP-SaNPF6.3 reached the plasma membrane of yeast cells. In S. altissima plants grown in media with either low (0.5 mM) or high (15 mM) NO₃⁻; concentrations, SaNPF6.3 was expressed at various ontogenetic stages in different organs, with the highest expression levels in roots, pointing to an important role of SaNPF6.3 in nitrate uptake. SaNPF6.3 expression was induced in roots of nitrate-deprived plants in response to raising the nitrate concentration in the medium and was suppressed when the plants were transferred from sufficient nitrate to the lower concentration. When NaCl concentration in the nutrient solution was elevated, the SaNPF6.3 transcript abundance in the roots increased at the low nitrate concentration and decreased at the high one. We also determined nitrate and chloride concentrations in the xylem sap excreted by detached S. altissima roots as a function of their concentrations in the root medium. Based on a linear increase in Cl⁻ concentrations in the xylem exudate as the external Cl⁻ concentration increased and the results of SaNPF6.3 expression experiments, we hypothesize that SaNPF6.3 is involved in chloride transport along with nitrate transport in S. altissima plants.

Researchers are often interested in proteins that are present in cells in small ratios compared to the total amount of proteins. These proteins include transcription factors, hormones and specific membrane proteins. However, sufficient amounts of well-purified protein preparations are required for functional and structural studies of these proteins, including the creation of artificial proteoliposomes and the growth of protein 2D and 3D crystals. This aim can be achieved by the expression of the target protein in a heterologous system. This review describes the applications of yeast heterologous expression systems in studies of plant membrane proteins. An initial brief description introduces the widely used heterologous expression systems of the baker’s yeast Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris. S. cerevisiae is further considered a convenient model system for functional studies of heterologously expressed proteins, while P. pastoris has the advantage of using these yeast cells as factories for producing large quantities of proteins of interest. The application of both expression systems is described for functional and structural studies of membrane proteins from plants, namely, K⁺- and Na⁺-transporters, various ATPases and anion transporters, and other transport proteins.

CLC family genes, comprising anion channels and anion/H⁺ antiporters, are widely represented in nearly all prokaryotes and eukaryotes. CLC proteins carry out a plethora of functions at the cellular level. Here the coding sequences of the SaCLCa2 and SaCLCc2 genes, homologous to Arabidopsis thaliana CLCa and CLCc, were cloned from the euhalophyte Suaeda altissima (L.) Pall. Both the genes cloned belong to the CLC family as supported by the presence of the key conserved motifs and glutamates inherent for CLC proteins. SaCLCa2 and SaCLCc2 were heterologously expressed in Saccharomyces cerevisiae GEF1 disrupted strain, Δgef1, where GEF1 encodes the only CLC family protein, the Cl⁻ transporter Gef1p, in undisrupted strains of yeast. The Δgef1 strain is characterized by inability to grow on YPD yeast medium containing Mn²⁺ ions. Expression of SaCLCa2 in Δgef1 cells growing on this medium did not rescue the growth defect phenotype of the mutant. However, a partial growth restoration occurred when the Δgef1 strain was transformed by SaCLCa2(C544T), the gene encoding protein in which proline, specific for nitrate, was replaced with serine, specific for chloride, in the selectivity filter. Unlike SaCLCa2, expression of SaCLCc2 in Δgef1 resulted in a partial growth restoration under these conditions. Analysis of SaCLCa2 and SaCLCc2 expression in the euhalophyte Suaeda altissima (L.) Pall by quantitative real-time PCR (qRT-PCR) under different growth conditions demonstrated stimulation of SaCLCa2 expression by nitrate and stimulation of SaCLCc2 expression by chloride. The results of yeast complementation assay, the presence of both the “gating” and “proton” glutamates in aa sequences of both the proteins, as well results of the gene expression in euhalophyte Suaeda altissima (L.) Pall suggest that SaCLCa2 and SaCLCc2 function as anion/H⁺ antiporters with nitrate and chloride specificities, respectively. The general bioinformatic overview of seven CLC genes cloned from euhalophyte Suaeda altissima is given, together with results on their expression in roots and leaves under different levels of salinity.