Proteins of the chloride channel family CLC (ChLoride Channel) are found in prokaryotes and eukaryotes. The family includes anion channels and anion/proton antiporters. In plants, the CLC proteins are found in various tissues and diverse intracellular membranes. They perform multiple functions and play important physiological roles, such as nitrate and chloride homeostasis at the cellular and whole plant levels, the regulation of transmembrane electrical potential and lumenal pH in organelles, the distribution of newly synthesized proteins among intracellular compartments, and the formation of plant resistance to biotic and abiotic stress factors. This review describes the currently known CLC proteins of plant origin whose genes are identified at the molecular-genetic and functional levels; the properties of CLC proteins from Arabidopsis thaliana (L.) Heynh. and other plant species are discussed with emphasis on the results obtained in the last decade. The functional and structural aspects of plant CLC proteins, as well as their physiological roles under normal and stressful conditions, are considered. A hypothesis is put forward that the endosomes and the proteins of the CLC family localized in endosomal membranes are involved in maintaining the anionic balance in the cytoplasm. The prospects of further studies on plant CLC proteins are briefly considered.

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.

Coding sequences of the CLC family genes SaCLCd, SaCLCf, and SaCLCg, the putative orthologs of Arabidopsis thaliana AtCLCd, AtCLCf, and AtCLCg genes, were cloned from the euhalophyte Suaeda altissima (L.) Pall. The key conserved motifs and glutamates inherent in proteins of the CLC family were identified in SaCLCd, SaCLCf, and SaCLCg amino acid sequences. SaCLCd and SaCLCg were characterized by higher homology to eukaryotic (human) CLCs, while SaCLCf was closer to prokaryotic CLCs. Ion specificities of the SaCLC proteins were studied in complementation assays by heterologous expression of the SaCLC genes in the Saccharomyces cerevisiae GEF1 disrupted strain Δgef1. GEF1 encoded the only CLC family protein, the Cl⁻ transporter Gef1p, in undisrupted strains of this organism. Expression of SaCLCd in Δgef1 cells restored their ability to grow on selective media. The complementation test and the presence of both the “gating” and “proton” conservative glutamates in SaCLCd amino acid sequence and serine specific for Cl⁻ in its selectivity filter suggest that this protein operates as a Cl⁻/H⁺ antiporter. By contrast, expression of SaCLCf and SaCLCg did not complement the growth defect phenotype of Δgef1 cells. The selectivity filters of SaCLCf and SaCLCg also contained serine. However, SaCLCf included only the “gating” glutamate, while SaCLCg contained the “proton” glutamate, suggesting that SaCLCf and SaCLCg proteins act as Cl⁻ channels. The SaCLCd, SaCLCf, and SaCLCg genes were shown to be expressed in the roots and leaves of S. altissima. In response to addition of NaCl to the growth medium, the relative transcript abundances of all three genes of S. altissima increased in the leaves but did not change significantly in the roots. The increase in expression of SaCLCd, SaCLCf, and SaCLCg in the leaves in response to increasing salinity was in line with Cl⁻ accumulation in the leaf cells, indicating the possible participation of SaCLCd, SaCLCf, and SaCLCg proteins in Cl⁻ sequestration in cell organelles. Generally, these results suggest the involvement of SaCLC proteins in the response of S. altissima plants to increasing salinity and possible participation in mechanisms underlying salt tolerance.

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.

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.