The aim of this study was to elucidate whether the membrane nanodomain protein AtFlot1 is involved in vesicular transport pathways and regulation of the P-type H⁺-ATPase content in plasma membrane of A. thaliana under salt stress. Transmission electron microscopy revealed changes in the endosomal system of A. thaliana root cells due to knockout mutation SALK_205125C (Atflot1ko). Immunoblotting of the plasma membrane-enriched fractions isolated from plant organs with an antibody to the H⁺-ATPase demonstrated changes in the H⁺-ATPase content in plasma membrane in response to the Atflot1ko mutation and salt shock. Expression levels of the main H⁺-ATPase isoforms, PMA1 and PMA2, as well as endocytosis activity of root cells determined by endocytic probe FM4-64 uptake assay, were unchanged in the Atflot1ko mutant. We have shown that AtFlot1 participates in regulation of the H⁺-ATPase content in the plasma membrane. We hypothesized that AtFlot1 is involved in both exocytosis and endocytosis, and, thus, contributes to the maintenance of cell ion homeostasis under salt stress. The lack of a pronounced Atflot1ko phenotype under salt stress conditions may be due to the assumed ability of Atflot1ko to switch vesicular transport to alternative pathways. Functional redundancy of AtFlot proteins may play a role in the functioning of these alternative pathways.

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.

Flotillin membrane proteins are involved in many cellular processes and physiological functions. However, these proteins’ participation in plant response to stresses remains poorly understood. In the present report, the possible involvement of flotillin Flot1 in Na⁺ and K⁺ homeostasis in cells under sodium chloride salinization of the medium was studied in Arabidopsis thaliana (L.) Heynh. For this purpose, the Flot1 gene transcription was analyzed in the roots and leaves of wild-type (WT) and flot1 mutant plants with a T-DNA insertion in the promoter using the quantitative Real Time-RT PCR (qRT-PCR). Along with this, a mutant phenotype was studied (growth characteristics, content of Na⁺ and K⁺ ions in organs, and ultrastructure of root cells) under normal conditions and in the presence of 100 mM NaCl in the culture medium. The mutation led to the activation of AtFlot1 expression, which was more noticeable in the roots during salinization. Under these conditions, the mutants had larger organ mass, lower Na⁺ content, and higher K⁺ content in organs than that of WT. The study of the ultrastructure of A. thaliana root cells in mutant plants showed more intensive formation of post-Golgi vesicles and multivesicular bodies (MVB) in the cytoplasm. In both mutant and WT plants, the presence of sodium chloride in the nutrient solution stimulated the formation of MVB and microvacuoles in the cytoplasm and fusion of the latters into larger structures. It is assumed that changes in the ultrastructure of root cells that are caused by mutation and salinization reflect the stimulation, respectively, of vesicular trafficking and biogenesis of vacuoles—processes involved in maintaining Na⁺ and K⁺ cell homeostasis.