Flotillins, membrane-associated proteins, belonging to a SPFH protein superfamily are essential constituents of the membrane microdomains and involved in many vital processes. In a number of studies, the flotillin-associated endocytosis has been shown to play an important role in plant response to biotic and abiotic stresses by regulating abundance of key to resistance proteins in plasma membrane (PM) and activating signaling cascades. Here, we report the identification of two flotillin genes, SaFlot1 and SaFlot2, in the euhalophyte Suaeda altissima, a species exhibiting high tolerance to NaCl and bacterial infections, the cloning of their full-length coding sequences, and the effects of an abiotic (NaCl) and a biotic (immune response elicitor, bacterial peptide fls22) stress-factors on their expression. Treatment of S. altissima plants with flg22 stimulated transcription, while NaCl lowered transcript levels of both SaFlot1 and SaFlot2. We hypothesis that observed changes in the levels of SaFlot1 and SaFlot2 transcripts reflect cell needs under stress conditions to modulate certain PM protein internalization accomplished by the flotillin-dependent (microdomain-associated) endocytic pathway. The A. thaliana plasma membrane receptor FLS2, a protein playing a signaling role at bacterial infections, forms the flg22-FLS2 complex that undergoes internalization into the cytoplasm. The flg22-induced stimulation of SaFlot1 and SaFlot2 expression may be associated with need to activate flg22-AtFLS2 homolog signaling by the flotillin-dependent endocytosis. NaCl-induced decrease in SaFlot1 and SaFlot2 transcription may be due to the cell need under salt stress conditions to keep in the plasma membrane the important for response to NaCl proteins, such as H+-ATPase, inward K+ channel AKT1 and some others, reducing flotillin-dependent endocytic activity.

Moderate concentrations of Na+ ions in the growth medium have positive effects on growth and performance characteristics of the C4 euhalophyte Suaeda altissima, optimizing functioning of both, stomata and photosynthetic apparatus. Effects of salinity on the morphology, ion relations, and gas exchange of the leaves in the euhalophyte Suaeda altissima (L.) Pall. were investigated with emphasis on the guard and epidermal cells. The presence of NaCl in the nutrient solution (NS) at both growth-stimulating (250 mM) and growth-inhibiting (750 mM) concentrations resulted in increased leaf succulence, net photosynthetic rate (Pn), and instantaneous water use efficiency (WUEi), and reduced stomatal conductance (gs), stomatal density on the leaf surface, and transpiration rate (E). X-ray microanalyses revealed Na and Cl accumulation in the guard and epidermal cells of leaves under salinity conditions. However, Na and Cl contents differed by not much in plants grown at 250 and 750 mM NaCl, indicating a mechanism preventing accumulation of Na+ and Cl− ions in cells paving leaf surface at high NaCl concentrations. Examination of gs and E as functions of CO2 concentration in the leaf gas exchange chamber revealed better ability to regulate these parameters in 250 mM NaCl-grown plants than in 750 mM NaCl-grown or control plants. The study of Pn dependent on CO2 concentration in leaf intercellular space revealed direct stimulating effect of NaCl on photosynthesis. We hypothesize that S. altissima, a species having anatomy and ultrastructure features of C4 plants, improves its performance characteristics under saline conditions, optimizing not only the functioning of the stomata complex but also the process of CO2 assimilation, including the C4 fixation pathway.

Разнообразные молекулы патогенного происхождения, такие как бактериальный флагеллин (flg22), распознаются растениями через рецепторы плазматической мембраны и индуцируют как местные, так и системные иммунные реакции. При этом везикулярный транспорт является ключевым в обеспечении быстрого и точного реагирования. При взаимодействии с патогенами, локализованными на поверхности клеток, иммунные рецепторы подвергаются эндоцитозу по общему эндосомальному пути. Какой из везикулярных путей патогены используют для проникновения в ткани и какое значение имеет в этом процессе flot1 остается не до конца изученным. Данное исследование посвящено влиянию биотических факторов стресса на иммунную реакцию растений Arabidopsis thaliana дикого типа и его нокаут-мутанта Atflot1ko. Изучены процессы эндоцитоза под действием разных агентов: 1-нафталинлуксусной кислоты и метил-ß-циклодекстрина. С помощью трансмиссионной электронной микроскопии выявлены различия в ответных реакциях клеток на стресс, индуцированный flg22. Показано, что биотический стресс у нокаут-мутантов активирует секреторный путь (экзоцитоз), необходимый для защиты клеток от действиях патогена на поверхности клетки, тогда как у растений дикого типа активируется эндоцитоз, направленный на перемещение патогена в вакуоль. Полученные данные показали, что обработка мутантов Atflot1ko бактериальным пептидом сохраняет активность работы комплекса Гольджи и способность этой структуры формировать ранние эндосомы, принимающие непосредственное участие в транспорте защитных белков к месту проникновения патогена.

Cloning of the SaSLAH3 gene (OQ271227.1), an AtSLAH3 ortholog from the slow anion channels family ( SLAC/SLAH ), that was originally identified in Arabidopsis thaliana , has been carried out from the euhalophyte Suaeda altissima . Under hydroponic conditions, this species related to Amaranthaceae/Chenopodiaceae is capable to complete life cycle on nutrient solutions containing NaCl at high concentrations up to 1 M. Cloning of SaSLAH3 was performed based on a putative similarity of SaSLAH3 with the SLAH3 homologs from the related species Suaeda glauca and Suaeda fruticosa , using transcriptomes assembled previously. Membrane topology, analyzed by in silico approach, the 3-D structure, presence of conservative phenylalanine-bearing motives and as well as MEME-generated sequence motif patterns of SaSLAH3, all of these features were found typical for the SLAC/SLAH channels and had validated SaSLAH3 belonging to this family. Analysis of SLAC/SLAH phylogenetic relationship demonstrated the evolutionary divergence of SLAC-like proteins into three distinct groups characteristic of this family with location of SaSLAH3 in SLAH2/3 clade. Investigation of the expression profile by quantitative Real-Time PCR revealed increasing SaSLAH3 expression in roots and leaves upon elevation of NaCl concentration in nutrient solution under conditions of both sufficient nitrate supply and nitrate deficiency. SaSLAH3 is suggested to play an important role in enhancing chloride loading into root xylem and, thereafter in chloride delivery to shoot under salinity. Another physiological role of SaSLAH3 may be attributed to regulation of nitrate concentration in cytoplasm to ensure proper nitrogen nutrition of S. altissima plants under conditions of stiff competition of nitrate with chloride.

The content and localization of Rubisco large subunit (RbcL), as well as the activities of NADP-MDH and NADP-ME enzymes involved in the C4 carbon-concentrating mechanism (CCM) were studied under elevated (800 ppm, eCO2) CO2 concentration at optimal (25°C) and elevated (32°C, eT) temperatures in the leaves of C4-NADP species Kochia prostrata. In control plants Rubisco was revealed not only in the bundle sheath cells (BS), typical of C4 plants but also in the mesophyll cells (M), indicating a C4-like type of photosynthesis. In addition, the presence of starch grains in the BS and the adjacent layer of mesophyll cells (M(I)) suggests the Calvin cycle activity. eT and/or eCO2 markedly reduced the number of starch grains in these cells, probably as a result of decreased activity of C4 CCM (NADP-MDH and NADP-ME activities). NADP-MDH activity was sensitive to temperature, while that of NADP-ME decreased under eT or eCO2 but most conspicuously when they acted together. Under eCO2, regardless of temperature, the appearance of starch grains was observed in the cells of the second mesophyll layer (M(II)), which also indicated Calvin cycle activity. Thus, K. prostrata exhibits the signs of C4-like photosynthesis and, possibly, an active C3 cycle in the M(II) cells under eCO2 alone or combined with eT.

A variety of molecules of pathogenic origin, such as bacterial flagellin (flg22), are recognized by plants through plasma membrane receptors and induce both local and systemic immune responses. In this case, vesicular transport is key to ensuring a rapid and accurate response. When interacting with pathogens localized on the cell surface, immune receptors undergo endocytosis along the common endosomal pathway. Which vesicular pathway pathogens are used to penetrate tissues and the importance of flot1 in this process remains incompletely understood. This study is devoted to the influence of biotic stress factors on the immune response of wild type Arabidopsis thaliana plants and its knockout mutant Atflot1ko. The processes of endocytosis under the influence of various agents have been studied: 1-naphthaleneacetic acid and methyl-ß-cyclodextrin. Transmission electron microscopy revealed differences in cell responses to stress induced by flg22. It has been shown that biotic stress in knockout mutants activates the secretory pathway (exocytosis), which is necessary to protect cells from the actions of the pathogen on the cell surface, while endocytosis aimed at moving the pathogen into the vacuole is activated in wild-type plants. The obtained data showed that treatment of Atflot1ko mutants with bacterial peptide preserves the activity of the Golgi complex and the ability of this structure to form early endosomes, which are directly involved in the transport of protective proteins to the site of pathogen penetration.

The effects of silver nanoparticles (AgNPs), both alone and in combination with mineral nutrients, on the growth and photosynthesis of Solanum lycopersicum plants during ontogeny were studied. The experiment involved weekly applications of 10 μmol of AgNPs for 15 weeks in a greenhouse over a summer period. A comprehensive characterization of the AgNPs was performed via TEM, ESI/EELS, and zeta potential measurements before and throughout the experiment. The activity of PSII, stomatal conductivity, photosynthesis, transpiration and respiration rates were measured, and the photosynthetic pigments, chloroplast ultrastructure, and dry and fresh masses of leaves, roots, and fruits were assessed. The results indicated that combining AgNPs with mineral nutrients increased PSII activity and the photosynthesis rate and altered the chloroplast ultrastructure. However, the use of mineral nutrients or AgNPs alone did not induce these changes. Atomic absorption spectrometry detected AgNPs in all the plant organs except the fruits. The highest fruit yield was associated with Veni Prisma®, a commercial product containing colloidal silver, which also caused desynchronized fruit maturation. This study hypothesizes that the synergistic effect of AgNPs and mineral nutrients enhances silver accumulation in chloroplasts, improving light utilization and photosynthetic efficiency, particularly under low light, thus increasing fruit quantity and dry mass. Conversely, long-term use of AgNPs alone was accompanied by silver accumulation outside the chloroplasts and did not lead to increased photosynthesis or an increase in fresh fruit mass.

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.

Marchantia polymorpha is a convenient model for studying light of different spectral compositions on various physiological and biochemical processes because its photoreceptor system is vastly simplified. The influence of red light (RL, 660 nm), far-red light (FRL, 730 nm), blue light (BL, 450 nm), and green light (GL, 525 nm) compared to white light (high-pressure sodium light (HPSL), white LEDs (WL 450 + 580 nm) and white fluorescent light (WFL) on photosynthetic and transpiration rates, photosystem II (PSII) activity, photomorphogenesis, and the expression of light and hormonal signaling genes was studied. The ultrastructure of the chloroplasts in different tissues of the gametophyte M. polymorpha was examined. FRL led to the formation of agranal chloroplasts (in the epidermis and the chlorenchyma) with a high starch content (in the parenchyma), which led to a reduced intensity of photosynthesis. BL increased the transcription of genes for the biosynthesis of secondary metabolites - chalcone synthase (CHS), cellulose synthase (CELL), and L-ascorbate peroxidase (APOX3), which is consistent with the increased activity of low-molecular weight antioxidants. FRL increased the expression of phytochrome apoprotein (PHY) and cytokinin oxidase (CYTox) genes, but the expression of the phytochrome interacting factor (PIF) gene decreased, which was accompanied by a significant change in gametophyte morphology. Analysis of crosstalk gene expression, and changes in morphology and photosynthetic activity was carried out.

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.

Plants are subjected to various stress factors within their lifespan. In this respect, the plasma membrane is a principal cell compartment responsible for plant adaptations to stresses. It is capable of remodeling its protein composition by means of endocytosis. In the plants, the main mode of this process is a clathrin-mediated endocytosis. Several clathrin-independent pathways are also known; these alternative mechanisms involve Flot1 protein. In the present research, the role of Flot1 in the endocytosis process was examined in seedling roots of a wild type and an Atflot1ko knockout mutant of Arabidopsis thaliana (L.) Heynh. Light microscopy with an FM4-64 lipophilic probe and transmission electron microscopy were used. It was found that endocytosis was arrested in the root cells of the wild type after a simultaneous treatment of the roots with an inhibitor of clathrin-mediated endocytosis (1-naphthylacetic acid) and the agent depleting the plasma membrane of sterols (methyl-β-cyclodextrin). In this case, such morphological change as reduction in cytoplasm vesiculation (including the early endosomes, the small vesicles originated from the agranular ER, the microvacuoles from its fragments, and the clathrin vesicles) was observed. The vesiculation was diminished in both the control and the stressed plants (exposed to 100 mM NaCl). In the Atflot1ko mutant, the cisterns of the Golgi complex closed up to a ring, and the process of formation of the early endosomes was completely abolished under these conditions. It is suggested that, in the roots of A. thaliana exposed to the inhibitors, the microdomain-associated Flot1 protein of the plasma membrane conserves the structure of the Golgi complex and its capacity to build early endosomes on the trans -side. In addition, the protein appears to participate in formation of the early endosomes from the trans -Golgi network.

The relationship between the endocytosis, ion homeostasis, and salt tolerance in Arabidopsis thaliana was studied using mutant plants with the insertion in the ARA7/AtRabF (AT4G19640) gene, encoding a small Rab5 GTPase that represents one of the key regulators of the vesicular transport. This mutation is characterized by an increased expression of the AtARA7 protein. A 14-nucleotide deletion in the 5'-untranslated region (5'-UTR) and the loss of the first three nucleotides at the 5'-end of mRNA were revealed in the mutant allele. Though these sequence changes slightly decreased the relative level of ARA7 transcripts in leaves of mutant plants under normal conditions, an increased content of the corresponding ARA7 protein in leaves was observed. This, in turn, resulted in significant phenotypic changes in plants grown on a NaCl-containing growth media. Electron microscopy studies revealed some changes on ultrastructural level in the root cells of mutant plants. The most significant differences between the mutant and wild-type (WT) plants included the presence of electron dense deposits on the tonoplast and increase in (a) the degree of vacuolization and vesiculation of the cytosol, (b) the content of fusing microvacuoles, and (c) the content of multivesicular bodies and autophagic structures that indicated changes in the endocytosis pathway and the vacuole formation dynamics. At the whole plant level, the mutant phenotype was characterized by a positive response of mutant plants to a long-term salt stress compared to WT plants. Mutant plants also had an increased mass of roots and leaves, heightened content of K+, and decreased average content of Na+. This indicates that mutants developed a higher degree of salt tolerance when compared to WT plants. Mutation-induced changes in the cell ultrastructure probably reflect perturbations in vesicular transport and autophagy resulting in changes in ion homeostasis and salt tolerance of plants.

The technique that enables simultaneous evaluation of water potential in the substomatal cavity of the intact leaf (ψwа) and rate of its photosynthetic CO2/H2O gas exchange was applied to a halophyte Suaeda altissima (L.) Pall. Formation of a water-potential gradient in a whole plant, rate of CO2 uptake, rate of transpiration, and concentrations of Na+ and Cl– ions in the plant organs were determined under conditions of chloride–sodium salinization of a nutrient solution. It was found that the salinization decreases not only biological productivity of the plants but also their capacity to accumulate Cl– in the amounts equivalent to Na+ accumulation. High salinity also diminished the gradient of water potential between a nutrient solution and the apoplast of the cells in the leaf substomatal cavity due to the increase in ψwа and, respectively, decrease in the water stream from roots to leaves and the rate of CO2/H2O gas exchange of the leaf. It was shown that the decrease in the water potential in the interface between liquid and gaseous phases in the apoplast of the substomatal cavity (expressed in the ψwа value) plays an essential role in the regulation of water uptake under salinization conditions. It is supposed that the NaCl-induced increase in ψwа is a consequence of the suppression of photosynthesis and a resultant osmolyte shortage together with the decrease in plant productivity due to the stress impact exerted by NaCl.

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 plants from two populations (P1 and P2) of xero-halophyte Sedobassia sedoides (Pall.) Freitag & G. Kadereit (Chenopodiaceae) with С3–С4 intermediate type of photosynthesis were examined. Morphophysiological parameters were determined: dry biomass of the above-ground organs, maximum quantum yield of photosystem II (PSII), ultra- and mesostructure of the leaf, carbon isotope discrimination (δ13С) in plants grown under normal conditions and exposed to moderate salinization (0 and 200 mM NaCl). By the values of δ13С and efficiency of PSII, we did not detect significant differences between the populations. Under control conditions, the chloroplasts and mitochondria were located in Kranz-like cells of the bundle sheath of both populations in the region adjacent to vascular bundle, which is characteristic of С2 type of photosynthesis (with photorespiratory СО2 concentrating mechanism). P2 plants were notable for a greater volume of Kranz-like cells of the bundle sheath and more numerous, larger chloroplasts and mitochondria than in P1 plants. By structural leaf parameters, P1 plants may be attributed to a proto-Kranz type of photosynthesis (intermediate between С3 and С2 types) and P2 plants to С2 photosynthesis. Under salinization, the accumulation of dry biomass was reduced in both populations but more considerably in P1. The ultrastructure of organelles in both populations showed different responses to salinization, which was especially pronounced in Kranz-like cells of the bundle sheath. In P1 plants, the area of chloroplasts and mitochondria rose, whereas the area and number of chloroplasts under stress did not change in P2 plants but the area and number of mitochondria decreased. In the cells of plants from both populations, signs of degradation were observed (more pronounced in P1 plants); they were shown in chloroplasts twisting and a disturbance of their granal stacking. In P2 plants, Kranz-like cells of the bundle sheath preserved safer organelles but there occurred the cells with complete degradation of a vacuolar type. Thus, intraspecific ultra- and mesostructural differences were detected in plants from two populations of С3–С4 intermediate species S. sedoides, which reflect different stages of development of photorespiratory СО2 concentrating mechanism. Under salinization, the plants from two different populations showed unlike strategies of adaptation on the level of leaf ultra- and mesostructure.

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.