Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.     

Halophytic NHXs confer salt tolerance by altering cytosolic and vacuolar K<Superscript>+</Superscript> and Na<Superscript>+</Superscript> in <Emphasis Type="Italic">Arabidopsis</Emphasis> root cell

While the role of the vacuolar NHX Na⁺/H⁺ exchangers in plant salt tolerance has been demonstrated on numerous occasions, their control over cytosolic ionic relations has never been functionally analysed in the context of subcellular Na⁺ and K⁺ homeostasis. In this work, PutNHX1 and SeNHX1 were cloned from halophytes Puccinellia tenuiflora and Salicornia europaea and transiently expressed in Arabidopsis wild type Col-0 and the nhx1 mutant. Phylogentic analysis, topological prediction, analysis of evolutionary conservation, the topology structure and analysis of hydrophobic or polar regions of PutNHX1 and SeNHX1 indicated that they are unique tonoplast Na⁺/H⁺ antiporters with characteristics for salt tolerance. As a part of the functional assessment, cytosolic and vacuolar Na⁺ and K⁺ in different root tissues and ion fluxes from root mature zone of Col-0, nhx1 and their transgenic lines were measured. Transgenic lines sequestered large quantity of Na⁺ into root cell vacuoles and also promoted high cytosolic and vacuolar K⁺ accumulation. Expression of PutNHX1 and SeNHX1 led to significant transient root Na⁺ uptake in the four transgenic lines upon recovery from salt treatment. In contrast, the nhx1 mutant maintained a prolonged Na⁺ efflux and the nhx1:PutNHX1 and nhx1:SeNHX1 lines started to actively pump Na⁺ out of the cell. Overall, our findings suggest that PutNHX1 and SeNHX1 improve Na⁺ sequestration in the vacuole and K⁺ retention in the cytosol and vacuole of root cells of Arabidopsis, and that they interact with other regulatory mechanisms to provide a highly orchestrated regulation of ionic relations among intracellular cell compartments.     

Comparative genomic analysis of <Emphasis Type="Italic">Geosporobacter ferrireducens</Emphasis> and its versatility of anaerobic energy metabolism

Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H⁺ and Na⁺ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na⁺-dependent F-type ATPase and H⁺-dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

Proton and calcium pumping P-type ATPases and their regulation of plant responses to the environment

Plant plasma membrane H⁺-ATPases and Ca²⁺-ATPases maintain low cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, and are essential for plant growth and development. These low concentrations allow plasma membrane H⁺-ATPases to function as electrogenic voltage stats, and Ca²⁺-ATPases as “off” mechanisms in Ca²⁺-based signal transduction. Although these pumps are autoregulated by cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, they are also subject to exquisite regulation in response to biotic and abiotic events in the environment. A common paradigm for both types of pumps is the presence of terminal regulatory (R) domains that function as autoinhibitors that can be neutralized by multiple means, including phosphorylation. A picture is emerging in which some of the phosphosites in these R domains appear to be highly, nearly constantly phosphorylated, whereas others seem to be subject to dynamic phosphorylation. Thus, some sites might function as major switches, whereas others might simply reduce activity. Here, we provide an overview of the relevant transport systems and discuss recent advances that address their relation to external stimuli and physiological adaptations.

The regulation of plasma membrane H^+-ATPases and autoinhibited Ca2^+-ATPases exhibits a complex and dynamic network of posttranslational regulation. The regulation of plasma membrane H⁺-ATPases and autoinhibited Ca²⁺-ATPases exhibits a complex and dynamic network of posttranslational regulation.

P-type ATPases are found in all domains of life and constitute a large superfamily of membrane-bound pumps that share a common machinery, including a reaction cycle that involves catalytic phosphorylation of an Asp, resulting in a phosphorylated intermediate (reviewed in Palmgren and Nissen, 2011; (hence the name P-type; Box 1). The catalytic phosphoryl-aspartate intermediate is not to be confused with regulatory phosphorylation, which occurs on Ser, Thr, and Tyr residues. Five major families of P-type ATPases have been characterized (P1–5), each of which is divided into a number of subfamilies (named with letters). Plasma membrane H⁺-ATPases are classified as P3A ATPases, whereas Ca²⁺ pumps constitute P2A and P2B ATPases. In plants, these pumps are best characterized in the model plant Arabidopsis thaliana (Arabidopsis).Box 1Enzymology of P-type ATPases.P-type ATPases (reviewed in Palmgren and Nissen, 2011) alternate between two extreme conformations during their catalytic cycle: a high-affinity (with respect to ATP and the ion to be exported) Enzyme1 (E1) state, and a low-affinity Enzyme2 (E2) state. Many P-type ATPases are autoinhibited by built-in molecular constraints, namely their C- and N-terminal (for plasma membrane H⁺-ATPases; Palmgren et al., 1999) or N-terminal (for P2B Ca²⁺-ATPases; Malmström et al., 1997) regulatory (R) domains of approximately 100 amino acid residues, which act as brakes by stabilizing the pumps in a low-affinity conformation (Palmgren and Nissen, 2011), most likely E2. Neutralizing the R domain results in a shift in conformational equilibrium towards a high-affinity state, likely E1. In this way, the R domains of plasma membrane H⁺-ATPases and Ca²⁺-ATPases allow posttranslational modification events to control the turnover numbers of these pumps. A structure of a plasma membrane H⁺-ATPase (from the distantly related yeast S. cerevisiae) in its autoinhibited state has been solved (Heit et al., 2021). Its R domain is situated adjacent to the P domain, which would suggest that the R domain functions to restrict the conformational flexibility of the pump. Normally, the hydrolysis of ATP and transport are tightly coupled in P-type ATPases. Therefore, P-type ATPases hydrolyze bound ATP as soon as their ligand-binding site(s) in the membrane region are occupied, but not before. Thus, increasing the ligand affinity of an ATPase simultaneously increases its turnover number, provided that the concentration of ATP is not limiting, which is rarely the case in cells. A specific feature of plasma membrane H⁺-ATPases is that in the autoinhibited state, ATP hydrolysis is only loosely coupled to H⁺ pumping, whereas pump activation results in tight coupling, with one H⁺ pumped per ATP split (Pedersen et al., 2018).In response to internal and/or external cues, plasma membrane H⁺-ATPase and Ca²⁺-ATPase activities are controlled by intracellular concentrations of H⁺ and Ca²⁺, respectively, via interacting proteins, through posttranslational modification by phosphorylation, and by regulated trafficking of the pump to and from the plasma membrane. Their regulation sometimes involves changes in gene expression and turnover, although this is rare, perhaps because both processes are time- and energy-consuming (Haruta et al., 2018).     

Physiological adaptation and gene expression analysis of <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> under salt stress

Casuarina equisetifolia is widely planted in coastal areas of tropical and subtropical regions as windbreaks or to stabilize dunes against wind erosion due to its high salt tolerance and nitrogen-fixing ability. To investigate the mechanisms responsible for its salt tolerance, we examined growth, mineral composition, expression of genes for sodium (Na⁺) and potassium (K⁺) transport proteins, and antioxidant responses under NaCl treatments. Increasing NaCl concentrations inhibited lateral root elongation and decreased plant height, length of internodes, and numbers of branches and twigs. The Na⁺ content significantly increased whereas the K⁺ content significantly decreased in both shoots and roots with increasing external NaCl concentration, resulting in a significant increase in Na⁺/K⁺ ratio. Most of the Na⁺/H⁺ antiporter genes (NHXs) were obviously upregulated in roots after 24 and 168 h of salt stress, and NHX7 was especially induced after 168 h. Almost all salt overly sensitive (SOS) genes were induced after 168-h treatment. Additionally, activities of superoxide dismutase, glutathione peroxidase, and catalase were significantly changed in shoots and roots under salt stress. Hence, we conclude that salinity tolerance of C. equisetifolia mainly relied on sequestering excess Na⁺ into vacuoles and on induced expression of NHX and SOS genes in roots and thus the maintenance of sufficient K⁺ content in shoots.     

Improved salt tolerance in a wheat stay-green mutant <Emphasis Type="Italic">tasg1</Emphasis>

Salt stress inhibited the growth of both tasg1 and wild-type (WT) wheat seedlings, but the inhibition in tasg1 plants was relatively weaker than that of WT. Compared to the WT, the chlorophyll content, thylakoid membrane polypeptides, Hill reaction activity, actual photochemical efficiency of PSII (ΦPSII), and Mg²⁺- and Ca²⁺-ATPase activities were higher in tasg1 under salt stress. At the same time, the photosynthetic activity of the tasg1 was significantly higher than that of WT. In addition, tasg1 plants displayed relatively less accumulation of reactive oxygen species and oxidative damage accompanied by higher activity of some antioxidant enzymes, and the up-regulation of antioxidant genes further demonstrated the improvement of antioxidant activity in tasg1 under salt stress. Furthermore, tasg1 plants also showed relatively weaker Na⁺ fluorescence and lower Na⁺ content, but relatively higher content of K⁺ in their roots and shoots, and then, the roots of tasg1 plants enhanced net outward Na⁺ flux and a correspondingly increased net inward K⁺ flux during salt stress. This might be associated with the relatively higher activity of H⁺-ATPase in tasg1 plants. These results suggest that the improved antioxidant competence and Na⁺/K⁺ ion homeostasis play an important role in the enhanced salinity tolerance of tasg1 plants.     

Significance of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> for Sustained Hydration of Organ Tissues in Ecologically Distinct Halophytes of the Family Chenopodiaceae

The contents of Na⁺, K⁺, water, and dry matter were measured in leaves and roots of euhalophytes Salicornia europaea L. and Climacoptera lanata (Pall.) Botsch featuring succulent and xeromorphic cell structures, respectively, as well as in saltbush Atriplex micrantha C.A. Mey, a halophyte having bladder-like salt glands on their leaves. All three species were able to accumulate Na⁺ in their tissues. The Na⁺ content in organs increased with elevation of NaCl concentration in the substrate, the concentrations of Na⁺ being higher in leaves than in roots. When these halophytes were grown on a NaCl-free substrate, a trend toward K⁺ accumulation was observed and was better pronounced in leaves than in roots. Particularly high K⁺ concentrations were accumulated in Salicornia leaves. There were no principal differences in the partitioning of Na⁺ and K⁺ between organs of three halophyte species representing different ecological groups. At all substrate concentrations of NaCl, the total content of Na⁺ and K⁺ in leaves was higher than in roots. This distribution pattern persisted in Atriplex possessing salt glands, as well as in euhalophytes Salicornia and Climacoptera. The physiological significance of such universal pattern of ion accumulation and distribution among organs in halophytes is related to the necessity of water absorption by roots, its transport to shoots, and maintenance of sufficient cell water content in all organs under high soil salinity.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

Microsatellite based linkage disequilibrium analyses reveal <Emphasis Type="Italic">Saltol</Emphasis> haplotype fragmentation and identify novel QTLs for seedling stage salinity tolerance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A set of 84 diverse rice genotypes were assessed for seedling stage salt tolerance and their genetic diversity using 41 polymorphic SSR markers comprising of 19 Saltol QTL linked and 22 random markers. Phenotypic screening under hydroponics identified three indica landraces (Badami, Shah Pasand and Pechi Badam), two Oryza rufipogon accessions (NKSWR2 and NKSWR17) and one each of Basmati rice (Seond Basmati) and japonica cultivars (Tompha Khau) as salt tolerant, having similar tolerance as of Pokkali and FL478. Among the salt tolerant genotypes, biomass showed positive correlation with shoot fresh weight and negative association with root and shoot Na⁺ content. The results indicated repression of Na⁺ loading within the tolerant plants. Linkage disequilibrium (LD) of the Saltol linked markers was weak, suggestive of high fragmentation of Pokkali haplotype, a result of evolutionary active recombination events. Poor haplotype structure of the Saltol region, may reduce its usefulness in marker assisted breeding programmes, if the target foreground markers chosen are wide apart. LD mapping identified eight robust marker-trait associations (QTLs), of which RM10927 was found linked to root and shoot Na⁺ content and RM10871 with shoot Na⁺/K⁺ ratio. RM271 on chromosome 10, an extra Saltol marker, was found associated to root Na⁺/K⁺ ratio. This marker showed a distinct allele among O. rufipogon accessions. There were also other novel loci detected on chromosomes 2, 5 and 10 influencing salt tolerance in the tested germplasm. Although Saltol remained as the key locus, the role of other genomic regions cannot be neglected in tailoring seedling stage salt tolerance in rice.     

Salt oversensitivity derived from mutation breeding improves salinity tolerance in barley <Emphasis Type="Italic">via</Emphasis> ion homeostasis

Salt stress imposes a major environmental threat to agriculture, therefore, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any breeding strategy. In the present study, the expression profile of genes involved in ion homeostasis including salt overly sensitive (HvSOS1, HvSOS2, HvSOS3), vacuolar Na⁺/H⁺ antiporter (HvNHX1), and H⁺-ATPase (HVA) along with ion content measurement were investigated in two genotypes of Hordeum vulgare under 300 mM NaCl. The gene expressions were measured in the roots and shoots of a salt-tolerant mutant genotype M4-73-30 and in its wild-type cv. Zarjou by real-time qPCR technique. The critical differences between the salt-tolerant mutant and its wild-type were observed in the expressions of HvSOS1 (105-fold), HvSOS2 (24-fold), HvSOS3 (31-fold), and HVA (202-fold) genes in roots after 6-h exposure to NaCl. The parallel early up-regulation of these genes in root samples of the salt-tolerant mutant genotype indicated induction of Na⁺/H⁺ antiporters activity and Na+ exclusion into apoplast and vacuole. The earlier up-regulation of HvSOS1, HVA, and HvNHX1 genes in shoot of the wild-type genotype corresponded to the relative accumulation of Na⁺ which was not observed in salt-tolerant mutant genotype because of efficient inhibitory role of the root in Na+ transport to the shoot. In conclusion, the lack of similarity in gene expression patterns between the two genotypes with similar genetic background may confirm the hypothesis that mutation breeding could change the ability of salt-tolerant mutant genotype for efficient ion homeostasis via salinity oversensitivity response.     

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na⁺/K⁺-ATPase or Ca²⁺-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.