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.     

The K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter <Emphasis Type="Italic">AhNHX1</Emphasis> improved tobacco tolerance to NaCl stress by enhancing K<Superscript>+</Superscript> retention

High salinity is the one of important factors limiting plant growth and crop production. Many NHX-type antiporters have been reported to catalyze K⁺/H⁺ exchange to mediate salt stress. This study shows that an NHX gene from Arachis hypogaea L. has an important role in K⁺ uptake and transport, which affects K⁺ accumulation and plant salt tolerance. When overexpressing AhNHX1, the growth of tobacco seedlings is improved with longer roots and a higher fresh weight than the wild type (WT) under NaCl treatment. Meanwhile, when exposed to NaCl stress, the transgenic seedlings had higher K⁺/H⁺ antiporter activity and their roots got more K⁺ uptake. NaCl stress could induce higher K⁺ accumulation in the roots, stems, and leaves of transgenic tobacco seedlings but not Na⁺ accumulation, thus, leading to a higher K⁺/Na⁺ ratio in the transgenic seedlings. Additionally, the AKT1, HAK1, SKOR, and KEA genes, which are involved in K⁺ uptake or transport, were induced by NaCl stress and kept higher expression levels in transgenic seedlings than in WT seedlings. The H⁺-ATPase and H⁺-PPase activities were also higher in transgenic seedlings than in the WT seedlings under NaCl stress. Simultaneously, overexpression of AhNHX1 increased the relative distribution of K⁺ in the aerial parts of the seedlings under NaCl stress. These results showed that AhNHX1 catalyzed the K⁺/H⁺ antiporter and enhanced tobacco tolerance to salt stress by increasing K⁺ uptake and transport.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> and K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporters AtNHX1 and AtNHX3 from <Emphasis Type="Italic">Arabidopsis</Emphasis> improve salt and drought tolerance in transgenic poplar

The tonoplast and plasma membrane localized sodium (potassium)/proton antiporters have been shown to play an important role in plant resistance to salt stress. In this study, AtNHX1 and AtNHX3, two tonoplast Na⁺(K⁺)/H⁺ antiporter encoding genes from Arabidopsis thaliana, were expressed in poplar to investigate their biological functions in the resistance to abiotic stresses in woody plants. Transgenic poplar plants expressing either gene exhibited increased resistance to both salt and water-deficit stresses. Compared to the wild type (WT) plants, transgenic plants accumulated more sodium and potassium ions in the presence of 100 mM NaCl and showed reduced electrolyte leakage in the leaves under water stress. Furthermore, the proton-translocating and cation-dependent H⁺ (Na⁺/H⁺ or K⁺/H⁺) exchange activities in the tonoplast vesicles isolated from the leaves of transgenic plants were higher than in those isolated from WT plants. Therefore, constitutive expression of either AtNHX1 or AtNHX3 genetically modified the salt and water stress tolerance of transgenic poplar plants, providing a potential tool for engineering tree species with enhanced resistance to multiple abitotic stresses.     

Enhanced salinity stress tolerance in transgenic chilli pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) plants overexpressing the wheat antiporter (<Emphasis Type="Italic">TaNHX2</Emphasis>) gene

Transgenic chilli pepper (Capsicum annuum L.) plants tolerant to salinity stress were produced by introducing the wheat Na⁺/H⁺ antiporter gene (TaNHX2) via Agrobacterium-mediated transformation. Cotyledonary explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBin438 that contains a wheat antiporter (TaNHX2) gene driven by the double CaMV 35S promoter and NPT II gene as a selectable marker. PCR and semiquantitative RT-PCR analysis confirmed that the TaNHX2 gene had been integrated and expressed in the T₁ generation of transgenic pepper plants as compared to the non-transformed plants. Southern blot analysis further verified the integration and presence of TaNHX2 gene in the genome of chilli pepper plants. Biochemical assays of these transgenic plants revealed enhanced levels of proline, chlorophyll, superoxide dismutase, ascorbate peroxidase, relative water content, and reduced levels of hydrogen peroxide (H₂O₂), malondialdehyde compared to wild-type plants under salt stress conditions. The present investigation clearly showed that overexpression of the TaNHX2 gene enhanced salt stress tolerance in transgenic chilli pepper plants.     

<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.     

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.     

Effect of the vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter transgene in a rice landrace and a commercial rice cultivar after its insertion by crossing

The vacuolar Na⁺/H⁺ antiporter is known to alleviate saline stress by sequestering Na⁺ in both wild-type Arabidopsis and rice and when over-expressed in many transgenic plants. Here we report on the effect of the NHX1 transgene on the salt tolerance properties it confers to a rice landrace and a commercial cultivar suitable for the dry winter season, but which suffers loss due to seasonal stresses, particularly in the coastal areas. The Nipponbare Na⁺/H⁺ antiporter 1.9 kb cDNA was cloned into pCAMBIA1305.1 under the control of the CaMV35S promoter and transformed into tissue-culture-responsive rice landrace Binnatoa (BA). The best-expressing transgenic line at T₂ was found to be significantly tolerant at the seedling stage and was advanced to T₃. The transgene was then transferred to the tissue-culture recalcitrant farmer-popular commercial rice genotype, BRRIdhan 28 (BR28) by crossing. The data generated both from semi-quantitative RT-PCR and western blot hybridization revealed that the transgene showed similar expression in the crossbred BR28 plants and BA transgenic line. Comparative stress tolerance tests, however, revealed that the BR28 crossbred lines were significantly less tolerant than its transgenic parent BA at both seedling and reproductive stages. A single successful transgenic event may therefore not show the same performance in the recipient genetic background, if introgressed by crossing.     

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.     

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.     

Isolation,Functional Characterization,and Expression Pattern of a Vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">TrNHX1</Emphasis> from <Emphasis Type="Italic">Trifolium repens</Emphasis> L.

Plant vacuolar Na⁺/H⁺ antiporter plays an important role in salt tolerance. A vacuolar Na⁺/H⁺ antiporter gene TrNHX1 was cloned from Trifolium repens L., a forage legume, by RT-PCR and RACE methods using degenerate oligonucleotide primers. The TrNHX1 sequence contains 2,394 nucleotides and an open-reading frame of 1,626 nucleotides that encodes a protein of 541 amino acids with a deduced molecular mass of 59.5 kDa. The deduced amino acid sequence of TrNHX1 is 78% identical to that of a vacuolar Na⁺/H⁺ antiporter of Arabidopsis thaliana, AtNHX1, and contains the consensus amiloride-binding domain. TrNHX1 could partially complement the NaCl-sensitive phenotypes of yeast mutants Δnhx1 and Δena1-4Δnhx1, and a similar complementation was also observed in the presence of LiCl and KCl. In addition, it was found that TrNHX1 suppressed the hygromycin-sensitive phenotype of yeast mutant Δena1-4Δnhx1. The expression of TrNHX1 in T. repens increased in the presence of 150 mM NaCl, and this result accords with that of Na⁺ contents determination under the same treatment. These results suggest that TrNHX1 functions as a vacuolar Na⁺/H⁺ antiporter and plays an important role in salt tolerance and ion homeostasis in T. repens.     

New isoform HvNHX3 of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter in barley: Expression and immunolocalization

The gene HvNHX3 encoding a new isoform of vacuolar Na⁺/H⁺-antiporter was identified in barley. This gene is expressed in roots and leaves of barley seedlings, and it encodes a protein consisting of 541 amino acid residues with pre-dicted molecular weight 59.7 kDa. It was found that by its amino acid sequence HvNHX3 is closest to the Na⁺/H⁺-antiporter HbNHX1 of wild type from Hordeum brevisibulatum that grows on salt-marsh (solonchak) soils (95% homology). The expression of HvNHX3 during salt stress is increased several-fold in roots and leaves of barley seedlings. At the same time, the amount of HvNHX3 protein in roots does not change, but in leaves it increases significantly. It was shown using HvNHX3 immunolocalization in roots that this protein is present in all tissues, but in control plants it was clustered and in experimental plants after salt stress it was visualized as small granules. It has been proposed that HvNHX3 is converted into active form during declusterization. The conversion of HvNHX3 into its active form along with its quantitative increase in leaves during salt stress activates Na⁺/H⁺-exchange across the vacuolar membrane and Na⁺ release from cytoplasm, and, as a consequence, an increase of salt stress tolerance.     

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.