An Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene from wheat plays an important role in stress tolerance

A vacuole Na⁺/H⁺ antiporter gene TaNHX2 was obtained by screening the wheat cDNA library and by the 5′-RACE method. The expression of TaNHX2 was induced in roots and leaves by treatment with NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA). When expressed in a yeast mutant (Δnhx1), TaNHX2 suppressed the salt sensitivity of the mutant, which was deficient in vacuolar Na⁺/H⁺ antiporter, and caused partial recovery of growth of Δnhx1 in NaCl and LiC1 media. The survival rate of yeast cells was improved by overexpressing the TaNHX2 gene under NaCl, KCl, sorbitol and freezing stresses when compared with the control. The results imply that TaNHX2 might play an important role in salt and osmotic stress tolerance in plant cells.     

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.     

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.     

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.     

<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.     

Role of sodium ion transporters and osmotic adjustments in stress alleviation of <Emphasis Type="Italic">Cynodon dactylon</Emphasis> under NaCl treatment: a parallel investigation with rice

Comparative analyses of the responses to NaCl in Cynodon dactylon and a sensitive crop species like rice could effectively unravel the salt tolerance mechanism in the former. C. dactylon, a wild perennial chloridoid grass having a wide range of ecological distribution is generally adaptable to varying degrees of salinity stress. The role of salt exclusion mechanism present exclusively in the wild grass was one of the major factors contributing to its tolerance. Salt exclusion was found to be induced at 4 days when the plants were treated with a minimum conc. of 200 mM NaCl. The structural peculiarities of the salt exuding glands were elucidated by the SEM and TEM studies, which clearly revealed the presence of a bicellular salt gland actively functioning under NaCl stress to remove the excess amount of Na⁺ ion from the mesophyll tissues. Moreover, the intracellular effect of NaCl on the photosynthetic apparatus was found to be lower in C. dactylon in comparison to rice; at the same time, the vacuolization process increased in the former. Accumulation of osmolytes like proline and glycine betaine also increased significantly in C. dactylon with a concurrent check on the H₂O₂ levels, electrolyte leakage and membrane lipid peroxidation. This accounted for the proper functioning of the Na⁺ ion transporters in the salt glands and also in the vacuoles for the exudation and loading of excess salts, respectively, to maintain the osmotic balance of the protoplasm. In real-time PCR analyses, CdSOS1 expression was found to increase by 2.5- and 5-fold, respectively, and CdNHX expression increased by 1.5- and 2-fold, respectively, in plants subjected to 100 and 200 mM NaCl treatment for 72 h. Thus, the comparative analyses of the expression pattern of the plasma membrane and tonoplast Na⁺ ion transporters, SOS1 and NHX in both the plants revealed the significant role of these two ion transporters in conferring salinity tolerance in Cynodon.     

Salt-induced changes in photosynthetic activity and growth in a potential medicinal plant Bishop’s weed (<Emphasis Type="Italic">Ammi majus</Emphasis> L.)

Sixty seven-days-old plants of Ammi majus L. were subjected for 46 d to sand culture at varying concentrations of NaCl, i.e. 0 (control), 40, 80, 120, and 160 mM. Increasing salt concentrations caused a significant reduction in fresh and dry masses of both shoots and roots as well as seed yield. However, the adverse effect of salt was more pronounced on seed yield than biomass production at the vegetative stage. Calculated 50 % reduction in shoot dry mass occurred at 156 mM (ca.15.6 mS cm^?1), whereas that in seed yield was at 104 mM (ca.10.4 mS cm^?1). As in most glycophytes, Na⁺ and Cl^? in both shoots and roots increased, whereas K⁺ and Ca²⁺ decreased consistently with the successive increase in salt level of the growth medium. Plants of A. majusmaintained markedly higher K⁺/Na⁺ ratios in the shoots than those in the roots, and the ratio remained more than 1 even at the highest external salt level (160 mM). Net photosynthetic (P_N) and transpiration (E) rates remained unaffected at increasing NaCl, and thus these attributes had a negative association with salt tolerance of A. majus. Proline content in the shoots increased markedly at the higher concentrations of salt. Essential oil content in the seed decreased consistently with increase in external salt level. Overall, A. majusis a moderately salt tolerant crop whose response to salinity is associated with maintenance of high shoot K⁺/Na⁺ ratio and accumulation of proline in shoots, but P_N had a negative association with the salt tolerance of this crop.     

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.     

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.     

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.     

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.     

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.