Molecular Cloning and Functional Analysis of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">ThNHX1</Emphasis> from a Halophytic Plant <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Na⁺/H⁺ exchanger catalyzes the countertransport of Na⁺ and H⁺ across membranes. Using the rapid amplification of cDNA ends method, a Na⁺/H⁺ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (⁸⁷LFFIYLLPPI⁹⁶) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na⁺/Li⁺-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na⁺/H⁺ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na⁺/H⁺ antiporter and play an important role in salt tolerance of T. halophila. Chunxia Wu, Xiuhua Gao, and Xiangqiang Kong contributed equally to this work.     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

Molecular characterization and functional analysis of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene (<Emphasis Type="Italic">HcNHX1</Emphasis>) from <Emphasis Type="Italic">Halostachys caspica</Emphasis>

According to sequences of several vacuolar Na⁺/H⁺ antiporter genes from Xinjiang halophytic plants, a new vacuolar Na⁺/H⁺ antiporter gene (HcNHX1) from the halophyte Halostachys caspica was obtained by RACE and RT-PCR using primers corresponding to conserved regions of the coding sequences. The obtained HcNHX1 cDNA was 1,983 bp and contained a 1,656 bp open reading frame encoding a deduced protein of 551 amino acid residues. The deduced amino acid sequence showed high identity with other NHX1 we have cloned previously from halophyte in Xinjiang desert area. The phylogenetic analysis showed that HcNHX1 formed a clade with NHX homologs of Chenopodiaceae. Expression profiles under salt treatment and ABA induction were investigated, and the results revealed that expression of HcNHX1 was induced by NaCl and ABA. To compare the degree of salt tolerance, we over-expressed HcNHX1 in Arabidopsis. Two transgenic lines grew more vigorously than the wild type (WT) under salt stress. The analysis of ion contents indicated that under salt stress, the transgenic plants compartmentalized more Na⁺ in the leaves compared with wild-type plants. Together, these results suggest that the products of the novel gene HcNHX1 from halophyte Halostachys caspica is a functional tonoplast Na⁺/H⁺ antiporter.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

Transformation of tomato with the <Emphasis Type="Italic">BADH</Emphasis> gene from <Emphasis Type="Italic">Atriplex</Emphasis> improves salt tolerance

Glycinebetaine is an important quaternary ammonium compound that is produced in response to salt and other osmotic stresses in many organisms. Its synthesis requires the catalysis of betaine aldehyde dehydrogenase encoded by BADH gene that converts betaine aldehyde into glycinebetaine in some halotolerant plants. We transformed the BADH gene, cloned from Atriplex hortensis and controlled by two 35S promoters of the cauliflower mosaic virus, into a salt-sensitive tomato cultivar, Bailichun, using Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBin438, and using a leaf regeneration system. Polymerase chain reaction and Southern hybridization analyses demonstrated that the BADH gene had integrated into the genome of tomato. Transgenic tomato plants showed significantly higher levels of mRNA and BADH enzyme activity than wild-type plants. Observations on rooting development and relative electronic conductivity suggested that the transgenic plants exhibited tolerance to salt stress, with these plants growing normally at salt concentrations up to 120 mM.     

Cloning and Functional Characterization of a Novel Glutathione <Emphasis Type="Italic">S</Emphasis>-Transferase Gene from <Emphasis Type="Italic">Limonium bicolor</Emphasis>

Plant glutathione S-transferases (GSTs) are involved in protecting plants against both diverse biotic and abiotic stresses. In the present study, a novel GST gene (LbGST1) was cloned from Limonium bicolor (Bunge) Kuntze (Plumbaginaceae). To characterize its function in salt tolerance, tobacco lines transformed with LbGST1 were generated. Compared with wild-type (WT) tobacco, transgenic plants overexpressing LbGST1 exhibited both GST and glutathione peroxidase activities. Moreover, superoxide dismutase, peroxidase (POD), and catalase activities in transgenic plants were significantly higher than those in WT plants, particularly when grown under conditions of salt stress. Similarly, levels of proline in transgenic plants were also higher than those in WT plants grown under NaCl stress conditions. Whereas, Malondialdehyde contents in transgenic plants were lower than those in WT plants under NaCl conditions. Furthermore, Na⁺ content in transgenic plants was lower than that in WT plants under these stress conditions. Subcellular localization analysis revealed that the LbGST1 protein was localized in the nucleus. These results suggested that overexpression of LbGST1 gene can affect many physiological processes associated with plant salt tolerance. Therefore, we hypothesize that LbGST1 gene can mediate many physiological pathways that enhance stress resistance in plants.     

Overexpression of a <Emphasis Type="Italic">Malus</Emphasis> vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene (<Emphasis Type="Italic">MdNHX1</Emphasis>) in apple rootstock M.26 and its influence on salt tolerance

Soil salinity is a major factor limiting apple production in some areas. Tonoplast Na⁺/H⁺ antiporters play a critical role in salt tolerance. Here, we isolated MdNHX1, a vacuolar Na⁺/H⁺ antiporter from Luo-2, a salt-tolerant rootstock of apple (Malus × domestica Borkh.), and introduced it into apple rootstock M.26 by Agrobacterium-mediated transformation. PCR and DNA gel blot analyses confirmed successful integration of MdNHX1. RT-PCR analysis indicated that the gene was highly expressed in transgenic plants, but the degree of this expression varied among lines. Its overexpression conferred high tolerance to salt stress. Analysis of ion contents showed that, when exposed to salinity stress, the transgenics compartmentalized more Na⁺ in the roots and also maintained a relatively high K⁺/Na⁺ ratio in the leaves compared with non-transformed plants. Under normal conditions, however, amounts of potassium and sodium did not differ significantly between transgenic and control plants.     

Enhanced salinity tolerance and improved yield properties in Bangladeshi rice Binnatoa through <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of <Emphasis Type="Italic">PgNHX1</Emphasis> from <Emphasis Type="Italic">Pennisetum glaucum</Emphasis>

Rice yield is severely affected by high-salt concentration in the vicinity of the plant. In an effort to engineer rice for improved salt tolerance Agrobacterium-mediated transformation of rice cv. Binnatoa was accomplished with the Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter gene (PgNHX1) under the constitutive CaMV35S promoter. For the molecular analysis of putative transgenic plants, PCR and RT-PCR were performed. Transgenic rice plants expressing PgNHX1 showed better physiological status and completed their life cycle by setting flowers and seeds in salt stress, while wild-type plants exhibited rapid chlorosis and growth inhibition. Moreover, transgenic rice plants produced higher grain yields than wild-type plants under salt stress. Assessment of the salinity tolerance of the transgenic plants at seedling and reproductive stages demonstrated the potential of PgNHX1 for imparting enhanced salt tolerance capabilities and improved yield.     

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.     

Regulation of expression of Na<Superscript>+</Superscript>-translocating NADH:quinone oxidoreductase genes in <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na⁺-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na⁺-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accession number: EF394942 (Vibrio harveyi arcB gene, partial cds).     

The Effect of Salinity Pretreatment on Cd Accumulation and Cd-Induced Stress in <Emphasis Type="Italic">BADH</Emphasis><Emphasis Type="Italic">-</Emphasis>Transgenic and Nontransgenic Rice Seedlings

The influence of betaine aldehyde dehydrogenase (BADH) and salinity pretreatment on oxidative stress under cadmium (Cd) toxicity was investigated in rice cv. Xiushui 11 and its BADH-transgenic line Bxiushui 11. The results showed that plants previously treated with 4.25 and 8.5 mM NaCl, respectively, for 5 days each had higher Cd concentrations in both roots and shoots of the two rice genotypes compared with the controls. Malondialdehyde (MDA) content in both leaves and roots was increased by salinity pretreatment and was significantly lower in the salinity-pretreatment plants than in the controls when the plants were consequently exposed to Cd stress. Salinity pretreatment also increased proline content and the activities of superoxide dismutase (SOD) and peroxidase (POD) in both leaves and roots. It can be assumed that salinity pretreatment enhances the defensive ability of plants against oxidative stress through increasing activities of antioxidative enzymes. The BADH-transgenic line (Bxiushui 11) had lower Cd and MDA content, higher SOD and POD activities, and higher proline content than its wild type (Xiushui 11). The current results suggest that betaine, a product of BADH expression, improves the tolerance of rice plants to Cd stress through increasing the activities of antioxidative enzymes and osmoprotectant content.     

<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.     

The wheat gene <Emphasis Type="Italic">TaST</Emphasis> can increase the salt tolerance of transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

On the basis of the results of gene chip analysis of the salt-tolerant wheat mutant RH8706-49 under conditions of salt stress, we identified and cloned an unknown salt-induced gene TaST (Triticum aestivum salt-tolerant). Real-time quantitative PCR analysis showed that the expression of the gene was induced by salt stress. Transgenic Arabidopsis plants overexpressing the TaST gene showed higher salt tolerance than the wild-type controls. Subcellular localization studies revealed that the protein encoded by this gene was in the nucleus. In comparison with wild-type controls, transgenic Arabidopsis plants accumulated more Ca²⁺, soluble sugar, and proline and less Na⁺ under salt stress. Real-time quantitative PCR analysis showed that Arabidopsis plants overexpressing TaST also showed increased expression of many stress-related genes. All these findings indicated that TaST can enhance the salt tolerance of transgenic Arabidopsis plants.     

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.     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.     

Overexpression of <Emphasis Type="Italic">IrlVHA</Emphasis>-<Emphasis Type="Italic">c</Emphasis>, A Vacuolar-Type H<Superscript>+</Superscript>-ATPase c Subunit Gene from <Emphasis Type="Italic">Iris lactea</Emphasis>, Enhances Salt Tolerance in Tobacco

Vacuolar-type H⁺-ATPase (V-ATPase), a multi-subunit endomembrane proton pump, plays an important role in plant growth and response to environmental stresses. In the present study, transgenic tobacco that overexpressed the V-ATPase c subunit gene from Iris lactea (IrlVHA-c) was used to determine the function of IrlVHA-c. Quantitative PCR analysis showed that IrlVHA-c expression was induced by salt stress in I. lactea roots and leaves. Subcellular localization of green fluorescent protein (GFP) as marker combined with FM4-64 staining showed that the IrlVHA-c-GFP was localized to the endosomal compartment in tobacco cells. Compared with the wild-type, the IrlVHA-c transgenic tobacco plants exhibited greater seed germination rates, root length, fresh weight, and higher relative water content (RWC) of leaves under salt stress. Furthermore, the IrlVHA-c transgenic tobacco leaves have lower stomatal densities and larger stomatal apertures than wild-type. Under salt stress, superoxide dismutase (SOD) activity in the transgenic tobacco was significantly enhanced. Moreover, the level of malondialdehyde (MDA) in the transgenic tobacco was significantly lower than that in wild-type plants under salt stress. Taken together, these results suggested that the IrlVHA-c plays an important role in salt tolerance in transgenic tobacco by influencing stomatal movement and physiological changes.