A Novel Plant Vacuolar Na+/H+ Antiporter Gene Evolved by DNA Shuffling Confers Improved Salt Tolerance in Yeast

Plant vacuolar Na⁺/H⁺ antiporters play important roles in maintaining cellular ion homeostasis and mediating the transport of Na⁺ out of the cytosol and into the vacuole. Vacuolar antiporters have been shown to play significant roles in salt tolerance; however the relatively low V_max of the Na⁺/H⁺ exchange of the Na⁺/H⁺ antiporters identified could limit its application in the molecular breeding of salt tolerant crops. In this study, we applied DNA shuffling methodology to generate and recombine the mutations of Arabidopsis thaliana vacuolar Na⁺/H⁺ antiporter gene AtNHX1. Screening using a large scale yeast complementation system identified AtNHXS1, a novel Na⁺/H⁺ antiporter. Expression of AtNHXS1 in yeast showed that the antiporter localized to the vacuolar membrane and that its expression improved the tolerance of yeast to NaCl, KCl, LiCl, and hygromycin B. Measurements of the ion transport activity across the intact yeast vacuole demonstrated that the AtNHXS1 protein showed higher Na⁺/H⁺ exchange activity and a slightly improved K⁺/H⁺ exchange activity.     

Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants

Agropyron elongatum, a species in grass family, has a strong tolerance to salt stress. To study the molecular mechanism of Agropyron elongatum in salt tolerance, we isolated a homolog of Na⁺/H⁺ antiporters from the root tissues of Agropyron plants. Sequence analysis revealed that this gene encodes a putative vacuolar Na⁺/H⁺ antiporter and was designated as AeNHX1. The AeNHX1–GFP fusion protein was clearly targeted to the vacuolar membrane in a transient transfection assay. Northern analysis indicated that AeNHX1 was expressed in a root-specific manner. Expression of AeNHX1 in yeast Na⁺/H⁺ antiporter mutants showed function complementation. Further, overexpression of AeNHX1 promoted salt tolerance of Arabidopsis plants, and improved osmotic adjustment and photosynthesis which might be responsible for normal development of transgenic plants under salt stress. Similarly, AeNHX1 also functioned in transgenic Festuca plants. The results suggest that this gene might function in the roots of Agropyron plants, and its expression is involved in the improvement of salt tolerance.     

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.     

Protective effect of exogenous polyamines on root tonoplast function against salt stress in barley seedlings

Salinity stress is one of the most serious factors limiting the productivity of agricultural crops. A possible survival strategy of plants under saline conditions is to sequester excess Na⁺ in the vacuole by vacuolar Na⁺/H⁺ antiport using a pH gradient generated by H⁺-ATPasc (EC 3.6.1.35) and H⁺-Pyrophosphatase (H⁺-PPase; EC 3.6.1.1) to maintain a higher K⁺/Na⁺ ratio in cytoplasm. The effect of exogenously applied polyamines (PAs) in stabilizing root tonoplast integrity and function against salt stress in the barley (Hordeum vulgare L.) seedlings was investigated. The NaCl-induced reductions in the contents of phospholipids and PAs in tonoplast vesicles isolated from barely seedling roots, as well as the activities of H⁺-ATPase, H⁺-PPase and vacuolar Na⁺/H⁺ antiport were all partially restored by the application of 0.5 mM putrescine and 0.5 mM spermidine, especially the former. The above results indicated that one of the mechanisms involved in attenuating salt injury in barley seedlings by exogenous PAs application was to maintain tonoplast integrity and function under saline conditions. Moreover, the possible mechanism involved in counteracting detrimental effects of salt on the barley seedlings by the application of exogenous PAs was discussed.     

Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum)

Agriculture productivity is severely affected by soil salinity. One possible mechanism by which plants could survive salt stress is to compartmentalize sodium ions away from the cytosol. In the present work, transgenic buckwheat plants overexpressing AtNHX1, a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis thaliana, were regenerated after transformation with Agrobacterium tumefaciens. These plants were able to grow, flower and accumulate more rutin in the presence of 200 mmol/l sodium chloride. Moreover, the content of important nutrients in buckwheat was not affected by the high salinity of the soil. These results demonstrated the potential value of these transgenic plants for agriculture use in saline soil.     

Isolation and characterization of a Na+/H+ antiporter gene from the halophyte Atriplex gmelini

With a homologous gene region we successfully isolated a Na⁺/H⁺ antiporter gene from a halophytic plant, Atriplex gmelini, and named it AgNHX1. The isolated cDNA is 2607 bp in length and contains one open reading frame, which comprises 555 amino acid residues with a predicted molecular mass of 61.9 kDa. The amino acid sequence of the AgNHX1 gene showed more than 75% identity with those of the previously isolated NHX1 genes from glycophytes, Arabidopsis thaliana and Oryza sativa. The migration pattern of AgNHX1 was shown to correlate with H⁺-pyrophosphatase and not with P-type H⁺-ATPase, suggesting the localization of AgNHX1 in a vacuolar membrane. Induction of the AgNHX1 gene was observed by salt stress at both mRNA and protein levels. The expression of the AgNHX1 gene in the yeast mutant, which lacks the vacuolar-type Na⁺/H⁺ antiporter gene (NHX1) and has poor viability under the high-salt conditions, showed partial complementation of the NHX1 functions. These results suggest the important role of the AgNHX1 products for salt tolerance.     

Heterologous expression of vacuolar H+-PPase enhances the electrochemical gradient across the vacuolar membrane and improves tobacco cell salt tolerance

Summary. The vacuolar H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) uses pyrophosphate as substrate to generate the proton electrochemical gradient across the vacuolar membrane to acidify vacuoles in plant cells. The heterologous expression of H⁺-PPase genes (TsVP from Thellungiella halophila and AVP1 from Arabidopsis thaliana) improved the salt tolerance of tobacco plants. Under salt stress, the transgenic seedlings showed much better growth and greater fresh weight than wild-type plants, and their protoplasts had a normal appearance and greater vigor. The cytoplasmic and vacuolar pH in transgenic and wild-type cells were measured with a pH-sensitive fluorescence indicator. The results showed that heterologous expression of H⁺-PPase produced an enhanced proton electrochemical gradient across the vacuolar membrane, which accelerated the sequestration of sodium ions into the vacuole. More Na⁺ accumulated in the vacuoles of transgenic cells under salt (NaCl) stress, revealed by staining with the fluorescent indicator Sodium Green. It was concluded that the tonoplast-resident H⁺-PPase plays important roles in the maintenance of the proton gradient across the vacuolar membrane and the compartmentation of Na⁺ within vacuoles, and heterologous expression of this protein enhanced the electrochemical gradient across the vacuolar membrane, thereby improving the salt tolerance of tobacco cells. Correspondence: J.-R. Zhang, School of Life Science, Shandong University, 27 Shanda South Road, Jinan, People’s Republic of China 250100.     

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.     

RETRACTED ARTICLE: Analysis of the physiological mechanism of salt-tolerant transgenic rice carrying a vacuolar Na+/H+ antiporter gene from Suaeda salsa

Salt stress is one of the most serious factors limiting the productivity of agricultural crops. Increasing evidence has demonstrated that vacuolar Na⁺/H⁺ antiporters play a crucial role in plant salt tolerance. In the present study, we expressed the Suaeda salsa vacuolar Na⁺/H⁺ antiporter SsNHX1 in transgenic rice to investigate whether this can increase the salt tolerance of rice, and to study how overexpression of this gene affected other salt-tolerant mechanisms. It was found that transgenic rice plants showed markedly enhanced tolerance to salt stress and to water deprivation compared with non-transgenic controls upon salt stress imposition under outdoor conditions. Measurements of ion levels indicated that K⁺, Ca²⁺ and Mg²⁺ contents were all higher in transgenic plants than in non-transformed controls. Furthermore, shoot V-ATPase hydrolytic activity was dramatically increased in transgenics compared to that of non-transformed controls under salt stress conditions. Physiological analysis also showed that the photosynthetic activity of the transformed plants was higher whereas the same plants had reduced reactive oxygen species generation. In addition, the soluble sugar content increased in the transgenics compared with that in non-transgenics. These results imply that up-regulation of a vacuolar Na⁺/H⁺ antiporter gene in transgenic rice might cause pleiotropic up-regulation of other salt-resistance-related mechanisms to improve salt tolerance.Fengyun Zhao and Zenglan Wang contributed equally to this work.     

Functional Analysis of the Na+/H+ Antiporter Encoding Genes of the Cyanobacterium Synechocystis PCC 6803

The role of putative Na⁺/H⁺ antiporters encoded by nhaS1 (slr1727), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response and internal pH regulation of the cyanobacterium Synechocystis PCC 6803 was investigated. For this purpose the mutants (single, double, and triple) impaired in genes coding for Na⁺/H⁺ antiporters were constructed using the method of interposon mutagenesis. PCR analyses of DNA demonstrated that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and the mutants contained only inactivated copies of the corresponding genes. Na⁺/H⁺ antiporter encoded by nhaS3 was essential for viability of Synechocystis since no completely segregated mutants were obtained. The steady-state intracellular sodium concentration and Na⁺/H⁺ antiporter activities were found to be the same in the wild type and all mutants. No differences were found in the growth rates of wild type and mutants during their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes coding for Na⁺/H⁺ antiporters was studied. No induction of any Na⁺/H⁺ antiporter encoding gene expression was found in wild type or single mutant cells grown under high salt or at different pH values. Nevertheless, in cells of double and triple mutants adapted to high salt or alkaline pH some of the remaining Na⁺/H⁺ antiporter encoding genes showed induction. These results might indicate that some of Na⁺/H⁺ antiporters can functionally replace each other under stress conditions in Synechocystis cells lacking the activity of more than one antiporter.     

Enhanced V-ATPase activity contributes to the improved salt tolerance of transgenic tobacco plants overexpressing vacuolar Na(+)/H (+) antiporter AtNHX1

AtNHX1, a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis thaliana, was introduced into tobacco genome via Agrobacterium tumefaciens-mediated transformation to evaluate the role of vacuolar energy providers in plants salt stress response. Compared to the wild-type plants, over-expression of AtNHX1 increased salt tolerance in the transgenic tobacco plants, allowing higher germination rates of seeds and successful seedling establishment in the presence of toxic concentrations of NaCl. More importantly, the induced Na⁺/H⁺ exchange activity in the transgenic plants was closely correlated to the enhanced activity of vacuolar H⁺-ATPase (V-ATPase) when exposed to 200 mM NaCl. In addition, inhibition of V-ATPase activity led to the malfunction of Na⁺/H⁺ exchange activity, placing V-ATPase as the dominant energy provider for the vacuolar Na⁺/H⁺ antiporter AtNHX1. V-ATPase and vacuolar Na⁺/H⁺ antiporter thus function in an additive or synergistic way. Simultaneous overexpression of V-ATPase and vacuolar Na⁺/H⁺ antiporter might be appropriate for producing plants with a higher salt tolerance ability.     

Membrane-bound pyrophosphatase of human gut microbe Clostridium methylpentosum confers improved salt tolerance in Escherichia coli,Saccharomyces cerevisiae and tobacco

Membrane-bound pyrophosphatases (PPases) are involved in the adaption of organisms to stress conditions, which was substantiated by numerous plant transgenic studies with H⁺-PPase yet devoid of any correlated evidences for other two subfamilies, Na⁺-PPase and Na⁺,H⁺-PPase. Herein, we demonstrate the gene cloning and functional evaluation of the membrane-bound PPase (CmPP) of the human gut microbe Clostridium methylpentosum. The CmPP gene encodes a single polypeptide of 699 amino acids that was predicted as a multi-spanning membrane and K⁺-dependent Na⁺,H⁺-PPase. Heterologous expression of CmPP could significantly enhance the salt tolerance of both Escherichia coli and Saccharomyces cerevisiae, and this effect in yeast could be fortified by N-terminal addition of a vacuole-targeting signal peptide from the H⁺-PPase of Trypanosoma cruzi. Furthermore, introduction of CmPP could remarkably improve the salt tolerance of tobacco, implying its potential use in constructing salt-resistant transgenic crops. Consequently, the possible mechanisms of CmPP to underlie salt tolerance are discussed.     

Functional validation of a novel isoform of Na+/H+ antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice

Salt stress is an environmental factor that severely impairs plant growth and productivity. We have cloned a novel isoform of a vacuolar Na⁺/H⁺ antiporter from Pennisetum glaucum (PgNHX1) that contains 5 transmembrane domains in contrast to AtNHX1 and OsNHX1 which have 9 transmembrane domains. Recently we have shown that PgNHX1 could confer high level of salinity tolerance when overexpressed in Brassica juncea. Here, we report the functional validation of this antiporter in crop plant rice. Overexpression of PgNHX1 conferred high level of salinity tolerance in rice. Transgenic rice plants overexpressing PgNHX1 developed more extensive root system and completed their life cycle by setting flowers and seeds in the presence of 150 mM NaCl. Our data demonstrate the potential of PgNHX1 for imparting enhanced salt tolerance capabilities to salt-sensitive crop plants for growing in high saline areas.