Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/H+ antiporter

The function of vacuolar Na+/H+ antiporter(s) in plants has been studied primarily in the context of salinity tolerance. By facilitating the accumulation of Na+ away from the cytosol, plant cells can avert ion toxicity and also utilize vacuolar Na+ as osmoticum to maintain turgor. As many genes encoding these antiporters have been cloned from salt-sensitive plants, it is likely that they function in some capacity other than salinity tolerance. The wide expression pattern of Arabidopsis thaliana sodium proton exchanger 1 (AtNHX1) in this study supports this hypothesis. Here, we report the isolation of a T-DNA insertional mutant of AtNHX1, a vacuolar Na+/H+ antiporter in Arabidopsis. Vacuoles isolated from leaves of the nhx1 plants had a much lower Na+/H+ and K+/H+ exchange activity. nhx1 plants also showed an altered leaf development, with reduction in the frequency of large epidermal cells and a reduction in overall leaf area compared to wild-type plants. The overexpression of AtNHX1 in the nhx1 background complemented these phenotypes. In the presence of NaCl, nhx1 seedling establishment was impaired. These results place AtNHX1 as the dominant K+ and Na+/H+ antiporter in leaf vacuoles in Arabidopsis and also suggest that its contribution to ion homeostasis is important for not only salinity tolerance but development as well.     

盐碱胁迫下碱地肤Na+/H+逆向转运蛋白基因KsNHX1表达分析

利用RACE技术得到碱地肤KsNHX1的3'cDNA序列,分子系统进化分析显示,KsNHX1为液泡膜Na+/H+逆向转运蛋白编码基因.通过半定量RT-PCR检测了该基因在盐碱胁迫下的表达,结果表明:200 mmol·L-1 NaCl胁迫2～24h,KsNHX1在叶片中表达量持续增加;200 mmol·L-1 NaCl处理10 h,KsNHX1在根、茎、叶和花中的表达都上调;不同浓度NaCl处理下,叶片中KsNHX1表达上调,160 mmol·L-1时达到最高;低于400 mmol·L-1浓度下,根中该基因的表达也都上调.经不同浓度Na2CO3胁迫,根中KsNHX1的表达变化趋势与相应浓度NaCl胁迫下的变化相同;但叶片中除160 mmol·L-1 Na,CO3处理下KsNHX1表达略有上调外,其他浓度下KsNHX1的表达都低于对照.KsNHX1的表达模式暗示,在不同盐碱胁迫下,碱地肤能够维持体内相对稳定的K+/Na+,其耐盐特性可能与Na+/H+逆向转运蛋白的作用密切相关.     

Expression levels of the Na + /K + transporter OsHKT2;1 and vacuolar Na + /H + exchanger OsNHX1 , Na enrichment,maintaining the photosynthetic abilities and growth performances of indica rice seedlings under salt stress

Salt affected soil inhibits plant growth, development and productivity, especially in case of rice crop. Ion homeostasis is a candidate defense mechanism in the salt tolerant plants or halophyte species, where the salt toxic ions are stored in the vacuoles. The aim of this investigation was to determine the OsNHX1 (a vacuolar Na+/H+ exchanger) and OsHKT2;1 (Na+/K+ transporter) regulation by salt stress (200 mM NaCl) in two rice cultivars, i.e. Pokkali (salt tolerant) and IR29 (salt susceptible), the accumulation of Na+ in the root and leaf tissues using CoroNa Green® staining dye and the associated physiological changes in test plants. Na+ content was largely increased in the root tissues of rice seedlings cv. Pokkali (15 min after salt stress) due to the higher expression of OsHKT2;1 gene (by 2.5 folds) in the root tissues. The expression of OsNHX1 gene in the leaf tissues was evidently increased in salt stressed seedlings of Pokkali, whereas it was unchanged in salt stressed seedlings of IR29. Na+ in the root tissues of both Pokkali and IR29 was enriched, when subjected to 200 mM NaCl for 12 h and easily detected in the leaf tissues of salt stressed plants exposed for 24 h, especially in cv. Pokkali. Moreover, the overexpression of OsNHX1 gene regulated the translocation of Na+ from root to leaf tissues, and compartmentation of Na+ into vacuoles, thereby maintaining the photosynthetic abilities in cv. Pokkali. Overall growth performance, maximum quantum yield (Fv/Fm), photon yield of PSII (ΦPSII) and net photosynthetic rate (Pn) was improved in salt stressed leaves of Pokkali than those in salt stressed IR29.     

A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants

In this study we have identified the first plant K+/H+ exchanger, LeNHX2 from tomato (Lycopersicon esculentum Mill. cv. Moneymaker), which is a member of the intracellular NHX exchanger protein family. The LeNHX2 protein, belonging to a subfamily of plant NHX proteins closely related to the yeast NHX1 protein, is abundant in roots and stems and is induced in leaves by short term salt or abscisic acid treatment. LeNHX2 complements the salt- and hygromycin-sensitive phenotype caused by NHX1 gene disruption in yeast, but affects accumulation of K+ and not Na+ in intracellular compartments. The LeNHX2 protein co-localizes with Prevacuolar and Golgi markers in a linear sucrose gradient in both yeast and plants. A histidine-tagged version of this protein could be purified and was shown to catalyze K+/H+ exchange but only minor Na+/H+ exchange in vitro. These data indicate that proper functioning of the endomembrane system relies on the regulation of K+ and H+ homeostasis by K+/H+ exchangers.     

An Na+/H+ 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 (deltanhx1), TaNHX2 suppressed the salt sensitivity of the mutant,which was deficient in vacuolar Na+/H+ antiporter, and caused partial recovery of growth of delta nhx1 in NaCl and LiCl 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.     

Characterization of a novel Na+/H+ antiporter gene InNHX2 and comparison of InNHX2 with InNHX1, which is responsible for blue flower coloration by increasing the vacuolar pH in the Japanese morning glory

The reddish-purple buds of the wild-type Japanese morning glory (Ipomoea nil) change into blue open flowers, and the shift in the flower coloration correlates with an increase in the vacuolar pH of the flower epidermal cell. In the mutant deficient in the InNHX1 gene for the vacuolar Na(+)/H(+) antiporter, the vacuolar alkalization occurs only partially, and reddish-purple buds become purple open flowers. While most of the plant NHX genes characterized are generally expressed in leaves, stems and roots and induced by NaCl treatment, the InNHX1 gene is expressed predominantly in the flower limbs at around 12 h before flower opening. It is expressed very sparsly in leaves, stems and roots, and no induction occurs in response to NaCl treatment. Here, we identified a novel vacuolar Na(+)/H(+) antiporter gene InNHX2, which is expressed in leaves, stems and roots and is induced in response to NaCl treatment. In addition, relatively higher expression of InNHX2 was observed in the flower limbs shortly before flower opening. We also discovered that both the InNHX1 and InNHX2 proteins can catalyze both Na(+) and K(+) transport into vacuoles. These results suggest that InNHX2 performs dual functions: to confer salt tolerance on the plant and to promote partial vacuolar alkalization in the petals. The implication is that the InNHX2 protein is probably one of the components responsible for converting reddish-purple buds into purple open flowers by partially increasing the vacuolar pH in the absence of major InNHX1 activity.     

植物Na+/H+逆向转运蛋白研究进展

盐胁迫主要由Na 引起,过高的Na 浓度引起的离子毒害,渗透胁迫和K ／Na 比率的不平衡使植物新陈代谢异常,这是对大多数器官造成伤害的原因。植物抵御盐胁迫的主要方式是将细胞内过多的Na 从质膜向细胞外排放和将Na 在液泡中区隔化,这一过程是由Na ／H 逆向转运蛋白完成的。本文概述了植物中Na ／H 逆向转运蛋白的发现、特征、分子生物学方面的研究,以及Na ／H 逆向转运蛋白在植物耐盐性中的重要作用。     

植物Na+/H+逆向转运蛋白研究进展

盐胁迫主要由Na+引起,过高的Na+浓度引起的离子毒害,渗透胁迫和K+/Na+比率的不平衡使植物新陈代谢异常,这是对大多数器官造成伤害的原因。植物抵御盐胁迫的主要方式是将细胞内过多的Na+从质膜向细胞外排放和将Na+在液泡中区隔化,这一过程是由Na+/H+ 逆向转运蛋白完成的。本文概述了植物中Na+/H+ 逆向转运蛋白的发现、特征、分子生物学方面的研究,以及Na+/H+ 逆向转运蛋白在植物耐盐性中的重要作用。     

Effects of non-uniform root zone salinity on water use, Na+ recirculation, and Na+ and H+ flux in cotton

A new split-root system was established through grafting to study cotton response to non-uniform salinity. Each root half was treated with either uniform (100/100?mM) or non-uniform NaCl concentrations (0/200 and 50/150?mM). In contrast to uniform control, non-uniform salinity treatment improved plant growth and water use, with more water absorbed from the non- and low salinity side. Non-uniform treatments decreased Na(+) concentrations in leaves. The [Na(+)] in the '0' side roots of the 0/200 treatment was significantly higher than that in either side of the 0/0 control, but greatly decreased when the '0' side phloem was girdled, suggesting that the increased [Na(+)] in the '0' side roots was possibly due to transportation of foliar Na(+) to roots through phloem. Plants under non-uniform salinity extruded more Na(+) from the root than those under uniform salinity. Root Na(+) efflux in the low salinity side was greatly enhanced by the higher salinity side. NaCl-induced Na(+) efflux and H(+) influx were inhibited by amiloride and sodium orthovanadate, suggesting that root Na(+) extrusion was probably due to active Na(+)/H(+) antiport across the plasma membrane. Improved plant growth under non-uniform salinity was thus attributed to increased water use, reduced leaf Na(+) concentration, transport of excessive foliar Na(+) to the low salinity side, and enhanced Na(+) efflux from the low salinity root.     

Na+ transport in plants

The ability of plants to grow in high NaCl concentrations is associated with the ability of the plants to transport, compartmentalize, extrude, and mobilize Na(+) ions. While the influx and efflux at the roots establish the steady state rate of entry of Na(+) into the plant, the compartmentation of Na(+) into the cell vacuoles and the radial transport of Na(+) to the stele and its loading into the xylem establish the homeostatic control of Na(+) in the cytosol of the root cells. Removal of Na(+) from the transpirational stream, its distribution within the plant and its progressive accumulation in the leaf vacuoles, will determine the ability to deal with the toxic effects of Na(+). The aim of this review is to highlight and discuss the recent progress in understanding of Na(+) transport in plants.     

Na+/H+ exchange activity in the plasma membrane of Arabidopsis

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt.