The Vacuolar Na+/H+ Antiporter Gene SsNHX1 from the Halophyte Salsola soda Confers Salt Tolerance in Transgenic Alfalfa (Medicago sativa L.)

A putative vacuolar Na⁺/H⁺ antiporter gene (SsNHX1) was isolated from the halophyte Salsola soda using the rapid amplification of cDNA ends method. Highly conserved regions of plant vacuolar Na⁺/H⁺ antiporter, including amiloride-binding domain, NHE (Na⁺/H⁺ exchange) domain, and 12 transmembrane segments, were found in the deduced amino acid sequence of SsNHX1. Multiple alignments of vacuolar Na⁺/H⁺ antiporters showed that SsNHX1 shared high identity with other plant vacuolar Na⁺/H⁺ antiporters. Phylogenetic relationship analysis indicated that SsNHX1 was clustered into the vacuolar Na⁺/H⁺ antiporter group. Taken together, these results suggest that SsNHX1 is a new member of the vacuolar Na⁺/H⁺ antiporter family. The effective expression of SsNHX1 in alfalfa (Medicago sativa L.) enhanced the salt tolerance of transgenic alfalfa which could grow in high concentrations of NaCl (up to 400 mM) over 50 days. This was the highest level of salt tolerance reported in transgenic plants. A further analysis of the physiological characteristics of transgenic and wild-type plants, including the Na⁺ and K⁺ contents, superoxide dismutase activity, the rate of electrolyte leakage, and the proline content, showed that large amounts of Na⁺ in the cytoplasm of leaves were transported into vacuoles by the exogenous Na⁺/H⁺ antiporter, which averted the toxic effects of Na⁺ to the cell of transgenic alfalfa.     

Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1

Transgenic rice plants co-expressing the Suaeda salsa SsNHX1 (vacuolar membrane Na⁺/H⁺ antiporter) and Arabidopsis AVP1 (vacuolar H⁺-PPase) showed enhanced salt tolerance during 3 d of 300 mM NaCl treatment under outdoor growth conditions. These transgenic rice seedlings also grew better on MS medium containing 150 mM NaCl compared to SsNHX1-transformed lines and non-transformed controls. Measurements on isolated vacuolar membrane vesicles derived from the salt stressed SsNHX1+AVP1-transgenic plants demonstrated that the vesicles had increased V-PPase hydrolytic activity in comparison with the Ss-transgenics and non-transgenics. Moreover the V-PPase activity was closely related to the development period of the SA-transgenic seedlings and markedly higher in 3-week-old seedlings than in 5-week-old seedlings. Statistic analysis indicated that the SA-transgenic rice plants contained relatively more ions with higher K⁺/Na⁺ ratio in their shoots compared to the SsNHX1-transformed lines upon salt treatment. Furthermore, these SA-transformants also exhibited relatively higher level of photosynthesis and root proton exportation capacity whereas reduced H₂O₂ generation in the same plants. In general, these results supported the hypothesis that simultaneous expression of the SsNHX1 and AVP1 conferred greater performance to the transgenic plants than that of the single SsNHX1.Feng-Yun Zhao and Xue-Jie Zhang contributed equally to this work     

Molecular Cloning and Different Expression of a Vacuolar Na+/H+ antiporter gene in Suaeda salsa Under Salt Stress

A Na⁺/H⁺ antiporter catalyzes the transport of Na⁺ and H⁺ across the tonoplast membrane. We isolated a vacuolar Na⁺/H⁺ antiporter cDNA (SsNHX1) clone from a euhalophyte, Suaeda salsa. The nuclear sequence contains 2262 bp with an open reading frame of 1665 bp. The deduced amino acid sequence is similar to that of AtNHX1 and OsNHX1 in rice, with the highest similarities within the predicted transmembrane segments and an amiloride-binding domain. Northern blot analysis shows that the expression of the S. salsa gene was increased by salt stress. The results suggest that the SsNHX1 product is likely a Na⁺/H⁺ antiporter and may play important roles in the salt tolerance of S. salsa. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

Overexpression ofENA1 from yeast increases salt tolerance inArabidopsis

In yeast, the plasma membrane Na⁺/H⁺ antiporter and Na⁺-ATPase are key enzymes for salt tolerance.Saccharomyces cerevisiae Na⁺-ATPase (Enalp ATPase) is encoded by theENA1/PMR2A gene; expression ofENA1 is tightly regulated by Na⁺ and depends on ambient pH. Although Enalp is active mainly at alkaline pH values inS. cerevisiae, no Na⁺-ATPase has been found in flowering plants. To test whether this yeast enzyme would improve salt tolerance in plants, we introducedENA1 intoArabidopsis (cv. Columbia) under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on a medium containing kanamyin. Southern blot analyses confirmed thatENA1 was transferred into theArabidopsis genome and northern blot analyses showed thatENA1 was expressed in the transformants. Several transgenic homozygous lines and wild-type (WT) plants were evaluated for salt tolerance. No obvious morphological or developmental differences existed between the transgenic and WT plants in the absence of stress. However, overexpression ofENA1 inArabidopsis improved seed germination rates and salt tolerance in seedlings. Under saline conditions, transgenic plants accumulated a lower amount of Na⁺ than did the wild type, and fresh and dry weights of the former were higher. Other experiments revealed that expression ofENA1 promoted salt tolerance in transgenicArabidopsis under both acidic and alkaline conditions. These authors contributed equally to this article.     

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.     

Expression of a Novel Antiporter Gene from Brassica napus Resulted in Enhanced Salt Tolerance in Transgenic Tobacco Plants

Tobacco leaf discs were transformed with a plasmid pBIBnNHX1, containing the selectable marker neomycin phosphotransferase gene (nptII) and Na⁺/H⁺ vacuolar antiporter gene from Brassica napus (BnNHX1), via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the BnNHX1 gene had integrated into plant genome and Northern blot analysis revealed the transgene expression at various levels in transgenic plants. Transgenic plants expressing BnNHX1 had enhanced salt tolerance and could grow and produce seeds normally in the presence of 200 mM NaCl. Analysis for the T₁ progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T₁ lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T₁ progenies could express _BnNHX1 and had salt tolerance at levels comparable to their T₀ parental lines. This study implicates that the BnNHX1 gene represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.     

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.     

Overexpression of SeNHX1 improves both salt tolerance and disease resistance in tobacco

Recently, we found NHX1, the gene encoding a Na⁺/H⁺ exchanger, participated in plant disease defense. Although NHX1 has been confirmed to be involved in plant salt tolerance, whether the NHX1 transgenic plants exhibit both salt tolerance and disease resistance has not been investigated. The T1 progenies of Nicotiana tabacum L. lines expressing SeNHX1 (from Salicornia europaea) were generated for the present study. Compared with PBI-type control plants, SeNHX1 transgenic tobaccos exhibited more biomass, longer root length, and higher K⁺/Na⁺ ratio at post germination or seedling stage under NaCl treatment, indicating enhanced salt tolerance. The vacuolar H⁺ efflux in SeNHX1 transgenic tobacco was increased after treatment of NaCl with different concentration. Meanwhile, the SeNHX1 transgenic tobaccos showed smaller wilted spot area, less H₂O₂ accumulation in leaves after infection of Phytophthora parasitica var. nicotianae. Further investigation demonstrated a larger NAD(P)(H) pool in SeNHX1 transgenic tobacco. These evidences revealed that overexpression of SeNHX1 intensified the compartmentation of Na⁺ into vacuole under salt stress and improved the ability of eliminating ROS after pathogen attack, which then enhanced salt tolerance and disease resistance simultaneously in tobacco. Our findings indicate NHX1 has potential value in creating crops with both improved salt tolerance and disease resistance.     

短期NaCl胁迫对不同小麦品种幼苗K+吸收和Na+、K+积累的影响

为研究盐胁迫下小麦幼苗生长及Na⁺、K⁺的吸收和积累规律,以中国春、洲元9369和长武134等3种耐盐性不同小麦品种为材料,采用非损伤微测技术检测盐胁迫2 d后的根系K⁺离子流变化,并对植株体内的Na⁺、K⁺含量进行测定。结果表明:短期(2d)盐胁迫对小麦生长有抑制作用,且对根系的抑制大于地上部,耐盐品种下降幅度小于盐敏感品种。盐胁迫下,小麦根际的 K⁺大量外流,盐敏感品种中国春K⁺流速显著高于耐盐品种长武134,最高可达15倍。小麦幼苗地上部分和根系均表现为Na⁺积累增加,K⁺积累减少,Na⁺/K⁺比随盐浓度增加而上升。中国春限Na⁺能力显著低于长武134,Na⁺/K⁺则显著高于长武134。综上所述,盐胁迫下造成小麦组织器官中Na⁺/K⁺比上升的主要原因是根系K⁺大量外流和Na⁺的过量积累,耐盐性不同的小麦品种间差异显著,并认为根系对K⁺的保有能力可能是作物耐盐性评价的一个重要指标。     

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.     

长苞香蒲对人工盐碱湿地Na+和K+的吸收与转运特征

为揭示长苞香蒲(Typha domingensis)对盐生湿地生态系统中Na⁺和K⁺的吸收与转运特征,探讨长苞香蒲对盐生湿地的生态修复效果,该研究采用人工模拟盐生湿地的方法,设置CK(对照)、T1(浇灌100 mmol·L^-1盐水)、T2(浇灌200 mmol·L^-1盐水)及T3(浇灌300 mmol·L^-1盐水)4种不同盐浓度的人工湿地生态系统,并分别于5月5日(开始盐胁迫处理,S0)、5月30日(S1)、6月30日(S2)和7月30日(S3)测量其株高和干重、植株地上与地下部分Na⁺和K⁺的含量以及底泥和水体中Na⁺和K⁺的含量以分析长苞香蒲对盐碱湿地的脱盐作用。结果表明:(1)各处理的长苞香蒲的株高和干重随着处理时间的延长呈增加趋势,但与CK 相比,各处理生长量随盐浓度升高出现下降趋势。(2)高浓度盐处理(T3)使长苞香蒲的地上部分和地下部分的Na⁺分别增加了2.56倍和1.75倍,地上部分及地下部分的K⁺含量分别降低了34.1%和35.8%。(3)地上部分和地下部分的Na⁺/K⁺在处理和对照间均随处理时间延长呈增加的趋势,选择性转移系数与Na⁺和K⁺转移系数总体随处理时间延长呈降低的趋势。(4)在S0至S3期间,长苞香蒲对处理组土壤Na⁺和K⁺的去除率为10.6%～15.8%和2.3%～12.8%,对处理组水体Na⁺和K⁺的去除率为55.0%～65.1%和1.6%～67.0%。综上表明,盐胁迫能影响长苞香蒲体内的Na⁺和 K⁺平衡,长苞香蒲能够有效地吸收Na⁺,并在一定盐浓度下能通过K⁺的交换将Na⁺从根部吸收转运至地上部分。因此,长苞香蒲可通过离子转运的形式完成对盐离子的吸收,可作为盐碱湿地生态修复的优良植物。     

Ethylene and nitric oxide are involved in maintaining ion homeostasis in <Emphasis Type="Italic">Arabidopsis</Emphasis> callus under salt stress

In the present study, the role of ethylene in nitric oxide (NO)-mediated protection by modulating ion homeostasis in Arabidopsis callus under salt stress was investigated. Results showed that the ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than the wild type (WT). Under 100 mM NaCl, etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane (PM) H⁺-ATPase activity compared to WT callus. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) or sodium nitroprusside (SNP, a NO donor) alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus. The SNP actions in NaCl stress were attenuated by a specific NO scavenger or an ethylene biosynthesis inhibitor in WT callus. Under 100 mM NaCl, the NO accumulation and ethylene emission appeared at early time, and NO production greatly stimulated ethylene emission in WT callus. In addition, ethylene induced the expression of PM H⁺-ATPase genes under salt stress. The recovery experiment showed that NaCl-induced injury was reversible, as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus. Taken together, the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance, and ethylene may be a part of the downstream signal molecular in NO action.     

Enhancement of the salt tolerance of Triticum turgidum L. by the Kna1 locus transferred from the Triticum aestivum L. chromosome 4D by homoeologous recombination

Durum wheat, Triticum turgidum L. (2n= 4x=28, genome formula AABB) is inferior to bread wheat, T. aestivum L. (2n=6x=42, genome formula AABBDD), in the ability to exclude Na⁺ under salt strees, in the ratio of the accumulated K⁺ to Na⁺ in the leaves under salt stress, and in tolerance of salt stress. Previous work showed that chromosome 4D has a major effect on Na⁺ and K⁺ accumulation in the leaves of bread wheat. The 4D chromosome was recombined with chromosome 4B in the genetic background of durum wheat. The recombinants showed that Na⁺ exclusion and enhanced K⁺/Na⁺ ratio in the shoots were controlled by a single locus, Kna1, in the long arm of chromosome 4D. The recombinant families were grown in the field under non-saline conditions and two levels of salinity to determine whether Kna1 confers salt tolerance. Under salt stress, the Kna1 families had higher K⁺/Na⁺ ratios in the flag leaves and higher yields of grain and biomass than the Kna1 ^- families and the parental cultivars. Kna1 is, therefore, one of the factors responsible for the higher salt tolerance of bread wheat relative to durum wheat. The present work provides conceptual evidence that tolerance of salt stress can be transferred between species in the tribe Triticeae.