Coexpression of ScNHX1 and ScVP in Transgenic Hybrids Improves Salt and Saline-Alkali Tolerance in Alfalfa (Medicago sativa L.)

Salt and saline-alkali are major environmental factors limiting the growth and productivity of alfalfa, the most economically important forage legume worldwide. In this study, alfalfa plants transgenic for both ScNHX1 (encoding vacuolar membrane Na⁺/H⁺ antiporter from Suaeda corniculata) and ScVP (encoding vacuolar H⁺-PPase from S. corniculata) were produced by cross-pollination. Transgenic alfalfa plants coexpressing ScVP/ScNHX1 showed enhanced salt and saline-alkali tolerance to 300 or 200 mM NaCl with 100 mM NaHCO₃ treatments, compared with wild-type plants. In addition, ScVP/ScNHX1-coexpressing alfalfa plants accumulated more Na⁺ in leaves and roots than wild-type plants and showed increased tolerance to higher salt and saline-alkali stress. Using the fluorescent carboxy-SNARF probe, the intracellular pH was visualized in the transgenic and wild-type plants under salt and saline-alkali stress. The results showed that the overnight treatment caused a massive change in pH in ScVP/ScNHX1-coexpressing alfalfa plants and they showed that there was significantly higher vacuolar alkalization under salt stress compared with wild-type plants. However, saline-alkali treatment enhanced vacuolar acidification more in the wild-type plants than in transgenic plants. Taken together, our results indicate that coexpression of multiple, effective genes in transgenic plants can enhance resistance to salt and saline-alkali stress.     

Enhanced drought and salt tolerance by expression of AtGSK1 gene in poplar

A GSK3/shaggy-like kinase (AtGSK1) has been implicated in the regulation of drought and salt tolerance. We transferred AtGSK1 from Arabidopsis thaliana to a hybrid poplar (Populus alba × P. tremula var. grandulosa) to determine the effect of the transgene expression in the transgenic trees. The results from northern blot and RT-PCR analyses showed that the expression level varied among the transgenic lines. During their culture on tissue culture media, the transgenic poplars formed vigorous growing roots even in the presence of 125 mM NaCl and callus in the presence of 150 mM NaCl. When the transgenic poplars were growing in pots and provided with NaCl solution, they stayed much healthier than did nontransgenic poplars, showing higher rates of photosynthetic rates, stomatal conductance, and evaporation rates under the stress. Whereas the total level of leaf Na⁺ level increased dramatically in transgenic poplars under severe saline conditions (150 mM NaCl), that of leaf K⁺ decreased in the same plants under the same conditions. Total root Na⁺ level increased in nontransgenic poplars under severe saline conditions. In contrast, total root K⁺ level decreased in the same plants under the same conditions. The chloride content and relative electrical conductivity of the transgenic poplars after salt stress treatment were lower than those of nontransgenic poplars. The transgenic poplars were also tolerant to up to 20 % PEG remaining significantly healthy when compared with nontransgenic poplars with necrosis and chlorosis symptoms. Another dramatic feature of the transgenic poplars was wilting tolerance for prolonged drought treatment up to 2 weeks. The results provide evidence that the expression of AtGSK1 gene conferred drought and salt tolerance in the transgenic poplars.     

Co-expression of the Arabidopsis SOS genes enhances salt tolerance in transgenic tall fescue (Festuca arundinacea Schreb.)

Crop productivity is greatly affected by soil salinity; therefore, improvement in salinity tolerance of crops is a major goal in salt-tolerant breeding. The Salt Overly Sensitive (SOS) signal-transduction pathway plays a key role in ion homeostasis and salt tolerance in plants. Here, we report that overexpression of Arabidopsis thaliana SOS1+SOS2+SOS3 genes enhanced salt tolerance in tall fescue. The transgenic plants displayed superior growth and accumulated less Na⁺ and more K⁺ in roots after 350 mM NaCl treatment. Moreover, Na⁺ enflux, K⁺ influx, and Ca²⁺ influx were higher in the transgenic plants than in the wild-type plants. The activities of the enzyme superoxide dismutase, peroxidase, catalase, and proline content in the transgenic plants were significantly increased; however, the malondialdehyde content decreased in transgenic plants compared to the controls. These results suggested that co-expression of A. thaliana SOS1+SOS2+SOS3 genes enhanced the salt tolerance in transgenic tall fescue.     

The over-expression of Chrysanthemum crassum CcSOS1 improves the salinity tolerance of chrysanthemum

Soil salinity represents a major constraint on plant growth. Here, we report that the over-expression of the Chrysanthemum crassum plasma membrane Na⁺/H⁺ antiporter gene CcSOS1, driven by the CaMV 35S promoter, improved the salinity tolerance of chrysanthemum ‘Jinba’. In salinity-stressed transgenic plants, both the proportion of the leaf area suffering damage and the electrical conductivity of the leaf were lower in the transgenic lines than in salinity-stressed wild type plants. After a 6 day exposure to 200 mM NaCl, the leaf content of both chlorophyll (a+b) and proline was higher in the transgenic than in the wild type plants. The activity of both superoxide dismutase and peroxidase was higher in the transgenic than in the wild type plants throughout the period of NaCl stress. The transgenic plants had a stronger control over the ingress of Na⁺ into the plant, particularly with respect to the youngest leaves, and so maintained a more favorable K⁺/Na⁺ ratio. The result suggests that a possible strategy for improving the salinity tolerance of chrysanthemum could target the restriction of Na⁺ accumulation. This study is the first to report the transgenic expression of a Na⁺ efflux carrier in chrysanthemum.     

Improved yield,fruit quality,and salt resistance in tomato co-overexpressing LeNHX2 and SlSOS2 genes

The K⁺, Na⁺/H⁺ antiporter LeNHX2 and the regulatory kinase SlSOS2 are important determinants of salt tolerance in tomato plants and their fruit production ability. In this work, we have analyzed the effects of LeNHX2 and SlSOS2 co-overexpression on fruit production, quality in tomato plants (Solanum lycopersicum L. cv. MicroTom), and analyzed physiological parameters related to salt tolerance. Plants overexpressing LeNHX2, SlSOS2 or both were grown in greenhouse. They were treated with 125 mM NaCl or left untreated and their salt tolerance was analyzed in terms of plant biomass and fruit yield. Under NaCl cultivation conditions, transgenic tomato plants overexpressing either SlSOS2 or LeNHX2 or both grew better and showed a higher biomass compared to their wild-type plants. Proline, glucose and protein content in leaves as well as pH and total soluble solid (TSS) in fruits were analyzed. Our results indicate that salinity tolerance of transgenic lines is associated with an increased proline, glucose and protein content in leaves of plants grown either with or without NaCl. Salt treatment significantly reduced yield, pH and TSS in fruits of WT plants but increased yield, pH and TSS in fruits of transgenic plants, especially those overexpressing both LeNHX2 and SlSOS2. All these results indicate that the co-overexpression of LeNHX2 and SlSOS2 improve yield and fruit quality of tomato grown under saline conditions.     

<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.     

Induction of salt tolerance in Azolla microphylla Kaulf through modulation of antioxidant enzymes and ion transport

Azolla microphylla plants exposed directly to NaCl (13 dsm^-1) did not survive the salinity treatment beyond a period of one day, whereas plants exposed directly to 4 and 9 dsm^-1 NaCl were able to grow and produce biomass. However, plants pre-exposed to NaCl (2 dsm^-1) for 7 days on subsequent exposure to 13 dsm^-1 NaCl were able to grow and produce biomass although at a slow rate and are hereinafter designated as pre-exposed plants. The pre-exposed and directly exposed plants distinctly differed in their response to salt in terms of lipid peroxidation, proline accumulation, activity of antioxidant enzymes, such as SOD, APX, and CAT, and Na⁺/K⁺ ratio. Efficient modulation of antioxidant enzymes coupled with regulation of ion transport play an important role in the induction of salt tolerance. Results show that it is possible to induce salt adaptation in A. microphylla by pre-exposing them to low concentrations of NaCl.     

Relationship between Sodium Influx and Salt Tolerance of Nitrogen-Fixing Cyanobacteria

The relationship between sodium uptake and cyanobacterial salt (NaCl) tolerance has been examined in two filamentous, heterocystous, nitrogen-fixing species of Anabaena. During diazotrophic growth at neutral pH of the growth medium, Anabaena sp. strain L-31, a freshwater strain, showed threefold higher uptake of Na⁺ than Anabaena torulosa, a brackish-water strain, and was considerably less salt tolerant (50% lethal dose of NaCl, 55 mM) than the latter (50% lethal dose of NaCl, 170 mM). Alkaline pH or excess K⁺ (>25 mM) in the medium causes membrane depolarization and inhibits Na⁺ influx in both cyanobacteria (S. K. Apte and J. Thomas, Eur. J. Biochem. 154:395-401, 1986). The presence of nitrate or ammonium in the medium caused inhibition of Na⁺ influx accompanied by membrane depolarization. These experimental manipulations affecting Na⁺ uptake demonstrated a good negative correlation between Na⁺ influx and salt tolerance. All treatments which inhibited Na⁺ influx (such as alkaline pH, K⁺ above 25 mM, NO₃⁻, and NH₄⁺), enhanced salt tolerance of not only the brackish-water but also the freshwater cyanobacterium. The results indicate that curtailment of Na⁺ influx, whether inherent or effected by certain environmental factors (e.g., combined nitrogen, alkaline pH), is a major mechanism of salt tolerance in cyanobacteria.     

Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake

In this study, metagenomic DNA was screened for the Na⁺/H⁺ antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na⁺/H⁺ antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na⁺/H⁺ antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6 %) with a putative NhaD-type Na⁺/H⁺ antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na⁺/H⁺ antiporters from Halomonas elongata (20.8 %), Alkalimonas amylolytica (19.0 %), Vibrio parahaemolyticus (18.9 %) and Vibrio cholerae (18.7 %). pH-dependent Na⁺(Li⁺)/H⁺ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na⁺(Li⁺)/H⁺ antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na⁺/H⁺ antiporters. Also, the apparent K _m values of Hb_NhaD for Na⁺ and Li⁺ at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na⁺/H⁺ antiporter.     

GmHKT1;4, a novel soybean gene regulating Na+/K+ ratio in roots enhances salt tolerance in transgenic plants

Salt is an important factor affecting the growth and development of soybean in saline or alkaline soil. The aims of the present study were to identify and functionally analyse the soybean GmHKTs gene family, and to explore their roles under NaHCO₃ and NaCl stresses. The GmHKTs gene family were isolated from soybean using genome sequence information. The GmHKTs gene family were further analysed for the structure and phylogenetic relationship. The expression patterns of soybean GmHKTs genes under NaHCO₃ and NaCl stresses were analysed via quantitative real-time PCR. As a result, the expression level of GmHKT1;4 was extremely up regulated in root under each treatment. Overexpression of GmHKT1;4 significantly enhanced the tolerance of transgenic tobacco plants to NaHCO₃ and NaCl stresses, compared with null plants. The overexpressed transgenic plants of this gene accomulated more K⁺ and less Na⁺ under salt stress, compaired with null plants. Our findings suggest that GmHKT1;4 plays an important role for regulation Na⁺/K⁺ ratio in roots under alkaline (NaHCO₃) and saline (NaCl) stresses.     

Overexpression of NHX1s in transgenic Arabidopsis enhances photoprotection capacity in high salinity and drought conditions

In this study, we observed that transgenic plants overexpressing NHX1 from different organisms grew well in the presence of 200 mM NaCl and also under water deprivation, while the wild-type plants exhibited chlorosis and growth inhibition. The photosynthesis activity of five kinds of transgenic plants was higher than the wild-type plants. The leaf water potential was less negative for wild-type than for transgenic plants. Moreover, these transgenic plants accumulated more Na⁺ and K⁺ in their leaf tissue than the wild-type plants. The toxic effects of Na⁺ accumulation in the cytosol are reduced by its sequestration into the vacuole. In addition, the thermal dissipation and ROS scavenging components increased in all transgenic Arabidopsis plants compared with that in non-transgenics. The salt tolerance of transgenic plants was passed on to the offsprings to the T₅ generation. Furthermore, it should be noted that in transgenic Arabidopsis plants, overexpression of NHX1s from dicots showed higher salt and drought tolerance than that from wheat.     

Overexpression of AtNHX5 improves tolerance to both salt and water stress in rice (Oryza sativa L.)

Overexpression of NHX genes has been previously shown to improve salt tolerance of transgenic plants. In this study, transgenic rice plants overexpressing AtNHX5 showed not only high salt tolerance, but also high drought tolerance. Measurements of ion levels indicated that Na⁺ and K⁺ contents were all higher in AtNHX5 overexpressing shoots than in wild type (WT) shoots in high saline conditions. After exposure to water deficiency and salt stress, the WT plants all died, while the AtNHX5 overexpressing rice plants had a higher survival rate, dry weight, leaf water content, and leaf chlorophyll contents, accumulated more proline, and had less membrane damage than the WT plants. In addition, seeds of both transgenic and WT plants germinated on 1/2 MS medium supplemented with 250 mM mannitol, but overexpression of AtNHX5 improved the shoot growth of the seedlings. Taken together, the results indicate that AtNHX5 gene could enhance the tolerance of rice plants to multiple environmental stresses by promoting the accumulation of more effective osmolytes (ions or proline) to counter the osmotic stress caused by abiotic factors.     

Ectopic expression of PgRab7 in rice plants (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) results in differential tolerance at the vegetative and seed setting stage during salinity and drought stress

In this work, we have overexpressed a vesicle trafficking protein, Rab7, from a stress-tolerant plant, Pennisetum glaucum, in a high-yielding but stress-sensitive rice variety Pusa Basmati-1 (PB-1). The transgenic rice plants were tested for tolerance against salinity and drought stress. The transgenic plants showed considerable tolerance at the vegetative stage against both salinity (200 mM NaCl) and drought stress (up to 12 days after withdrawing water). The protection against salt and drought stress may be by regulating Na⁺ ion homeostasis, as the transgenic plants showed altered expression of multiple transporter genes, including OsNHX1, OsNHX2, OsSOS1, OsVHA, and OsGLRs. In addition, decreased generation and maintenance of lesser reactive oxygen species (ROS), with maintenance of chloroplast grana and photosynthetic machinery was observed. When evaluated for reproductive growth, 89–96 % of seed setting was maintained in transgenic plants during drought stress; however, under salt stress, a 33–53 % decrease in seed setting was observed. These results indicate that PgRab7 overexpression in rice confers differential tolerance at the seed setting stage during salinity and drought stress and could be a favored target for raising drought-tolerant crops.     

Overexpression of a wheat Na+/H+ antiporter gene (TaNHX2) enhances tolerance to salt stress in transgenic tomato plants (Solanum lycopersicum L.)

Soil salinity is a major environmental stress limiting plant productivity. Vacuole Na⁺/H⁺ antiporters play important roles for the survival of plants under salt stress conditions. We have developed salt stress tolerant transgenic tomato plants (Solanum lycopersicum cv. PED) by overexpression of the wheat Na⁺/H⁺ antiporter gene TaNHX2 using Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBin438 that contains the TaNHX2 gene under the control of double CaMV 35S promoter and npt II as a selectable marker. PCR and Southern blot analysis confirmed that TaNHX2 gene has been integrated and expressed in the T₁ generation transgenic tomato plants. When TaNHX2 expressing plants were exposed to 100 or 150 mM NaCl, they were found to be more tolerant to salt stress compared to wild type plants. Biochemical analyses also showed that transgenic plants have substantial amount of relative water content and chlorophyll content under salt stress conditions compared to wild type plants. The relative water content in transgenic and wild type plants ranged from 68 to 75 % and 46–73 % and chlorophyll content fall in between 1.8 to 2.4 mg/g fw and 1.0 to 2.4 mg/g fw, respectively, in all stress conditions. In the present study, we observed a better germination rate of T₁ transgenic seeds under salt stress conditions compared with wild type plants. Our results indicated that TaNHX2-transgenic tomato plants coped better with salt stress than wild type plants.     

NaCl increases the activity of the plasma membrane H+-ATPase in C3 halophyte Suaeda salsa callus

Suaeda salsa calli treated with different concentrations of NaCl were used to examine the response of the plasma membrane (PM) H⁺-ATPase to NaCl and its role in salt tolerance. The optimum concentration of NaCl for growth of the calli was 50 mM, while growth was significantly inhibited at 250 mM NaCl. The ion and organic solute contents of calli increased with increasing NaCl. Activity of the PM H⁺-ATPase increased when the calli were treated with NaCl over a certain concentration range (0–150 mM NaCl). However, the activity reached its maximum with 150 mM NaCl. Immunoblotting analysis of the PM H⁺-ATPase protein from calli cultures with anti-Zea mays H⁺-ATPase serum (monoclonal 46E5B11D5) identified a single polypeptide of ~90 kDa. The peptide levels increased in the calli treated with NaCl at 150 mM NaCl compared to control, but the increase at 50 mM NaCl was less pronounced. Northern blot analysis showed that the expression of the PM H⁺-ATPase also increased after the calli were treated with NaCl. These results suggest that the increase in PM H⁺-ATPase activity is due to both an increase in the amount of PM H⁺-ATPase protein and an up-regulation of the PM H⁺-ATPase gene, which is involved in the salt tolerance of S. salsa calli.     

SOS1 gene overexpression increased salt tolerance in transgenic tobacco by maintaining a higher K(+)/Na(+) ratio

Crop productivity is greatly affected by soil salinity, so improvement in salinity tolerance of crops is a major objective of many studies. We overexpressed the Arabidopsis thaliana SOS1 gene, which encodes a plasma membrane Na⁺/H⁺ antiporter, in tobacco (Nicotiana tabacum cv. Xanthi-nc). Compared with nontransgenic plants, seeds from transgenic tobacco had better germination under 120 mM (mmol L⁻¹) NaCl stress; chlorophyll loss in the transgenic seedlings treated with 360 mM NaCl was less; transgenic tobacco showed superior growth after irrigation with NaCl solutions; and transgenic seedlings with 150 mM NaCl stress accumulated less Na⁺ and more K⁺. In addition, roots of SOS1-overexpressing seedlings lost less K⁺ instantaneously in response to 50 mM NaCl than control plants. These results showed that the A. thaliana SOS1 gene potentially can improve the salt tolerance of other plant species.     

Cloning and characterization of Na+/H+ antiporter (LfNHX1) gene from a halophyte grass Leptochloa fusca for drought and salt tolerance

Abiotic stresses such as salinity and drought have adverse effects on plants. In the present study, a Na⁺/H⁺ antiporter gene homologue (LfNHX1) has been cloned from a local halophyte grass (Leptochloa fusca). The LfNHX1 cDNA contains an open reading frame of 1,623 bp that encodes a polypeptide chain of 540 amino acid residues. LfNHX1 protein sequence showed high similarity with NHX1 homologs reported from other halophyte plants. Amino acid and nucleotide sequence similarity, protein topology modeling and the presence of conserved functional domains in the LfNHX1 protein sequence classified it as a vacuolar NHX1 homolog. The overexpression of LfNHX1 gene under CaMV35S promoter conferred salt and drought tolerance in tobacco plants. Under drought stress, transgenic plants showed higher relative water contents, photosynthetic rate, stomatal conductance and membrane stability index as compared to wild type plants. More negative value of leaf osmotic potential was also observed in transgenic plants when compared with wild type control plants. Transgenic plants showed better germination and root growth at 2 mg L^?1 Basta herbicide and three levels (100, 200 and 250 mM) of sodium chloride. These results showed that LfNHX1 is a potential candidate gene for enhancing drought and salt tolerance in crops.     

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.     

Effects of salt stress and exogenous Ca2+ on Na+ compartmentalization,ion pump activities of tonoplast and plasma membrane in Nitraria tangutorum Bobr. leaves

Nitraria tangutorum Bobr. is a typical halophyte with superior tolerance to salinity. However, little is known about its physiological adaptation mechanisms to the salt environment. In the present study, N. tangutorum seedlings were treated with different concentrations of NaCl (100, 200, 300 and 400 mmol L^?1) combined with five levels of Ca²⁺ (0, 5, 10, 15 and 20 mmol L^?1) to investigate the effects of salt stress and exogenous Ca²⁺ on Na⁺ compartmentalization and ion pump activities of tonoplast and plasma membrane (PM) in leaves. Na⁺ and Ca²⁺ treatments increased the fresh weight and dry weight of N. tangutorum seedlings. The absorption of Na⁺ in roots, stems and leaves was substantially increased with the increases of NaCl concentration, and Na⁺ was mainly accumulated in leaves. Exogenous Ca²⁺ reduced Na⁺ accumulation in roots but promoted Na⁺ accumulation in leaves. The absorption and transportation of Ca²⁺ in N. tangutorum seedlings were inhibited under NaCl treatments. Exogenous Ca²⁺ promoted Ca²⁺ accumulation in the plant. Na⁺ contents in apoplast and symplast of leaves were also significantly increased, and symplast was the main part of Na⁺ intracellular compartmentalization. The tonoplast H⁺-ATPase and H⁺-PPase activities were significantly promoted under salt stress (NaCl concentrations ≤300 mmol L^?1). PM H⁺-ATPase activities gradually increased under salt stress (NaCl concentrations ≤200 mmol L^?1) followed by decreases with NaCl concentration increasing. The tonoplast H⁺-ATPase, H⁺-PPase and PM H⁺-ATPase activities increased first with the increasing exogenous Ca²⁺ concentration, reached the maximums at 15 mmol L^?1 Ca²⁺, and then decreased. The tonoplast and PM Ca²⁺-ATPase activities showed increasing trends with the increases of NaCl and Ca²⁺ concentration. These results suggested that certain concentrations of exogenous Ca²⁺ effectively enhanced ion pump activities of tonoplast and PM as well as promoted the intracellular Na⁺ compartmentalization to improve the salt tolerance of N. tangutorum.