Hydrogen sulfide is involved in maintaining ion homeostasis via regulating plasma membrane Na+/H+ antiporter system in the hydrogen peroxide-dependent manner in salt-stress Arabidopsis thaliana root

Hydrogen sulfide (H₂S) and hydrogen peroxide (H₂O₂) function as the signaling molecules in plants responding to salt stresses. The present study presents a signaling network involving H₂S and H₂O₂ in salt resistance pathway of the Arabidopsis root. Arabidopsis roots were sensitive to 100 mM NaCl treatment, which displayed a great increase in electrolyte leakage (EL) and Na⁺/K⁺ ratio under salt stress. The treatment of H₂S donors sodium hydrosulfide (NaHS) enhanced the salt tolerance by maintaining a lower Na⁺/K⁺ ratio. In addition, the inhibition of root growth under salt stress was removed by H₂S. Further studies indicated that H₂O₂ was involved in H₂S-induced salt tolerance pathway. H₂S induced the production of the endogenous H₂O₂ via regulating the activities of glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) NADPH oxidase, with the treatment with dimethylthiourea (DMTU, an ROS scavenger), diphenylene iodonium (DPI, a PM NADPH oxidase inhibitor), or glycerol (G6PDH inhibitor) removing the effect of H₂S. Treatment with amiloride (an inhibitor of PM Na⁺/H⁺ antiporter) and vanadate (an inhibitor of PM H⁺-ATPase) also inhibited the activity of H₂S on Na⁺/K⁺ ratio. Through an analysis of quantitative real-time polymerase chain reaction and Western blot, we found that H₂S promoted the genes expression and the phosphorylation level of PM H⁺-ATPase and Na⁺/H⁺ antiporter protein level. However, when the endogenous H₂O₂ level was inhibited by DPI or DMTU, the effect of H₂S on the PM Na⁺/H⁺ antiporter system was removed. Taken together, H₂S maintains ion homeostasis in the H₂O₂-dependent manner in salt-stress Arabidopsis root.     

H2O2 and cytosolic Ca2+ signals triggered by the PM H+‐coupled transport system mediate K+/Na+ homeostasis in NaCl‐stressed Populus euphratica cells

Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H₂O₂, cytosolic Ca²⁺ ([Ca²⁺]_cyt) and the PM H⁺‐coupled transport system in K⁺/Na⁺ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na⁺/H⁺ antiport was seen in salinized cells; however, NaCl stress caused a net K⁺ efflux, because of the salt‐induced membrane depolarization. H₂O₂ levels, regulated upwards by salinity, contributed to ionic homeostasis, because H₂O₂ restrictions by DPI or DMTU caused enhanced K⁺ efflux and decreased Na⁺/H⁺ antiport activity. NaCl induced a net Ca²⁺ influx and a subsequent rise of [Ca²⁺]_cyt, which is involved in H₂O₂‐mediated K⁺/Na⁺ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na⁺/H⁺ antiport system, the NaCl‐induced elevation of H₂O₂ and [Ca²⁺]_cyt was correspondingly restricted, leading to a greater K⁺ efflux and a more pronounced reduction in Na⁺/H⁺ antiport activity. Results suggest that the PM H⁺‐coupled transport system mediates H⁺ translocation and triggers the stress signalling of H₂O₂ and Ca²⁺, which results in a K⁺/Na⁺ homeostasis via mediations of K⁺ channels and the Na⁺/H⁺ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.     

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.     

Expression of a Suaeda salsa Vacuolar H+/Ca2+ Transporter Gene in Arabidopsis Contributes to Physiological Changes in Salinity

A cDNA (SsCAX1) encoding a tonoplast-localised Ca²⁺/H⁺ exchanger was isolated from a C₃ halophyte Suaeda salsa (L.). To clarify the role of SsCAX1 in plant salt tolerance, Arabidopsis plants expressing SsCAX1 were treated with NaCl. Transgenic Arabidopsis plants displayed decreased salt tolerance. Although Na⁺ content was close to wild-type plants, transgenic plants accumulated more Ca²⁺ and retained less K⁺ in leaves than the wild-type plants in salinity. Furthermore, transgenic lines held higher leaf membrane leakage than wild-type lines under NaCl treatment. In addition, transgenic plants showed a 23% increase in vacuolar H⁺-ATPase activity compared with wild-type plants in normal condition. But the leaf V-H⁺-ATPase activity had subtle changes in transgenic plants, while significantly increased in wild-type plants under saline condition. These results suggested that regulated expression of Ca²⁺/H⁺ antiport was critical for maintenance of cation homeostasis and activity of V-H⁺-ATPase under saline condition.     

Congruence between PM H<Superscript>+</Superscript>-ATPase and NADPH oxidase during root growth: a necessary probability

Plasma membrane (PM) H⁺-ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O₂˙^?, respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H⁺-ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H⁺-ATPase inhibitor). Conversely, H⁺-ATPase activity retarded in response to different ROS scavengers [CuCl₂, N, N’ –dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl₂ and diphenyleneiodonium (DPI)], while H₂O₂ promoted PM H⁺-ATPase activity at lower concentrations. Repressing effects of Ca⁺² antagonists (La⁺³ and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H⁺-ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H⁺-ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.     

Transduction mechanism(s) of Na-saccharin in the blowfly <Emphasis Type="Italic">Protophormia terraenovae</Emphasis>: evidence for potassium and calcium conductance involvement

The study on transduction mechanisms underlying bitter stimuli is a particularly intriguing challenge for taste researchers. The present study investigates, in the labellar chemosensilla of the blowfly Protophormia terraenovae, the transduction mechanism by which saccharin evokes the response of the “deterrent” cell, with particular attention to the contribution of K⁺ and Ca²⁺ current and the role of cyclic nucleotides, since second messengers modulate Ca²⁺, Cl⁻ and K⁺ currents to different extents. As assessed by extracellular single-sensillum recordings, our results show that the addition of a Ca²⁺ chelator such as EGTA or the Ca²⁺ current blockers SK&F-96365, Mibefradil, Nifedipine and W-7 decrease the response of the “deterrent” cell to saccharin. A similar decreasing effect was also obtained following the addition of 4-aminopyridine, a K⁺ current blocker. On the contrary, the membrane-permeable cyclic nucleotide 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP) activates this cell and shows an additive effect when presented mixed with saccharin. Our results are consistent with the hypothesis that in the labellar chemosensilla of the blowfly both Ca²⁺ and K⁺ ions are involved in the transduction mechanism of the “deterrent” cell in response to saccharin. Our results also suggest a possible pathway common to saccharin and 8Br-cGMP.     

The <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic"> cax3</Emphasis> mutants display altered salt tolerance,pH sensitivity and reduced plasma membrane H<Superscript>+</Superscript>-ATPase activity

Perturbing CAX1, an Arabidopsis vacuolar H⁺/Ca²⁺ antiporter, and the related vacuolar transporter CAX3, has been previously shown to cause severe growth defects; however, the specific function of CAX3 has remained elusive. Here, we describe plant phenotypes that are shared among cax1 and cax3 including an increased sensitivity to both abscisic acid (ABA) and sugar during germination, and an increased tolerance to ethylene during early seedling development. We have also identified phenotypes unique to cax3, namely salt, lithium and low pH sensitivity. We used biochemical measurements to ascribe these cax3 sensitivities to a reduction in vacuolar H⁺/Ca²⁺ transport during salt stress and decreased plasma membrane H⁺-ATPase activity. These findings catalog an array of CAX phenotypes and assign a specific role for CAX3 in response to salt tolerance.     

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.     

Role of Nitric Oxide and Hydrogen Peroxide During the Salt Resistance Response

Ion homeostasis is essential for plant cell resistance to salt stress. Under salt stress, to avoid cellular damage and nutrient deficiency, plant cells need to maintain adequate K nutrition and a favorable K to Na ratio in the cytosol. Recent observations revealed that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) act as signaling molecules to regulate K to Na ratio in calluses from Populus euphratica under salt stress. Evidence indicated that NO mediating H₂O₂ causes salt resistance via the action of plasma membrane H⁺-ATPase but that activity of plasma membrane NADPH oxidase is dependent on NO. Our study demonstrated the signaling transduction pathway. In this addendum, we proposed a testable hypothesis for NO function in regulation of H₂O₂ mediating salt resistance.Key Words: hydrogen peroxide, nitric oxide, signaling molecule, salt resistanceUnder salinity conditions, tolerant plant cells achieve ion homeostasis by extruding Na to the external medium and/or compartmentalizing into vacuoles, maintaining K uptake and high K and low Na in the cytosol.^1,2 Control of Na movement across the plasma membrane (PM) and tonoplast in order to maintain a low Na concentration in the cytoplasm is a key factor of cellular adaptation to salt stress.^3,4 Na transport across the PM is dependent on the electrochemical gradient created by the PM H⁺-ATPase.^5,6 It has been proven that the activity of the PM H⁺-ATPase is a key index of plant adaptation to salt stress.⁷ Therefore, the regulation of expression of the PM H⁺-ATPase may represent an important cellular mechanism for salt resistance. In contrast to our understanding of the regulation of PM H⁺-ATPase by other factors, the roles of NO and H₂O₂ act as signals under salt stress have been less known.Previous studies have shown that both NO and H₂O₂ function as stress signals in plants, mediating a range of resistance mechanisms in plants under stress conditions.^8–10 We have previously shown that NO serves as a signal in inducing salt resistance by increasing the K to Na ratio, which is dependent on the increased PM H⁺-ATPase activity in calluses from reed.¹¹ Although NO acts as a signal molecule under salt stress and induces salt resistance by increasing PM H⁺-ATPase activity, our research results also indicated NO can not activate purified PM H⁺-ATPase activity, at least in vitro. Subsequently, we set out to find the other signal molecules and factors between NO and PM H⁺-ATPase activity. Since our studies have indicated that NO can not induce salt resistance directly, what roles dose it play in salt resistance in tolerant cells under salt stress? We initially hypothesized ABA or H₂O₂ might be downstream signal molecules to regulate the activity of PM H⁺-ATPase. Further results indicated H₂O₂ content increased greatly under salt stress. Since H₂O₂ might be the candidate downstream signal molecule, we tested PM H⁺-ATPase activity and K to Na ratio in calluses by adding H₂O₂. The results suggested that H₂O₂ inducing an increased PM H⁺-ATPase activity resulted in an increased K to Na ratio. Summing up this new assay that allows us to speculate NO maybe regulate the H₂O₂ generation.Since H₂O₂ is involved in downstream signal molecule of NO, PM NADPH oxidase, the main source of H₂O₂ production, might be the regulated target of NO. We took a pharmacological approach to examine the speculation. The results indicated that PM NADPH oxidase is required for H₂O₂ accumulation and PM NADPH oxidase activity could attribute to NO in calluses under salt stress. These results also raised another question regarding what concentrations of NO can induce such effects. In our experiments, NO content was induced 1.6 times higher than the control values under salt treatment. We speculated there exists an effective balance point in NO signal system similar to previous reports by Delledonne et al.¹² in disease resistance.Further research work is required to decipher the mechanism through which NO and H₂O₂ acts and how K and Na elements uptake might be connected with salt resistance. We would like to propose a simple testable model that accounts for the results reported in this paper (Fig. 1). According to our model, H₂O₂ rather than NO is the major signaling molecular that mediated directly PM H⁺-ATPase under salt stress. Normally, NO generated from nitric oxide synthase (NOS) acts as a signal molecule to regulate other mechanisms. Under salt stress, accumulated NO activates PM NADPH oxidase activity. Then, a number of H₂O₂ is produced from PM NADPH oxidase. The PM H⁺-ATPase is activated greatly by the accumulated H₂O₂. Eventually, the transmembrane electrochemical gradient is created and K to Na ratio increases. The model we have proposed here is testable and should provide further insights into salt resistance mechanism regulated by NO and H₂O₂ signal molecules.Open in a separate windowFigure 1Hypothetical model for the potential function of NO and H₂O₂ as signaling molecules in inducing salt resistance. Salt stress activates a signal transduction cascade that leads to the increased activity of PM H⁺-ATPase, whose expression produces salt resistance. NO is generated by NOS, and H₂O₂ is produced by NADPH oxidase attributed to NO. The activity of PM H⁺-ATPase is regulated by H₂O₂ directly under salt stress. The model is based on the recent results in calluses from P. euphratica¹² and those previously reported on the NO function in reed.¹¹Research on roles of NO and H₂O₂ under stress conditions in plant is advancing rapidly. Further analysis of salt resistance mechanism with novel technology will certainly increase our knowledge in this field.     

NMR measurements of Ca2+ and H+ transport mediated by A23187 and reconstituted plasma membrane Ca2+-ATPase

NMR-based assays for measuring the fluxes of Ca²⁺, H⁺, and ATP in liposomal systems are presented. The ¹⁹F NMR Ca²⁺-chelating molecule 5,5-difluoro-1,2-bis(o-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) was trapped inside large unilamellar vesicles and used to monitor passive and A23187-mediated Ca²⁺ transport into them. The data were analyzed using progress curves of the transport reaction. They demonstrated the general applicability of 5FBAPTA as a ¹⁹F NMR probe of active Ca²⁺ transport. ³¹P NMR time-courses were used to monitor simultaneously the ATP hydrolysing activity of the reconstituted human erythrocyte Ca²⁺-ATPase and the concomitant acidification of the reaction medium in a suspension of small unilamellar vesicles. Using an estimate of the extraliposomal buffering capacity, the H⁺/ATP coupling stoichiometry, in the presence of A23187, was estimated from the NMR-derived data at steady state; it amounted to 1.4±0.3. This result is discussed with respect to the issue of molecular `slip' in the context of a non-equilibrium thermodynamics model of the pump (accompanying paper in this issue). Importantly, NMR, in contrast to optical detection methods, can potentially register all fluxes and (electro)chemical gradients involved in the Ca²⁺-ATPase-mediated H⁺/Ca²⁺counterport, in a single experiment. Received: 19 June 1997 / Accepted: 3 December 1997     

Cyclic mononucleotides modulate potassium and calcium flux responses to H2O2 in Arabidopsis roots

Cyclic mononucleotides are messengers in plant stress responses. Here we show that hydrogen peroxide (H₂O₂) induces rapid net K⁺-efflux and Ca²⁺-influx in Arabidopsis roots. Pre-treatment with either 10 μM cAMP or cGMP for 1 or 24 h does significantly reduce net K⁺-leakage and Ca²⁺-influx, and in the case of the K⁺-fluxes, the cell permeant cyclic mononucleotides are more effective. We also examined the effect of 10 μM of the cell permeant 8-Br-cGMP on the Arabidopsis microsomal proteome and noted a specific increase in proteins with a role in stress responses and ion transport, suggesting that cGMP is sufficient to directly and/or indirectly induce complex adaptive changes to cellular stresses induced by H₂O₂.     

The interaction between H<Subscript>2</Subscript>O<Subscript>2</Subscript> and NO,Ca<Superscript>2+</Superscript>, cGMP,and MAPKs during adventitious rooting in mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In the present study, we present a signaling network involving H₂O₂, nitric oxide (NO), calcium (Ca²⁺), cyclic guanosine monophosphate (cGMP), and the mitogen-activated protein kinase (MAPK) cascade during adventitious rooting in mung bean seedlings. Both exogenous H₂O₂ and the NO donor sodium nitroprussiate were capable of promoting the formation and development of adventitious roots. H₂O₂ and NO signaling pathways were elicited in parallel in auxin-induced adventitious rooting. Cytosolic Ca²⁺ was required for adventitious rooting, and Ca²⁺ served as a downstream component of H₂O₂, as well as cGMP or MAPK, signaling cascades. cGMP and MAPK cascades function downstream of H₂O₂ signaling and depend on auxin responses in adventitious root signaling processes.     

The plasma-membrane H+-ATPase from beet root is inhibited by a calcium-dependent phosphorylation

Several plasma-membrane proteins from beet root (Beta vulgaris L.) have been functionally incorporated into reconstituted proteoliposomes. These showed H⁺-ATPase activity, measured both as ATP hydrolysis and H⁺ transport. The proton-transport specific activity was 10 times higher than in plasma membranes, and was greatly stimulated by potassium and valinomycin. These proteoliposomes also showed calcium-regulated protein kinase activity. This kinase activity is probably due to a calmodulin-like domain protein kinase (CDPK), since two protein bands were recognized by antibodies against soybean and Arabidopsis CDPK. This kinase phosphorylated histone and syntide-2 in a Ca²⁺-dependent manner. Among the plasma-membrane proteins phosphorylated by this kinase, was the H⁺-ATPase. When the H⁺-ATPase was either prephosphorylated or assayed in the presence of Ca²⁺, both the ATP-hydrolysis and the proton-transport activities were slower. This inhibition was reversed by an alkaline-phosphatase treatment. A trypsin treatment (that has been reported to remove the C-terminal autoinhibitory domain from the H⁺-ATPase) also reversed the inhibition caused by phosphorylation. These results indicate that a Ca²⁺-dependent phosphorylation, probably caused by a CDPK, inhibits the H⁺-ATPase activities. The substrate of this regulatory phosphorylation could be the H⁺-ATPase itself, or a different protein influencing the ATPase activities. Received: 1 May 1997 / Accepted: 25 June 1997     

Putative involvement of cytosolic Ca2+ and GTP-binding proteins in cyclic-GMP-mediated induction of stomatal opening by auxin in Commelina communis L.

To investigate whether cyclic GMP (cGMP) would mediate, in an intracellular Ca²⁺ -dependent manner, coupling of auxin to stomatal opening, the stomatal opening responses to the auxin indolyl-3-butyric acid (IBA) and to the cGMP membrane-permeable derivative 8-bromoguanosine 3^′,5^′-cyclic monophosphate (8-Br-cGMP) were compared in epidermal strips of Commelina communis. In this comparison were studied possible effects of intracellular Ca²⁺ modulators, GTP-binding protein (G-protein) modulators and selective inhibitors of enzymatic reactions which use or generate cGMP. The stomatal response to IBA was almost similarly reversed by the Ca²⁺ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N^′,N^′-tetraacetic acid (BAPTA), the intracellular Ca²⁺-release inhibitors ruthenium red and procaine, the inactive cGMP analog Rp-8-bromoguanosine 3^′,5^′-cyclic monophosphorothioate (Rp-8-Br-cGMPS), the inhibitor of cGMP-producing guanylyl cyclase LY 83583, the G-protein inhibitor mas17 and the G-protein antagonist pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH₂. Comparison with stomatal opening in response to 8-Br-cGMP, which was almost completely suppressed by either BAPTA, ruthenium red, procaine or Rp-8-Br-cGMPS, strongly suggests that cGMP acts downstream of G-protein activation as a second messenger for IBA signal transduction and that the cGMP pathway likely depends on cytosolic Ca²⁺signaling. Received: 8 November 1997 / Accepted: 6 March 1998     

Increased cucumber salt tolerance by grafting on pumpkin rootstock and after application of calcium

Self-grafted and pumpkin rootstock-grafted cucumber plants were subjected to the following four treatments: 1) aerated nutrient solution alone (control), 2) nutrient solution with 10 mM Ca(NO₃)₂ (Ca), 3) nutrient solution with 90 mM NaCl (NaCl), and 4) nutrient solution with 90 mM NaCl + 10 mM Ca(NO₃)₂ (NaCl+Ca). The NaCl treatment decreased the plant dry mass and content of Ca²⁺ and K⁺ but increased the Na⁺ content in roots and shoots. Smaller changes were observed in pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ ameliorated the negative effects of NaCl on plant dry mass, relative growth rate (RGR), as well as Ca²⁺, K⁺, and Na⁺ content especially for pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ distinctly stimulated the plasma membrane (PM) H⁺-ATPase activity which supplies the energy to remove excess Na⁺ from the cells. The expressions of gene encoding PM H⁺-ATPases (PMA) and gene encoding a PM Na⁺/H⁺ antiporter (SOS1) were up-regulated when Ca(NO₃)₂ was applied. The pumpkin rootstock-grafted plants had higher PM H⁺-ATPase activity as well as higher PMA and SOS1 expressions than the self-grafted plants under NaCl + Ca treatment. Therefore, the addition of Ca²⁺ in combination with pumpkin rootstock grafting is a powerful way to increase cucumber salt tolerance.