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.     

Properties of plasma membrane H<Superscript>+</Superscript>-ATPase in salt-treated <Emphasis Type="Italic">Populus euphratica</Emphasis> callus

The plasma membrane (PM) vesicles from Populus euphratica (P. euphratica) callus were isolated to investigate the properties of the PM H⁺-ATPase. An enrichment of sealed and oriented right-side-out PM vesicles was demonstrated by measurement of the purity and orientation of membrane vesicles in the upper phase fraction. Analysis of pH optimum, temperature effects and kinetic properties showed that the properties of the PM H⁺-ATPase from woody plant P. euphratica callus were consistent with those from herbaceous species. Application of various thiol reagents to the reaction revealed that reduced thiol groups were essential to maintain the PM H⁺-ATPase activity. In addition, there was increased H⁺-ATPase activity in the PM vesicles when callus was exposed to NaCl. Western blotting analysis demonstrated an enhancement of H⁺-ATPase content in NaCl-treated P. euphratica callus compared with the control.     

The Arabidopsis Chaperone J3 Regulates the Plasma Membrane H+-ATPase through Interaction with the PKS5 Kinase

The plasma membrane H⁺-ATPase (PM H⁺-ATPase) plays an important role in the regulation of ion and metabolite transport and is involved in physiological processes that include cell growth, intracellular pH, and stomatal regulation. PM H⁺-ATPase activity is controlled by many factors, including hormones, calcium, light, and environmental stresses like increased soil salinity. We have previously shown that the Arabidopsis thaliana Salt Overly Sensitive2-Like Protein Kinase5 (PKS5) negatively regulates the PM H⁺-ATPase. Here, we report that a chaperone, J3 (DnaJ homolog 3; heat shock protein 40-like), activates PM H⁺-ATPase activity by physically interacting with and repressing PKS5 kinase activity. Plants lacking J3 are hypersensitive to salt at high external pH and exhibit decreased PM H⁺-ATPase activity. J3 functions upstream of PKS5 as double mutants generated using j3-1 and several pks5 mutant alleles with altered kinase activity have levels of PM H⁺-ATPase activity and responses to salt at alkaline pH similar to their corresponding pks5 mutant. Taken together, our results demonstrate that regulation of PM H⁺-ATPase activity by J3 takes place via inactivation of the PKS5 kinase.     

Salinity adaptation of plasma membrane H+-ATPase in the salt marsh plant Spartina patens: ATP hydrolysis and enzyme kinetics

Spartina patens, an intertidal C4 grass, grows in the upper salt marsh and tolerates coastal seawater salinity. The regulation of ion movement across the plasma membrane (PM) for plant salt tolerance is thought to be achieved by an electrochemical gradient generated by plasma membrane H⁺-ATPase. In this study, the change of PM H⁺-ATPase in response to NaCl was characterized for S. patens callus. Callus was cultured for 10 weeks under salinity levels of 0 mM, 170 mM, 340 mM, and 510 mM NaCl. Plasma membrane was isolated from a Dextran/PEG aqueous polymer two-phase system and the purity was demonstrated with membrane enzyme markers. There was a significant increase (up to 2-3 fold) of PM H⁺-ATPase activity when callus was grown on media containing NaCl. The incremental activation of PM H⁺-ATPase activity would enable the cell to tolerate higher cytoplasmic NaCl concentrations. PM H⁺-ATPase appeared to have a higher Vmax and a lower substrate concentration (Km to reach Vmax. When growth medium salinity increased from 0 mM to 170 and 340 mM, the Vmax of H⁺-ATPase increased from 0.64 to 1.00 and 1.73, respectively, while the Km decreased from 3.58 to 2.07 and 2.44 mM, respectively. In vitro NaCl inhibition kinetic data revealed a pattern of non-competitive inhibition by NaCl on PM H⁺-ATPase. The response of PM H⁺-ATPase in S. patens callus suggests that this species has evolved mechanisms that can regulate this important enzyme when cells are exposed to NaCl.     

Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

灵武长枣果实质膜和液泡膜H~+-ATPase和H~+-PPase活性与果实糖分积累

以不同发育时期灵武长枣(Ziziphus jujuba cv.Lingwuchangzao)的果实为材料,通过测定与分析果肉组织中细胞质膜、液泡膜H+-ATPase和H+-PPase活性、果实糖分含量变化,研究了灵武长枣果实质膜、液泡膜H+-ATPase和H+-PPase活性与糖积累特性的关系。结果表明:(1)果实第二次快速生长期之前主要积累葡萄糖和果糖,之后果实迅速积累蔗糖,葡萄糖和果糖含量则逐渐下降,成熟期果实主要积累蔗糖。(2)在果实发育的缓慢生长期S1,质膜H+-ATPase活性最低;第一次快速生长期,质膜H+-ATPase活性最高;缓慢生长期S2,其活性降低;第二次快速生长期,质膜H+-ATPase活性升至次高;完熟期,质膜H+-ATPase活性下降幅度较大。(3)在果实发育过程中,液泡膜H+-ATPase和H+-PPase活性的变化趋势相似。缓慢生长期S1,液泡膜H+-ATPase和H+-PPase活性较低;从缓慢生长期S1至第一次快速生长期缓慢下降至最低;从第一次快速生长期开始,液泡膜H+-ATPase和H+-PPase活性呈现为逐渐增高的变化趋势;除第二次快速生长期以外,液泡膜H+-PPase活性始终高于H+-ATPase。由此推测,质膜H+-ATPase和液泡膜H+-ATPase、H+-PPase对灵武长枣果实糖分的跨膜次级转运起到重要的调控作用。     

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.     

cGMP regulates hydrogen peroxide accumulation in calcium-dependent salt resistance pathway in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots

3′,5′-cyclic guanosine monophosphate (cGMP) is an important second messenger in plants. In the present study, roles of cGMP in salt resistance in Arabidopsis roots were investigated. Arabidopsis roots were sensitive to 100 mM NaCl treatment, displaying a great increase in electrolyte leakage and Na⁺/K⁺ ratio and a decrease in gene expression of the plasma membrane (PM) H⁺-ATPase. However, application of exogenous 8Br-cGMP (an analog of cGMP), H₂O₂ or CaCl₂ alleviated the NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and increasing the PM H⁺-ATPase gene expression. In addition, the inhibition of root elongation and seed germination under salt stress was removed by 8Br-cGMP. Further study indicated that 8Br-cGMP-induced higher NADPH levels for PM NADPH oxidase to generate H₂O₂ by regulating glucose-6-phosphate dehydrogenase (G6PDH) activity. The effect of 8Br-cGMP and H₂O₂ on ionic homeostasis was abolished when Ca²⁺ was eliminated by glycol-bis-(2-amino ethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, a Ca²⁺ chelator) in Arabidopsis roots under salt stress. Taken together, cGMP could regulate H₂O₂ accumulation in salt stress, and Ca²⁺ was necessary in the cGMP-mediated signaling pathway. H₂O₂, as the downstream component of cGMP signaling pathway, stimulated PM H⁺-ATPase gene expression. Thus, ion homeostasis was modulated for salt tolerance.     

Adenosine 5′-monophosphate decreases citrate exudation and aluminium resistance in Tamba black soybean by inhibiting the interaction between 14-3-3 proteins and plasma membrane H<Superscript>+</Superscript>-ATPase

Our previous study suggested that aluminium (Al) stress increased plasma membrane (PM) H⁺-ATPase activity and citrate secretion and simultaneously enhanced the interaction between 14-3-3 proteins and phosphorylated PM H⁺-ATPase in Al-resistant Tamba black soybean (RB). Adenosine 5′-monophosphate (AMP) is known as an inhibitor of the interaction between 14-3-3 proteins and PM H⁺-ATPases. To investigate the effects of AMP on Al resistance, PM H⁺-ATPase activity and citrate exudation, AMP was used to treat Al-stressed RB. The results showed that after treatment with either 100 μM AMP or 50 μM Al for 8 h, RB root growth was inhibited by approximately 50 and 30%, respectively. However, simultaneous treatment with 100 μM AMP and 50 μM Al for 8 h resulted in a 60% inhibition of RB root growth, indicating that the presence of AMP reduced Al tolerance in RB. The interaction of PM H⁺-ATPase and 14-3-3 proteins in the root tips of Al-treated RB was stronger than that in the untreated control. However, the interaction of the two proteins was greatly reduced (lower than that in the control) after co-treatment with Al and AMP, suggesting that the presence of AMP under Al stress reduced the Al-enhanced interaction between PM H⁺-ATPase and 14-3-3 proteins. Consequently, PM H⁺-ATPase activity decreased by approximately 50%, which led to a significant decrease in H⁺ efflux and citrate secretion in RB roots under Al stress. Collectively, these results indicate that AMP reduced citrate exudation and Al resistance in RB by inhibiting the interaction between 14-3-3 proteins and PM H⁺-ATPases under Al stress.     

Reduced Plasma Membrane H+-ATPase Isoform OsA7 Expression and Proton Pump Activity Decrease Growth Without Affecting Nitrogen Accumulation in Rice

Plasma membrane H⁺-ATPase (PM H⁺-ATPase, EC 3.6.1.3.) is a proton pump that is necessary to promote cell growth and ion fluxes across the plasma membrane. The main goal of this study was to evaluate the role of PM H⁺-ATPase isoform OsA7 expression in rice growth and nitrogen (N) accumulation using three genetically engineered lineages with artificial micro RNA (amiRNA) targeting OsA7 (osa7.1, osa7.2, and osa7.3). PM H⁺-ATPase isoform expression in rice shoots and roots (wild-type) revealed that OsA7 is highly expressed in roots and is the most highly expressed PM H⁺-ATPase isoform. The three osa7 lineages had lower fresh weight, grain yield, height, and 1000-grain weight compared to control IRS plants. The hydroponic experiment comprised three NO₃⁻ levels over 30 days: 0.2 mM NO₃⁻–N, 2.0 mM NO₃⁻–N, and NO₃⁻ starvation for 3 days. The three osa7 lineages had lower PM H⁺-ATPase and V-H⁺-PPase activity as compared to the IRS plants. The root and shoot fresh weights were lower in osa7 lineages. The root/shoot ratio was lower in the osa7 lineages cultivated without nitrogen for 3 days and with 0.2 mM of NO₃⁻–N as compared to IRS, and did not change in plants cultivated with 2.0 mM NO₃⁻–N. The total N concentration did not change in the three osa7 lineages as compared to IRS. Overall, the results indicate that OsA7 is important for rice growth, grain production, and root growth, but does not affect N accumulation, highlighting the importance of other PM H⁺-ATPase isoforms in N uptake.

     

Multiple Effects of Lysophosphatidylcholine on the Activity of the Plasma Membrane H+-ATPase of Radish Seedlings*

We analyzed the effect of lysophosphatidylcholine (lysoPC) on the activity of the plasma membrane (PM) H⁺-AT-Pase measured at pH 6.3 or 7.5 in inside-out PM vesicles isolated from germinating radish seeds. LysoPC stimulated PM H⁺-ATPase at both pHs, but the dependence of the effect on lysoPC concentration was different: at pH 6.3 maximal stimulation was observed with 40 to 200 μg ml^?1 lysoPC, while at pH 7.5 a sharp peak of activation was observed at about 50 μg ml^?1 lysoPC, higher concentrations becoming dramatically inhibitory; this inhibitory effect was considerably reduced in the presence of 10% (v/v) glycerol. In trypsin-treared PM lysoPC stimulated the H⁺-ATPase activity assayed at pH 6.3, but only marginally that assayed at pH 7.5. LysoPC increased both V_max (from 190 to 280nmol min^?1 mg^?1 prot) and apparent K_M (from 0.15 to 0.3 mM) of the H⁺-ATPase at pH 6.3, while it increased V_max (from 120 to 230 nmol min^?1 mg^?1 prot) and decreased apparent K_m (from 0.8 to 0.4 mM) at pH 7.5. Low concentrations of Nacetylimidazole (10 to 50 mM), which modifies tyrosine residues, abolished the stimulation by lysoPC of the PM H⁺-ATPase activity at pH 7.5, but not that observed at pH 6.3. These results indicate that lysoPC influences the PM H⁺-ATPase through different mechanisms, and that its effect can only partly be ascribed to its ability to hamper the inhibitory interaction of the regulatory C-terminal domain with the catalytic site. N-acety-limidazole did not affect the stimulation of PM H⁺-ATPase by controlled trypsin treatment or by fusicoccin, indicating that the requirement for the tyrosine residue(s) modified by low Nacetylimidazole concentrations is specific for lysoPC-induced displacement of the C-terminal domain.     

Adaptation of plasma membrane H+ ATPase and H+ pump to P deficiency in rice roots

The plasma membrane (PM) H⁺ ATPase is involved in the plant response to nutrient deficiency. However, adaptation of this enzyme in monocotyledon plants to phosphorus (P) deficiency lacks direct evidence. In this study, we detected that P deficient roots of rice (Oryza Sativa L.) could acidify the rhizosphere. We further isolated the PM from rice roots and analyzed the activity of PM H⁺ ATPase. In vitro, P deficient rice roots showed about 30% higher activity of PM H⁺ ATPase than the P sufficient roots at assay of pH 6.0. The P deficiency resulted in a decrease of the substrate affinity value (K _m ) of PM H⁺ ATPase. The proton pumping activity of membrane vesicles from the P deficient roots was about 70% higher than that from P sufficient roots. Western blotting analysis indicated that higher activity of PM H⁺ ATPase in P deficient roots was related to a slightly increase of PM H⁺ ATPase protein abundance in comparison with that in P sufficient roots. Taken together, our results demonstrate that the P deficiency enhanced activities of both PM H⁺-ATPase and H⁺ pump, which contributed to the rhizosphere acidification in rice roots.     

Plasma-membrane H+-ATPases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco

Nepenthes is a unique genus of carnivorous plants that can capture insects in trapping organs called pitchers and digest them in pitcher fluid. The pitcher fluid includes digestive enzymes and is strongly acidic. We found that the fluid pH decreased when prey accumulates in the pitcher fluid of Nepenthes alata. The pH decrease may be important for prey digestion and the absorption of prey-derived nutrients. To identify the proton pump involved in the acidification of pitcher fluid, plant proton-pump homologs were cloned and their expressions were examined. In the lower part of pitchers with natural prey, expression of one putative plasma-membrane (PM) H⁺-ATPase gene, NaPHA3, was considerably higher than that of the putative vacuolar H⁺-ATPase (subunit A) gene, NaVHA1, or the putative vacuolar H⁺-pyrophosphatase gene, NaVHP1. Expression of one PM H⁺-ATPase gene, NaPHA1, was detected in the head cells of digestive glands in the lower part of pitchers, where proton extrusion may occur. Involvement of the PM H⁺-ATPase in the acidification of pitcher fluid was also supported by experiments with proton-pump modulators; vanadate inhibited proton extrusion from the inner surface of pitchers, whereas bafilomycin A₁ did not, and fusicoccin induced proton extrusion. These results strongly suggest that the PM H⁺-ATPase is responsible for acidification of the pitcher fluid of Nepenthes. Received: 8 June 2000 / Accepted: 8 August 2000     

Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase

Plasma membrane (PM) H⁺-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg²⁺- and Mn²⁺-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H⁺-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H⁺-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H⁺-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H⁺-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H⁺-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H⁺-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H⁺-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H⁺-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H⁺-ATPase in guard cells.

Type 2C protein phosphatase clade D family members redundantly dephosphorylate the penultimate C-terminal threonine residue of plasma membrane H⁺-ATPase in guard cells to control stomatal movement.