Identification of P-type plasma membrane H^(+)-ATPases in common wheat and characterization of TaHA7 associated with seed dormancy and germination

The P-type plasma membrane(PM)H^(+)-ATPases(HAs)are crucial for plant development,growth,and defense.The HAs have been thoroughly characterized in many different plants.However,despite their importance,the functions of HAs in germination and seed dormancy(SD)have not been validated in wheat.Here,we identified 28 TaHA genes(TaHA1-28)in common wheat,which were divided into five subfamilies.An examination of gene expression in strong-and weak-SD wheat varieties led to the discovery of six candidate genes(TaHA7/-12/-14/-16/-18/-20).Based on a single nucleotide polymorphism(SNP)mutation(C/T)in the TaHA7 coding region,a CAPS marker(HA7)was developed and validated in 168 wheat varieties and 171 Chinese mini-core collections that exhibit diverse germination and SD phenotypes.We further verified the roles of the two allelic variations of TaHA7 in germination and SD using wheat mutants mutagenized with ethyl methane sulphonate(EMS)in‘Jimai 22’and‘Jing 411’backgrounds,and in transgenic Arabidopsis lines.TaHA7 appears to regulate germination and SD by mediating gibberellic acid(GA)and abscisic acid(ABA)signaling,metabolism,and biosynthesis.The results presented here will enable future research regarding the TaHAs in wheat.

Effect of Lysophosphatidylcholine on ATP and ρ-Nitrophenyl Phosphate Hydrolysis by the Plasma Membrane H^+-ATPase from Soybean Hypocotyls

The stimulatory effect of lysophosphatidylcholine (lyso_PC) on ATP and ρ_nitrophenyl phosphate (PNPP) hydrolysis by the plasma membrane H +_ATPase from soybean (Glycine max (L.) Merr.) hypocotyls was studied. Results showed that lyso_PC stimulated the hydrolysis of ATP; ATP hydrolysis was enhanced dramatically when lyso_PC was within 0-0.03%, and increased slightly when lyso_PC was higher than 0.03%. At the concentration of 0.03%, lyso_PC stimulated ATP hydrolysis by 80.5%. Kinetics analysis showed that V max increased from 0.46 μmol P i·mg -1 protein·min -1 to 0.87 μmol P i·mg -1 protein·min -1 while K m increased from 0.88 mmol/L to 1.15 mmol/L under lyso_PC treatment. The optimum pH of ATP hydrolysis was shifted from 6.5 to 7.0 . Moreover, it was found lyso_PC enhanced the inhibition of ATP hydrolysis by hydroxylamine. In the presence of 200 mmol/L hydroxylamine, ATP hydrolysis was inhibited by 74.4%, while it was inhibited by 84.4% when treated with lyso_PC. However, PNPP hydrolysis and the inhibitory effect of vanadate were not affected by lyso_PC. The above results indicated that the kinase domain might be an action site or regulatory region of the C_terminal autoinhibitory domain in the plant plasma membrane H +_ATPase.

质膜H^(+)-ATPase基因对铝胁迫下水稻硝态氮吸收的 调控作用

为探讨细胞质膜ATP酶(PM H^(+)-ATPase)基因对铝胁迫下水稻(Oryza sativa L.)吸收硝态氮(NO_(3)^(-)-N)的调控,本试验以2个水稻品种峰1A和峰1A优5为研究对象,以无铝处理幼苗为对照,分析铝胁迫下水稻根尖PM H^(+)-ATPase活性、H^(+)-泵活性、H^(+)通量、PM H^(+)-ATPase基因家族(OsA1~OsA10)表达水平、PM H^(+)-ATPase和14-3-3蛋白的互作水平以及NO_(3)^(-)-N吸收量。结果表明,与对照相比,铝胁迫下2个水稻品种根尖PM H^(+)-ATPase活性、H^(+)-泵活性和根尖H^(+)通量下降,PM H^(+)-ATPase和14-3-3蛋白的互作水平下降。铝胁迫抑制了OsA1、OsA5、OsA7和OsA8基因的表达,其中OsA7的表达水平显著下调,铝胁迫下峰1A和峰1A优5的OsA7表达量仅为对照组的21%和39%。NO_(3)^(-)-N吸收量较对照下降,分别为对照的81%和85%。综上,PM H^(+)-ATPase基因的表达水平影响水稻对NO_(3)^(-)-N的吸收能力,OsA1、OsA5、OsA7和OsA8在铝胁迫下水稻吸收NO_(3)^(-)-N过程中起关键的调控作用。本研究结果可为增强酸铝条件下水稻吸收NO_(3)^(-)-N的能力,促进水稻生长及增产提供理论依据。

Effects of salinity on activities of H^+-ATPase, H^+-PPase and membrane lipid composition in plasma membrane and tonoplast vesicles isolated from soybean(Glycine max L.) seedlings

The effects of NaCl stress on the H +-ATPase, H +-PPase activity and lipid composition of plasma membrane(PM) and tonoplast(TP) vesicles isolated from roots and leaves of two soybean cultivars(Glycine max L.) differing in salt tolerance(Wenfeng7, salt-tolerant; Union, salt-sensitive) were investigated. When Wenfeng7 was treated with 0.3%(W/V) NaCl for 3 d, the H +-ATPase activities in PM and TP from roots and leaves exhibited a reduction and an enhancement, respectively. The H +-PPase activity in TP from roots also increased. Similar effects were not observed in roots of Union. In addition, the increases of phospholipid content and ratios of phospholipid to galactolipid in PM and TP from roots and leaves of Wenfeng7 may also change membrane permeability and hence affect salt tolerance.

Involvement of Plasma Membrane H+-ATPase in Adaption of Rice to Ammonium Nutrient

The preference of paddy rice for NH4+ rather than NO3ˉ is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH4+ absorption.However,the adaptation of rice root to low pH has not been fully elucidated.The plasma membrane H+-ATPase is a universal electronic H+ pump,which uses ATP as energy source to pump H+ across the plasma membranes into the apoplast.The key function of this enzyme is to keep pH homeostasis of plant cells and generate a H+ electrochemical gradient,thereby providing the driving force for the active influx and efflux of ions and metabolites across the plasma membrane.This study investigated the acclimation of plasma membrane H+-ATPase of rice root to low pH.This mechanism might be partly responsible for the preference of rice plants to NH4+ nutrition.

Changes of Plasma Membrane H^+-ATPase Activities of Glycine max Seeds by PEG Treatment

The soybean （Glycine max） Heihe No. 23 is sensitive to imbibitional chilling injury. Polyethylene glycol （PEG） treatment can improve chilling tolerance of soybean seeds to a certain extent. The changes of hydrolytic ATPase in plasma membranes and H^＋-pumping responses in soybean seeds were investigated during PEG treatments. Effects of exogenous calcium and exogenous ABA on the hydrolytic ATPase were also examined in order to understand the mechanism of chilling resistance. Highly purified plasma membranes were isolated by 6.0% aqueous two-phase partitioning from soybean seeds, as judged by the sensitivity of hydrolytic ATPase to sodium vanadate. PEG treatment resulted in a slight increase of the hydrolytic ATPase activity in 12 h. Then the activity decreased gradually, but still higher than the control. The H^＋-pumping activity increased steadily during PEG treatment. Exogenous calcium had both activating and inhibiting effects on the hydrolytic ATPase, but the activity was inhibited in soybean seeds treated with exogenous ABA. Results suggested that PEG treatment, not the exogenous calcium and ABA, up-regulated H^＋-ATPase activities in soybean seeds.

Role of platelet plasma membrane Ca^(2+)-ATPase in health and disease

Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.

Effects of Calcium on the GABA_A-Coupled CI^-/HCO₃^--ATPase from Plasma Membrane of Rat Brain

The work is a study of the influence of Ca2+ (0.01 - 1 mM) on neuronal CI-, HCO3-, -ATPase complex: an enzyme that is a CI--pump which is functionally and structurally coupled to GABAA-receptors. It is found that influence of Ca2+ on the multifunctional complex starts at concentration of 50·M and at concentration of 0.1 mM, it reduces the “basal” one and increases the CI-, HCO3-, -stimulated Mg2+-ATPase activities. GABA (0.1 - 100μM) activates the “basal” Mg2+-ATPase activity in the ab-sence of calcium. The effect of GABA on the enzyme in the presence of 0.01 ·M Ca2+ does not change. At the same time, 1 mM Ca2+eliminates the GABA effect on the “basal” Mg2+-ATPase activity. Competitive blocker of GABAA-receptors bicuculline (5 - 20 μM) in the absence of Ca2+ ions elimi-nates the stimulation of the “basal” Mg2+-ATPase by anions. When 0.25 mM Ca2+ is added to the in-cubation medium the inhibitory bicuculline effect on the enzyme does not appear. We found that 0.1 mM o-vanadate (protein tyrosine phosphatase blocker) reduces the GABA-activated ATPase activity. At the same time, 0.1 mM genistein (a protein tyrosine kinase blocker) has no effect on enzyme activity. In the presence of Ca2+ (0.25 mM), the effect of o-vanadate on the “basal” and CI-, HCO3-, -ATPase activities does not appear. It is shown for the first time that high concentrations of Ca2+prevent the action of GABAA-ergic ligands on the study ATPase. It is assumed that there is the involvement of protein kinases and protein phosphatases in the modulation of the enzyme activity by calcium. The observed effect of calcium on the ATPase may play an important role in the study of the mechanisms of epileptogenesis and seizure activity.

Plasma membrane Ca^(2+)-ATPases:Targets of oxidative stress in brain aging and neurodegeneration

The plasma membrane Ca2+-ATPase(PMCA)pumps play an important role in the maintenance of precise levels of intracellular Ca2+[Ca2+]i,essential to the functioning of neurons.In this article,we review evidence showing age-related changes of the PMCAs in synaptic plasma membranes(SPMs).PMCA activity and protein levels in SPMs diminish progressively with increasing age. The PMCAs are very sensitive to oxidative stress and undergo functional and structural changes when exposed to oxidants of physiological relevance.The major signatures of oxidative modification in the PMCAs are rapid inactivation,conformational changes,aggregation, internalization from the plasma membrane and proteolytic degradation.PMCA proteolysis appears to be mediated by both calpains and caspases.The predominance of one proteolytic pathway vs the other,the ensuing pattern of PMCA degradation and its consequence on pump activity depends largely on the type of insult,its intensity and duration.Experimental reduction of PMCA expression not only alters the dynamics of cellular Ca2+ handling but also has a myriad of downstream conse-quences on various aspects of cell function,indicating a broad role of these pumps.Age-and oxidation-related down-regulation of the PMCAs may play an important role in compromised neuronal function in the aging brain and its several-fold increased susceptibility to neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease,and stroke.Therapeutic approaches that protect the PMCAs and stabilize[Ca2+]i homeostasis may be capable of slowing and/or preventing neuronal degeneration.The PMCAs are therefore emerging as a new class of drug targets for therapeutic interventions in various chronic degenerative disorders.

Activation of Plasma Membrane NADPH Oxidase and Generation of H_2O_2 Mediate the Induction of PAL Activity and Saponin Synthesis byEndogenous Elicitor in Suspension-Cultured Cells of Panax ginseng

Endogenous elicitor, termed cellulase-degraded cell wall (CDW), was prepared from the cell wall of suspension-cultured ginseng (Panax ginseng C.A. Meyer) cells via cellulase degradation. CDW activated the NADPH oxidase activity of isolated plasma membranes and stimulated in vivo H2O2 generation in ginseng cell suspensions. CDW also increased the activity of phenylalanine ammonia lyase (PAL), expression of a P. ginseng squalene epoxidase (sqe) gene and saponin synthesis. NADPH oxidase inhibitors inhibited both in vitro NADPH oxidase activity and in vivo H2O2 generation. Induction of PAL activity, saponin synthesis and sqe gene expression were all inhibited by such inhibitor treatments and reduced by incubation with catalase and HA scavengers. These data indicate that activation of NADPH oxidase and generation of H2O2 are essential signalling events mediating defence responses induced by the endogenous elicitor(s) present in CDW.

Influence of K + on the Coupling Between ATP Hydrolysis and Proton Transport by the Plasma Membrane H +_ATPase from Soybean Hypocotyls

The plasma membrane vesicles were purified from soybean (Glycine max L.) hypocotyls by two_phase partitioning methods. The stimulatory effects of K + on the coupling between ATP hydrolysis and proton transport by the plasma membrane H +_ATPase were studied. The results showed that the proton transport activity was increased by 850% in the presence of 100 mmol/L KCl, while ATP hydrolytic activity was only increased by 28.2%. Kinetic studies showed that K m of ATP hydrolysis decreased from 1.14 to 0.7 mmol/L, while V max of ATP hydrolysis increased from 285.7 to 344.8 nmol Pi·mg -1 protein·min -1 in the presence of KCl. Experiments showed that the optimum pH was 6.5 and 6.0 in the presence and absence of KCl, respectively. Further studies revealed that K + could promote the inhibitory effects of hydroxylamines and vanadates on the ATP hydrolytic activity. The above results suggested that K + could regulate the coupling between ATP hydrolysis and proton transport of the plasma membrane H +_ATPase through modulating the structure and function of the kinase and phosphatase domains of the plasma membrane H +_ATPase.

Involvement of Plasma Membrane Ca^(2+)/H^+ Antiporter in Cd^(2+) Tolerance

Cation exchangers （CAXs） belong to the cation/Ca2＋exchanger superfamily which have been extensively investigated in plant tonoplasts over the last decade. Recently, the roles of CAXs involved in heavy metal accumulation and tolerance in plants have been studied for phytoremediation and food security. In this mini review, we summarize the roles of the Ca2＋/H＋ antiporter in Ca2＋ signal transduction, maintaining ion homeostasis and sequestering heavy metals into the vacuole. Moreover, we present a possible role of the plasma membrane Ca2＋/H＋ antiporter in heavy metal detoxification.

Changes of plasma membrane ATPase activity, membrane potential and transmembrane proton gradient in Kandelia candel and Avicennia marina seedlings with various salinities

The salt-secreting mangrove, Avicennia marina, and non-salt-secreting mangrove, Kandelia candel were cultivated in sand with various salinities(0‰, 10‰, 20‰, 30‰, 40‰) for 60 d. Plasma membrane vesicles of high-purity in leaves and roots of A.marina and K. candel seedlings were obtained by two-phase partitioning. The function of the plasma membranes, the activity of ATPase, membrane potential and transmembrane proton gradient, at various salinities were investigated. The results showed that within a certain range of salinity(A. marina and roots of K. candel: 0—30‰; leaves of K.candel: 0—20‰), the activity of ATPase increased with increasing salinity, while high salinity(above 30‰ or 20‰) inhibited ATPase activity. In comparison with A. marina, K. candel appeared to be more sensitive to salinity. The dynamics of membrane potential and transmembrane proton gradient in leaves and roots of A. marina and K. candel seedlings were similar to that of ATPase. When treated directly by NaCl all the indexes were inhibited markedly: there was a little increase within 0—10‰(K. candel) or 0—20‰(A. marina) followed by sharp declining. It indicated that the structure and function of plasma membrane was damaged severely.

Protein and lipid characterization of wheat roots plasma membrane damaged by Fe and H₂O₂using ATR-FTIR method

In plant cells the plasma membrane is a highly elaborated structure that functions as the point of exchange with adjoining cells, cell walls and the external environment. In this study, we investigated the structure and function characteristic of wheat root plasma membrane (PM) as affected by H2O2 and Fe by using fluorescence spectroscopic and attenuated total reflectance infrared (ATR-IR) techniques. The results showed that these oxidant damaged induced an obviously reduced membrane fluidity were observed in the roots PM treated with the 200 μM H2O2, FeSO4, and FeCl3. Computer-aided software analyses of the FTIR spectrum indicated that the content of the α-helices decreased and β-sheet increased in the secondary structures of proteins after exposure to the oxidants of 200 μM H2O2, FeSO4, and FeCl3. The number of P=O and C=C bonds area declined rapidly in the lipids of the membrane under the oxidants stress. These structural alterations might explain the reason of the roots PM instability under most of the abiotic stress.

水稻根系细胞膜H^+-ATPase对铵硝营养的响应差异

用两相法分离了铵态氮(NH4+-N)和硝态氮(NO3--N)营养下水稻苗期根系的细胞膜,并测定了细胞膜上H+-ATPase的水解活性,以期阐明水稻根系细胞质膜上H+-ATPase对不同氮素形态的响应差异。两相法分离的细胞膜纯度达到95%以上。在离体条件下,NH4+-N营养的水稻根系细胞膜H+-ATPase的水解活性和H+-ATPase的Km和Vmax均显著高于NO3--N营养。NH4+-N营养的水稻根系细胞膜H+-ATPase最适pH值为6.0,而NO3--N营养的在pH6.2左右。Westernblot结果表明,NH4+-N营养的水稻根系细胞膜H+-ATPase浓度显著高于NO3--N营养的H+-ATPase。说明NH4+-N营养的水稻根系细胞膜H+-ATPase活性高是因为单位细胞膜上的H+-ATPase分子数量大于NO3--N营养,并且在NH4+-N营养的水稻根系细胞膜上可能存在着与NO3--N营养不同的H+-ATPase的同工酶。因此,NH4+-N营养的水稻根系细胞膜H+-ATPase活性高很可能是水稻根系对铵态氮营养的一种适应机制。

盐胁迫及La^(3+)对不同耐盐性水稻根中抗氧化酶及质膜H^+-ATPase的影响

用盐敏感的武运粳8号和强耐盐的韭菜青两个粳稻品种,比较了盐胁迫条件下根中抗氧化酶类和质膜H+-ATPase活性的变化以及La3+对其变化的影响。结果表明,两个品种根中SOD和APX活性无显著区别,但耐盐品种的CAT和POD活性强于盐敏感品种。盐胁迫不同程度地提高了两者抗氧化酶活性。La3+对其影响最显著的差异出现在高盐条件下(200~300mmolL-1NaCl),对耐盐品种添加La3+可以提高上述4种酶的活力,而对盐敏感品种,La3+的作用不明显。对盐敏感品种,盐胁迫导致其质膜H+-ATPase活性持续下降,添加La3+明显提高其H+-ATPase活性,而对耐盐品种,盐胁迫导致其质膜H+-ATPase活性呈现先降后升的趋势,La3+对其活性影响不大。进一步分析表明,上述H+-ATPase活性变化及La3+对其影响均发生在转录水平上。据此推测,以上2个品种可能具有完全不同的盐胁迫响应机制。

NaCl胁迫下枸杞愈伤组织活性氧产生与质膜H^+-ATPase活性的关系

以枸杞愈伤组织为材料,研究了盐胁迫对活性氧伤害和质膜H+-ATPase活性的影响。结果表明,NaCl浓度为100mmol/L时超氧阴离子(O2-·)和过氧化氢(H2O2)含量上升,质膜H+-ATPase活性先升后降;质膜相对透性与丙二醛(MDA)含量呈显著正相关;超氧阴离子(O2-·)和过氧化氢(H2O2)含量与质膜H+-ATPase活性在重度胁迫下呈显著负相关,说明盐胁迫下活性氧积累可能是加速伤害质膜功能的主要原因之一。

铜对小麦根质膜H^+-ATPase活性的影响

用两相法提取小麦根质膜微囊,研究了铜对质膜H+-ATPase活性的影响.硫酸铜(CuSO4)抑制小麦根质膜H+-ATPase的活性,其作用具有浓度依赖性.金属离子螯合剂乙二胺四乙酸二钠(EDTA-Na2,200μmol.L-1)可以完全消除CuSO4对H+-ATPase活性的抑制效应.检测硫代巴比妥酸反应物质(TBARS)的相对含量,结果显示Cu2+的加入使质膜微囊产生了较多的TBARS,表明Cu2+作用导致细胞膜发生较强的氧化损害.实验结果表明,CuSO4可以减弱小麦根质膜H+-ATPase的活性,此效应是由Cu2+引起的;此外,Cu2+导致质膜微囊产生较强的膜脂过氧化.

质膜H^+-ATPase与环境胁迫

植物根系质膜H+-ATPase在调节细胞内pH值,促进养分吸收、同化物运输等方面具有重要作用。对质膜H+-ATPase的结构、功能和分子机制进行综述,并讨论了质膜H+-ATPase在信号传递过程及植物适应环境胁迫中的作用,最后就植物质膜H+-ATPase的研究及应用提出几点看法。

盐胁迫下植物质膜H^+-ATPase研究进展

质膜H+-ATPase在植物细胞的跨膜物质转运、胞内pH值调节以及植物对环境胁迫的响应等诸多方面具有重要作用,是植物生命活动过程的主宰酶.盐胁迫是影响植物生长发育的主要环境因子,植物质膜H+-ATPase活性的调节是植物适应盐环境、提高耐盐性的重要细胞机理.综述了植物质膜H+-ATPase活性变化及其调节机理对盐胁迫响应的研究状况,对质膜H+-ATPase活性调节机理研究中仍未解决的问题进行了展望.