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.

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.

Maintenance of mesophyll potassium and regulation of plasma membrane H^+-ATPase are associated with physiological responses of tea plants to drought and subsequent rehydration

Drought stress is one of the main factors limiting yield in tea plants. The plant cell's ability to preserve K^+homeostasis is an important strategy for coping with drought stress. Plasma membrane H^+-ATPase in the mesophyll cell is important for maintaining membrane potential to regulate K^+transmembrane transport. However, no research to date has investigated the possible relationship between plasma membrane H^+-ATPase and mesophyll K^+retention in tea plants under drought and subsequent rehydration conditions. In our experiment, drought stress inhibited plasma membrane H^+-ATPase activities and induced net H^+influx, leading to membrane potential depolarization and inducing a massive K^+efflux in tea plant mesophyll cells. Subsequent rehydration increased plasma membrane H^+-ATPase activity and induced net H^+efflux, leading to membrane potential hyperpolarization and thus lowering K^+loss. A first downregulated and then upregulated plasma membrane H^+-ATPase protein expression level was also observed under drought and subsequent rehydration treatment, a finding in agreement with the change of measured plasma membrane H^+-ATPase activities. Taken together, our results suggest that maintenance of mesophyll K^+in tea plants under drought and rehydration is associated with regulation of plasma membrane H^+-ATPase activity.

Plasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways

Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular freecalcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulindependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.

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.

Plasma Membrane Calcium ATPase Expression in Human Lens Epithelium Cell Lines

Purpose:To study the expression of four plasma membrane calcium ATPase (PMCA) isoforms in human lens epithelium cell lines (HLE-B3 cells) both on mRNA and protein levels.Methods:Both total mRNA and membrane protein samples were collected,after HLE-B3 cells were cultured to 90% confluency.Reverse Transcription Polymerase Chain Reaction (RT-PCR) were used to detect mRNAs of PMCA isoform 1,2,3,and 4 by using corresponding PMCA isoform 1,2,3,and 4 primers.Western Blot analysis was employed to detect PMCA isoform 1,2,3,and 4 protein using corresponding anti-PMCA1,2,3,and 4 antibodies.Results:A 420 bp fragment was amplified with PMCA1 primer.A 550 bp fragment was amplified with PMCA2 primer.A 840 bp fragment was amplified with PMCA4 primer.No fragment was amplified with PMCA3 primer.Western Blotting confirmed that the expected ～153 kDa,～125 kDa and ～147 kDa protein were recognized by anti PMCA1,2 and 4 antibodies respectively.No protein was recognized by PMCA3 antibody.Conclusion:This is the first study showing only PMCA1,2,and 4 gene are expressed in HLE-B3 cells on both mRNA and protein level.PMCA3 is not expressed in HLE-B3 cells.The PMCA isoforms expression pattern in HLE-B3 cell lines is different from that in the lens of other species.PMCA2 may play a more important role over other isoforms.

Effects of Calcium on ATPase Activity and Lipid omposition of Plasma Membranes from Wheat Roots Under Aluminum Stress

Effects of calcium on ATPase activities, lipid contents, and fatty acid compositions of plasma membrane from wheat roots were assayed under aluminum stress. The results showed that the increase of calcium concentration in the nutrient solution increased the activity of H + ATPase and the phospholipid content, decreased the activity of Ca 2+ ATPase and the galactolipid of plasma membrane. Owing to the decrease of linolenic acid content, the index of unsaturated fatty acid (IUFA) and index of double bond (DBI) decreased in Altas66. The IUFA and DBI of plasma membrane from Scout66 roots increased because its linolenic acid content increased obviously and its palmitic acid content decreased apparently.

Plasma membrane Ca^(2+)-ATPases in the nervous system during development and ageing

Calcium signaling is used by neurons to control a variety of functions,including cellular differentiation,synaptic maturation,neurotransmitter release,intracellular signaling and cell death.This review focuses on one of the most important Ca2+regulators in the cell,the plasma membrane Ca2+-ATPase(PMCA),which has a high affinity for Ca2+and is widely expressed in brain.The ontogeny of PMCA isoforms,linked to specific requirements of Ca2+ during development of different brain areas,is addressed, as well as their function in the adult tissue.This is based on the high diversity of variants in the PMCA family in brain,which show particular kinetic differences possibly related to specific localizations and functions of the cell. Conversely,alterations in the activity of PMCAs could lead to changes in Ca2+homeostasis and,consequently,to neural dysfunction.The involvement of PMCA isoforms in certain neuropathologies and in brain ageing is also discussed.

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.

Contribution of plasma membrane Ca^(2+) ATPase to cerebellar synapse function

The cerebellum expresses one of the highest levels of the plasma membrane Ca2+ATPase,isoform 2 in the mammalian brain.This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex;i.e. the Purkinje neurons(PNs) .Here we review recent evidence,including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2(PMCA2) knockout mouse,to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour.These studies have also revealed that deletionof PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development,they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.

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.

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.

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.

Plasma membrane calcium pumps and their emerging roles in cancer

Alterations in calcium signaling and/or the expression of calcium pumps and channels are an increasingly recognized property of some cancer cells.Alterations in the expression of plasma membrane calcium ATPase(PMCA) isoforms have been reported in a variety of cancer types,including those of breast and colon,with some studies of cancer cell line differentiation identifying specific PMCA isoforms,which may be altered in some cancers.Some studies have also begun to assess levels of PMCA isoforms in clinical tumor samples and to address mechanisms of altered PMCA expression in cancers.Both increases and decreases in PMCA expression have been reported in different cancer types and in many cases these alterations are isoform specific.In this review,we provide an overview of studies investigating the expression of PMCA in cancer and discuss how both the overexpression and reduced expression of a PMCA isoform in a cancer cell could bestow a growth advantage,through augmenting responses to proliferative stimuli or reducing sensitivity to apoptosis.

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。