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.

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 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.

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.

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.

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.

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.

Ca^(2+)-dependent TaCCD1 cooperates with TaSAUR215 to enhance plasma membrane H^(+)-ATPase activity and alkali stress tolerance by inhibiting PP2C-mediated dephosphorylation of TaHA2 in wheat

Alkali stress is a major constraint for crop production in many regions of saline-alkali land.However,little is known about the mechanisms through which wheat responds to alkali stress.In this study,we identified a calcium ion-binding protein from wheat,TaCCD1,which is critical for regulating the plasma membrane(PM)H^(+)-ATPase-mediated alkali stress response.PM H+-ATPase activity is closely related to alkali tolerance in the wheat variety Shanrong 4(SR4).We found that two D-clade type 2C protein phosphatases,TaPP2C.D1 and TaPP2C.D8(TaPP2C.D1/8),negatively modulate alkali stress tolerance by dephosphorylating the penultimate threonine residue(Thr926)of TaHA2 and thereby inhibiting PM H+-ATPase activity.Alkali stress induces the expression of TaCCD1 in SR4,and TaCCD1 interacts with TaSAUR215,an early auxin-responsive protein.These responses are both dependent on calcium signaling triggered by alkali stress.TaCCD1 enhances the inhibitory effect of TaSAUR215 on TaPP2C.D1/8 activity,thereby promoting the activity of the PM H^(+)-ATPase TaHA2 and alkali stress tolerance in wheat.Functional and genetic analyses verified the effects of these genes in response to alkali stress,indicating that TaPP2C.D1/8 function downstream of TaSAUR215 and TaCCD1.Collectively,this study uncovers a new signaling pathway that regulates wheat responses to alkali stress,in which Ca^(2+)-dependent TaCCD1 cooperates with TaSAUR215 to enhance PM H+-ATPase activity and alkali stress tolerance by inhibiting TaPP2C.D1/8-mediated dephosphorylation of PM H+-ATPase TaHA2 in wheat.

Plasma membrane calcium pump regulation by metabolic stress

The plasma membrane Ca2+-ATPase(PMCA)is an ATPdriven pump that is critical for the maintenance of low resting[Ca2+]i in all eukaryotic cells.Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism,has the capacity to cause ATP depletion and thus inhibit PMCA activity.This has potentially fatal consequences,particularly for non-excitable cells in which the PMCA is the major Ca2+efflux pathway.This is because inhibition of the PMCA inevitably leads to cytosolic Ca2+ overload and the consequent cell death.However,the relationship between metabolic stress,ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted.There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion.In particular,there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function.Moreover, membrane phospholipids,mitochondrial membrane potential,caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA.The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca2+ overload and cytotoxicity.

A critical review on natural compounds interacting with the plant plasma membrane H^(+)-ATPase and their potential as biologicals in agriculture

The plant plasma membrane(PM)H^(+)-ATPase is an essential enzyme controlling plant growth and development.It is an important factor in response to abiotic and biotic stresses and is subject to tight regulation.We are in demand for new sustainable natural growth regulators and as a key enzyme for regulation of transport into the plant cell the PM H^(+)-ATPase is a potential target for these.In this review,we have evaluated the known non-protein natural compounds with regulatory effects on the PM H^(+)-ATPase,focusing on their mechanism of action and their potential as biologicals/growth regulators in plant production of future sustainable agriculture.

Testing the polar auxin transport model with a selective plasma membrane H^(+)-ATPase inhibitor

Auxin is unique among plant hormones in that its function requires polarized transport across plant cells.A chemiosmotic model was proposed to explain how polar auxin transport is derived by the H^(+)gradient across the plasma membrane(PM)established by PM H^(+)-adenosine triphosphatases(ATPases).However,a classical genetic approach by mutations in PM H^(+)-ATPase members did not result in the ablation of polar auxin distribution,possibly due to functional redundancy in this gene family.To confirm the crucial role of PM H^(+)-ATPases in the polar auxin transport model,we employed a chemical genetic approach.Through a chemical screen,we identified protonstatin-1(PS-1),a selective small-molecule inhibitor of PM H^(+)-ATPase activity that inhibits auxin transport.Assays with transgenic plants and yeast strains showed that the activity of PM H^(+)-ATPases affects auxin uptake as well as acropetal and basipetal polar auxin transport.We propose that PS-1 can be used as a tool to interrogate the function of PM H^(+)-ATPases.Our results support the chemiosmotic model in which PM H^(+)-ATPase itself plays a fundamental role in polar auxin transport.

The fungal endophyte Epichloëgansuensis increases NaCltolerance in Achnatherum inebrians through enhancing the activity of plasma membrane H^(+)-ATPase and glucose-6-phosphate dehydrogenase

Salt stress negatively affects plant growth,and the fungal endophyte Epichloëgansuensis increases the tolerance of its host grass species,Achnatherum inebrians,to abiotic stresses.In this work,we first evaluated the effects of E.gansuensis on glucose-6-phosphate dehydrogenase(G6PDH)and plasma membrane(PM)H^(+)-ATPase activity of Achnatherum inebrians plants under varying NaCl concentrations.Our results showed that the presence of E.gansuensis increased G6PDH,PMH^(+)-ATPase,superoxide dismutase and catalase activity to decrease O2•^(–),H_(2)O_(2)and Na^(+)contents in A.inebrians under NaCl stress,resulting in enhanced salt tolerance.In addition,the PM NADPH oxidase activity and NADPH/NADP+ratios were all lower in A.inebrians with E.ganusensis plants than A.inebrians plants without this endophyte under NaCl stress.In conclusion,E.gansuensis has a positive role in improving host grass yield under NaCl stress by enhancing the activity of G6PDH and PM H^(+)-ATPase to decrease ROS content.This provides a new way for the selection of stress-resistant and high-quality forage varieties by the use of systemic fungal endophytes.

Identification of H^+/Ca^(2+) Antiporter in Barley Root Plasma Membrane

On the basis of two types of calcium transport system detected in the barley root plasma membrane,the mechanisms of the calcium transport have been further studied.Ionophore CCCP has been found to inhibit Mg^(2+) -dependent calcium transport by 20%.In contrast,Mg^(2+) -independent calcium trans- port is insensitive to CCCP.The Mg^(2+) -dependent calcium transport following the collapse of H^+ gradient across the plasma membrane could be driven by the H^+ gradient either set up by ATP or imposed artificially. Any relation between Mg^(2+) -independent calcium transport and H^+ gradient has not been observed.These results indicate that Mg^(2+) -dependent calcium transport is accompanied by the decrease of H^+ gradient,and Mg^(2+) -independent calcium transport has nothing to do with the H^+ gradient.It is therefore suggested that the calcium transport across the barley root plasma membrane is driven by ATPase that is independent of Mg^(2+),and H^+/Ca^(2+) antiporter that is dependent on Mg^(2+).

Kinetics of the H＋-ATPase from Dry and 5-Hours- Imbibed Maize Embryos in Its Native, Solubilized, and Reconstituted Forms

Membranes undergo recovery upon rehydration in seed germination. Previous work has described that the plasma membrane H＋-ATPase from maize embryos adopts two different forms at 0 and 5 h of imbibition. We investigated how the kinetics of these two forms could be affected by alterations in the plasma membrane （PM）. In comparison to the O-h, PMs from the 5-h imbibed embryos showed changes in glycerophospholipid composition, decrease in leakage, and increase in fluidity. Kinetics of the PM H^-ATPase from 0 and 5-h imbibed embryos showed negative cooperativity, With the removal of the membrane environment, the activity of the enzymes shifted to a more complex kinetics, displaying two enzyme components. Lipid reconstitution produced one component with positive cooperativity. In all cases, enzymes from 0 and 5-h imbibed embryos presented similar kinetics with some quantitative differences. These results indicate that the two enzyme forms have the potential ability to respond to changes in the membrane enyiror^rpent, but the fact that they do not show differences in the native membranes at 0 or 5 h implies that modifications in the membrane are not drastic enough to alter their kinetics, or that they are able to preserve their boundary lipids or associated proteins and thus retain the same kinetic behavior.

GABA_A-Coupled Cl-/ HCO₃<sup style="margin-left:-10px">-</sup>-ATPase from Plasma Membrane of the Rat Brain: Role of HCO₃<sup style="margin-left:-10px">-</sup>in the Enzyme Activation

This work examines the influence of Cl- (2.5 - 125 mM) and HCO3- (2 - 30 mM) on the Cl-/HCO3- - ATPase complex of the neuronal membrane and this enzyme is a Cl--pump that is coupled to GABAA receptors. The greatest (44%) activating effect on the enzyme is found with HCO3- (20 - 30 mM), while the maximum activity occurs in the presence of a ratio of ~25 mM HCO3- /~5mM Cl-. Blockers of the GABAA receptor, namely bicuculline (10 - 50 μM) and picrotoxin (50 - 100 μM), inhibit this anion activation, whereas the HCO3- -ATPase activity is not sensitive to these ligands. Autoradiographic analysis of the spectrum of the partially purified enzyme phosphorylated with [γ-32P]ATP allowed us to distinguish three major 32P-labeled protein whose molecular weight are about 57, 53, and 48 kDa. In the presence of 5 mM Cl-/25mM HCO3- and 100 μM picrotoxin, the intensity of the phosphorylation of bands significantly decreased, thereby confirming the assumption about coupled of binding sites for anions and GABAA-ergic ligands. It was suggested scheme of Cl--transport through the plasma membrane by utilizing neuronal Cl-/ -HCO3- ATPase in the low (5 mM) Cl- and high (25 mM) HCO3- concentrations. The data demonstrated for the first time that the GABAA-coupled Cl-/ HCO3- -ATPase from rat brain neuronal membranes is maximally activated at a Cl-/HCO3- ratio of 1:5 and it remains stable at high concentrations of substrate and buffer.