Plasmalemma ATPase Research Articles

Silencing of ascorbate oxidase results in reduced growth, altered ascorbic acid levels and ripening pattern in melon fruit

Ascorbate oxidase (AO, EC 1.10.3.3) is a copper-containing enzyme localized at the apoplast, where it catalyzes the oxidation of ascorbic acid (AA) to dehydroascorbic acid (DHA) via monodehydroascorbic acid (MDHA) intermediate. Despite it has been extensively studied, no biological roles have been definitively ascribed. To understand the role of AO in plant metabolism, fruit growth and physiology, we suppressed AO expression in melon (Cucumis melo L.) fruit. Reduction of AO activity increased AA content in melon fruit, which is the result of repression of AA oxidation and simultaneous induction of certain biosynthetic and recycling genes. As a consequence, ascorbate redox state was altered in the apoplast. Interestingly, transgenic melon fruit displayed increased ethylene production rate coincided with elevated levels of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO, EC 1.14.17.4) activity and gene expression, which might contribute to earlier ripening. Moreover, AO suppressed transgenic melon fruit exhibited a dramatic arrest in fruit growth, due to a simultaneous decrease in fruit cell size and in plasmalemma (PM) ATPase activity. All the above, support for the first time, the in vivo AO participation in the rapid fruit growth of Cucurbitaceae and further suggest an alternative route for AA increase in ripening fruit.

Effects of calcium application on the salt tolerance and sodium excretion from salt glands in zoysiagrass (Zoysia japonica)

AbstractZoysiagrass (Zoysia japonica Steud.), a native grass in Japan, is a warm‐season turfgrass displaying high salt tolerance, mainly derived from salt excretion through salt glands. The present study focused on how external calcium (Ca) affects sodium (Na) excretion from salt glands to relieve salt stress, including response to oxidative stress, and discussed the physiological mechanisms possibly involved. Treatments using 0, 25, 50, 75, 100, 125 and 150 mmol/L sodium chloride (NaCl), with or without applying 2 or 10 mmol/L calcium chloride (CaCl2), were conducted for seven days. Growth inhibition and an abrupt increase in Na excretion from salt glands occurred above 100 mmol/L NaCl treatment, while Na concentrations in shoot and root tissues increased with increasing NaCl concentration from 0 to 75 mmol/L treatment. Above 100 mmol/L NaCl treatment, pectic acid‐binding Ca and glycinebetaine concentrations decreased in shoot and root tissues, but free proline concentration and the activities of plasmalemma ATPase, catalase (CAT) and ascorbate peroxidase (APX) increased. Calcium application alleviated growth inhibition when added to high level of NaCl treatments (over 100 mmol/L NaCl). Compared to the treatment without Ca, Na concentration in shoot and root tissues and ATPase activity in the root decreased, but Na exclusion from salt glands, pectic acid‐binding Ca concentration in shoot and root tissues, and ATPase, CAT and APX activities in the shoot increased. Thus, Na excretion through salt glands seems to occur when Na concentration in the plant exceeds its accumulation limit, and this salt tolerance reaction involving salt glands proceeds in two steps: the grass absorbs and accumulates Na in the shoot and root up to a threshold, above which Na is removed through salt glands. Calcium application promoted Na excretion through salt glands, especially under growth‐inhibiting Na concentrations, which might be induced by the increase in extracellular excretion of Na by plasmalemma ATPase.

How Plants Cope with Foreign Compounds. Translocation of xenobiotic glutathione conjugates in roots of barley (Hordeum vulgare) (9 pp)

Numerous herbicides and xenobiotic organic pollutants are detoxified in plants to glutathione conjugates. Following this enzyme catalyzed reaction, xenobiotic GS-conjugates are thought to be compartmentalized in the vacuole of plant cells. In the present study, evidence is presented from experiments with roots of barley (Hordeum vulgare cv. Cherie) that part of these conjugates will undergo long range transport in plants, rather than be stored in the vacuole. To our knowledge, this is the first report about the unidirectional long-range transport of xenobiotic conjugates in plants and the exsudation of a glutathione conjugate from the root tips. This could mean that plants possess an excretion system for unwanted compounds giving them similar advantages as animals. Barley plants (Hordeum vulgare) were grown in Petri dishes soaked with tap water in the greenhouse. Fluorescence Microscopy. Roots of barley seedlings were cut under water, and the end at which the pesticides were applied was fixed in an aperture with a thin latex foil and transferred into a drop of water on a cover slide. The cover slide was fixed in a measuring chamber on the stage of an inverse fluorescence microscope (Zeiss Axiovert 100). Monobromo- and Monochlorobimane, two model xenobiotics that are conjugated rapidly in plant cells with glutathione, hereby forming fluorescent metabolites, were used as markers. Their transport in the root could be followed with high time resolution. Spectrometric enzyme assay. Glutathione S-transferase (GST) activity was determined in the protein extracts following established methods. Aliquots of the enzyme extract were incubated with 1-chloro-2,4-dinitrobenzene (CDNB), or monochlorobimane. Controls lacking enzyme or GSH were measured. Pitman chamber experiments. Ten days old barley plants or detached roots were inserted into special incubation chambers, either complete with tips or decapitated, as well as 10 days old barley plants without root tips. Compartment A was filled with a transport medium and GSH conjugate or L-cysteine conjugate. Compartments B and C contained sugar free media. Samples were taken from the root tip containing compartment C and the amount of conjugate transported was determined spectro-photometrically. Results. The transport in roots is unidirectional towards the root tips and leads to exsudation of the conjugates at rates between 20 and 200 nmol min(-1). The microscopic studies have been complemented by transport studies in small root chambers and spectroscopic quantification of dinitrobenzene-conjugates. The latter experiments confirm the microscopic studies. Furthermore it was shown that glutathione conjugates are transported at higher rates than cysteine conjugates, despite of their higher molecular weights. This observation points to the existence of glutathione specific carriers and a specific role of glutathione in the root. It can be assumed that long distance transport of glutathione conjugates within the plant proceeds like GSH or amino acid transport in both, phloem and xylem. The high velocity of this translocation of the GS-X is indicative of an active transport. For free glutathione, a rapid transport-system is essential because an accumulation of GSH in the root tip inhibits further uptake of sulfur. Taking into account that all described MRP transporters and also the GSH plasmalemma ATPases have side activities for glutathione derivatives and conjugates, co-transport of these xenobiotic metabolites seems credible. On the other hand, when GS-B was applied to the root tips from the outside, no significant uptake was observed. Thus it can be concluded that only those conjugates can be transported in the xylem which are formed inside the root apex. Having left the root once, there seems to be no return into the root vessels, probably because of a lack of inward directed transporters. Plants seem to possess the capability to store glutathione conjugates in the vacuole, but under certain conditions, these metabolites might also undergo long range transport, predominantly into the plant root. The transport seems dependent on specific carriers and is unidirectional, this means that xenobiotic conjugates from the rhizosphere are not taken up again. The exudation of xenobiotic metabolites offers an opportunity to avoid the accumulation of such compounds in the plant. The role of glutathione and glutathione related metabolites in the rhizosphere has not been studied in any detail, and only scattered data are available on interactions between the plant root and rhizosphere bacteria that encounter such conjugates. The final fate of these compounds in the root zone has also not been addressed so far. It will be interesting to study effects of the exsuded metabolites on the biology of rhizosphere bacteria and fungi.

Variations in salt tolerance of reed canarygrass (Phalaris arundinacea L.) plants grown at sites with different degrees of cattle urine contamination

Variations in the salt tolerance of reed canarygrass (RCG) plants grown at sites with different degrees of cattle urine contamination were investigated. Cattle urine overflowed the urine reservoir and invaded the grassland. Soils and RCG plants were collected from sampling spots selected along the urine flowing line from 0 (contaminated site beside the urine reservoir) to 100 m. With increasing distance from the reservoir, the contents of K, Na, Ca, Mg and NO3-N in the soil and Na, K and NO3-N in the collected RCG plants decreased. The RCG plants were grown in a standard solution to obtain a large number of clones and seeds for the next generation. After the RCG clones and their next generation were subjected to water culture to the fifth leaf stage, NaCl was applied to the solution to investigate their salt tolerance. The salt tolerance, which was the highest in the RCG clones and their next generation for the plants collected from the site beside the reservoir, decreased with increasing distance from the reservoir. Under saline water culture conditions, Na and free-proline contents in shoots, K content in shoots and roots, water potential of leaves and plasmalemma ATPase activity of roots increased in the RCG clones and their next generation along with a higher salt tolerance. In conclusion, responses to saline conditions were similar between the RCG clones and the next generation that were cross-pollinated. It was considered that, even within a confined range of the grassland, salt-tolerant RCG plants that had adapted to the urine-contaminated soils were selected, and that high salt tolerance was genetically controlled. It appears likely that the maintenance of the water potential and K-absorbing function under NaCl stress contributed to the high salt tolerance.

Comparison of Ca Effect on the Salt Tolerance of Suspension Cells and Intact Plants of Tobacco (Nicotiana tabacum L., cv. Bright Yellow-2)

Salt tolerance and the effect of Ca application on the salt tolerance were compared between suspension culture cells and intact plants of tobacco (Nicotiana tabacum L., cv. Bright Yellow-2). The suspension culture cells and intact plants of tobacco were grown in a culture medium without NaCl and with 100 mM NaCl containing 0.3 or 10 mM CaCl2. At 7 d after the treatment, the dry weight of the cells and intact plants grown in the medium containing 100 mM NaCl with 0.3 mM CaCl2 decreased and the decreasing rate of the weight was much higher in the intact plants. In the medium containing 100 mM NaCl with 10 mM CaCl2, the growth of the cells and intact plants was improved, and the improvement of the growth by Ca application was much more significant in the cultured cells. Sodium content in the intact plants markedly increased with the increase of the NaCl concentration, compared to that in the cells. Calcium application decreased the Na content both in the cells and intact plants and the decreasing rate was higher in the cells than in the intact plants. Thus, it was suggested that the growth inhibition and the increase of the Na content under 100 mM NaCl conditions were more pronounced in intact plants, and that the alleviatory effect of Ca on the growth inhibition was much more conspicuous in the cells. Although the contents of total Ca and pectic acid-binding Ca in the cells and plant roots decreased with the increase of the NaCl concentration, they increased by Ca application. The activity of plasmalemma ATPase of the cells and plant roots decreased in the medium containing 100 mM NaCl with 0.3 mM CaCl2, but increased by Ca application, regardless of the NaCl concentration. In summary, Ca application to the culture medium containing 100 mM NaCl alleviated the growth inhibition, and the difference in the growth inhibition induced by 100 mM NaCl between the suspension culture cells and intact plants was closely related to that in the Na content. It appears that the alleviatory effect of Ca on the growth inhibition might be caused mainly by the enhancement of the Na-excluding function. The high content of pectic acid-binding Ca on the cell wall also contributed moderately to the alleviatory effect of Ca through the decrease of apoplasmic Na translocation.

Does H+ pumping by plasmalemma ATPase limit leaf growth of maize (Zea mays) during the first phase of salt stress?

AbstractIn the first phase of salt stress, growth of plants is impaired mainly by osmotic stress. To elucidate the effect of NaCl salinity on elongation growth of maize leaves in the first phase of salt stress, we investigated the effect of NaCl on gene expression and activity of the plasmalemma H+ ATPase of elongating leaves of maize (Zea mays L.). Treatment of maize plants with 125 mM NaCl for 3 d decreased leaf growth relative to control plants (1 mM NaCl). Whereas H+ ATPase hydrolytic activity was unaffected, the ability of the H+ ATPase to establish a pH gradient was strongly reduced. Total mRNA of plasmalemma H+ ATPase was slightly increased. However, mRNA of the ATPase isoform MHA1 was significantly reduced and ATPase isoform MHA4 was strongly increased at the mRNA level. Synthesis of total H+ ATPase protein was unchanged as revealed by western blot. The results indicate that reduced pumping of H+ ATPase in leaf plasmalemma under salt stress may be caused by a switch to gene expression of the specific isoform MHA4, which shows inferior H+‐pumping efficiency in comparison to isoforms expressed under control conditions. We propose that reduced H+ pumping of plasmalemma H+ ATPase is involved in the reduction of leaf growth of maize during the first phase of salt stress.

Energy-dependent solute transport from the apoplast into the symplast of leaves during transpiration

Inorganic and organic salts, amino acids, sugars, and phosphate esters (concentrations usually 25 mM) were fed via the transpiration stream through the petiole into detached leaves of Lepidium sativum and Solanum tuberosum. While water was lost by transpiration, solutes did not accumulate in the apoplast. Uptake into leaf cells was indicated by stimulation of respiration and by changes of membrane potential and apoplastic pH. Apoplastic alkalinization (followed by transient acidification) and membrane depolarization (followed by repolarization) indicated energization of transport at the expense of the proton motive force (PMF) across the plasma membrane in all examined cases. Loss of ATP in the symplast during proton extrusion into the apoplast by the plasmalemma ATPase is thought to be responsible for stimulation of respiration. Even unphysiological solutes such as β-morpholinoethane sulfonate (Mes), or potentially toxic salts such as CdCl2 or AlCl3, and metabolites involved in energy conservation such as AMP and NAD, were readily transported into leaf cells at the expense of metabolic energy. At the maximum stimulation of CO2 release by D-serine (which is unlikely to be metabolized) respiration exceeded basal respiration by an average of 33%. Occasionally, and with other solutes, basal respiration was almost doubled. The ratio of transported solute to released extra CO2 was 6.9 ± 1.1 (n = 11) in the case of D-serine. From this, maximum energized transport of D-serine was calculated to be close to 500 nmol/(m2 leaf area s). Solute/CO2 ratios similar to those observed with D-serine were also obtained for sucrose. Lower ones were observed with organic solutes such as L-glycine, pyruvate, malate or citrate where secondary metabolic conversions may contribute to CO2 release.

Changes in Parameters of the Plasmalemma ATPase during Peach Vegetative Bud Dormancy

Plant dormancy and dormancy breaking depend, at least partially, on close relationships between buds and tissues underlying bud (bud stands). In Prunus persica, the dormancy was related to high nutrient absorption in bud stands linked to high plasmalemma ATPase (EC 3.6.1.3) activity. Two plasmalemma fractions was isolated from peach vegetative buds and bud stands using aqueous phase partitioning and ultracentrifugation. Results of markers enzyme assays indicated that both plasmalemma enriched fractions obtained were highly purified. During the dormancy period plasma membrane ATPase amount and activity were higher in bud stands than in buds. Moreover, assays performed at different temperatures (4, 18, 30 °C) indicated modifications of kinetic parameters (Km, Vm) in both tissues during dormancy release. In buds, from November to February, Km declined at 4°C and increased at 30 °C whereas no changes was measured at 18 °C and Vm increased at all temperature. In bud stands, no changes of Km was measured at 4 °C and 18 °C whereas an increase occurred at 30 °C and Vm decreased at all temperature. According to the results, it can be postulated that dormancy release in peach-tree could be related to modifications of plasma membrane ATPase properties, in buds and bud stands, during winter time.

Adaptation of Active Proton Pumping and Plasmalemma ATPase Activity of Corn Roots to Low Root Medium pH1

Corn (Zea mays L.) root adaptation to pH 3.5 in comparison with pH 6. 0 (control) was investigated in long-term nutrient solution experiments. When pH was gradually reduced, comparable root growth was observed irrespective of whether the pH was 3.5 or 6.0. After low-pH adaptation, H+ release of corn roots in vivo at pH 5.6 was about 3 times higher than that of control. Plasmalemma of corn roots was isolated for investigation in vitro. At optimum assay pH, in comparison with control, the following increases of the various parameters were caused by low-pH treatment: (a) hydrolytic ATPase activity, (b) maximum initial velocity and Michaelis constant (c) activation energy of H+-ATPase, (d) H+-pumping activity, (e) H+ permeability of plasmalemma, and (f) pH gradient across the membranes of plasmalemma vesicles. In addition, vanadate sensitivity remained unchanged. It is concluded that plasmalemma H+-ATPase contributes significantly to the adaptation of corn roots to low pH. A restricted net H+ release at low pH in vivo may be attributed to the steeper pH gradient and enhanced H+ permeability of plasmalemma but not to deactivation of H+-ATPase. Possible mechanisms responsible for adaptation of plasmalemma H+-ATPase to low solution pH during plant cultivation are discussed.

Absorption and efflux of glyphosate by cell suspensions

The absorption and efflux of [14C]-glyphosate (N-[phosphonomethyl]glycine) was studied in maize (Zea mays L. cv. Aussie) and soybean (Glycine max L. Merr. cv. Maple Arrow) cell suspensions. Glyphosate absorption was complex: at low external herbicide concentrations (3–250 (μM) there was evidence for a single active uptake system with an apparent Km of 31 μM and Vmax of 11 nmol g−1 fr. wt. 2 h−1. The system was inhibited by carbonylcyanide m-chlorophenyl hydrazone (CCCP), orthovanadate, diethylstilbestrol (DES), phosphate, and phosphonoformic acid (PFA) suggesting the glyphosate carrier to be a phosphate transporter energized by the plant plasmalemma ATPase. At higher external glyphosate concentrations the operation of this carrier was masked as passive diffusion became the dominant absorption mechanism. Any non-specific binding of glyphosate to the cell surface during absorption was low (0.01–0.02 nmol g−1 fr. wt). Efflux kinetics of [14C]-glyphosate suggests the herbicide to be located in the cells in three kinetically distinct compartments: after 24 h uptake of radiolabelled herbicide, 71% of absorbed glyphosate was found in the slow compartment (t½, 162 h), 19% in the medium (t½, 185 min) and 10% in the fast (t½, 27 min). The implications of these results in relation to the delivery of glyphosate to its subcellular target site and subsequent phytotoxicity are discussed.

Effects of salicylic acid on sugar and amino acid uptake

of organic compounds such as hexoses, sucrose and amino acids (Sakr et al., 1993). Uptake rates of these nutrients Ageing of sugar beet (Beta vulgaris L.) leaf discs depends on the activity of their respective H+-cotransinduces a large stimulation of sugar and amino acid porters and on the plasmalemma ATPase which energizes uptake. This may result from an hormonal imbalance. this transport. Stimulation of sucrose uptake by ageing The effects of various hormones added during the results from an increased activity of the plasma membrane ageing period were studied on sugar and amino acid H+-ATPase energizing the sucrose transporter, whereas uptake. Salicylic acid (SA) inhibited both valine and stimulation of hexose and valine uptake involves both an sucrose uptake in a concentration-dependent manner increased activity of the ATPase and of the corresponding from 10 mM to 200 mM. This inhibition was specific for hexose and amino acid transporter (Noubhani et al., SA: various mono- and di-substituted analogues of 1996; Sakr et al., 1997). benzoic acid did not significantly affect uptake, except The eVects induced by ageing may result from excision for 2,4-dichlorobenzoic acid. SA inhibited the active of leaf tissues, removal of the lower epidermis and cutting component of uptake, and the inhibition was reversible of discs which provokes wounding responses in the tissues upon its removal. SA did not affect the amounts of and may aVect the hormonal balance. Volatile methyl transcripts encoding the sucrose transporter and the jasmonate can be released from plant tissues, especially amounts of the sucrose transporter protein. SA if wounded (Albrecht et al., 1993); the levels of aminodecreased medium acidification due to the activity of 1-cyclopropane carboxylic acid synthase transcripts level the plasma membrane H+-ATPase, without affecting increase largely between 1 h and 4 h after wounding in the amounts of ATPase transcripts and ATPase protein. sliced tomato fruit (Li et al., 1992), and it is well known SA significantly decreased the energy charge of the that auxins and cytokinins are involved in the formation tissues during ageing. It is suggested that SA may of wound-induced callus. Therefore, the increase of nutriaffect the uptake of sugars and amino acids by indirect ent uptake which develops with ageing may be aVected inhibition of the plasma membrane H+-ATPase ener- by hormones. This hypothesis was tested in sugar beet gizing this process. leaf discs using several phytohormones or hormone-like substances.

Ultrastructure and some plasma membrane characteristics of ozone‐exposed loblolly pine needles

Tropospheric ozone is a widespread and phytotoxic air pollutant in the industrialized world and causes reduced growth in many tree species. It is therefore important that, for example, the responses of the economically important loblolly pine to ozone are determined thoroughly. The objective of the study was to determine changes in ultrastructure, the vanadate‐sensitive ATPase activity of the plasma membrane, the fatty acids of plasma membrane phospholipids, visible injury, and growth in loblolly pine (Pinus taeda L.) needles exposed to different concentrations of ozone in open‐top chambers. The treatments were charcoal filtered air (CF), nonfiltered air (NF), or NF‐air with ozone added for 12 h daily at 1.5‐ or 2‐fold ambient ozone concentrations from May to October, 1993. Visible injury was more severe in the high than in the low ozone treatments. Growth of needles of the first flush of 1993 was significantly reduced in the highest ozone treatment. Two types of ultrastructural injury, characterized as either acute or chronic, were observed in mesophyll cells under elevated ozone. The acute injury lead to cell collapse and death. The chronic injury, characterized by several symptoms, e.g. decreased chloroplast size and increased density of the stroma, was also found in the NF ozone treatment. Increased density of chloroplast stroma and swelling of thylakoids were transient symptoms, suggesting partial recovery as ozone concentrations decreased in fall. Ozone induced decreases in the specific activity of vanadate‐sensitive ATPase of plasmalemma and in the degree of unsaturation in phospholipid fatty acids. The detected reduced needle growth, ultrastructural injury and perturbations in the function and composition of the plasma membrane indicate susceptibility of loblolly pine to ozone. Changes in the plasma membrane phospholipids may have contributed to the decrease in ATPase activity. Injury to the key enzyme of the plasma membrane can directly affect intracellular processes. In the long‐term, decreased viability of needles can lead to reductions in loblolly pine productivity.

Plasmalemma ATPase activity modifications induced by traumatisms inBidens pilosa

It has previously been shown that cotyledonary pricks induced modifications of ion levels (H + and K + ) in hypocotyl cells of Bidens pilosa. These modifications differed according to the light quality : H + levels increased and K + levels decreased in white light (WL), whereas H + levels decreased and K + levels increased in blue light (BL). In this study, in order to determine the mechanism responsible for these ionic modifications, plasma membrane vesicles have been isolated and characterized from hypocotyl cells. The effects of light quality and cotyledonary pricks on plasmalemma ATPase activity (EC 3.6.1.3) were studied. Cotyledonary pricks induced, in WL, rapid (5 min) and transient (restoration in 60 min) inhibition of plasmalemma ATPase activity. Conversely, in BL, a rapid and transient stimulation was observed. These results suggest that, in Bidens pilosa, plasmalemma ATPase is involved in 'short-term' ionic level modifications induced by traumatisms.

The Tonoplast H+-ATPase of Acer pseudoplatanus Is a Vacuolar-Type ATPase That Operates with a Phosphoenzyme Intermediate.

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure included size-exclusion and ion-exchange chromatography. The H+-ATPase consists of at least eight subunits, of 95, 66, 56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis thaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the vacuolar H+-ATPase of oat roots. The functional molecular size of the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+-ATPase was rapidly phosphorylated by [[gamma]-32P]ATP in vitro. The complete loss of radio-activity in the 66-kD subunit after a short pulse-chase experiment with unlabeled ATP reflected a rapid turnover, which characterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to hydroxylamine and alkaline pH. These results lead us to suggest that the tonoplast H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate with a plasmalemma-type ATPase catalytic mechanism.

Characterization of the Plasmalemma ATPase from the Cyanobacteria Synechococcus PCC 6311 and PCC 7942

Biochemical properties of the ATPase from the plasma membrane of the cyanobacteria Synechococcus PCC 6311 and PCC 7942 were examined. ATPase activity associated with purified plasma membrane vesicles was strongly inhibited by 100 μM vanadate (87%), 100 μM diethyistilbestrol (70%) and 100 mM fluoride ions (83%). No inhibition was observed in the presence of dicyclohexylcarbodiimide, nitrate, azide, or molybdate. A 50% activation was observed in the presence of 50 mM KCl but none was observed in the presence of NaCl or NH 4Cl. This ATPase was able to form a pH gradient, the amplitude of which was decreased by the presence of 100 μM vanadate. On Western blot of the plasmalemma proteins, no labeling was observed with a monoclonal antibody against the β subunit of the F 0-F 1 ATPase, although staining was observed with the 55-kDa subunit of the thylakoid membrane ATPase. After phosphorylation of plasmalemma vesicles, by [γ- 32P]ATP, the autoradiograms of the electrophoreses, performed under acid conditions, exhibited labeling of a 110-kDa protein. The results indicated that the Synechococcus plasma membrane ATPase can be classified as a H + translocating P-type ATPase and compared to the plant plasmalemma ATPase.

ATP-and NADH-dependent membrane potential generation in plasmalemma enriched vesicles from parenchyma of dormant and non-dormant jerusalem artichoke tubers

Using a membrane potential probe, Oxonol VI, it was possible to demonstrate generation of ATP- and NADH-dependent membrane potential across the plasmalemma, with membrane vesicles derived from parenchyma cells of Jerusalem artichoke tubers(Helianthus tuberosus L.). It was shown that ATP- and NADH-dependent membrane potential generation was higher in dormant material than in non-dormant tissue and that the effects of ATP and NADH on membrane potential generation were additive. ATP-dependent potential generation was sensitive to vanadate, an inhibitor of plasmalemma ATPase activity. The results are discussed in relation to the properties of the different enzymes bound to the plasma membrane, the morphogenetic potentialities of tuber buds and the hypothesis that tuber dormancy could be an extreme case of nutrient deficiency induced by short-distance intercellular relationships.

Importance of plasmalemma ATPase in the retention and exclusion of inorganic ions

AbstractThe experiments were focused on the question whether the plasmalemma ATPase activity (proton pump) has an influence on the efflux of major inorganic ion species. Efflux from roots of intact Trifolium pratense, Hordeum vulgare, Glycine max, and Zea mays was examined into a solution containing 100 μM CaCl2 and 500 μM NH4+ as sulfate in the control solution and 100 μM CaCl2 and 500 μM NH4+ as vanadate in the test solution. Vanadate being an inhibitor of the plasmalemma ATPase depressed significantly the H+ secretion of roots into the outer solution but had no major impact on the efflux of cation species. In the presence of vanadate significantly higher amounts of sulfate, phosphate, and nitrate were released into the outer solution by roots of soya and maize as compared with the control treatment (no vanadate). In the absence of vanadate, virtually no nitrate was released by all species examined whereas in the vanadate treatment significant amounts of NO3− were released. Vanadate inhibited the uptake of Cl− in barley and maize and increased the uptake of Ca2+ in soya. It is concluded that the plasmalemma ATPase activity plays a major rule in the “ionic stat” of cells in providing protons to the apoplast for the reabsorption of sulfate, phosphate, and particularly nitrate which have leaked out of the cytosol.

Inhibition of Malate Synthesis by Vanadate: Implications for the Cytosolic Alkalinization Induced by Vanadate Concentrations Partially Inhibiting the Plasmalemma H+Pump

The effects of Na-orthovanadate, at concentrations only partially inhibiting net H+ extrusion, were determined on vacuolar and cytosolic pH by the weak base and weak acid distribution at equilibrium. Treatment with vanadate induces in Elodea densa leaves and in Arabidopsis thaliana seedlings a moderate acidification of both cell sap and vacuole. Conversely, it induces an alkalinization of cytosol, this effect being in apparent contrast with a condition of reduced activity of the H+-transporting plasmalemma ATPase, which should be associated with a cytosolic acidification. In Arabidopsis seedlings treated with vanadate, the increase in pH of both cytosol and external medium is associated with a decrease in cell sap buffer capacity, more evident for higher vanadate concentrations, and particularly marked in the pH range between 3·5 and 5·5. In these conditions, the malate content is strongly reduced, its decrease almost completely accounting for the decrease in cell sap buffer capacity. An in vitro analysis of the vanadate effect on phosphoenolpyruvate carboxylase indicates that the decrease in malate content seems substantially due to an inhibiting effect of vanadate on this enzyme. These results stress that the in vivo use of vanadate as an inhibitor of the plasmalemma H+-ATPase must be taken with caution; in particular, for studying the correlations between changes in net H+ extrusion and changes in cytosolic pH and related processes.

Vanadate-sensitive ATPase in the plasmalemma of Acetabularia: biochemical and kinetic characterization

The electrogenic Cl(-)-pump in the plasmalemma of the marine alga Acetabularia acetabulum (L.) Silva has been suggested to be an unusual type of ATPase (Gradmann, 1989, Methods Enzymol. 174, 490). For a biochemical treatment of this issue, a plasmalemma-rich membrane fraction from Acetabularia has been prepared by phase-partitioning. About 80% of the ATPase activity in this material is inhibited by vanadate (K I50=1-2 μM). The phosphohydrolytic properties of the corresponding enzyme were further investigated. Its primary substrate MgATP(2-) (K m about 270 μM). Compared with other plasmalemma ATPases, it has an extremely alkaline pH optimum (pH 8-8.5), a weak sensitivity to diethyl-stilbestrol and to N,N'-dicyclohexylcarbodiimide, a strong selectivity for Mg(2+) over alternative divalent cations, and a weak selectivity for ATP over other phosphohydrolytic substrates. It is insensitive to KCl at concentrations up to 200 mM. Both ATP(4-) and Mg2ATP inhibit the ATPase, satisfying a relationship for competitive inhibition by 2ATP(4-) (K IATP=1.56 mM) and noncompetitive inhibition by Mg2ATP (K IMg2ATP=1.35 mM). Since no transport experiments are reported in this study, the ion species (H(+) or Cl(-)) that is transferred by this ATPase is not identified.

The modulating effects of pH and benzyladenine on the Ca2+– and Mg2+‐ATPases in the plasmalemma of wheat root cells

Plant cells frequently and rapidly have to respond to environmental changes for survival. Regulation of transport and other energy‐requiring processes in the plasmalemma of root cells is therefore one important aspect of the ecological adaptation of plants. Wheat (Triticum aestivum L. cv. Drabant) was grown hydroponically, with or without 50 nM benzyladenine in the medium, and plasma membranes from root cells of 8‐day‐old plants were prepared by aqueous polymer two‐phase partitioning. The influence of Ca2+ and Mg2+ on the plasmalemma ATPase activities was investigated. The presence of benzyladenine during growth increased the ATPase activity, that dependent upon Ca2+ more than that elicited by Mg2+. As a general characteristic, ATP was the preferred substrate, but all nucleotide tri‐ and diphosphates could be accepted with activities in plasma membranes from control plants of 7‐36% (Mg2+) and 40‐86% (Ca2+) and in plasma membranes from benzyladenine‐treated plants of 12‐47% (Mg2+) and 53‐102% (Ca2+) as compared with activities obtained with ATP. Nucleotidemonophosphates were not hydrolyzed by the preparations. In preparations from benzyladenine‐treated plants one peak of Ca2+‐ATPase at pH 5.2–5.6, with a tail from pH 6 and upwards, and one peak of Mg2+‐ATPase at pH 6.0–6.5 were observed in the presence of EDTA in the assay media. In preparations from control plants, the addition of EDTA to the assays resulted in a wide optimum between pH 6 and 7 for Mg2+‐ATPase and low Ca2+‐ATPase activity with no influence of pH in the range 4.5 to 8. Analysis of the pH dependence in the presence of both Ca2+ and Mg2+ indicates that the control plants mainly contain Mg2+‐ATPase corresponding to the proton pump. Preparations from benzyladenine‐treated wheat roots show, in addition, activation by Ca2+, which, in the slightly alkaline pH range may correspond to a Ca2+‐extruding (Ca2++ Mg2+)‐ATPase. In the acidic range, the responses are more complicated: the Mg2+‐ATPase is inhibited by vanadate, while the Ca2+‐ATPase is insensitive, and benzyladenine added during growth influences the interaction between Ca2+ and Mg2+ in a way that parallels the effect of high salt medium.