Abscisic acid does not mediate NaCl-induced accumulation of 70-kDa subunit tonoplast H+ -ATPase message in tomato

There is an increased accumulation of message for the catalytic (70-kDa) subunit of the tonoplast H⁺-ATPase in leaves of tomato (Lycopersicon esculentum L.) plants responding to NaCl. To determine if abscisic acid (ABA) mediates this response, message accumulation was examined in treatments designed to separate exposure to NaCl from increases in endogenous ABA. Under three different experimental conditions, salt-induced changes in the accumulation of 70-kDa message were unrelated to any change in endogenous ABA. The results were as follows: (i) under drought stress, plants accumulated levels of ABA similar to those measured in salt-treated plants; however, no increase in 70-kDa subunit message was observed; (ii) the ABA-deficient mutant sitiens exhibited an increased accumulation of message despite the absence of NaCl-induced accumulation of ABA; and (iii) the inhibitor of general isoprenoid biosynthesis, Lovastatin, blocked NaCl-induced accumulation of ABA but did not alter NaCl-induced accumulation of message. In addition to these three experimental responses, application of exogenous ABA increased endogenous ABA levels without any comparable increase in message accumulation. Based on these results, it is concluded that ABA does not mediate the NaCl-induced accumulation of 70-kDa subunit tonoplast H⁺ -ATPase message accumulation in tomato.     

Changes in membrane-associated H+-ATPase activities and amounts in young grape plants during the cross adaptation to temperature stresses

Proton pumps make a critical contribution to the physiology of plants, although it remains unclear whether or not membrane-associated H⁺-ATPase is involved in the cross adaptation to different temperature stresses. This experiment investigated the changes in membrane-associated H⁺-ATPase activities that associated with chilling-treated plants after heat acclimation (HA, 38 °C/10 h) and with heat-treated plants after cold acclimation (CA, 8 °C/2.5 days) in annual young grape plants (Vitis vinifera L. cv. Jingxiu) using biochemical and electron microscopic cytochemical assay methods in which cerium trichloride (CeCl₃) precipitation was adopted. The results indicated that plasma membrane H⁺-ATPase activity increased as a result of both pretreatments, while V-type and F-type H⁺-ATPase activity hardly changed. Under subsequent cross temperature stresses, however, the three H⁺-ATPase types did maintain higher activity levels than that of the control. This finding suggests that either a HA or CA pretreatment may promote stability in membrane-associated H⁺-ATPase. A western-blotting assay of the plasma membrane H⁺-ATPase (P-H⁺-ATPase) indicated that the immuno-signal intensity of a 100 kDa peptide was visibly stronger in the HA and CA pretreated plants than in the control both before and after stress. This suggests that the HA- or CA-induced P-type H⁺-ATPase activation can be partly attributed to a new synthesis of the enzyme protein. Further, the results also suggested that membrane-associated H⁺-ATPase was involved in the HA-induced chilling resistance and the CA-induced thermo-tolerance in grape plants and that they had a similar regulating mechanism.     

Subunit E of the vacuolar H+-ATPase of Hordeum vulgare L.: cDNA cloning,expression and immunological analysis†

A tonoplast protein of 31 kDa apparent molecular mass (TpP 31) was isolated from two-dimensional gels. Amino acid sequences were determined from LysC endoproteinase-peptide fragments. Using degenerate oligonucleotides, a corresponding cDNA clone of 1034 bp was isolated from a barley leaf cDNA library. It encodes for subunit E of the vacuolar H⁺-ATPase, the first one identified in plants so far. The open reading frame extends over 681 bp, encoding a gene product of 227 amino acids and a calculated molecular weight of 26 228 g mol^?1. Northern and Western blot analysis indicates constitutive expression of subunit E in all plant organs with only small effects of salt stress. Localization of TpP 31 at the tonoplast was confirmed in fractions of purified vacuolar membrane obtained by free-flow electrophoresis. Immunoprecipitation of newly synthesized ³⁵S-labelled membrane proteins with anti-TpP 31 gave two additional bands with apparent molecular masses of about 53 and 62 kDa. Gel filtration after mild solubilization showed co-purification of TpP 31 with the 55 kDa subunit of the H⁺-ATPase. Both results provide evidence beyond the sequence homology that TpP 31 is a structural component of the vacuolar H⁺-ATPase.     

Molecular cloning and characterisation of gamma subunit of H+-ATPase inLactobacillus acidophilus MG2-9

The acid tolerance is considered as one of the most important properties of lactic acid bacteria as probiotics. H⁺-ATPase is one of the genes associated with the ability of acid tolerance. We have cloned and sequenced full length cDNA of γ subunit of H⁺-ATPase gene inLactobacillus acidophilus MG2-9. The cDNA sequence consists of 975 nucleotides; the putative protein has 320 amino acids.     

Subunit Composition and Organization of the Vacuolar H-ATPase from Oat Roots

The vacuolar H⁺-translocating ATPase (H⁺-ATPase), originally reported to consist of three major subunits, has been further purified from oat roots (Avena sativa var Lang) to determine the complete subunit composition. Triton-solubilized ATPase activity was purified by gel filtration on Sephacryl S400 and ion-exchange chromatography (Q-Sepharose). ATP hydrolysis activity of purified preparations was inhibited by 100 nanomolar bafilomycin A₁, a specific vacuolar-type ATPase inhibitor. The purified oat H⁺-ATPase (relative molecular weight = 650,000) was composed of polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. To analyze the organization of the H⁺-ATPase subunits, native vacuolar membranes were treated with KI and MgATP to dissociate peripheral proteins. Release of 70, 60, 44, 42, 36, and 29 kilodalton polypeptides from the membrane was accompanied by a loss of ATP hydrolysis and ATP-dependent H⁺-pumping activities. Five of the peripheral subunits were released from the membrane as a large complex of 540 kilodaltons. Vesicles that had lost the peripheral sector of the ATPase could hold a pH gradient generated by the proton-translocating pyrophosphatase, suggesting that the integral sector of the ATPase did not form a H⁺-conducting pathway. Negative staining of native vesicles revealed knob-like structures of 10 to 12 nanometers in dense patches on the surface of vacuolar membranes. These structures were removed by MgATP and KI, which suggested that they were the peripheral sectors of the H⁺-ATPase. These results demonstrate that the vacuolar H⁺-ATPase from oat roots has 10 different subunits. The oat vacuolar ATPase is organized as a large peripheral sector and an integral sector with a subunit composition similar, although not identical to, other eukaryotic vacuolar ATPases. Variations in subunit composition observed among several ATPases support the idea that distinct types of vacuolar H⁺-ATPases exist in plants.     

Crosstalk between blue-light- and aba-signaling pathways in stomatal guard cells

We recently established an immunohistochemical method for the detection of blue light (BL)-induced and phototropin-mediated phosphorylation of plasma-membrane H⁺-ATPase in stomatal guard cells of Arabidopsis thaliana. This technique makes it possible to detect the phosphorylation/activation status of guard-cell H⁺-ATPase in the epidermis of a single rosette leaf, without the need to prepare guard-cell protoplasts (GCPs) from a large number of plants. Moreover, it can detect guard-cell responses under more natural and stress-free conditions compared to using GCPs. Taking advantage of these properties, we examined the effect of abscisic acid (ABA) on BL-induced phosphorylation of guard-cell H⁺-ATPase by using ABA-insensitive mutants. This revealed inhibition of BL-induced phosphorylation of guard-cell H⁺-ATPase via the early ABA-signaling components PYR/PYL/RCAR-PP2Cs-SnRK2s, which are known to be early ABA-signaling components for a wide range of ABA responses in plants.      

Cold stress causes rapid but differential changes in properties of plasma membrane H-ATPase of camelina and rapeseed

Camelina (Camelina sativa) and rapeseed (Brassica napus) are well-established oil-seed crops with great promise also for biofuels. Both are cold-tolerant, and camelina is regarded to be especially appropriate for production on marginal lands. We examined physiological and biochemical alterations in both species during cold stress treatment for 3 days and subsequent recovery at the temperature of 25 °C for 0, 0.25, 0.5, 1, 2, 6, and 24 h, with particular emphasis on the post-translational regulation of the plasma membrane (PM) H⁺-ATPase (EC3.6.3.14). The activity and translation of the PM H⁺-ATPase, as well as 14-3-3 proteins, increased after 3 days of cold stress in both species but recovery under normal conditions proceeded differently. The increase in H⁺-ATPase activity was the most dramatic in camelina roots after recovery for 2 h at 25 °C, followed by decay to background levels within 24 h. In rapeseed, the change in H⁺-ATPase activity during the recovery period was less pronounced. Furthermore, H⁺-pumping increased in both species after 15 min recovery, but to twice the level in camelina roots compared to rapeseed. Protein gel blot analysis with phospho-threonine anti-bodies showed that an increase in phosphorylation levels paralleled the increase in H⁺-transport rate. Thus our results suggest that cold stress and recovery in camelina and rapeseed are associated with PM H⁺-fluxes that may be regulated by specific translational and post-translational modifications.     

Immunocytochemical localization of H+?K+-ATPase in the rat colon

Summary The presence and distribution of gastric-type H⁺−K⁺-ATPase were investigated in the rat colon using a monoclonal antibody raised against hog gastric H⁺−K⁺-ATPase. Rat stomach was used as positive control. Rat kidney and ileum, in both of which H⁺−K⁺-ATPase has been reported in the past, were also studied. In stomach, very strong staining was found confined to the parietal cells, and a strong band atM _r∼94 kDa on the immunoblots. In colon a moderate staining was found in the supranuclear region of the epithelial cells, with similar intensity and distribution of staining of the surface and deep mucosa of the crypts, throughout the length of the colon. Another monoclonal antibody, specific to the 31 kDa subunit of H⁺-ATPase, used as a negative control, or omission of the primary antibody, resulted in lack of any staining in either colon or stomach. On immunoblots of homogenates of colonic mucosa, no specific band could be identified, either due to very low expression of the H⁺−K⁺-ATPase or loss of antigenicity of the epitope during the processing steps. No positive staining was observed in rat kidney and ileum, suggesting that they contain isoforms that are structurally different.     

NaCl increases the activity of the plasma membrane H+-ATPase in C3 halophyte Suaeda salsa callus

Suaeda salsa calli treated with different concentrations of NaCl were used to examine the response of the plasma membrane (PM) H⁺-ATPase to NaCl and its role in salt tolerance. The optimum concentration of NaCl for growth of the calli was 50 mM, while growth was significantly inhibited at 250 mM NaCl. The ion and organic solute contents of calli increased with increasing NaCl. Activity of the PM H⁺-ATPase increased when the calli were treated with NaCl over a certain concentration range (0–150 mM NaCl). However, the activity reached its maximum with 150 mM NaCl. Immunoblotting analysis of the PM H⁺-ATPase protein from calli cultures with anti-Zea mays H⁺-ATPase serum (monoclonal 46E5B11D5) identified a single polypeptide of ~90 kDa. The peptide levels increased in the calli treated with NaCl at 150 mM NaCl compared to control, but the increase at 50 mM NaCl was less pronounced. Northern blot analysis showed that the expression of the PM H⁺-ATPase also increased after the calli were treated with NaCl. These results suggest that the increase in PM H⁺-ATPase activity is due to both an increase in the amount of PM H⁺-ATPase protein and an up-regulation of the PM H⁺-ATPase gene, which is involved in the salt tolerance of S. salsa calli.     

Essential sulfhydryl groups in the catalytic center of the tonoplast H+-ATPase from coleoptiles ofZea mays L. as demonstrated by the biotin-streptavidin-peroxidase system

Functional properties and the localization of essential SH-groups of the tonoplast H⁺-ATPase fromZea mays L. were studied. In contrast to the pyrophosphate-dependent H⁺-translocation activity of the tonoplast, the H⁺-ATPase activity was inhibited by SH-blocking agents, such as N-ethylmaleimide and iodoacetic acid. In the case ofp-hydroxymercuribenzoate, HgCl₂ and oxidized glutathione, the inhibition could be reversed by adding reduced glutathione or dithiothreitol. Incubation of tonoplast vesicles with oxidized glutathione or N-ethylmaleimide in the presence of Mg·ADP—a competitive inhibitor of the ATP-dependent H⁺ pump—avoided the inhibition of the H⁺-pumping activity. This effect is an indication for the occurrence of essential SH-groups at the catalytic site of the H⁺-ATPase. In order to characterize the active center these thiols were specifically labeled with maleimidobutyrylbiocytin. Subsequently, the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an immobilizing membrane. The maleimidobutyrylbiocytin-labeled active-center protein was detected by a biotin-streptavidin-peroxidase staining system and was shown to be a 70-kDa subunit of the tonoplast H⁺-ATPase. It is suggested that the oxidation state of the critical sulfhydryl groups within the active center of the enzyme and their reversible blocking by endogenous compounds might be of great importance for the regulation of the enzyme activity in vivo.     

Wheat V-H+-ATPase Subunit Genes Significantly Affect Salt Tolerance in Arabidopsis thaliana

Genes for V-H⁺-ATPase subunits were identified and cloned from the salt-tolerant wheat mutant RH8706-49. Sequences of these genes are highly conserved in plants. Overexpression of these genes in Arabidopsis thaliana improved its salt tolerance, and increased the activities of V-H⁺-ATPase and Na⁺/H⁺ exchange, with the largest increase in plants carrying the c subunit of V-H⁺-ATPase. Results from quantitative RT-PCR analysis indicated that the mRNA level of each V-H⁺-ATPase subunit in the Arabidopsis increased under salt stress. Overall, our results suggest that each V-H⁺-ATPase subunit plays a key role in enhancing salt tolerance in plants.     

Active Detergent-solubilized H+,K+-ATPase Is a Monomer

The H⁺,K⁺-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a β-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H⁺,K⁺-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C₁₂E₈, followed by exchange of C₁₂E₈ with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the β-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s_20,w = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H⁺,K⁺-ATPase α,β-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,β-protomer with 0.9–1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α₂,β₂ dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H⁺,K⁺-ATPase. Thus, α,β H⁺,K⁺-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca²⁺-ATPase and Na⁺,K⁺-ATPase.