Bacterial phytotoxins, syringomycin, syringostatin and syringotoxin, exert their effect on the plasma membrane H+-ATPase partly by a detergent-like action and partly by inhibition of the enzyme

Syringostatin is a newly discovered phytotoxin produced by a phytopathogenic bacterium Pseudomonas syringae pv. syringae lilac isolate. The effects of syringostatin and the similar phytotoxins, syringomycin and syringotoxin, on H⁻-ATPase activity were investigated using cultured mung bean ( Vigna radiata L. cv. Ryokuto) cells or plasma membrane vesicles isolated from mung bean hypocotyls. ³¹P-NMR analysis of cultured cells treated with syringostatin revealed that the cytoplasmic pH was decreased. When plasma membrane was prepared by a two-step method (Dextran gradient followed by a sucrose gradient). syringostatin, syringomycin and syringotoxin inhibited the H⁺-ATPase activity in a dose-dependent manner. In contrast, these toxins stimulated H⁺-ATPase activity when plasma membrane was prepared by a one-step method (sucrose gradient). While these toxins inhibited the H⁺-ATPase activity of inside-out plasma membrane vesicles, the H⁺-ATPase activity of right-side-out vesicles was stimulated. The detergent. Triton X-100, abolished this stimulatory effect of the toxins on the H⁺-ATPase of right-side-out vesicles and of one-step purified plasma membrane. The toxins also inhibited the activity of the plasma membrane H⁺-ATPase solubilized with deoxycholate and Zwittergent 3–14. Taken together, these results indicate that these toxins exert their effects partly by a detergent-like action on the plasma membrane and partly by inhibition of the enzyme.     

Synergistic activation of plasma membrane H+– ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin

The regulation of the H⁺-ATPase of plasma membrane is a crucial point in the integration of transport processes at this membrane. In this work the regulation of H⁺-ATPase activity induced by changes in turgor pressure was investigated and compared with the stimulating effect of fusicoccin (FC). The exposure of cultured cells of Arabidopsis thaliana L. (ecotype Landsberg 310–14-2) to media containing mannitol (0. 15 or 0. 3 M ) or polyethylene glycol 6000 (PEG) (15. 6% or 22% w/v) resulted in a decrease in the turgor pressure of the cells and in a strong stimulation of H⁺ extrusion in the incubation medium. The osmotica-induced H⁺ extrusion was (1) inhibited by the inhibitor of plasma membrane H⁺-ATPase, erythrosin B (EB), (2) dependent on the external K⁺ concentration, (3) associated with a net K⁺ influx, and (4) lead to an increase of cellular malate content. These results show that the reduction of external osmotic potential stimulates the activity of plasma membrane H⁺-ATPase
The effect of mannitol was only partially inhibited by treatments with cycloheximide (CH) and cordycepin, which block protein and mRNA synthesis, respectively. All the effects of osmotica were qualitatively and quantitatively similar to those induced by 5 μ M FC. However, when FC and mannitol (or PEG) were fed together, their effects on H⁺ extrusion appeared synergistic, irrespective of whether FC was present at suboptimal or optimal concentrations. This behaviour suggests that the modes of action of FC and of the osmotica on H⁺-ATPase activity differ at least in some step(s)     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots

The effect of boron excess and deficiency on H⁺ efflux from excised roots from sunflower ( Heliarahus annuus L. cv. Enano) seedlings and on plasma membrane H⁺-ATPase (EC 3.6.1.35) in isolated KI-washed microsomes has been investigated. When seedlings were grown in media with toxic levels of H₃BO₃ (5 m M ) or without added boron and exposed to light conditions, an inhibition of the capacity for external acidification by excised roots was observed as compared to roots from seedlings grown with optimal H₃BO₃ concentration (0.25 m M ). Toxic and deficient boron conditions also inhibited the vanadate-sensitive H⁺-ATPase of microsomes isolated from the roots. The mechanism of boron toxicity was investigated in vitro with microsorne vesicles. A strong effect of boron on the vanadate-sensitive, ATP-dependent H⁺ transport was found, but the vanadate-sensitive phospho-bydrolase activity was not affected. These results suggest that boron could exert an effect on the plasma membrane properties, directly or indirectly regulating, proton transport.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Plasma membrane H-ATPase activity is involved in adaptation of tomato calli to NaCl

A tomato ( Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 m M NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 m M NaCl. Analysis of callus ion content showed a strong increase in Na⁺ and Cl⁻ both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H⁺ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H⁺ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H⁺-ATPase (EC 3.6.1.35) and ATP-dependent H⁺ transport, while the stimulation of H⁺ extrusion by cells tolerant to 50 m M NaCl was correlated with an increase in plasma membrane ATP-dependent H⁺ transport. The increase of ATP-dependent H⁺ extrusion in plasma membranes isolated from 50 m M NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H⁺. Relative to NaCl-sensitive calli, plasma membrane H⁺-ATPase from calli tolerant to 50 m M NaCl showed a lower K_m for Mg²⁺-ATP. Our results indicate that tolerance of tomato calli to 50 m M NaCl increases the affinity of plasma membrane H⁺-ATPase for the substrate ATP and stimulates the H⁺-pumping activity of this enzyme without modifying its phosphohydrolytic activity.     

Aquaporin functionality in relation to H+-ATPase activity in root cells of Capsicum annuum grown under salinity

As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H⁺-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H⁺-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na⁺, K⁺ or Cl⁻) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca²⁺. Similar results were obtained for cell hydraulic conductivity, Lp_c, and H⁺-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca²⁺. However, fusicoccin (an activator of H⁺-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg²⁺ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H⁺-ATPase and aquaporin activities may not be related in pepper plants.     

Immunopurification of H+-ATPase-containing lipid-protein particles from the cytosol of carnation petals

Lipid-protein particles originating from the plasma membrane were immunopurified from the cytosol of carnation petal cells ( Dianthus caryophyllus L. cv. Improved White Sim) using antibodies raised against the central hydrophilic domain of the H⁺-ATPase. The immunopurified particles are enriched in lipid metabolites, in particular free fatty acids and steryl/wax esters, by comparison with corresponding microsomal membranes, and the lipids of the particles are more saturated than those of microsomal membranes. Proteolytic catabolites of the H⁺-ATPase, a protein associated with the plasma membrane, but not the native H⁺-ATPase protein, are also present in the immunopurified cytosolic particles. Osmiophilic particles were discernible in the cytosol of carnation petal cells by transmission electron microscopy, and the association of H⁺-ATPase catabolites with a subpopulation of these particles was confirmed by immunogold labelling with H⁺-ATPase antiserum. Cross-reaction of the H⁺-ATPase antiserum with elements of the cytosol was also evident by immunofluorescent light microscopy. These observations collectively indicate that lipid-protein particles of plasma membrane origin are present in the cytosol of carnation petal cells and that their formation may serve as a means of removing lipid and protein metabolites from the plasma membrane which would otherwise destabilize its structure.     

Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe

The plasma membrane H⁺-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H⁺-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae , even after deletion of the enzyme's carboxy terminus. Functional chimerical H⁺-ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site-directed mutagenesis of the H⁺-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H⁺-ATPase. The reverse Ala-598 →Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H⁺-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells. ATPase activity showed a more alkaline pH optimum, lower K _m values for MgATP and decreased V _max compared with wild-type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S. pombe H⁺-ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- 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. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

NaCl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato

NaCl-induced changes in the accumulation of message for the 70 kDa subunit of the tonoplast H⁺-ATPase and plasma membrane H⁺-ATPase were studied in hydroponically grown plants of Lycopersicon esculentum Mill. cv. Large Cherry Red. There was increased accumulation of message for the 70 kDa (catalytic) subunit of the tonoplast H⁺-ATPase in expanded leaves of tomato plants 24 h after final NaCl concentrations were attained. This was a tissue-specific response; levels of this message were not elevated in roots or in young, unexpanded leaves. The NaCl-induced accumulation of this message was transient in the expanded leaves and returned to control levels within 7 days. The temporal and spatial patterns of NaCl-induced accumulation of message for the plasma membrane H⁺-ATPase differed from the patterns associated with the 70 kDa subunit of the tonoplast H⁺-ATPase. NaCl-induced accumulation of the plasma membrane H⁺-ATPase message occurred in both roots and expanded leaves. Initially accumulation of the plasma membrane H⁺-ATPase message was greater in root tissue than in expanded leaves, but increased to higher levels in expanded leaves after 7 days. These results suggest that increased expression of the tonoplast H⁺-ATPase is an early response to salinity stress and may be associated with survival mechanisms, rather than with long-term adaptive processes.     

Enhanced H+/ATP coupling ratio of H+-ATPase and increased 14-3-3 protein content in plasma membrane of tomato cells upon osmotic shock

Modulation of proton extrusion and ATP-dependent H⁺ transport through the plasma membrane in relation to the presence of 14-3-3 proteins in this membrane in response to osmotic shock was studied in tomato ( Lycopersicon esculentum Mill. cv. Pera) cell cultures. In vivo H⁺ extrusion by cells was activated rapidly and significantly after adding 100 m M NaCl, 100 m M KCl, 50 m M Na₂SO₄, 1.6% sorbitol or 2 µ M fusicoccin to the medium. The increase in H⁺ extrusion by cells treated with 100 m M NaCl was correlated with an increase of H⁺ transport by the plasma membrane H⁺-ATPase (EC 3.6.1.35), but not with changes in ATP hydrolytic activity of this enzyme, suggesting an increased coupling ratio of the enzyme. Immunoblot experiments showed increased amounts of 14-3-3 proteins in plasma membrane fractions isolated from tomato cells treated with 100 m M NaCl as compared to control cells without changing the amount of plasma membrane H⁺-ATPase. Together, these data indicate that in tomato cells an osmotic shock could enhance coupling between ATP hydrolysis and proton transport at the plasma membrane through the formation of a membrane 14-3-3/H⁺-ATPase complex.     

Effects of salt stress on H+- ATPase and H+-PPase activities of tonoplast-enriched vesicles isolated from sunflower roots

The control of ion concentration in the cytosol and the accumulation of ions in vacuoles are thought to be key factors in salt tolerance. These processes depend on the establishment in vacuolar membranes of an electrochemical H⁺ gradient generated by two distinct H⁺-translocating enzymes: a H⁺-PPase and a H⁺-ATPase. H⁺-lrans locating activities were characterized in tonoplast-enriched membrane fractions isolated by sucrose gradient centrifugation from sunflower ( Helianthus annuus L.) roots exposed for 3 days to different NaCl regimes. The 15/32% sucrose interface was enriched in membrane vesicles possessing a vacuolar-type H⁺-ATPase and a H⁺-PPase, as indicated by inhibitor sensitivity, pH optimum, substrate specificity, ion effects kinetic data and immunolabelling with specific antibodies. Mild and severe stress did not alter the pH profile, ion dependence, apparent K_m nor the amount of antigenic protein of either enzyme. Saline treatments slightly increased K⁺-stimulaied PPase activity with no change in ATPase activity, while both PP_i-dependent and NO₃-sensitive ATP-dependent H⁺ transport activities were strongly stimulated. These results are discussed in terms of an adaptative mechanism of the moderately tolerant sunflower plants to salt stress.     

Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast

Transport across the plasma membrane is driven by an electrochemical gradient of H⁺ ions generated by the plasma membrane proton pump (H⁺-ATPase). Random mutants of Arabidopsis H⁺-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H⁺-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H⁺ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H⁺ extrusion and in their ability to sustain an electrochemical H⁺ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H⁺-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H⁺ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters.     

Inhibition of Glutamate Uptake and Proton Pumping in Synaptic Vesicles by S-Nitrosylation

Abstract: Nitric oxide (NO; including NO^•, NO⁺, and NO⁻) was found to inhibit glutamate uptake by isolated synaptic vesicles of rat brain. This was observed when two unrelated NO donors, S -nitrosogluthathione and S -nitroso- N -acetylpenicillamine, were used. The primary target of NO is the H⁺-ATPase found in the synaptic vesicles, which leads to dissipation of the electrochemical proton gradient and inhibition of glutamate uptake. Oxyhemoglobin (12 µ M ) and, to a much lesser extent, methemoglobin protected the vacuolar H⁺-ATPase from inhibition. Inhibition of H⁺ pumping by NO was reversed by addition of 0.5 m M dithiothreitol. The results indicate that the vacuolar H⁺-ATPase from synaptic vesicles is inhibited by NO by a mechanism that involves S -nitrosylation of critical sulfhydryl groups in the enzyme. The interaction of NO with synaptic vesicles might be of importance for the understanding of the multiple effects of NO in neurotransmission.     

Plasma membrane ATPase activity during pepino (Solanum muricatum) ripening

Plasma membrane-enriched samples were extracted from pepino fruit (cv. El Camino) by phase partitioning. H⁺-ATPase (EC 3.6.1.35) activity in these samples increased during late fruit development (immediately before the onset of ripening) and western blotting confirmed there was an increase in enzyme abundance at this time. H⁺-ATPase activity decreased during early ripening and then increased again in the final phase of ripening. Immunolocalisation showed the plasma membrane H⁺-ATPase was most abundant in the outer cell layers of the fruit, which are considered to have a major role in determining fruit texture. Fruit softening was not accelerated by harvest and there was no stimulation of H⁺-ATPase activity by harvest. An in vitro tensile test using fruit rings showed tissue softening proceeded faster at low apoplastic pH (4.5) than at pH 6.5; and tissue buffered at pH 6.5 softened less than unbuffered rings. Erythrosin B, an inhibitor of the plasma membrane H⁺-ATPase, also retarded softening in vitro. These data suggest that plasma membrane H⁺-ATPase activity may contribute to the onset of pepino softening through a reduction in apoplastic pH.     

Proton-ATPase activity in cells of lactobacilli grown in the presence of propionate

Lactobacillus helveticus ATCC 15009 and CRL 581, and Lact. casei LC3 were grown in a complex medium with and without 15 mmol 1^-1 of neutralized propionic acid and assayed for proton-translocating ATPase activity. The enzyme activity was higher when the medium contained fatty acid than in its absence for all strains studied. Characteristics of this increased ATPase were identical to those of the enzyme located on the membrane of normal cells. The substrate consumption rate of resting cells was increased by propionate. This effect was reverted by the specific H⁺-ATPase inhibitor N,N '-dicyclohexylcarbodiimide indicating that the increment of fermentative activity was related to the H⁺-ATPase activity. These results suggest that the amplification of H⁺-ATPase activity could be involved in the inhibition of lactobacilli growth in cultures where propionic acid is unavoidably present, such as some mixed cultures with propionibacteria.