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.     

Intracellular localisation of PPI1 (proton pump interactor, isoform 1), a regulatory protein of the plasma membrane H+-ATPase of Arabidopsis thaliana

PPI1 (proton pump interactor isoform 1) is a novel protein able to interact with the C-terminal autoinhibitory domain of the Arabidopsis thaliana plasma membrane (PM) H⁺-ATPase. In vitro, PPI1 binds the PM H⁺-ATPase in a site different from the known 14-3-3 binding site and stimulates its activity. In this study, we analysed the intracellular localisation of PPI1. The intracellular distribution was monitored in A. thaliana cultured cells by immunolocalisation using an antiserum against the PPI1 N-terminus and in Vicia faba guard cells and epidermal cells by transient expression of a GFP::PPI1 fusion. The results indicate that the bulk of PPI1 is localised at the endoplasmic reticulum, from which it might be recruited to the PM for interaction with the H⁺-ATPase in response to as yet unidentified signals.     

A Phosphopeptide Corresponding to the Cytosolic Stretch Connecting Transmembrane Segments 8 and 9 of the Plasma Membrane H+-ATPase Binds 14-3-3 Proteins and Inhibits Fusicoccin-Induced Activation of the H+-ATPase

Abstract: A putative consensus domain for binding of 14-3-3 proteins to the plasma membrane (PM) H⁺-ATPase was identified in the highly-conserved sequence RSR(p)SWSF [where (p)S is Ser776 of the maize isoform MHA2], localized in the cytosolic stretch connecting transmembrane segments 8 and 9. A 15 amino acid biotinylated phosphopeptide comprising this motif: i) bound a recombinant 14-3-3 protein, ii) inhibited fusicoccin-induced stimulation of the PM H⁺-ATPase activity both in PM isolated from germinating radish ( Raphanus sativus L.) seedlings and in ER isolated from Saccharomyces cerevisiae expressing AHA1 (an isoform of Arabidopsis thaliana PM H⁺-ATPase), and iii) inhibited fusicoccin binding to PM isolated from germinating radish seedlings. The corresponding non-phosphorylated peptide was inactive in all the performed assays. Together, these results suggest that the cytosolic strand connecting transmembrane segments 8 and 9 of the PM H⁺-ATPase is a 14-3-3 binding site which might cooperate with the C-terminal domain of the'enzyme in generating a stable association between the H⁺-ATPase and 14-3-3 protein.     

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.     

Evidence for an indirect coupling mechanism for the nitrate-sensitive proton pump from corn root tonoplast membranes

The nitrate-sensitive proton-translocating adenosine triphosphatase (H⁺-ATPase) of tonoplast membranes plays an important role in regulating the flow of nutrients and metabolic waste between the cytoplasm and vacuole in the cells of plant roots. Relatively little information is available regarding the coupling between ATP hydrolysis and proton pumping by the nitrate-sensitive, tonoplast H⁺-ATPase. The coupling may be achieved either directly, i. e. the two reaction pathways share at least one common molecular step, or indirectly, i. e. the two reaction pathways do not share an intermediate step. These coupling mechanisms may be differentiated by the responses of the two events to external perturbation. The effects of the presence of nitrate in the assay medium on the rates of ATP hydrolysis and proton transport catalyzed by the tonoplast H⁺-ATPase from maize ( Zea mays L. cv. FRB 73) were investigated. The presence of nitrate inhibited proton transport activity of the tonoplast H⁺-ATPase to a much greater degree than ATP hydrolysis. This differential response of the two activities to nitrate is the basis for a proposed reaction model for the tonoplast H⁺-ATPase that features an indirect coupling mechanism between ATP hydrolysis and proton transport.     

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.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

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.     

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.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

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.     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage.To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS).The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.     

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.     

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.