Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H+-ATPase in stomatal guard cells

Blue light (BL) receptor phototropins activate the plasma membrane H(+)-ATPase in guard cells through phosphorylation of a penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H?-ATPase, mediating stomatal opening. To date, detection of the phosphorylation level of the guard cell H?-ATPase has been performed biochemically using guard cell protoplasts (GCPs). However, preparation of GCPs from Arabidopsis for this purpose requires >5,000 rosette leaves and takes >8 h. Here, we show that BL-induced phosphorylation of guard cell H?-ATPase is detected in the epidermis from a single Arabidopsis rosette leaf via an immunohistochemical method using a specific antibody against the phosphorylated penultimate threonine of H?-ATPase. BL-induced phosphorylation of the H?-ATPase was detected immunohistochemically in the wild type, but not in a phot1-5 phot2-1 double mutant. Moreover, we found that physiological concentrations of the phytohormone ABA completely inhibited BL-induced phosphorylation of guard cell H?-ATPase in the epidermis, and that inhibition by ABA in the epidermis is more sensitive than in GCPs. These results indicate that this immunohistochemical method is very useful for detecting the phosphorylation status of guard cell H?-ATPase. Thus, we applied this technique to ABA-insensitive mutants (abi1-1, abi2-1 and ost1-2) and found that ABA had no effect on BL-induced phosphorylation in these mutants. These results indicate that inhibition of BL-induced phosphorylation of guard cell H?-ATPase by ABA is regulated by ABI1, ABI2 and OST1, which are known to be early ABA signaling components for a wide range of ABA responses in plants.     

Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte

The plasma membrane H⁺-ATPase provides the driving force for solute transport via an electrochemical gradient of H⁺ across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H⁺-ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H⁺-ATPase (pT H⁺-ATPase) and non-pT H⁺-ATPase as in the green algae, and that pT H⁺-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H⁺-ATPase genes, designated PpHA (Physcomitrella patens H⁺-ATPase). Six isoforms are the pT H⁺-ATPase; a remaining isoform is non-pT H⁺-ATPase. An apparent 95-kD protein was recognized by anti-H⁺-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H⁺-ATPase. Furthermore, we could not detect the pT H⁺-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H⁺-ATPase most likely appeared for the first time in bryophyte.     

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.     

Phytohormone effects on hydrolytic activity of phosphohydrolases in red beet (Beta vulgaris L.) tonoplasts

The effects of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA₃) and kinetin on the hydrolytic activity of proton pumps (adenosine triphosphatase, H⁺-ATPase, pyrophosphatase, H⁺-PPase) of tonoplasts isolated from stored red beet (Beta vulgaris L. cv. Bordo) roots were studied. Results suggest that the phytohormones can regulate the hydrolytic activities of H⁺-ATPase and H⁺-PPase of the vacuolar membrane. Each of the proton pumps of the tonoplast has its own regulators in spite of similar localization and functions. IAA and kinetin seem to be regulators of the hydrolytic activity for H⁺-PPase whereas for H⁺-ATPase it may be GA₃. Stimulation of enzyme activity by all hormones occurred at concentrations of 10^–6 to 10^–7 M.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA₃ gibberellic acid - H⁺-ATPase adenosine triphosphatase - H⁺-PPase pyrophosphatase - ATP adenosine triphosphate - Tris Tris (hydroxymethyl)-aminomethane - MES (2[N-Morpholino]) ethane sulfonic acid - EDTA ethylene diamine tetraacetic acid - P_i inorganic phosphate     

Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast

Nitric oxide (NO), an endogenous signaling molecule in animals and plants, mediates responses to abiotic and biotic stresses. Our previous work demonstrated that 100 μM sodium nitroprusside (SNP, an NO donor) treatment of maize seedlings increased K⁺ accumulation in roots, leaves and sheathes, while decreasing Na⁺ accumulation (Zhang et al. in J Plant Physiol Mol Biol 30:455–459, 2004b). Here we investigate how NO regulates Na⁺, K⁺ ion homeostasis in maize. Pre-treatment with 100 μM SNP for 2 days improved later growth of maize plants under 100 mM NaCl stress, as indicated by increased dry matter accumulation, increased chlorophyll content, and decreased membrane leakage from leaf cells. An NO scavenger, methylene blue (MB-1), blocked the effect of SNP. These results indicated that SNP-derived NO enhanced maize tolerance to salt stress. Further analysis showed that NaCl induced a transient increase in the NO level in maize leaves. Both NO and NaCl treatment stimulated vacuolar H⁺-ATPase and H⁺-PPase activities, resulting in increased H⁺-translocation and Na⁺/H⁺ exchange. NaCl-induced H⁺-ATPase and H⁺-PPase activities were diminished by MB-1. 1-Butanol, an inhibitor of phosphatidic acid (PA) production by phospholipase D (PLD), reduced NaCl- and NO-induced H⁺-ATPase activation. In contrast, applied PA stimulated H⁺-ATPase activity. These results suggest that NO acts as a signal molecule in the NaCl response by increasing the activities of vacuolar H⁺-ATPase and H⁺-PPase, which provide the driving force for Na⁺/H⁺ exchange. PLD and PA play an important role in this process.     

V H-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin (I₅₀ = 38.4 nM) than Golgi and vacuole pumps (I₅₀ = 0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.     

Identification and biochemical characterization of the plasma-membrane H+-ATPase in guard cells of Vicia faba L.

The plasma-membrane H⁺-pump in guard cells generates the driving force for the rapid ion fluxes required for stomatal opening. Since our electrophysio-logical studies revealed a two fold higher pump-current density in guard cells than in mesophyll cells of Vicia faba L. we elucidated the biochemical properties of this proton-translocating ATPase in plasma-membrane vesicles isolated from both cell types. The capability of the H⁺ —ATPase to create an H⁺ gradient is maintained in plasma-membrane vesicles derived from purified guard cells via blender maceration, high-pressure homogenization and polymer separation. The H⁺-pumping activity of these vesicles coincides with the presence of two polypeptides of approx. 100 and 92 kDa which are recognized by a monoclonal antibody raised against the plasma-membrane H⁺-ATPase from Zea mays L. coleoptiles. Comparison of H⁺-pumping activities of isolated membranes revealed an approximately two fold higher activity in guard cells than in mesophyll cells with respect to the total membrane protein content. Furthermore, we demonstrated by western blotting that the difference in pump activities resulted from a higher abundance of the electroenzyme per unit membrane protein in guard-cell plasma membranes. We suggest that the high H⁺-pump capacity is necessary to enable guard cells to respond to sudden changes in the environment by a change in stomatal aperture.     

Cloning and Characterization of MxVHA-c, a Vacuolar H+-ATPase Subunit C Gene Related to Fe Efficiency from Malus xiaojinensis

The vacuolar H⁺-ATPase plays a crucial role in secondary transport and in plant response to environmental stress. In this study, a vacuolar H⁺-ATPase (MxVHA-c) gene, consisting of an ORF of 498 base pairs and 165 amino acid residues, has been cloned from the iron-efficient genotype of Malus xiaojinensis. Subsequently, this gene has been targeted to the tonoplast using transient expression analysis. Quantitative real-time (qRT) PCR results reveal that the MxVHA-c gene is expressed in both roots and leaves of Fe-deficient plants; however, it is sensitive to iron stress in roots. This suggests that MxVHA-c expression in roots may mediate iron-dependent responses. MxVHA-c expression is up-regulated following exogenous treatment with abscisic acid (ABA) and down-regulated following treatment with CaCl₂. Overexpression of the MxVHA-c gene in yeast strains has revealed that MxVHA-c transiently alleviated cadmium toxicity via the Cd²⁺/H⁺ antiport protein. H⁺-ATPase activity is slightly increased in yeast overexpressing the MxVHA-c gene compared to that in yeast transformed with an empty vector. In addition, this transgenic yeast strain can grow in a liquid medium containing 40???M ferrozine. These findings may provide useful information in elucidating molecular mechanisms that mediate resistance to iron deficiency.     

Phosphatidic Acid Inhibits Blue Light-Induced Stomatal Opening via Inhibition of Protein Phosphatase 1

Stomata open in response to blue light under a background of red light. The plant hormone abscisic acid (ABA) inhibits blue light-dependent stomatal opening, an effect essential for promoting stomatal closure in the daytime to prevent water loss. However, the mechanisms and molecular targets of this inhibition in the blue light signaling pathway remain unknown. Here, we report that phosphatidic acid (PA), a phospholipid second messenger produced by ABA in guard cells, inhibits protein phosphatase 1 (PP1), a positive regulator of blue light signaling, and PA plays a role in stimulating stomatal closure in Vicia faba. Biochemical analysis revealed that PA directly inhibited the phosphatase activity of the catalytic subunit of V. faba PP1 (PP1c) in vitro. PA inhibited blue light-dependent stomatal opening but did not affect red light- or fusicoccin-induced stomatal opening. PA also inhibited blue light-dependent H⁺ pumping and phosphorylation of the plasma membrane H⁺-ATPase. However, PA did not inhibit the autophosphorylation of phototropins, blue light receptors for stomatal opening. Furthermore, 1-butanol, a selective inhibitor of phospholipase D, which produces PA via hydrolysis of phospholipids, diminished the ABA-induced inhibition of blue light-dependent stomatal opening and H⁺ pumping. We also show that hydrogen peroxide and nitric oxide, which are intermediates in ABA signaling, inhibited the blue light responses of stomata and that 1-butanol diminished these inhibitions. From these results, we conclude that PA inhibits blue light signaling in guard cells by PP1c inhibition, accelerating stomatal closure, and that PP1 is a cross talk point between blue light and ABA signaling pathways in guard cells.Stomatal guard cells in the epidermis of aerial plants regulate gas exchange between leaves and the atmosphere, allowing the uptake of CO₂ for photosynthesis and the loss of water by transpiration. Guard cells integrate a wide variety of stimuli such as light, humidity, temperature, CO₂, and plant hormones to prevent excessive water loss and optimize plant growth under changing environmental conditions (Vavasseur and Raghavendra, 2005; Shimazaki et al., 2007). Among them, blue light and abscisic acid (ABA) represent key factors that promote stomatal opening and closure, respectively (Assmann and Shimazaki, 1999; Hetherington, 2001; Schroeder et al., 2001; Roelfsema and Hedrich, 2005). Blue light induces H⁺ pumping by activation of the plasma membrane H⁺-ATPase, which causes membrane hyperpolarization and drives K⁺ uptake into guard cells via inward-rectifying K⁺ channels (Assmann et al., 1985; Shimazaki et al., 1986; Schroeder et al., 1987). By contrast, ABA activates the anion channels, thereby causing membrane depolarization and promoting K⁺ efflux from guard cells via outward-rectifying K⁺ channels (Schroeder et al., 1987). There is cross talk between the opening and closure systems, and ABA inhibits blue light-induced activation of the H⁺-ATPase (Shimazaki et al., 1986; Goh et al., 1996; Roelfsema et al., 1998). Such inhibition of H⁺-ATPase by ABA is crucial to maintain the plasma membrane depolarization and supports efficient stomatal closure of open stomata. For example, when H⁺-ATPase is kept in the active state, as was found in the open stomata2 mutants, plants lost the stomatal closure response to ABA, which brought about the wilty phenotype even under well-watered conditions (Merlot et al., 2002, 2007). Although the regulation of the stomatal opening system by ABA is important for plant survival, the mechanism by which ABA inhibits the activation of H⁺-ATPase by blue light is largely unknown.Blue light is required for the activation of phototropins, plant-specific Ser/Thr autophosphorylating kinases, and the activated phototropins transmit the signal to the plasma membrane H⁺-ATPase for its activation (Kinoshita et al., 2001; Christie, 2007). Activation of the H⁺-ATPase is caused by the phosphorylation of a Thr residue in the C terminus with subsequent binding of a 14-3-3 protein to the Thr residue (Kinoshita and Shimazaki, 1999; Emi et al., 2001). Since phototropins are Ser/Thr protein kinases, it might be possible that phototropins directly phosphorylate the H⁺-ATPase. However, this has been shown not to be the case. Recently, we demonstrated that protein phosphatase 1 (PP1), a major member of the PPP family of Ser/Thr protein phosphatases, mediates the signaling between phototropins and H⁺-ATPase in guard cells (Takemiya et al., 2006). Therefore, ABA is likely to inhibit the signaling molecule(s), including phototropins, PP1, H⁺-ATPase, and other unidentified components.In guard cells, ABA induces the production of phosphatidic acid (PA), and PA has been implicated in stimulating stomatal closure and inhibiting light-induced stomatal opening (Jacob et al., 1999; Zhang et al., 2004a; Mishra et al., 2006). PA has also been shown to interact with the catalytic subunit of human PP1 (PP1c) and decreases its phosphatase activity (Kishikawa et al., 1999; Jones and Hannun, 2002). It is thus conceivable that PA also functions as an inhibitor of plant PP1c and suppresses the blue light signaling of guard cells.In this study, we investigated the effect of PA on blue light responses of stomata from Vicia faba. We found that PA inhibited the phosphatase activity of PP1c in vitro, suppressed blue light-dependent H⁺ pumping and phosphorylation of H⁺-ATPase, and did not affect the autophosphorylation of phototropins in guard cells.     

CPK12: A Ca2+-dependent protein kinase balancer in abscisic acid signaling

Ca²⁺ is believed to be a critical second messenger in ABA signal transduction. Ca²⁺-dependent protein kinases (CDPKs) are the best characterized Ca²⁺ sensors in plants. Recently, we identified an Arabidopsis CDPK member CPK12 as a negative regulator of ABA signaling in seed germination and post-germination growth, which reveals that different members of the CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction. We observed that both RNA interference and overexpression of CPK12 gene resulted in ABA-hypersensitive phenotypes in seed germination and post-germination growth, suggesting a high complexity of the CPK12-mediated ABA signaling pathway. CPK12 stimulates a negative ABA-signaling regulator (ABI2) and phosphorylates two positive ABA-signaling regulators (ABF1 and ABF4), which may partly explain the ABA hypersensitivity induced by both downregulation and upregulation of CPK12 expression. Our data indicate that CPK12 appears to function as a balancer in ABA signal transduction in Arabidopsis.     

Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H+K+-ATPase

Summary Membrane fragments containing the H⁺K^–-ATPase from parietal cells have been adsorbed to a planar lipid membrane. The transport activity of the enzyme was determined by measuring electrical currents via the capacitive coupling between the membrane sheets and the planar lipid film. To initiate the pump currents by the ATPase a light-driven concentration jump of ATP from caged ATP was applied as demonstrated previously for Na⁺K⁺-ATPase (Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985.EMBO J. 4:3079–3085). Since H⁺K⁺-ATPase is an electroneutrally working enzyme no stationary pump currents were observed in the presence of K⁺. By separation of the H⁺ and K⁺ transport steps of the reaction cycle, however, the electrogenic step of the phosphorylation could be measured. This was achieved in the absence of K⁺ or at low concentrations of K⁺. The observed transient current is ATP dependent which can be assigned to the proton movement during the phosphorylation. From this it was conclueded that the K⁺ transport during dephosphorylation is electrogenic, too, in contrast to the Na⁺K⁺-ATPase where the K⁺ step is electroneutral. The transient current was measured at different ionic conditions and could be blocked by vanadate and by the H⁺K⁺-ATPase specific inhibitor omeprazole. An alternative mechanism for activation of this inhibitor is discussed.     

Purification and characterization of the ouabain-sensitive H/K-ATPase from guinea-pig distal colon

Distal colon absorbs K⁺ through a Na⁺-independent, ouabain-sensitive H⁺/K⁺-exchange, associated to an apical ouabain-sensitive H⁺/K⁺-ATPase. Expression of HKα2, gene associated with this ATPase, induces K⁺-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K⁺-ATPase activities have been described in colonocytes. However, native H⁺/K⁺-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H⁺/K⁺-ATPase from guinea-pig distal colon is described. H⁺/K⁺-ATPase is Mg²⁺-dependent and activated by K⁺, Cs⁺ and NH₄⁺ but not by Na⁺ or Li⁺, independently of K⁺-accompanying anion. H⁺/K⁺-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [³²P]-γ-ATP, forming an acyl-phosphate bond, in an Mg²⁺-dependent, vanadate-sensitive process. K⁺ inhibited phosphorylation, effect blocked by ouabain. H⁺/K⁺-ATPase is an α/β-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKα2 and Na⁺/K⁺-ATPase β1-subunit, respectively. Thus, colonic ouabain-sensitive H⁺/K⁺-ATPase is a distinctive P-type ATPase.     

Characterization of Syrian hamster gastric mucosal H+,K+-ATPase

Employing a simple one-step sucrose gradient fractionation method, gastric mucosal membrane of Syrian hamster was prepared and demonstrated to be specifically enriched in H⁺,K⁺-ATPase activity. The preparation is practically devoid of other ATP hydrolyzing activity and contains high K⁺-stimulated ATPase, activity of at least 4–5 fold compared to basal ATPase activity. The H⁺,K⁺-ATPase showed hydroxylamine-sensitive phosphorylation and K⁺-dependent dephosphorylation of the phospho-enzyme, characteristic inhibition by vanadate, omeprazole and SCH 28080, and nigericin-reversible K⁺-dependent H⁺-transport — properties characteristic of gastric proton pump One notable difference with H⁺,K⁺-ATPase of other species has been the observation of valinomycin-independent H⁺ transport in such membrane vesicles. It is proposed that such H⁺,K⁺-ATPase-rich hamster gastric mucosal membrane preparation might provide a unique model to study physiological aspects of H⁺,K⁺-ATPase-function in relation to HCl secretion.     

Inhibition of light-induced stomatal opening and of guard-cell-protoplast swelling in Vicia faba L. by tentoxin,an inhibitor of photophosphorylation

The fungal phytotoxin tentoxin and its natural derivative dihydrotentoxin impair light-induced stomatal opening in epidermal strips of broad bean (Vicia faba L.) incubated in a potassium-rich medium. Swelling of guard-cell protoplasts (GCPs) of the same species is inhibited in the presence of both substances. Swollen GCPs shrink after tentoxin or dihydrotentoxin treatment and these effects cannot be fully compensated by the phytoeffector fusicoccin. A comparison with the potassium carrier valinomycin shows that tentoxin acts in a different manner, because it is effective in the light only, whereas valinomycin causes shrinkage of GCPs also in the dark. Determination of adenine nucleotides in GCPs indicates a reduced ATP content and an enhanced ADP level after addition of tentoxin. At the same time, tentoxintreated GCPs contain more NADPH and less NAD⁺ than the control (NADP⁺ and NADH content does not differ). The results presented are consistent with the hypothesis that tentoxin closes stomata as a consequence of its inhibitory action on photophosphorylation.Abbreviations FC fusicoccin - GCP guard-cell protoplast - KIDA potassium iminodiacetate     

Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase

Plasma membrane (PM) H⁺-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg²⁺- and Mn²⁺-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H⁺-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H⁺-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H⁺-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H⁺-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H⁺-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H⁺-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H⁺-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H⁺-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H⁺-ATPase in guard cells.

Type 2C protein phosphatase clade D family members redundantly dephosphorylate the penultimate C-terminal threonine residue of plasma membrane H⁺-ATPase in guard cells to control stomatal movement.     

Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.     

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Eukaryotic P-type plasma membrane H⁺-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H⁺-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H⁺-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules.