Testing the polar auxin transport model with a selective plasma membrane H+-ATPase inhibitor

Auxin is unique among plant hormones in that its function requires polarized transport across plant cells. A chemiosmotic model was proposed to explain how polar auxin transport is derived by the H⁺ gradient across the plasma membrane (PM) established by PM H⁺-adenosine triphosphatases (ATPases). However, a classical genetic approach by mutations in PM H⁺-ATPase members did not result in the ablation of polar auxin distribution, possibly due to functional redundancy in this gene family. To confirm the crucial role of PM H⁺-ATPases in the polar auxin transport model, we employed a chemical genetic approach. Through a chemical screen, we identified protonstatin-1 (PS-1), a selective small-molecule inhibitor of PM H⁺-ATPase activity that inhibits auxin transport. Assays with transgenic plants and yeast strains showed that the activity of PM H⁺-ATPases affects auxin uptake as well as acropetal and basipetal polar auxin transport. We propose that PS-1 can be used as a tool to interrogate the function of PM H⁺-ATPases. Our results support the chemiosmotic model in which PM H⁺-ATPase itself plays a fundamental role in polar auxin transport.     

Effect of auxin concentration and growth phase on the plasma membrane H-ATPase of tobacco calli

The activity of plasma membrane H⁺-ATPase increases up to 3 fold during the growth of tobacco calli for 6 weeks. In medium with optimal auxin this activity is always greater than in medium with suboptimal hormone. The amount of H⁺-ATPase measured with specific antibody only experiences small changes under the same conditions. Therefore, auxin level and growth phase mostly influence the molecular activity of the enzyme and not its amount. Despite the increase in H⁺-ATPase activity, there is a decrease in K⁺ content and ths decrease is greater in medium with suboptimal auxin. Apparently, there is a progressive deenergization of the plasma membrane which is only partially compensated by an auxin-dependent increase in activity of plasma membrane H⁺-ATPase.     

不同生境两种生态型芦苇叶片质膜H+-ATPase的比较

利用两相法化纯化质膜微囊,研究了分布西北沙地区的两种生态型芦苇(Phragmites communis trih.)水生芦苇和重度盐化草甸芦苇,分别简称为水芒和盐芦)叶片质膜H - ATPase的部分性质.结果显示,与水芦相比,盐芦质膜H -ATPase的ATP水解活性升高,Km值由1.27mmol\l降至Vmax没有显著差异.并且该酶活性对温度的敏感必玫PH谱型也发生了变化.以对硝基苯磷酸盐为底物,低浓度时盐芦的的质膜H -ATPase水解活性有差异.钡酸盐抑制实验表明,两种生态的质膜H -ATPase磷酸-酶区的催化性质不同.胰酶对质膜H -ATPase活性的活化谱型也存在差异,说明该酶C末端的结构或性质发生了变化.此外,与水芦相比,盐芦质膜H -ATPase的质子泵活性的耦联程度也升高了.以上结果明,当芦苇从水生环境向盐渍环境过渡时,质膜H -ATPase的催化性质发生了变化,这些变化可能是由酶结构的修饰和不同的同工酬酶谱引起的.H -ATPase催化性质的变化可能是对盐渍生境的适应性反应.     

磷饥饿对番茄幼苗H+分泌及Pi吸收的影响

磷饥饿条件下番茄幼苗的H⁺分泌速率明显提高。质膜质子泵专一性抑制剂钒酸盐能显著抑制番茄幼苗的H⁺分泌,也能显著抑制其Pi吸收。此结果表明,磷饥饿时番茄幼苗Pi吸收速率的变化与H⁺分泌速率的变化之间可能具有一定的相关性,并进一步暗示质膜H⁺-ATPase可能参与其中。本文结果还表明,Pi/H⁺的准量关系约为1:1。     

Changes in plasmalemma K+Mg2+ -ATPase dephosphorylating activity and H+ transport in relation to seasonal growth and freezing tolerance of Festuca pratensis Huds

Changes in plasmalemma K⁺Mg²⁺-ATPase dephosphorylating activity and H⁺ transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and ΔμH⁺ were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K⁺Mg²⁺-ATPase activity was found throughout the seasonal growth of the plants (i). The K⁺Mg²⁺-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K⁺Mg²⁺-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K⁺Mg²⁺-ATPase activity than in that of H⁺ transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4–2 °C) and at freezing (−8 °C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed.     

拟南芥AtJ3通过核质转运调控植物质膜H~+-ATPase活性与ABA响应

拟南芥AtJ3(Arabidopsis thaliana Dna J homolog 3)为一蛋白分子伴侣,在植物体内可通过与PKS5(SOS2-like protein kinase 5)蛋白激酶形成复合物来抑制PKS5的活性;同时AtJ3-PKS5复合物可对质膜上H~+-ATPase质子转运活性进行正向调节,并参与对外源ABA的响应。为揭示AtJ3-PKS5复合物参与质膜H~+-ATPase活性调节及对外源ABA响应中的作用,本研究以拟南芥AtJ3、PKS5不同突变体为材料,在盐及ABA共同处理下对AtJ3-PKS5复合物的功能及作用机制进行了探讨。结果显示,在2种因素共同处理下,AtJ3-PKS5复合物可同时对处理因素进行响应。即AtJ3-PKS5复合物可对质膜上H~+-ATPase质子转运活性进行调节,并使细胞内p H值发生变化,同时还可诱导ABI5下游ABA响应基因的表达;外源ABA可引起AtJ3从细胞核向细胞质的转运,从而增强了AtJ3对H~+-ATPase活性的调节。说明AtJ3-PKS5复合物在对H~+-ATPase活性调节及对外源ABA响应的交互代谢途径中起着关键调节子的作用。     

Efficient iron plaque formation on tea(Camellia sinensis) roots contributes to acidic stress tolerance

Tea plants grow in acidic soil, but to date, their intrinsic mechanisms of acidic stress tolerance have not been elucidated. Here, we assessed the tea plant response to growth on NHt4 nutrient media having different p H and iron levels. When grown in standard NHt4 nutrient solution(iron insufficient, 0.35 mg Là1 Fe2t), tea roots exhibited significantly lower nitrogen accumulation, plasma membrane Ht-ATPase activity, and protein levels; net Htefflux was lower at pH 4.0 and 5.0 than at pH 6.0. Addition of30 mg Là1 Fe2t(iron sufficient, mimicking normal soil Fe2tconcentrations) to the NHt4 nutrient solution led to more efficient iron plaque formation on roots and increased root plasma membrane Ht-ATPase levels and activities at p H 4.0 eland 5.0, compared to the p H 6.0 condition. Furthermore,plants grown at pH 4.0 and 5.0, with sufficient iron,exhibited significantly higher nitrogen accumulation than those grown at pH 6.0. Together, these results support the hypothesis that efficient iron plaque formation, on tea roots, is important for acidic stress tolerance. Furthermore,our findings establish that efficient iron plaque formation is linked to increased levels and activities of the tea root plasma membrane Ht-ATPase, under low pH conditions.     

盐地碱蓬叶片液泡膜H+-ATPase B亚基的克隆及盐胁迫下表达分析

植物液泡膜H -ATPase在建立跨液泡膜质子梯度、促进液泡Na 区域化、提高植物耐盐性方面发挥着重要作用.本实验从盐生植物盐地碱蓬(Suaeda salsa L.)cDNA文库分离到碱蓬叶片液泡膜H -ATPase B亚基cDNA克隆.测序表明该基因长达1 974 bp,开放阅读框有1 470 bp编码489个氨基酸,含有一个保守的ATP结合位点,其蛋白分子量约为54.29 kD.Northem及Western印迹表明盐地碱蓬液泡膜H -ATPase B亚基表达明显受NaCl胁迫诱导,并且在NaCl胁迫下,B亚基在转录及翻译水平上与液泡膜H -ATPase c亚基存在协同作用.盐胁迫下,盐地碱蓬液泡H -ATPase B亚基与c亚基的协同表达增加了液泡H -ATPase的数量,从而提高了液泡H -ATPase活性,为碱蓬叶片液泡Na 区域化提供了动力,最终提高了碱蓬植株的耐盐性.     

硫化氢对低温胁迫下甜樱桃柱头和子房线粒体功能的影响

为探索低温胁迫下外源硫化氢(H₂S)对甜樱桃花的柱头和子房线粒体功能的影响,本研究以甜樱桃品种‘早大果’花枝为试材,在-2 ℃低温下喷施0.05 mmol·L^-1硫氢化钠(NaHS,H₂S供体)和15 μmmol·L^-1 次牛磺酸(HT、H₂S清除剂),测定柱头和子房线粒体中活性氧、抗氧化酶和线粒体膜通透性转换孔(MPTP)开放程度、膜流动性、膜电位和细胞色素(Cyt c/a)比值变化。结果表明: 低温胁迫导致线粒体内过氧化氢(H₂O₂)和丙二醛(MDA)含量显著增加,线粒体MPTP明显增大,膜流动性降低,膜电位和线粒体Cyt c/a吸光度比值、膜H⁺-ATPase活性显著下降,线粒体结构受到损伤。低温胁迫下,外施0.05 mmol·L^-1 NaHS可显著降低低温胁迫下柱头和子房线粒体H₂O₂和MDA含量,在较长时间内维持较高的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性,减小线粒体MPTP开放程度,增强线粒体膜流动性,提高线粒体膜电位、Cyt c/a值和膜H⁺-ATPase活性;NaHS清除剂HT则抵消NaHS对上述参数的影响。综上所述,外源H₂S可以提高低温胁迫下甜樱桃柱头和子房线粒体抗氧化酶活性,减少H₂O₂和MDA积累,提高膜H⁺-ATPase活性,稳定线粒体膜结构和功能,进而缓解低温胁迫对花器官的伤害。     

Phosphorylation-dephosphorylation of membrane proteins controls the microsomal H-ATPase activity of corn roots

The Mg²⁺-dependent H⁺-ATPase activity of a sealed microsomal vesicle fraction isolated from corn (Zea mays L.) roots appears to be controlled by a phosphorylation-dephosphorylation cycle. Phosphorylation of the microsomal fraction is carried out by a Ca²⁺/calmodulin (CaM)-stimulated process. The H⁺-ATPase activity decreases with increasing phosphorylation of the membranes and becomes only slightly uncoupled by ionophores and less inhibited by dicyclohexylcarbodiimide (DCCD), diethylstilbestrol (DES), NO₃⁻ and vanadate. The inhibitory effect of phosphorylation is greater on the NO₃⁻-sensitive H⁺-ATPase activity than on the vanadate-sensitive activity. Restoration of H⁺-ATPase activity is achieved by allowing the phosphorylated membranes to dephosphorylate either in the absence or presence of exogenous alkaline phosphatase. Moreover, the presence of fluphenazine during the Ca²⁺/CaM-stimulated treatment inhibits membrane phosphorylation and protects the H⁺-ATPase activity from inhibition.     

Stimulation of plant plasma membrane Ca2+-ATPase activity by acidic phospholipids

The effect of phospholipids on the activity of the plasma membrane (PM) Ca²⁺-ATPase was evaluated in PM isolated from germinating radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) seeds after removal of endogenous calmodulin (CaM) by washing the PM vesicles with EDTA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4,5-diphosphate (PIP2)≈phosphatidylinositol 4-monophosphate>phosphatidylinositol≈phosphatidylserine≈phosphatidic acid. Neutral phospholipids as phosphatidylcholine and phosphatidylethanolamine were essentially ineffective. When the assays were performed in the presence of optimal free Ca²⁺ concentrations (10 μ M ) acidic phospholipids did not affect the Ca²⁺-ATPase activated by CaM or by a controlled trypsin treatment of the PM, which cleaved the CaM-binding domain of the enzyme. Analysis of the dependence of Ca²⁺-ATPase activity on free Ca²⁺ concentration showed that acidic phospholipids increased V_max and lowered the apparent K_m for free Ca²⁺ below the value measured upon tryptic cleavage of the CaM-binding domain; in particular, PIP2 was shown to lower the apparent K_m for free Ca²⁺ of the Ca²⁺-ATPase also in trypsin-treated PM. These results indicate that acidic phospholipids activate the plant PM Ca²⁺-ATPase through a mechanism only partially overlapping that of CaM, and thus involving a phospholipid-binding site in the Ca²⁺-ATPase distinct from the CaM-binding domain. The physiological implications of these results are discussed.     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H+-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响.结果表明,0.1 mmol/L SNP处理显著缓解了1 50 mmol/L NaCl胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性.进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜H -ATPase和焦磷酸酶活性诱导有关.此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响.应用外源CaSO4和EGTA处理也证实,Ca2 可能在NO诱导的质膜H -ATPase和焦磷酸酶活性的提高过程中起信号作用.另外,分析盐胁迫下小麦幼苗根部Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一.     

Influence of ATPase activity on PPi dependent H-transport in tonoplast vesicles of Acer pseudoplatanus

Tonoplast H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) were previously characterized in Acer pseudoplatanus cells (A. Pugin et al., Plant Sci., 73 (1991) 23–34; A. Fraichard et al., Plant Physiol. Biochem., 31 (1993) 349–359). The present study concerns the relationships between these two enzymes in vitro. ATP and PPi hydrolysis were additive and the inhibition of one did not affect the activity of the second one. ATP and PPi H⁺-transports were also additive. The H⁺ -PPase inhibition did not change ATP-dependent H⁺-transport but H⁺-ATPase inhibition inhibited the PPi dependent H⁺-transport. Because H⁺-PPase was reported to transport H⁺ and K⁺ into the vacuole (Davies et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 11701–11705), these results led us to suggest that the inhibition of the H⁺-ATPase activity could modify the H⁺/K⁺ stoichiometry for the benefit of K⁺-transport.     

Calmodulin/Ca2+-ATPase interaction at the Arabidopsis thaliana plasma membrane is dependent on calmodulin isoform showing isoform-specific Ca2+ dependencies

Arabidopsis thaliana plasma membrane (PM) Ca²⁺-ATPase is a type IIB P-type ATPase, which binds calmodulin (CaM) to an autoinhibitory N-terminal domain. Here, we took advantage of the fact that PM isolated from cultured cells mainly contains At -ACA8, the first cloned A. thaliana PM Ca²⁺-ATPase, to analyse its interaction with CaM in detail. Analysis of the ability of different peptides designed from At -ACA8 N-terminus to compete with the native protein for binding of bovine brain CaM (bbCaM) showed that peptide ⁴¹I-T⁶³ had the same affinity of the native protein [apparent dissociation constant (KD) at 10 µ M free Ca²⁺ about 25 n M ], thus localizing At -ACA8 CaM-binding site within this sequence. The interaction of At -ACA8 N-terminus with bbCaM, as determined by surface plasmon resonance, was rapid, and slowly but was fully reversible. Analysis of Ca²⁺-ATPase activation as a function of the concentration of different isoforms of A. thaliana CaM showed that Ca²⁺-ATPase is activated to similar extent by bbCaM and by different isoforms of homologous CaM. However, the affinity for the divergent A. thaliana isoform CaM8 was lower than that for canonical CaM isoforms such as A. thaliana CaM2, CaM4 and CaM6 or bbCaM. The apparent KD for CaM isoforms of the native enzyme increased with the decrease of free Ca²⁺ concentration, suggesting that enzyme conformation is affected by Ca²⁺. Binding of CaM isoforms to At -ACA8 N-terminus was affected differently by free Ca²⁺ concentration, suggesting that plant CaMs may have different affinities for Ca²⁺.     

Topology and target interaction of the fusicoccin-binding 14-3-3 homologs of Commelina communis

Upon binding to a high-affinity plasma membrane (PM) protein (a member of the 14-3-3 family of regulatory proteins), the fungal phytotoxin fusicoccin (FC) activates the H⁺- ATPase by hindering the inhibitory interaction of the enzyme's C-terminus with its catalytic site. Protease protection experiments carried out with sealed PM vesicles of different orientation proved that the FC-binding site faces the cytoplasmic surface of the membrane. The in vivo induced activation of the H⁺-ATPase by FC was retained during solubilization of PM proteins. Two-dimensional gel systems combining a native separation of membrane protein complexes with a denaturing dimension as well as high-performance anion-exchange chromatography proved the existence of a labile ATPase:14-3-3 complex in plasma membranes. Stabilization of this complex could be achieved by FC treatment in vivo or in vitro . Mild proteolytic removal of the C-terminal auto-inhibitory domain of the H⁺ATPase liberated apparent hydrophobic 14-3-3 isoforms from the membrane in soluble form. During size exclusion chromatography of the proteolytically released proteins, co-elution of 14-3-3 dimers, protein-bound FC and the C-terminus of the H⁺ATPase was observed. Moreover, the data suggest that 14-3-3 dimers themselves are not able to bind FC. Based on these results, it is proposed that the 'FC receptor' is represented by a labile complex between a 14-3-3 dimer and the H⁺-ATPase whose formation is part of a mechanism regulating ATPase-activity under physiological conditions. In our working model, binding of FC stabilizes this labile complex, thus leading to a strong and persistent activation of the H⁺-ATPase in vivo . The possibility that the C-terminus of the enzyme represents the binding domain for 14-3-3 homologs is discussed.     

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10'-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

Plasma membrane H+-ATPase-dependent citrate exudation from cluster roots of phosphate-deficient white lupin

White lupin ( Lupinus albus L.) is able to grow on soils with sparingly available phosphate (P) by producing specialized structures called cluster roots. To mobilize sparingly soluble P forms in soils, cluster roots release substantial amounts of carboxylates and concomitantly acidify the rhizosphere. The relationship between acidification and carboxylate exudation is still largely unknown. In the present work, we studied the linkage between organic acids (malate and citrate) and proton exudations in cluster roots of P-deficient white lupin. After the illumination started, citrate exudation increased transiently and reached a maximum after 5 h. This effect was accompanied by a strong acidification of the external medium and alkalinization of the cytosol, as evidenced by in vivo nuclear magnetic resonance (NMR) analysis. Fusicoccin, an activator of the plasma membrane (PM) H⁺-ATPase, stimulated citrate exudation, whereas vanadate, an inhibitor of the H⁺-ATPase, reduced citrate exudation. The burst of citrate exudation was associated with an increase in expression of the LHA1 PM H⁺-ATPase gene, an increased amount of H⁺-ATPase protein, a shift in pH optimum of the enzyme and post-translational modification of an H⁺-ATPase protein involving binding of activating 14-3-3 protein. Taken together, our results indicate a close link in cluster roots of P-deficient white lupin between the burst of citrate exudation and PM H⁺-ATPase-catalysed proton efflux.     

The effect of assay composition, detergent solubilization and reconstitution on red beet (Beta vulgaris L.) plasma membrane H-ATPase kinetic properties

Modification of the salt concentration, composition and/or buffer type in the assay of plasma membrane ATPase activity caused substantial changes in the K_m and slight changes in the temperature dependence of this enzyme. The K_m and temperature dependence were also affected by detergent solubilization of the ATPase and its subsequent reconstitution into liposomes. Modulation of kinetic properties by assay composition and hydrophobic state reflect the sensitivity of the plasma membrane H⁺-ATPase to its immediate environment. This may indicate a possible regulatory mechanism for this important plant enzyme.