Seasonal changes of plasma membrane H(+)-ATPase and endogenous ion current during cambial growth in poplar plants

The plasma membrane H(+)-ATPase (PM H(+)-ATPase), potassium ions, and endogenous ion currents might play a fundamental role in the physiology of cambial growth. Seasonal changes of these parameters were studied in twigs of Populus nigra and Populus trichocarpa. Monoclonal and polyclonal antibodies against the PM H(+)-ATPase, x-ray analysis for K(+) localization and a vibrating electrode for measurement of endogenous ion currents were used as probes. In dormant plants during autumn and winter, only a slight immunoreactivity against the PM H(+)-ATPase was found in cross sections and tissue homogenates, K(+) was distributed evenly, and the density of endogenous current was low. In spring during cambial growth, strong immunoreactivity against a PM H(+)-ATPase was observed in cambial cells and expanding xylem cells using the monoclonal antibody 46 E5 B11 F6 for fluorescence microscopy and transmission electron microscopy. At the same time, K(+) accumulated in cells of the cambial region, and strong endogenous current was measured in the cambial and immature xylem zone. Addition of auxin to dormant twigs induced the formation of this PM H(+)-ATPase in the dormant cambial region within a few days and an increase in density of endogenous current in shoot cuttings within a few hours. The increase in PM H(+)-ATPase abundance and in current density by auxin indicates that auxin mediates a rise in number and activity of an H(+)-ATPase in the plasma membrane of cambial cells and their derivatives. This PM H(+)-ATPase generates the necessary H(+)-gradient (proton-motive force) for the uptake of K(+) and nutrients into cambial and expanding xylem cells.     

A major isoform of the maize plasma membrane H(+)-ATPase: characterization and induction by auxin in coleoptiles.

The plasma membrane (PM) H(+)-ATPase has been proposed to play important transport and regulatory roles in plant physiology, including its participation in auxin-induced acidification in coleoptile segments. This enzyme is encoded by a family of genes differing in tissue distribution, regulation, and expression level. A major expressed isoform of the maize PM H(+)-ATPase (MHA2) has been characterized. RNA gel blot analysis indicated that MHA2 is expressed in all maize organs, with highest levels being in the roots. In situ hybridization of sections from maize seedlings indicated enriched expression of MHA2 in stomatal guard cells, phloem cells, and root epidermal cells. MHA2 mRNA was induced threefold when nonvascular parts of the coleoptile segments were treated with auxin. This induction correlates with auxin-triggered proton extrusion by the same part of the segments. The PM H(+)-ATPase in the vascular bundies does not contribute significantly to auxin-induced acidification, is not regulated by auxin, and masks the auxin effect in extracts of whole coleoptile segments. We conclude that auxin-induced acidification in coleoptile segments most often occurs in the nonvascular tissue and is mediated, at least in part, by increased levels of MHA2.     

Salt-tolerant ATPase activity in the plasma membrane of the marine angiosperm Zostera marina L

Plasma membrane (PM) H(+)-ATPase and H(+) transport activity were detected in PM fractions prepared from Zostera marina (a seagrass), Vallisneria gigantea (a freshwater grass) and Oryza sativa (rice, a terrestrial plant). The properties of Z. marina PM H(+)-ATPase, specifically, the optimal pH for ATPase activity and the result of trypsin treatment, were similar to those of authentic PM H(+)-ATPases in higher plants. In V. gigantea and O. sativa PM fractions, vanadate-sensitive (P-type) ATPase activities were inhibited by the addition of NaCl. In contrast, activity in the Z. marina PM fraction was not inhibited. The nitrate-sensitive (V-type) and azide-sensitive (F-type) ATPase activities in the Z. marina crude microsomal fraction and the cytoplasmic phosphoenolpyruvate carboxylase activity, however, were inhibited by NaCl, indicating that not all enzyme activities in Z. marina are insensitive to salt. Although the ratio of Na(+) to K(+) (Na(+)/K(+)) in seawater is about 30, Na(+)/K(+) in the Z. marina cells was about 1.0. The salt-tolerant ATPase activity in the plasma membrane must play an important role in maintaining a low Na(+) concentration in the seagrass cells.     

Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway.     

Expression of a constitutively activated plasma membrane H+-ATPase alters plant development and increases salt tolerance

The plasma membrane proton pump ATPase (H(+)-ATPase) plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. Its activity is regulated by a C-terminal autoinhibitory domain that can be displaced by phosphorylation and the binding of regulatory 14-3-3 proteins, resulting in an activated enzyme. To better understand the physiological consequence of this activation, we have analyzed transgenic tobacco (Nicotiana tabacum) plants expressing either wild-type plasma membrane H(+)-ATPase4 (wtPMA4) or a PMA4 mutant lacking the autoinhibitory domain (DeltaPMA4), generating a constitutively activated enzyme. Plants showing 4-fold higher expression of wtPMA4 than untransformed plants did not display any unusual phenotype and their leaf and root external acidification rates were not modified, while their in vitro H(+)-ATPase activity was markedly increased. This indicates that, in vivo, H(+)-ATPase overexpression is compensated by down-regulation of H(+)-ATPase activity. In contrast, plants that expressed DeltaPMA4 were characterized by a lower apoplastic and external root pH, abnormal leaf inclination, and twisted stems, suggesting alterations in cell expansion. This was confirmed by in vitro leaf extension and curling assays. These data therefore strongly support a direct role of H(+)-ATPase in plant development. The DeltaPMA4 plants also displayed increased salt tolerance during germination and seedling growth, supporting the hypothesis that H(+)-ATPase is involved in salt tolerance.     

Aluminum inhibits the H(+)-ATPase activity by permanently altering the plasma membrane surface potentials in squash roots

Although aluminum (AL) toxicity has been widely studied in monocotyledonous crop plants, the mechanism of Al impact on economically important dicotyledonous plants is poorly understood. Here, we report the spatial pattern of Al-induced root growth inhibition, which is closely associated with inhibition of H(+)-ATPase activity coupled with decreased surface negativity of plasma membrane (PM) vesicles isolated from apical 5-mm root segments of squash (Cucurbita pepo L. cv Tetsukabuto) plants. High-sensitivity growth measurements indicated that the central elongation zone, located 2 to 4 mm from the tip, was preferentially inhibited where high Al accumulation was found. The highest positive shifts (depolarization) in zeta potential of the isolated PM vesicles from 0- to 5-mm regions of Al-treated roots were corresponded to pronounced inhibition of H(+)-ATPase activity. The depolarization of PM vesicles isolated from Al-treated roots in response to added Al in vitro was less than that of control roots, suggesting, particularly in the first 5-mm root apex, a tight Al binding to PM target sites or irreversible alteration of PM properties upon Al treatment to intact plants. In line with these data, immunolocalization of H(+)-ATPase revealed decreases in tissue-specific H(+)-ATPase in the epidermal and cortex cells (2--3 mm from tip) following Al treatments. Our report provides the first circumstantial evidence for a zone-specific depolarization of PM surface potential coupled with inhibition of H(+)-ATPase activity. These effects may indicate a direct Al interaction with H(+)-ATPase from the cytoplasmic side of the PM.     

Changes in the level and activation state of the plasma membrane H(+)-ATPase during aging of red beet slices.

The effect of aging on the plasma membrane (PM) H(+)-ATPase of red beet (Beta vulgaris L.) parenchyma discs was analyzed in PM purified by aqueous two-phase partitioning. Aging increased both the activity in the amount of immunodetectable H(+)-ATPase in the PM. The activity assayed at slightly alkaline pH values increased earlier and more strongly than that assayed at acidic pH values, so that the pH curve of the enzyme from aged beet discs was shifted toward more alkaline values. Aging decreased the stimulation of the PM H(+)-ATPase activity by controlled trypsin treatments or by lysophosphatidylcholine. After trypsin treatment the pH dependence of H(+)-ATPase from dormant or aged beet discs became equal. These results indicate that aging not only increases the level of H(+)-ATPase in the PM, but also determines its activation, most likely by modifying the interaction between the autoinhibitory carboxyl-terminal domain and the catalytic site. When the PM H(+)-ATPase activity was assayed at a slightly alkaline pH, the tyrosine modifier N-acetylimidazole inhibited the H(+)-ATPase in the PM from dormant beet discs much less than in the PM from aged discs, suggesting that modification of a tyrosine residue may be involved in the activation of the PM H(+)-ATPase induced by aging. The results are discussed with regard to aging-induced development of transmembrane transport activities.     

Modulation of H+-ATPase Activity by Fusicoccin in Plasma Membrane Vesicles from Oat (Avena sativa L.) Roots (A Comparison of Modulation by Fusicoccin,Trypsin, and Lysophosphatidylcholine)

The fungal phytotoxin fusicoccin affects various transport processes in the plasma membrane of plant cells. The plasma membrane (PM) H+-ATPase (EC 3.6.1.35) seems to be the primary target of fusicoccin action. The kinetics of the stimulation of the PM H+-ATPase by fusicoccin was studied in PM vesicles isolated from oat (Avena sativa cv Adamo) roots by aqueous two-phase partitioning. Considerable stimulation of activity was observed only when roots were treated with fusicoccin prior to the PM isolation. Fusicoccin treatment shifted the pH optimum of the ATPase toward more alkaline values and increased Vmax. No effects on Km were observed. Treatment with trypsin resulted in stimulation of ATPase activity in control vesicles but not in the fusicoccin-treated vesicles. The characteristics of stimulation by trypsin in control vesicles were comparable with those of stimulation by fusicoccin. This result and the change of the polypeptide pattern on western blots suggest the involvement of the C-terminal inhibitory domain in the fusicoccin signal transduction chain. On the other hand, stimulation by lyso-PC demonstrated other characteristics than stimulation by fusicoccin. Lyso-PC was able to stimulate ATPase activity at both acidic and alkaline pH values. Kinetic analysis of the pH dependency curves revealed different mechanisms for activation by fusicoccin and by lyso-PC. Whereas fusicoccin shifted the pH dependency of formation of phosphorylated intermediate to more alkaline values, lyso-PC seemed to increase dephosphorylation independently of pH.     

Plasma membrane H+‐ATPase regulation is required for auxin gradient formation preceding phototropic growth

Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H⁺‐ATPases that are required to control apoplastic pH. Our results show that H⁺‐ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H⁺‐ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH.     

Acid secretion by mitochondrion-rich cells of medaka (Oryzias latipes) acclimated to acidic freshwater

In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/immunocytochemistry, the Na(+)/H(+) exchanger 3 (NHE3) and H(+)-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H(+)-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H(+)-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation; however, the mRNA level of NHE3 was remarkably higher than that of H(+)-ATPase. A scanning ion-selective electrode technique was used to measure H(+), Na(+), and NH(4)(+) transport by individual MR cells in larval skin. Results showed that Na(+) uptake and NH(4)(+) excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na(+) uptake/NH(4)(+) excretion mechanism plays a critical role in acidic equivalent (H(+)/NH(4)(+)) excretion by MR cells of the freshwater medaka.     

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.     

Identification and partial purification of a cytosolic activator of vacuolar H(+)-ATPases from mammalian kidney.

A factor that activates affinity-purified vacuolar H(+)-ATPase from bovine kidney microsomes was identified and partially purified from bovine kidney cytosol. The activator is a heat-stable, trypsin-sensitive acidic protein with a Mr by gel filtration of approximately 35,000. The activator increased the activity of renal microsomal and brush border H(+)-ATPase by over 60% but stimulated lysosomal H(+)-ATPase activity by only 28%; it had little or no activity against the remaining N-ethylmaleimide-insensitive ATPase in kidney microsomes and other transport ATPases. Stimulation of ATPase activity appeared to result from binding of the activator to the H(+)-ATPase. Activation was saturable, with a Hill coefficient of 1 at low protein concentrations. Both activator binding and stimulation of H(+)-ATPase activity were enhanced at pH values less than or equal to 6.5. The activator has selective effects on different H(+)-ATPases and is poised to activate the enzyme at low physiologic values of cytosolic pH; this newly identified cytosolic proteins may participate in the physiologic regulation of the vacuolar H(+)-ATPase.     

Blue light inactivates plasma membrane H(+)-ATPase in pulvinar motor cells of Phaseolus vulgaris L

Unilateral blue light irradiation induces bending of pulvini of Phaseolus vulgaris towards the source of light. The pulvinar bending is caused by a decrease in turgor pressure of motor cells that are irradiated with blue light. Decrease in the turgor pressure is caused by the net efflux of K(+) and counter anions, accompanying membrane depolarization. In the present study the effect of blue light on the activity of plasma membrane H(+)-ATPase was studied in relation to the membrane depolarization. The activity of the plasma membrane H(+)-ATPase was measured using protoplast suspensions prepared from laminar pulvini from primary leaves. A pulse of blue light under continuous red light irradiation induced both a transient increase in the external pH and transient inhibition of the vanadate-sensitive ATPase. Continuous blue light irradiation under continuous red light irradiation induced both a sustained increase in the external pH and sustained inhibition of the vanadate-sensitive ATPase. These results show that blue light inhibits the activity of the plasma membrane H(+)-ATPase. Inactivation of the plasma membrane H(+)-ATPase supports the membrane depolarization induced by the blue light irradiation.     

Characteristics of adventitious root formation in cotyledon segments of mango (Mangifera indica L. cv. Zihua): two induction patterns, histological origins and the relationship with polar auxin transport

Cotyledon segments derived from zygote embryos of mango (Mangifera indica L. cv. Zihua) were cultured on agar medium for 28 days. Depending on different pre-treatments with plant growth regulators, two distinct patterns of adventitious roots were observed. A first pattern of adventitious roots was seen at the proximal cut surface, whereas no roots were formed on the opposite, distal cut surface. The rooting ability depended on the segment length and was significantly promoted by pre-treatment of embryos with indol-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for 1 h. A pre-treatment with the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) completely inhibited adventitious root formation on proximal cut surfaces. A second pattern of roots was observed on abaxial surfaces of cotyledon segments when embryos were pre-treated with 2,700 μM 1-naphthalenacetic acid (NAA) for 1 h. Histological observations indicated that both patterns of adventitious roots originated from parenchymal cells, but developmental directions of the root primordia were different. A polar auxin transport assay was used to demonstrate transport of [³H] indole-3-acetic acid (IAA) in cotyledon segments from the distal to the proximal cut surface. In conclusion, we suggest that polar auxin transport plays a role in adventitious root formation at the proximal cut surface, whereas NAA levels (influx by diffusion; carrier mediated efflux) seem to control development of adventitious roots on the abaxial surface of cotyledon segments.     

低温、高pH胁迫对水稻幼苗根系质膜、液泡膜ATP酶活性的影响

以耐冷性不同的两个水稻品种为材料,比较研究了幼苗根系质膜、液泡膜ATP酶对低温(8℃)及高pH(8.0)胁迫的反应。结果表明水稻根细胞质膜和液泡膜上均存在Ca3+-ATP酶,但活性远低于H+-ATP酶。耐冷品种武育粳3号经低温(8℃)处理2d,根系质膜和液泡膜H+-ATP酶、Ca2+-ATP酶活性均明显升高,至冷处理12d,H+-ATP酶、Ca2+-ATP酶活性有所下降,但仍与对照相近;而冷敏感品种汕优63经低温(8℃)处理2d,根系质膜H+-ATP酶活性略有升高,而质膜Ca2+-ATP酶以及液泡膜H+-ATP酶、Ca2+-ATP酶活性已明显下降;至冷处理12d,4种酶活性均明显低于对照。高pH胁迫使质膜和液泡膜H+-ATP酶活性下降,而使Ca2+-ATP酶活性上升。高pH胁迫会加剧低温冷害。结果表明,耐冷品种质膜、液泡膜ATP酶比冷敏感品种对低温胁迫有更强的适应能力。     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

Altered plasma membrane H(+)-ATPase from the Dio-9-resistant pma1-2 mutant of Schizosaccharomyces pombe.

The pma1-2 mutation affecting the plasma membrane H(+)-ATPase of Schizosaccharomyces pombe has been selected for resistance to the antibiotic Dio-9. In membrane fractions purified from glucose-starved cells, the mutant ATPase activity is reduced by 96%, is insensitive to inhibition by vanadate and has a pH profile displaced in the acidic pH range when compared to the wild type. The maximum velocity of the H(+)-ATPase activity of plasma membranes from glucose-activated pma1-2 cells is activated 20-fold. This is in striking contrast with the wild-type ATPase activity, the maximal velocity of which is not affected by glucose. However, similar to the wild-type enzyme, glucose activation of the pma1-2 mutant H(+)-ATPase reduces the Km for MgATP 9-2 mM and shifts the optimal pH from 4.8 to 6.0-6.5. The pma1-2 mutation modifies Lys250 to a threonine, which is highly conserved in fungal and plant H(+)-ATPases. These results, compared to those reported for mutations of neighbour residues in yeast or mammalian P-type ATPases, suggest that Lys250 could play a significant role, not only in phosphate binding and/or in the E1P-E2P conformational isomerisation, but also in glucose activation of the H(+)-ATPase.     

磷饥饿时番茄幼苗根系质膜H+-ATP酶活性

磷饥饿提高了番茄幼苗质膜H+-ATP酶活性并促进了番茄幼苗根部的H+分泌。动力学分析表明,磷饥饿使番茄幼苗根部质膜H+-ATP酶的Km值明显降低,亦即提高了该酶对其底物的亲和力,但对该酶的Vmax影响不大。另外,磷饥饿并不改变ATP酶的最适pH值(最适pH值为6.5)。钒酸盐显著抑制番茄幼苗根部质膜ATP酶的活性以及H+分泌,也显著抑制番茄幼苗的Pi吸收。与对照相比,上述抑制作用在饥饿处理的植物中表现得更强。以上结果表明,磷饥饿时高亲和性Pi传递系统的诱导很可能包含质膜H+-ATP酶的参与。     

Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio)

In the skin of zebrafish embryo, the vacuolar H(+)-ATPase (V-ATPase, H(+) pump) distributed mainly in the apical membrane of H(+)-pump-rich cells, which pump internal acid out of the embryo and function similarly to acid-secreting intercalated cells in mammalian kidney. In addition to acid excretion, the electrogenic H(+) efflux via the H(+)-ATPases in the gill apical membrane of freshwater fish was proposed to act as a driving force for Na(+) entry through the apical Na(+) channels. However, convincing molecular physiological evidence in vivo for this model is still lacking. In this study, we used morpholino-modified antisense oligonucleotides to knockdown the gene product of H(+)-ATPase subunit A (atp6v1a) and examined the phenotype of the mutants. The H(+)-ATPase knockdown embryos revealed several abnormalities, including suppression of acid-secretion from skin, growth retardation, trunk deformation, and loss of internal Ca(2+) and Na(+). This finding reveals the critical role of H(+)-ATPase in embryonic acid -secretion and ion balance, as well.