V-PPase Activity Research Articles

BnVP1, a novel vacuolar H+ pyrophosphatase gene from Boehmeria nivea confers cadmium tolerance in transgenic Arabidopsis.

Plants have developed precise defense mechanisms against cadmium (Cd) stress, with vacuolar compartmentalization of Cd2+ being a crucial process in Cd detoxification. The transport of Cd into vacuoles by these cation / H+ antiporters is powered by the pH gradient created by proton pumps. In this study, the full-length cDNA of a vacuolar H+-pyrophosphatase (V-PPase) gene from Boehmeria nivea (ramie), BnVP1, was isolated using the rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of BnVP1 is 2292 bp, encoding a 763 amino acid V-PPase protein with 15 predicted transmembrane domains. Sequence alignment and phylogenetic analysis revealed that BnVP1 belongs to the Type I V-PPase family. Quantitative RT-PCR assays demonstrated that BnVP1 expression was significantly higher in ramie roots than in shoots. Cd treatments markedly induced BnVP1 expression in both roots and leaves of ramie seedlings, with a more pronounced effect in roots. Additionally, BnVP1 expression was significantly upregulated by the plant hormone methyl jasmonate (MeJA). Heterologous expression of BnVP1 in transgenic Arabidopsis significantly enhanced V-PPase activity in the roots. The growth performance, root elongation, and total chlorophyll content of transgenic plants with high tonoplast H+-PPase (V-PPase) activity were superior to those of wild-type plants. Overexpression of BnVP1 reduced membrane lipid peroxidation and ion leakage, and significantly increased Cd accumulation in the roots of transgenic Arabidopsis seedlings. This study provides new genetic resources for the phytoremediation of Cd-contaminated farmland.

Targeted mutagenesis of the vacuolar H+ translocating pyrophosphatase gene reduces grain chalkiness in rice.

Grain chalkiness is a major concern in rice production because it impacts milling yield and cooking quality, eventually reducing market value of the rice. A gene encoding vacuolar H+ translocating pyrophosphatase (V-PPase) is a major quantitative trait locus in indica rice, controlling grain chalkiness. Higher transcriptional activity of this gene is associated with increased chalk content. However, whether the suppression of V-PPase could reduce chalkiness is not clear. Furthermore, natural variation in the chalkiness of japonica rice has not been linked with V-PPase. Here, we describe promoter targeting of the japonica V-PPase allele that led to reduced grain chalkiness and the development of more translucent grains. Disruption of a putative GATA element by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 suppressed V-PPase activity, reduced grain chalkiness and impacted post-germination growth that could be rescued by the exogenous supply of sucrose. The mature grains of the targeted lines showed a much lower percentage of large or medium chalk. Interestingly, the targeted lines developed a significantly lower chalk under heat stress, a major inducer of grain chalk. Metabolomic analysis showed that pathways related to starch and sugar metabolism were affected in the developing grains of the targeted lines that correlated with higher inorganic pyrophosphate and starch contents and upregulation of starch biosynthesis genes. In summary, we show a biotechnology approach of reducing grain chalkiness in rice by downregulating the transcriptional activity of V-PPase that presumably leads to altered metabolic rates, including starch biosynthesis, resulting in more compact packing of starch granules and formation of translucent rice grains.

Proteomics analysis of the effects for different salt ions in leaves of true halophyte Sesuvium portulacastrum

Sesuvium portulacastrum is a true halophyte and shows an optimal development under moderate salinity with large amounts of salt ions in its leaves. However, the specific proteins in response to salt ions are remained unknown. In this study, comparative physiological and proteomic analyses of different leaves subject to NaCl, KCl, NaNO3 and KNO3 were performed. Chlorophyll content was decreased under the above four kinds of salt treatments. Starch and soluble sugar contents changed differently under different salt treatments. A total of 53 differentially accumulated proteins (DAPs) were identified by mass spectrometry. Among them, 13, 25, 26 and 25 DAPs were identified after exposure to KCl, NaCl, KNO3, and NaNO3, respectively. These DAPs belong to 47 unique genes, and 37 of them are involved in protein-protein interactions. These DAPs displayed different expression patterns after treating with different salt ions. Functional annotation revealed they are mainly involved in photosynthesis, carbohydrate and energy metabolism, lipid metabolism, and biosynthesis of secondary metabolites. Genes and proteins showed different expression profiles under different salt treatments. Enzyme activity analysis indicated P-ATPase was induced by KCl, NaCl and NaNO3, V-ATPase was induced by KCl and NaCl, whereas V-PPase activity was significantly increased after application of KNO3, but sharply inhibited by NaCl. These results might deepen our understanding of responsive mechanisms in the leaves of S. portulacastrum upon different salt ions.

Loss-of-function of vacuolar-type H+ pyrophosphatase gene lead to reduce in stomatal aperture and density

Transgenic plants over-expressing vacuolar H+ type pyrophosphatase (V-PPase) gene were reported to display drought resistance, reduced vacuolar pH and raised stomatal conductance. To further understand the role of V-PPase on stomatal regulation, loss- and gain-of-function approaches were combined for analysing relationship between stomatal aperture and V-PPase gene expression. Homozygous mutants of this gene were isolated by polymerase chain reaction (PCR) method. BCECF-AM fluorescence probe was used for detecting cellular pH. The result here indicated that Arabidopsis plant lines over-expressing of V-PPase gene displayed raised stomatal aperture. Both of stomatal aperture and density of homozygous vpp mutants were less than that of control plants. In addition, cellular pH of guard cells in vpp mutants was higher than control evidently. In general, our results suggested that V-PPase activity regulates stomatal aperture by changing guard cell pH.

Overexpression of a proton pumping gene OVP1 enhances salt stress tolerance, root growth and biomass yield by regulating ion balance in rice (Oryza sativa L.)

Oryza sativa vacuolar H+-pyrophosphatase 1 (OVP1) regulates plant development and osmotic stress response through its function as an electrogenic proton (H+) pump in the vacuolar membrane. Here, we show that OVP1-overexpressing transgenic (OTG) rice plants display greater resistance to salt stress and root growth than wild-type (WT) rice plants. This improved performance of OTG plants under salt stress was attributed to increases in vacuolar pyrophosphatase (V-PPase) activity, ion uptake, and vacuolar cation ion antiporter gene expression. OTG plants had higher sodium levels in the vacuole than in the cytosol. Under natural paddy field conditions, OTG plants outperformed WT plants and produced more extensive root systems. OTG plants also had higher tiller number, V-PPase activity, chlorophyll content, membrane stability, and ion uptake than WT plants. Transcript profiling analyses revealed that the expression levels of vacuolar H+-pump genes (ATPase and OVP1) and ion antiporter genes (CAX, MHX, and NHX) were higher in OTG plants than in WT plants. These combined results suggest that OVP1 overexpression affects salt stress responses, root development, and biomass yield by regulating ion balance in rice.

The vacuolar proton pyrophosphatase gene (SbVPPase) from the Sorghum bicolor confers salt tolerance in transgenic Brahmi [Bacopa monnieri (L.) Pennell

Plants overcome the effect of Na+ toxicity either by excluding Na+ at the plasma membrane or bysequestering them into the vacuoles. Influx of Na+ ions into the plant vacuoles is usually driven by H+ generated by vacuolar-type H+-ATPase as well as vacuolar proton pyrophosphatse (VPPase). In the present study, we have developed Bacopa monnieri transgenics via Agrobacterium tumefaciens containing the recombinant vector pCAMBIA2300-SbVPPase gene. Transformants were produced using nodal explants. Transformants were confirmed by PCR and DNA blot analysis. qPCR analysis showed higher transcript levels of SbVPPase compared to untransformed control (UC). Higher VPPase activity was recorded in transgenics compared to UC. Under 150mM salt stress, transgenic shoots showed enhanced Na+ accumulation with better biomass production, increased glycine betaine content, and total soluble sugar levels than UC. Transgenic shoots showed 2.9-3.8-folds lower levels of malondialdehyde content indicating lesser membrane damage. Increase in antioxidant enzyme activities (1.4-3.2-folds) was observed in transgenics compared to UC. Transgenics also displayed 7.3-9.0-folds enhanced accumulation of the medicinally important compound bacoside A. Increased biomass production, accumulation of Na+, osmolytes (glycine betaine, sugars etc.), and elevated antioxidant enzyme activities indicate better osmotic adjustment in transgenics by compartmentalization of Na+ into the vacuoles under salt stress conditions. Thus, overexpression of SbVPPase in Bacopa alleviated salt stress by sequestering Na+.

The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na+ sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H+-ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H+-PPase (V-PPase) and vacuolar Na+/H+ antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H+-PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na+ staining showed the reduced vacuolar Na+ sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na+ sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH.

Nitrate reutilization mechanisms in the tonoplast of two Brassica napus genotypes with different nitrogen use efficiency

Nitrate (NO3−) can accumulate in high concentrations in plant cell vacuoles if it is not reduced, reutilized or transported into the cytoplasm. Such accumulation of NO3− in the vacuole occurs when mechanisms for NO3− assimilation in the cytoplasm are saturated. Moreover, other processes such as efflux across the plasma membrane might affect NO3− accumulation in the vacuole. These are the main reasons limiting nitrogen use efficiency (NUE) in plants. This study elucidates mechanisms for NO3− transport from the cytoplasm to vacuoles by the V-proton pump (V-ATPase and V-PPase) and their relationship with different NUE in four Brassica napus genotypes. Pot experiments were conducted in a greenhouse under normal (15.0 mmol L−1) and limited N (7.5 mmol L−1) concentrations of nitrate using B. napus genotypes that demonstrated either high (742 and Xiangyou 15) or low (814 and H8) NUE (g g−1). Specific inhibitors of V-ATPase and V-PPase increased nitrate reductase (NR) activity, resulting in greatly decreased NO3− in plant tissues. Nitrate reductase activity and NO3− content correlated more highly to V-PPase activity than they did to V-ATPase activity, and correlation between V-PPase activity and NO3− content was significantly higher than it was to V-ATPase. Genotypes with high NUE had significantly lower activities of V-ATPase and V-PPase than those with low NUE. In the high-NUE plants, lower activities of V-proton pump underlie mechanisms that result in significantly lower NO3− content in plant tissues of the high-NUE genotypes than those found in plant tissues of the low-NUE genotypes. Our results show that the tonoplast proton pumps V-PPase and V-ATPase strongly negatively affect NR activity and positively affect NO3− content. V-PPase contributed more to this regulatory mechanism than did V-ATPase.

Vacuolar proton pumps regulation during development of Vigna unguiculata seedlings under salt stress

Global climatic changes as high temperatures and low precipitation contribute to increase cultivated areas affected by high salt soil content. Soil salinity is well known to reduce the ability of plants to take up water and this quickly causes reduction in their growth rate. V-ATPase (EC 3.6.3.14) and V-PPase (EC 3.6.1.1) hydrolytic and proton transport activities, and gene expression were evaluated in hypocotyls of 3-, 5-, 7-day-old Vigna unguiculata (L.) Walp cv. Vita 3 germinated in 100 mM NaCl in order to highlight their differential regulation and activity modulation under salt stress. Semi-quantitative RT-PCR revealed that both genes were up-regulated by salt stress in all salt exposition times studied. Up-regulation was correlated with the increase in protein content at 5 and 7-day-old seedlings. Co-expression between A and E V-ATPase subunits was also observed. The hydrolytic and proton transport activities showed that these enzymes presented a differential modulation of their activities in the presence of 100 mM NaCl. These results suggest that V-ATPase and V-PPase activities are modulated by salt stress and a multi-step regulation is exerted in order to re-establish homeostasis.

V-ATPase and V-PPase at the Tonoplast Affect NO3 − Content in Brassica napus by Controlling Distribution of NO3 − Between the Cytoplasm and Vacuole

Nitrate, once taken up by plants, can either be stored in vacuoles or reduced by nitrate reductase in the cytoplasm. High accumulation of NO3− in the vacuole occurs when assimilation into the cytoplasm is saturated. This study elucidates how proton pumps at the tonoplast (V-ATPase and V-PPase) affect the NO3− content of Brassica napus by controlling the distribution of NO3− between the cytoplasm and vacuole. Pot experiments were conducted in a greenhouse under normal N (15.0 mM nitrate) conditions using B. napus genotypes that demonstrated either high (Xiangyou15) or low (814) nitrogen use efficiency (NUE). The NO3− content of the high NUE genotype was significantly lower than that of the low NUE genotype, whereas the total N per plant of the two genotypes was almost the same, suggesting that the different NUE between the two genotypes is not due to the difference of NO3− uptake. The relative expression levels of V-ATPase (vha-a3) and V-PPase (avp1) genes in the high NUE genotype were significantly lower than in the low NUE genotype, resulting in lower V-ATPase and V-PPase activities in the high NUE genotype. The transport of NO3− and protons from the cytoplasm to the vacuole is powered by V-ATPase and V-PPase, so their lower activities increase H+ efflux from and reduce NO3− influx into the vacuoles of the high NUE genotype. We conclude that the lower activity of proton pumps at the tonoplast is the main reason the high NUE genotype possesses lower NO3− content and higher N-use efficiency.

Fe deficiency differentially affects the vacuolar proton pumps in cucumber and soybean roots

Iron uptake in dicots depends on their ability to induce a set of responses in root cells including rhizosphere acidification through H+ extrusion and apoplastic Fe(III) reduction by Fe(III)-chelate reductase. These responses must be sustained by metabolic rearrangements aimed at providing the required NAD(P)H, ATP and H+. Previous results in Fe-deficient cucumber roots showed that high H+ extrusion is accompanied by increased phosphoenolpyruvate carboxylase (PEPC) activity, involved in the cytosol pH-stat; moreover 31P-NMR analysis revealed increased vacuolar pH and decreased vacuolar [inorganic phosphate (Pi)]. The opposite was found in soybean: low rhizosphere acidification, decreased PEPC activity, vacuole acidification, and increased vacuolar [Pi]. These findings, highlighting a different impact of the Fe deficiency responses on cytosolic pH in the two species, lead to hypothesize different roles for H+ and Pi movements across the tonoplast in pH homeostasis. The role of vacuole in cytosolic pH-stat involves the vacuolar H+-ATPase (V-ATPase) and vacuolar H+-pyrophosphatase (V-PPase) activities, which generating the ΔpH and ΔΨ, mediate the transport of solutes, among which Pi, across the tonoplast. Fluxes of Pi itself in its two ionic forms, H2PO4- predominating in the vacuole and HPO42- in the cytosol, may be involved in pH homeostasis owing to its pH-dependent protonation/deprotonation reactions. Tonoplast enriched fractions were obtained from cucumber and soybean roots grown with or without Fe. Both V-ATPase and V-PPase activities were analyzed and the enrichment and localization of the corresponding proteins in root tissues were determined by Western blot and immunolocalization. V-ATPase did not change its activity and expression level in response to Fe starvation in both species. V-PPase showed a different behavior: in cucumber roots its activity and abundance were decreased, while in Fe-deficient soybean roots they were increased. The distinct role of the two H+ pumps in Pi fluxes between cytoplasm and vacuole in Fe-deficient cucumber and soybean root cells is discussed.

Differential regulation of vacuolar H+-ATPase and H+-PPase in Cucumis sativus roots by zinc and nickel

Zinc and nickel, as micronutrients, are essential for all organisms. We investigated the effect of 10 and 100μM Zn and Ni on two tonoplast proton pumps, vacuolar H+-ATPase (V-ATPase) (EC 3.6.3.14) and vacuolar H+-pyrophosphatase (V-PPase) (EC 3.6.1.1), in cucumber roots. ATP-dependent proton transport as well as ATP hydrolysis, catalyzed by V-ATPase, decreased in roots after exposure of plants to both Zn and Ni under all the examined conditions. In contrast, V-PPase activities, measured as PPi hydrolysis and PPi-driven H+ transport, were stimulated by lower concentration of metals. However, at higher metal concentration, hydrolytic activity of V-PPase remained unchanged, while PPi-dependent proton pumping into the tonoplast vesicles was reduced. When heavy metals were introduced into the enzyme reaction medium, both V-ATPase and V-PPase activities were lowered by Zn and Ni in a similar manner. As the gene expression and immunoblot analyses depicted, observed changes in the activity of both tonoplast proton pumps in response to zinc and nickel were not due to the modification in the expression of the CsVHA-A, CsVHA-c and CsVP genes encoding V-ATPase subunit A and c, and V-PPase, respectively, in cucumber roots or in amounts of enzyme proteins. Moreover, Zn as well as Ni ions did not enhance the lipid peroxidation in the root tonoplast fractions. Comparison of ATP and pyrophosphate contents in the control roots and roots treated with heavy metals revealed that Zn and Ni do not affect the ATP amount but reduce the PPi level.

Stress-mediated alteration in V-ATPase and V-PPase of Butea monosperma

The activity and subunit amounts of V-ATPase and V-PPase in various plants of Butea monosperma Taub. (Fabaceae) (ver. Dhak; Palas) growing as a natural inhabitant in varying stress conditions in southeast Rajasthan were studied. Western blot analysis followed by immunological quantification of V-ATPase subunits using specific polyclonal antibodies showed that the subunits A, B, D, E, and c are clearly detectable in all plants, with A, B, and c appearing as intense bands. The other subunits of V-ATPase, viz., C, a, and d, were also detected in majority of the plants. Various subunits exhibited variations in their protein amount in different plants. Besides, a few other clear bands were also detected. Of these, the 30- and 29-kD bands may possibly be Di and Ei. Furthermore, a clear band of V-PPase corresponding to 67-70 kD was also detected. A comparison of the V-ATPase and V-PPase activity revealed that Butea plants in the upper region of the study site showed 70% and 39% higher activity, respectively. Furthermore, the immunological quantification showed that the V-ATPase and V-PPase protein amounts are also higher in the upper Butea plants which have drought stress and, moreover, are also exposed to stronger light intensities for relatively longer duration.

Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation

The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar H(+)-ATPase (V-ATPase) and the vacuolar H(+)-pyrophosphatase (V-PPase); however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar Zn(2+)/H(+)-antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar Na(+)/H(+)-antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vha-a2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth.

Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1

Transgenic rice plants co-expressing the Suaeda salsa SsNHX1 (vacuolar membrane Na+/H+ antiporter) and Arabidopsis AVP1 (vacuolar H+-PPase) showed enhanced salt tolerance during 3 d of 300 mM NaCl treatment under outdoor growth conditions. These transgenic rice seedlings also grew better on MS medium containing 150 mM NaCl compared to SsNHX1-transformed lines and non-transformed controls. Measurements on isolated vacuolar membrane vesicles derived from the salt stressed SsNHX1+AVP1-transgenic plants demonstrated that the vesicles had increased V-PPase hydrolytic activity in comparison with the Ss-transgenics and non-transgenics. Moreover the V-PPase activity was closely related to the development period of the SA-transgenic seedlings and markedly higher in 3-week-old seedlings than in 5-week-old seedlings. Statistic analysis indicated that the SA-transgenic rice plants contained relatively more ions with higher K+/Na+ ratio in their shoots compared to the SsNHX1-transformed lines upon salt treatment. Furthermore, these SA-transformants also exhibited relatively higher level of photosynthesis and root proton exportation capacity whereas reduced H2O2 generation in the same plants. In general, these results supported the hypothesis that simultaneous expression of the SsNHX1 and AVP1 conferred greater performance to the transgenic plants than that of the single SsNHX1.

Enhancement of Vacuolar H+-ATPase and H+-pyrophosphatase Expression by Phytohormones in Pear Fruit

Vacuolar H+-pumps, vacuolar H+-ATPase and vacuolar H+-pyrophosphatase (V-ATPase and V-PPase), have important roles in the accumulation of sugars in vacuoles, in which their expression seems to be associated with changes in phytohormones. We investigated how phytohormones affect the expression of vacuolar H+-pumps by using immature fruit of pear (Pyrus communis L. var. sativa DC, ‘La France’) that actively accumulates sugars. Abscisic acid (ABA), gibberellic acid (GA), indole acetic acid (IAA) or 6-benzyladenine (BA) treatments increased markedly the levels of V-ATPase subunit proteins (A subunit (65 kDa), B subunit (55 kDa), and c subunit (16 kDa)). The V-ATPase activity increased by 28% over the control in disc tissues incubated with 10 μM ABA, accompanied by an increase in A subunit mRNA level. GA increased the V-ATPase activity by 48% over the control, but not the mRNA level. IAA and BA had no effect on the activity. Treatment with 100 μM GA increased the V-PPase protein level, the V-PPase activity by 20% over the control, and mRNA. IAA and BA also increased V-PPase protein but not the activity. These results show that the expression of vacuolar H+-pumps is complicated; it is controlled at each level of mRNA, protein, or activity, and that the increase in protein does not always lead to an increase in activity.

Changes in acidity and in proton transport at the tonoplast of grape berries during development.

As in many fruits, the induction of grape berry (Vitis vinifera L.) ripening results in intense breakdown of malic acid. Using membrane fractions, we tested the hypothesis that changes in acidity resulted from malate vacuolar decompartmentation. The hydrolytic activities of the two primary vacuolar pumps inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) and vacuolar ATPase (V-ATPase; EC 3.6.1.3) increased throughout development with an acceleration during ripening, as confirmed by Western blotting and analysis of transcript expression. The ratio of V-PPase activity to V-ATPase activity was always in favour of V-PPase and reached its maximum value at véraison. The rate of anion transport strongly increased during ripening. Before ripening, tonoplast passive permeability was low, but rose during ripening. Our data indicate that tonoplast leakage dramatically increased during ripening. This leakage is probably the prime cause of malate decompartmentation, amplified by the incapacity of oxidative phosphorylation to face increased energy demand.

Partial characterization of H-translocating inorganic pyrophosphatase from 3 citrus varieties differing in vacuolar pH.

Vacuolar pyrophosphatase (V-PPase) from juice cells of 3 citrus varieties (differing in their vacuolar pH) were partially characterized using purified tonoplast vesicles. Total V-PPase activity was highest in vesicle samples from sweet limes with vacuolar pH of 5.0, while samples from acid limes (with lowest vacuolar pH of 2.0) had the minimal total V-PPase activity. Samples from 'Valencia' orange had intermediate V-PPase levels. When assayed at equal V-PPase activity (measured as Pi production), V-PPase was not able to generate a pH gradient (DeltapH) in vesicles from acid lime, despite its capacity to form a DeltapH in the presence of ATP. Vesicles from sweet lime and 'Valencia' orange were able to form similar DeltapHs in the presence of PPi and ATP supplied together or separately. Antibodies raised against a peptide corresponding to the catalytic site of mung bean V-PPase reacted with samples from all varieties, coinciding with their capacity to hydrolyze PPi. However, antibodies raised against the entire V-PPase polypeptide from mung bean recognized V-PPase from sweet lime and 'Valencia' orange, but did not recognize acid lime samples even at elevated protein concentrations. The structural differences highlighted by antibody recognition, substrate affinity and proton-pumping reactions of V-PPase presented here may reflect evolutionary adaptations related to its reduced function under in vivo conditions and are in agreement with our understanding of acid, sugar accumulation and vacuolar pH changes during the development and maturation of citrus fruits.

The Fluorescent pH-Indicator BCECF: An Inhibitor of the Vacuolar ATPase from Chenopodium rubrum

Summary Selective vacuolar accumulation of the fluorescent pH-indicator 2′, 7′-bis (2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF) may allow to study vacuolar acidification in vivo (Brauer et ai., 1995), which in plant cells is driven by two distinct proton pumps: the V-ATPase (EC 3.6.1.3) and the V-PPase (EC 3.6.1.1). The use of BCECF may help to investigate these proton pumps in vivo . A necessary precondition for such a study is that both enzymes are not affected by the indicator. This was investigated using tonoplast enriched membrane vesicles from Chenopodium rubrum L. suspension cells. We found that BCECF was a strong inhibitor of the V-ATPase. Half-maximal inhibition of the V-ATPase was observed with 10 μmol/L BCECF. The Hill constant (n H = 1.3) for inhibition of the V-ATPase insdicates the presence of two binding sites for BCECF. BCECF was less inhibitory to the vacuolar PPase than to the vacuolar ATPase; half-maximal inhibition was obtained with 500 μmol/L BCECF (n H = 0.5). Moreover, we found that BCECF (2 μmol/L) increased the affinity of the V-ATPase for its specific inhibitor bafilomycin, i.e. it decreased the concentration for half-maximal inhibition from 2 nmol/L to 0.17 nmol/L bafilomycin. We suggest positive cooperation between binding of BCECF and bafilomycin to the V-ATPase. Fluorescein acid and its derivatives 5-carboxyfluorescein, 6-carboxyfluorescein, BCECF and BCECF-AM (acetoxymethyl ester of BCECF) gave similar inhibition of the V-ATPase, indicating that the chromophore fluorescein interacts with the enzyme. Finally, living cells were incubated for 1 h in BCECF-AM (5 μmol/L), a cell-permeable ester derivative of BCECF that is hydrolyzed by cytosolic esterases to yield intracellularly trapped BCECF. Tonoplast vesicles were isolated by sucrose density gradient centrifugation from cells washed free from the loading solution. V-ATPase and V-PPase activity of these tonoplast vesicles showed a decrease of 18 % and 45 %, respectively. Together, these data indicate that the widely used in vivo pH-indicator BCECF alters V-ATPase activity and properties and can therefore interfere with the pH of cellular compartments acidified or alkalized by the V-ATPase, whereas BCECF-AM added to living cells inhibits the V-PPase by an interaction with cellular mechanisms regulating its activity.