Reappearance of hydrolytic activities and tonoplast proteins in the regenerated vacuole of evacuolated protoplasts

Mesophyll protoplasts of tobacco (Nicotiana tabacum L. cv. Xanthi) were evacuolated by centrifugation in a density gradient. Evacuolation resulted in the quantitative loss of vacuolar hydrolytic activities. The evacuolated miniprotoplasts were cultivated under different conditions, and the regeneration of the central vacuole was investigated by light and electron microscopy as well as by the determination of activities of vacuolar marker enzymes. Vacuoles and hydrolytic activities, as well as cell wall material reappeared faster when the cells were cultivated at low osmotic strength. A newly synthesized tonoplast polypeptide could be detected using a polyspecific serum raised against tonoplast proteins of barley (Hordeum vulgare L.). Both vacuolar proton pumps, the ATPase as well as the pyrophosphatase appear to be newly synthesized during the regeneration of the vacuole.Abbreviations GAP-DH NADP-dependent glyceraldehyde 3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PPi pyrophosphate - PPase pyrophosphatase We thank Dr. Ernst Wehrli, Labor für Elektronenmikroskopie I, ETH Zürich, for taking micrographs. Esther Vogt assisted in the determination of the hydrolases. Bafilomycin was kindly provided by Professor Altendorf, Osnabrück FRG. This work was supported by the Swiss National Foundation grant No. 31-25196.88.     

NaCl胁迫对高粱根、叶鞘和叶片液泡膜ATP酶和焦磷酸酶活性的影响

NaCl胁迫初期 ,Na 主要在根和叶鞘中积累。相应地 ,根和叶鞘液泡膜ATP酶和焦磷酸酶水解活性、依赖ATP和PPi的质子泵活性及Na /H 逆向转运活性均明显增加 ,根和叶鞘的生长没有受到抑制。NaCl胁迫后期 ,Na 开始向地上部分运输并在叶片中积累。此时 ,叶片液泡膜质子泵和Na /H 逆向转运活性开始增加 ,根和叶鞘的Na/K比增加 ,其液泡膜ATP酶和焦磷酸酶水解活性、质子泵活性和Na /H 逆向转运活性下降。相应地 ,根和叶鞘的生长也下降。当保温介质中Na/K比超过 1时 ,液泡膜微囊ATP酶和焦磷酸酶活性均随Na/K比的增加而下降。表明非盐生植物液泡膜质子泵在盐胁迫的初期对Na 在液泡内的积累及其耐盐性起重要作用     

Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and Pyrophosphatase : Evidence for a Hydrophobic l-Amino Acid Carrier System

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K_m-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V_{max app} was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 10⁶ vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H⁺-translocating enzyme activities revealed K_{m app} values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH⁺ while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.     

The lysolipid sphingosine modulates pyrophosphatase activity in tonoplast vesicles and isolated vacuoles from a heterotrophic cell suspension culture of Chenopodium rubrum

The proton pumping activity of the tonoplast (vacuolar membrane) H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) has been studied on a tonoplast-enriched microsomal fraction and on intact vacuoles isolated from a heterotrophic cell suspension culture of Chenopodium rubrum L. in the presence of the lysosphingolipids D-sphingosine, psychosine (galactosylsphingosine) and lysosulfatide (sulfogalactosyl-sphingosine). Sphingosine strongly stimulates (K_a= 0.16 μ M ) the PPase activity, assayed both as ΔpH formation across the tonoplast vesicle membrane, and as reversible clamp current measured by the whole-vacuolar mode of the patch-clamp technique. Psychosine showed a minor, and lysosulfatide no stimulatory effect. No effect upon the ATPase activity has been observed. No sphingosine-induced change could be observed in the affinity of the PPase for its substrate (apparent K_m= 10 μ M MgPPi). We tentatively conclude that sphingosine, which is known as a potent inhibitor of the protein kinase C in animal cells, may be a regulator of the plant vacuolar PPase.     

Vacuolar respiration of nitrate coupled to energy conservation in filamentous Beggiatoaceae

We show that the nitrate storing vacuole of the sulfide‐oxidizing bacterium Candidatus Allobeggiatoa halophila has an electron transport chain (ETC), which generates a proton motive force (PMF) used for cellular energy conservation. Immunostaining by antibodies showed that cytochrome c oxidase, an ETC protein and a vacuolar ATPase are present in the vacuolar membrane and cytochrome c in the vacuolar lumen. The effect of different inhibitors on the vacuolar pH was studied by pH imaging. Inhibition of vacuolar ATPases and pyrophosphatases resulted in a pH decrease in the vacuole, showing that the proton gradient over the vacuolar membrane is used for ATP and pyrophosphate generation. Blockage of the ETC decreased the vacuolar PMF, indicating that the proton gradient is build up by an ETC. Furthermore, addition of nitrate resulted in an increase of the vacuolar PMF. Inhibition of nitrate reduction, led to a decreased PMF. Nitric oxide was detected in vacuoles of cells exposed to nitrate showing that nitrite, the product of nitrate reduction, is reduced inside the vacuole. These findings show consistently that nitrate respiration contributes to the high proton concentration within the vacuole and the PMF over the vacuolar membrane is actively used for energy conservation.     

Anion-sensitive ATPase activity and proton transport in isolated vacuoles of species of the CAM genus Kalanchoë

Vacuoles were isolated from leaves of Kalanchoë daigremontiana Hamet et Perrier de la Bathie, and the ionic sensitivity of the vacuolar ATPase was studied in vacuole homogenates desalted on Sephadex G-25. The ATPase activity was dependent on the presence of divalent cations (Mg²⁺≥ Mn²⁺≥ Ca²⁺, Co²⁺; Zn²⁺ had no effect). Mg²⁺-dependent ATPase activity was stimulated by anions (Cl^? > malate²⁺, HCO^?₃), with maximal stimulation at concentrations above 50 mM. Mg²⁺-Dependent activity was inhibited by NO^?₃ above 2 mM, but no saturation was observed up to 100 mM. No stimulation by K⁺ or Na⁺ was detected; stimulation by NH⁺₄ was abolished by 0.01% (w/v) Triton X-100, suggesting that the NH⁺₄ effect was due to the permeability of vacuolar membrane vesicles to NH₃. Trans-tonoplast electrical potentials (Δψ) and intra-vacuolar pH were measured with glass microelectrodes and antimony covered glass micro-pH-electrodes, respectively. Free vacuofes isolated from Kalanchoë tubiflora (Harv.) Hamet were slightly positive with respect to the suspension medium. This Δψ was insensitive to the protonophore FCCP and depolarized by about 4 mV on addition of 50 mM KCl, still remaining about +5 mV. Upon addition of 7 mM Mg-ATP, vacuoles showed an FCCP-sensitive increase of Δψ from +9.2 ± 2.8 (13) to +17.8 ± 3.7 (12) mV [given as x?± sd (n)] and an internal acidification from pH 5.4 ± 0.2 (11) to pH 4.3 ± 0.4 (12). Mg-ADP and ATP without Mg²⁺ had no effect on Δψ. It is concluded that the H4 pumping at the tonoplast is due to the functioning of the anion-sensitive vacuolar ATPase and that this is an essential part of the mechanism of nocturnal acid accumulation in CAM.     

Alternative photophosphorylation,inorganic pyrophosphate synthase and inorganic pyrophosphate

This minireview in memory of Daniel I. Arnon, pioneer in photosynthesis research, concerns properties of the first and still only known alternative photophosphorylation system, with respect to the primary phosphorylated end product formed. The alternative to adenosine triphosphate (ATP), inorganic pyrophosphate (PPi), was produced in light, in chromatophores from the photosynthetic bacterium Rhodospirillum rubrum, when no adenosine diphosphate (ADP) had been added to the reaction mixture (Baltscheffsky H et al. (1966) Science 153: 1120–1122). This production of PPi and its capability to drive energy requiring reactions depend on the activity of a membrane bound inorganic pyrophosphatase (PPase) (Baltscheffsky M et al. (1966) Brookhaven Symposia in Biology, No. 19, pp 246–253); (Baltscheffsky M (1967) Nature 216: 241–243), which pumps protons (Moyle J et al. (1972) FEBS Lett 23: 233–236). Both enzyme and substrate in the PPase (PPi synthase) are much less complex than in the case of the corresponding adenosine triphosphatase (ATPase, ATP synthase). Whereas an artificially induced proton gradient alone can drive the synthesis of PPi, both a proton gradient and a membrane potential are required for obtaining ATP. The photobacterial, integrally membrane bound PPi synthase shows immunological cross reaction with membrane bound PPases from plant vacuoles (Nore BF et al. (1991) Biochem Biophys Res Commun 181: 962–967). With antibodies against the purified PPi synthase clones of its gene have been obtained and are currently being sequenced. Further structural information about the PPi synthase may serve to elucidate also fundamental mechanisms of electron transport coupled phosphorylation. The existence of the PPi synthase is in line with the assumption that PPi may have preceded ATP as energy carrier between energy yielding and energy requiring reactions.     

Vacuolar compartmentalization as indispensable component of heavy metal detoxification in plants

Plant cells orchestrate an array of molecular mechanisms for maintaining plasmatic concentrations of essential heavy metal (HM) ions, for example, iron, zinc and copper, within the optimal functional range. In parallel, concentrations of non‐essential HMs and metalloids, for example, cadmium, mercury and arsenic, should be kept below their toxicity threshold levels. Vacuolar compartmentalization is central to HM homeostasis. It depends on two vacuolar pumps (V‐ATPase and V‐PPase) and a set of tonoplast transporters, which are directly driven by proton motive force, and primary ATP‐dependent pumps. While HM non‐hyperaccumulator plants largely sequester toxic HMs in root vacuoles, HM hyperaccumulators usually sequester them in leaf cell vacuoles following efficient long‐distance translocation. The distinct strategies evolved as a consequence of organ‐specific differences particularly in vacuolar transporters and in addition to distinct features in long‐distance transport. Recent molecular and functional characterization of tonoplast HM transporters has advanced our understanding of their contribution to HM homeostasis, tolerance and hyperaccumulation. Another important part of the dynamic vacuolar sequestration syndrome involves enhanced vacuolation. It involves vesicular trafficking in HM detoxification. The present review provides an updated account of molecular aspects that contribute to the vacuolar compartmentalization of HMs.     

Regulation of pyrophosphate levels by H+-PPase is central for proper resumption of early plant development

The synthesis of DNA, RNA, and de novo proteins is fundamental for early development of the seedling after germination, but such processes release pyrophosphate (PPi) as a byproduct of ATP hydrolysis. The over-accumulation of the inhibitory metabolite PPi in the cytosol hinders these biosynthetic reactions. All living organisms possess ubiquitous enzymes collectively called inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of PPi into two orthophosphate (Pi) molecules. Defects in PPase activity cause severe developmental defects and/or growth arrest in several organisms. In higher plants, a proton-translocating vacuolar PPase (H⁺­PPase) uses the energy of PPi hydrolysis to acidify the vacuole. However, the biological implications of PPi hydrolysis are vague due to the widespread belief that the major role of H⁺­PPase in plants is vacuolar acidification. We have shown that the Arabidopsis fugu5 mutant phenotype, caused by a defect in H⁺­PPase activity, is rescued by complementation with the yeast cytosolic PPase IPP1. In addition, our analyses have revealed that increased cytosolic PPi levels impair postgerminative development in fugu5 by inhibiting gluconeogenesis. This led us to the conclusion that the role of H⁺­PPase as a proton-pump is negligible. Here, we present further evidence of the growth-boosting effects of removing PPi in later stages of plant vegetative development, and briefly discuss the biological role of PPases and their potential applications in different disciplines and in various organisms.     

Transport processes of solutes across the vacuolar membrane of higher plants

The central vacuole is the largest compartment of a mature plant cell and may occupy more than 80% of the total cell volume. However, recent results indicate that beside the large central vacuole, several small vacuoles may exist in a plant cell. These vacuoles often belong to different classes and can be distinguished either by their contents in soluble proteins or by different types of a major vacuolar membrane protein, the aquaporins. Two vacuolar proton pumps, an ATPase and a PPase energize vacuolar uptake of most solutes. The electrochemical gradient generated by these pumps can be utilized to accumulate cations by a proton antiport mechanism or anions due to the membrane potential difference. Uptake can be catalyzed by channels or by transporters. Growing evidence shows that for most ions more than one transporter/channel exist at the vacuolar membrane. Furthermore, plant secondary products may be accumulated by proton antiport mechanisms. The transport of some solutes such as sucrose is energized in some plants but occurs by facilitated diffusion in others. A new class of transporters has been discovered recently: the ABC type transporters are directly energized by MgATP and do not depend on the electrochemical force. Their substrates are organic anions formed by conjugation, e.g. to glutathione. In this review we discuss the different transport processes occurring at the vacuolar membrane and focus on some new results obtained in this field.     

Nitrate enhancement of CAM activity in two Kalanchoë species is associated with increased vacuolar proton transport capacity

Among species that perform CAM photosynthesis, members of the genus Kalanchoë have been studied frequently to investigate the effect of environmental factors on the magnitude of CAM activity. In particular, different nitrogen sources have been shown to influence the rate of nocturnal CO₂ fixation and organic‐acid accumulation in several species of Kalanchoë. However, there has been little investigation of the interrelationship between nitrogen source (nitrate versus ammonium), concentration and the activity of the vacuolar proton pumps responsible for driving nocturnal organic‐acid accumulation in these species. In the present study with Kalanchoë laxiflora and Kalanchoë delagoensis cultivated on different nitrogen sources, both species were found to show highest total nocturnal organic‐acid accumulation and highest rates of ATP‐ and PPi‐dependent vacuolar proton transport on 2.5 mM nitrate, whereas plants cultivated on 5.0 mM ammonium showed the lowest values. In both species malate was the principal organic‐acid accumulated during the night, but the second‐most accumulated organic‐acid was fumarate for K. laxiflora and citrate for K. delagoensis. Higher ATP‐ and PPi‐dependent vacuolar proton transport rates and greater nocturnal acid accumulation were observed in K. delagoensis compared with K. laxiflora. These results show that the effect of nitrogen source on CAM activity in Kalanchoë species is reflected in corresponding differences in activity of the tonoplast proton pumps responsible for driving sequestration of these acids in the vacuole of CAM‐performing cells.     

Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings

Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata) were successfully isolated using Ficoll gradient and two-phase (polyethylene glycol-dextran) partition. The ER-enriched vesicles contained inorganic pyrophosphate (PPi) hydrolysis and its associated proton translocating activities. Antiserum prepared against vacuolar H+-pyrophosphatase (V-PPase, EC 3.6.1.1) did not inhibit this novel pyrophosphatase-dependent proton translocation, excluding the possible contamination of tonoplast vesicles in the ER-enriched membrane preparation. The optimal ratios of Mg2+/PPi (inorganic pyrophosphate) for enzymatic activity and PPi-dependent proton translocation of ER-enriched vesicles were higher than those of vacuolar membranes. The PPi-dependent proton translocation of ER-enriched vesicles absolutely required the presence of monovalent cations with preference for K+, but could be inhibited by a common PPase inhibitor, F-. Furthermore, ER H+-pyrophosphatase exhibited some similarities and differences to vacuolar H+-PPases in cofactor/substrate ratios, pH profile, and concentration dependence of F-, imidodiphosphate (a PPi analogue), and various chemical modifiers. These results suggest that ER-enriched vesicles contain a novel type of proton-translocating PPase distinct from that of tonoplast from higher plants.     

Comparative studies on the electrical properties of the H+ translocating ATPase and pyrophosphatase of the vacuolar-lysosomal compartment.

The electrical properties of the vacuolar-lysosomal H+ pumps were studied by direct measurement of the pump currents using the whole-cell configuration of the patch-clamp technique. Both pumps, the proton-translocating ATPase and pyrophosphatase, when activated by MgATP or inorganic Mg pyrophosphate (MgPP(i)), transport protons into the vacuole and polarize the membrane potential (positive inside the vacuole). Accumulation of protons in the lumen of vacuole vesicles was monitored by absorbance changes of the pH probe, acridine orange. The electrochemical gradient provided by both the ATPase and pyrophosphatase stimulates effectively the uptake of various metabolites such as malate, citrate and sucrose. The maximal current density produced by the ATPase was about 2.5 microA/cm2 and about 0.5 microA/cm2 for the pyrophosphatase. K(m)ATP was 0.6 mM; K(m)PPi was 15-20 microM with progressive inhibition above 150 microM. At a cytoplasmic pH of 7.5 both enzymes were capable of pumping protons against a 10,000-fold concentration gradient (pH 3.5 inside the vacuole). Proton current produced by the ATPase was blocked reversibly by extravacuolar NO(3)- only.     

Differential pH restoration after ammonia-elicited vacuolar alkalisation in rice and maize root hairs as measured by fluorescence ratio

Changes of vacuolar pH in hair cells of young rice (Oryza sativa L.) and maize (Zea mays L.) roots were measured after ammonia application at various levels of external pH. After loading the pH-sensitive, fluorescent dye Oregon green 488 carboxylic acid 6-isomer into the vacuoles of root hairs, ratiometric pH data of high statistical significance were obtained from root hair populations comprising hundreds of cells. The pH of the vacuole at external pH 5.0 was 5.32 ± 0.08 (±SD, n= 15) and 5.41 ± 0.13 (±SD, n= 15) in rice and maize, respectively. A moderate external ammonia concentration of 2 mM led to vacuolar alkalisation at both, low (pH 5.0) and high (pH 7.0–9.0) external pH, presumably due to NH₃ permeation into the vacuole. With increasing external pH, ammonia application did not cumulatively increase vacuolar pH. In rice, the increase in vacuolar pH ranged from 0.1–0.8 pH units; in maize a more constant increase of 0.5 pH units was observed. The vacuolar pH increase was efficiently depressed in rice (especially at high external pH), but not in maize. Inhibition of the tonoplast H⁺-ATPase by concanamycin A raised vacuolar pH and increased the ammonia-elicited vacuolar alkalisation in both species, proving that vacuolar H⁺-ATPase activity counters the ammonia-elicited alkalisation effect. However, even under conditions of vacuolar H⁺-ATPase inhibition, rice was still able to restore an ammonia-elicited pH increase. High vacuolar pH levels as found in maize under conditions of high NH₃ influx may derive from inefficient cytosolic ammonia assimilation and tonoplast proton pumping. Thus, in maize, prolonged reduction of the proton gradient between the cytosol and the vacuole may play an important role in NH₃ toxicity. Received: 12 September 1997 / Accepted: 19 January 1998     

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Effects of pH on proton transport by vacuolar pumps from maize roots

Protons pumps of the tonoplast may be involved in the regulation of cytosolic pH, but the effects of pH on the coupled activities of these transporters are poorly understood. The effects of pH on the activities of the H⁺-translocating pyrophosphatase (PP_iase) and vacuolar-type H⁺-translocating adenosine triphosphatase (H⁺-ATPase) from maize ( Zea mays L. cv. FRB 73) root membranes were assessed by model that simultaneously considers proton transport by the pump and those processes that reduce net transport. The addition of either pyrophosphate or ATP to either microsomal or tonoplast membranes generated a pH gradient. The pH gradient generated in the presence of both substrates was not the sum of the gradients produced by the two substrates added separately. When membranes were separated by sucrose density gradient centrifugation, pyrophosphate (PP_i)-dependent proton transport was associated with light density membranes having tonoplast H⁺-ATPase activity. These results indicate that some portion of the PP_iase was located on the same membrane system as the tonoplast ATPase; however, tonoplast vesicles may be heterogeneous, differing slightly in the ratio of ATP- to PP_i-dependent transport. Proton transport by both the PP_iase and ATPase had maximal activity at pH 7.0 to 8.0 Decreases in proton transport by the ATPase at pH above the optimum were associated with increases in the processes that reduce net transport. Such an association was not observed at pH values below the optimum. These results are discussed in terms of in situ regulation of cytoplasmic pH by the two pumps.     

TRANSPARENT TESTA 13 is a tonoplast P3A‐ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P‐type H⁺‐ATPases in the plasma membrane, and multimeric vacuolar‐type H⁺‐ATPases (V‐ATPases) and vacuolar H⁺‐pyrophosphatases (H⁺‐PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P_3A‐ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA‐filled central vacuoles as observed in the wild‐type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P‐type H⁺‐ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H⁺‐PPase VHP1. Our findings indicate that the P_3A‐ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12‐mediated transport of PA precursors to the vacuole.     

Chilling induces a decrease in pyrophosphate-dependent H+-accumulation associated with a DeltapH(vac)-stat in mung bean, a chill-sensitive plant

Chilling leads to cytoplasmic acidification in chill-sensitive plants. A possible explanation for this observation is that a ΔpH-stat between the cytosol and vacuole (ΔpH_vac-stat) is perturbed by chilling. To understand the nature of this ΔpH_vac-stat, the effect of temperature, between 20 and 0 °C, on pyrophosphate (PPi)- or ATP-dependent acidification of vacuolar vesicles, isolated from mung bean hypocotyls, was determined. Over the temperature range investigated, the H⁺-influx mediated by PPase was balanced with the H⁺-efflux, which was PPi-dependently suppressed, and consequently a constant pH in vesicles (pH_in) of ca. 5 was maintained against temperature changes. However, the ΔpH_in driven by ATP decreased as the temperature dropped. Thus, the PPi-dependent H⁺-accumulation may function as an essential factor to form a ΔpH_vac-stat against temperature changes. Next, to study the chilling sensitivity of PPi-dependent H⁺-accumulation, vacuolar vesicles were isolated from control seedlings or from seedlings chilled at 0 °C for 1 d. Chilling treatment resulted in a decrease in the H⁺-accumulation rate and in the steady-state ΔpH_in formed by PPi, the causes of which were enhanced by PPi-dependent H⁺-efflux and reduced by H⁺-influx driven by PPase. Together, the results suggest that the decrease of PPi-dependent H⁺-accumulation associated with the ΔpH_vac-stat could result in cytoplasmic acidification.