Transport Processes in Vacuoles of Higher Plants

The vacuole is the largest compartment of a mature plant cell and serves as an internal reservoir of metabolites and nutrients. In the last years transport of solutes across the tonoplast has been intensively investigated. It was shown that two different proton pumps reside in the tonoplast. These pumps generate an electrochemical gradient which can be used as an energy-source to accumulate solutes. Cation uptake is driven by an H⁺ antiport mechanism. Anions are accumulated in response to the inside positive membrane potential. In addition, the existence of ion channels was shown using the patch clamp technique. The aim of this review is to compare and to discuss the present state of our knowledge of solute transport across the tonoplast.     

Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2

The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole.     

Flavone glucoside uptake into barley mesophyll and Arabidopsis cell culture vacuoles. Energization occurs by H(+)-antiport and ATP-binding cassette-type mechanisms

In many cases, secondary plant products accumulate in the large central vacuole of plant cells. However, the mechanisms involved in the transport of secondary compounds are only poorly understood. Here, we demonstrate that the transport mechanisms for the major barley (Hordeum vulgare) flavonoid saponarin (apigenin 6-C-glucosyl-7-O-glucoside) are different in various plant species: Uptake into barley vacuoles occurs via a proton antiport and is competitively inhibited by isovitexin (apigenin 6-C-glucoside), suggesting that both flavone glucosides are recognized by the same transporter. In contrast, the transport into vacuoles from Arabidopsis, which does not synthesize flavone glucosides, displays typical characteristics of ATP-binding cassette transporters. Transport of saponarin into vacuoles of both the species is saturable with a K(m) of 50 to 100 microM. Furthermore, the uptake of saponarin into vacuoles from a barley mutant exhibiting a strongly reduced flavone glucoside biosynthesis is drastically decreased when compared with the parent variety. Thus, the barley vacuolar flavone glucoside/H(+) antiporter could be modulated by the availability of the substrate. We propose that different vacuolar transporters may be responsible for the sequestration of species-specific/endogenous and nonspecific/xenobiotic secondary compounds in planta.     

Plant cation/H+ exchangers (CAXs): biological functions and genetic manipulations

Inorganic cations play decisive roles in many cellular and physiological processes and are essential components of plant nutrition. Therefore, the uptake of cations and their redistribution must be precisely controlled. Vacuolar antiporters are important elements in mediating the intracellular sequestration of these cations. These antiporters are energized by the proton gradient across the vacuolar membrane and allow the rapid transport of cations into the vacuole. CAXs (for CAtion eXchanger) are members of a multigene family and appear to predominately reside on vacuoles. Defining CAX regulation and substrate specificity have been aided by utilising yeast as an experimental tool. Studies in plants suggest CAXs regulate apoplastic Ca(2+) levels in order to optimise cell wall expansion, photosynthesis, transpiration and plant productivity. CAX studies provide the basis for making designer transporters that have been used to develop nutrient enhanced crops and plants for remediating toxic soils.     

Recycling und Entsorgung in der Pflanzenzelle: Die Vakuole

The vacuole as a typical feature of plant cells plays an important role within intracellular digestion, storage of solutes, plant defence as well as signalling. For all these purposes, the surrounding biomembrane, the tonoplast, is equipped with different carriers allowing an efficient sequestration of a variety of solutes into the vacuolar lumen. They range from translocators for facilitated diffusion, proton symporters up to primarily energized pumps. They are sed in dependence of the existing concentration and electrical gradient of the respective compound. Some of them are known to respond to environmental changes as salt surplus or variable nutrient supply. During the past two decades, research has led to a better although by far not sufficient understanding of the unique functions of the vacuole at a variety of levels, including stress responses and herbizide detoxification.     

基于液泡膜质子泵的硝态氮再利用研究进展

全面掌握洛川果园的土壤水分环境特征,不仅可为苹果的园址选择、砧穗组合和改进土壤水分管理措施提供理论依据,而且对我国苹果产区果园提质增效具有借鉴价值.采用定点土壤水分连续监测法,对洛川苹果园的总体土壤水分环境以及不同生长年限、不同立地类型和乔、矮化果园的土壤水分分异特征进行分析.结果表明： 苹果树根际区 (0～200 cm)土壤水分普遍亏欠,且0～60 cm土层的水分亏欠小于60～200 cm土层;生长季0～60 cm土层贮水量与降水量的变化一致,土壤相对含水量大多<60%,季节性旱象严重;果园剖面土壤含水量变异系数随土壤深度加深而递减;随果园生长年限的增大,土壤剖面贮水量下降;在栽培密度一致的条件下,矮化果园5 m土层土壤含水量均高于乔化果园,而栽培密度大的矮化果园的土壤贮水量低于栽培密度小的乔化果园;塬地成龄果园的土壤水分含量最高,川地次之,台地相对较低.密度对果园土壤水分含量有很大影响,在栽培密度一致的条件下,采用矮化栽培能减少土壤水分消耗,显著提高果园土壤含水量;挖株降低栽培密度是维持苹果园土壤水分平衡、实现可持续发展的有效途径.     

Vacuolar transporters and their essential role in plant metabolism

Following the unequivocal demonstration that plants contain at least two types of vacuoles, scientists studying this organelle have realized that the plant 'vacuome' is far more complex than they expected. Some fully developed cells contain at least two large vacuoles, with different functions. Remarkably, even a single vacuole may be subdivided and fulfil several functions, which are supported in part by the vacuolar membrane transport systems. Recent studies, including proteomic analyses for several plant species, have revealed the tonoplast transporters and their involvement in the nitrogen storage, salinity tolerance, heavy metal homeostasis, calcium signalling, guard cell movements, and the cellular pH homeostasis. It is clear that vacuolar transporters are an integrated part of a complex cellular network that enables a plant to react properly to changing environmental conditions, to save nutrients and energy in times of plenty, and to maintain optimal metabolic conditions in the cytosol. An overview is given of the main features of the transporters present in the tonoplast of plant cells in terms of their function, regulation, and relationships with the microheterogeneity of the vacuome.     

Na+/H+逆向转运蛋白和植物耐盐性

Na^ /H^ 逆向转运蛋白对植物耐盐起着重要作用,它利用质膜H^ -ATPase或液泡膜H^ -ATPase及PPiase泵H^ 产生的驱动力把Na^ 排出细胞或在液泡中区隔化以消除Na^ 的毒害。主要讨论植物中Na^ /H^ 逆向转运蛋白研究在分子水平的最新进展。     

Sodium transport in plant cells

Salinity limits plant growth and impairs agricultural productivity. There is a wide spectrum of plant responses to salinity that are defined by a range of adaptations at the cellular and the whole-plant levels, however, the mechanisms of sodium transport appear to be fundamentally similar. At the cellular level, sodium ions gain entry via several plasma membrane channels. As cytoplasmic sodium is toxic above threshold levels, it is extruded by plasma membrane Na(+)/H(+) antiports that are energized by the proton gradient generated by the plasma membrane ATPase. Cytoplasmic Na(+) may also be compartmentalized by vacuolar Na(+)/H(+) antiports. These transporters are energized by the proton gradient generated by the vacuolar H(+)-ATPase and H(+)-PPiase. Here, the mechanisms of sodium entry, extrusion, and compartmentation are reviewed, with a discussion of recent progress on the cloning and characterization, directly in planta and in yeast, of some of the proteins involved in sodium transport.     

Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells

The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.     

Saccharomyces cerevisiae lacking Btn1p modulate vacuolar ATPase activity to regulate pH imbalance in the vacuole

The vacuolar H(+)-ATPase (V-ATPase) along with ion channels and transporters maintains vacuolar pH. V-ATPase ATP hydrolysis is coupled with proton transport and establishes an electrochemical gradient between the cytosol and vacuolar lumen for coupled transport of metabolites. Btn1p, the yeast homolog to human CLN3 that is defective in Batten disease, localizes to the vacuole. We previously reported that Btn1p is required for vacuolar pH maintenance and ATP-dependent vacuolar arginine transport. We report that extracellular pH alters both V-ATPase activity and proton transport into the vacuole of wild-type Saccharomyces cerevisiae. V-ATPase activity is modulated through the assembly and disassembly of the V(0) and V(1) V-ATPase subunits located in the vacuolar membrane and on the cytosolic side of the vacuolar membrane, respectively. V-ATPase assembly is increased in yeast cells grown in high extracellular pH. In addition, at elevated extracellular pH, S. cerevisiae lacking BTN1 (btn1-Delta), have decreased V-ATPase activity while proton transport into the vacuole remains similar to that for wild type. Thus, coupling of V-ATPase activity and proton transport in btn1-Delta is altered. We show that down-regulation of V-ATPase activity compensates the vacuolar pH imbalance for btn1-Delta at early growth phases. We therefore propose that Btn1p is required for tight regulation of vacuolar pH to maintain the vacuolar luminal content and optimal activity of this organelle and that disruption in Btn1p function leads to a modulation of V-ATPase activity to maintain cellular pH homeostasis and vacuolar luminal content.     

V-ATPase, ScNhx1p and yeast vacuole fusion

Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.     

Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum

The mechanism of nickel uptake into vacuoles isolated from leaf tissue of Alyssum lesbiacum was investigated to help understand the ability of this species to hyperaccumulate Ni. An imaging system was designed to monitor Ni uptake by single vacuoles using the metal-sensitive fluorescent dye, Newport Green. Nickel uptake into isolated vacuoles from leaf tissue of A. lesbiacum was enhanced by the presence of Mg/ATP, presumably via energisation of the vacuolar H(+)-ATPase (V-ATPase). This ATP-stimulated Ni uptake was abolished by bafilomycin (a diagnostic inhibitor of the V-ATPase) and by dissipation of the transmembrane pH difference with an uncoupler. These observations are consistent with Ni(2+)/nH(+) antiport activity at the tonoplast driven by a proton electrochemical gradient established by the V-ATPase, which would provide a mechanism for secondary active transport of Ni(2+) into the vacuole. This study provides insights into the molecular basis of Ni tolerance in Alyssum, and may aid in the identification of genes involved in Ni hyperaccumulation.     

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.     

The ABC-like vacuolar transporter for rye mesophyll flavone glucuronides is not species-specific

In many cases, the vacuolar uptake of secondary metabolites has been demonstrated to be strictly specific for a given compound and plant species. While most plants contain glycosylated secondary substances, few cases are known where flavonoids may also carry negative charges, e.g. as glucuronide conjugates. Vacuolar transport of glucosylated phenylpropanoid derivatives has been shown to occur by proton substrate antiport mechanisms (Klein, M., Weissenb?ck. G., Dufaud, A., Gaillard, C., Kreuz, K., Martinoia, E., 1996. Different energization mechanisms drive the vacuolar uptake of a flavonoid glucoside and a herbicide glucoside. J. Biol. Chem. 271, 29,666-29,671). In contrast, flavone glucuronides appearing specifically in rye mesophyll vacuoles are taken up by direct energisation utilising MgATP, strongly arguing for the presence of an ATP-binding cassette (ABC) transporter belonging to the subfamily of multidrug resistance-associated proteins (MRP) on the rye vacuolar membrane (Klein, M., Martinoia, E., Hoffmann-Thoma, G., Weissenb?ck, G., 2000. A membrane-potential dependent, ubiquitous ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides regulation of glucturonide uptake by glutathione and its conjugates. Plant Journal 21, 289-304). MRPs are known to transport negatively charged organic anions. Results presented here suggest that the vacuolar directly energised MRP-like glucuronate pump for plant-specific flavone glucuronides is ubiquitously present in diverse plant species since rye flavone glucuronides are taken up into vacuoles isolated from the barley mesophyll or from the broccoli stalk parenchyma representing two species which do not synthesise glucuronidated secondary compounds. According to the transport characteristics and inhibition profile observed we propose the existence of a high-capacity, uncoupler-insensitive vacuolar ABC transporter for flavone glucuronides and possibly other negatively charged organic compounds -- plant-born or xenobiotic -- irrespective of the plant's capability to endogenously produce glucuronidated compounds.     

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.     

The effect of dihydroquercetin on active and passive ion transport systems in plant vacuolar membrane

The effect of dihydroquercetin (DHQ) on proton pumps of the vacuolar membrane (H⁺-ATPase and H⁺-pyrophosphatase), slow vacuolar (SV) channel, lipid peroxidation, and stability of isolated vacuoles was studied. The results of experiments showed that DHQ affected active and passive transport systems of the vacuolar membrane. The mechanism of action of DHQ may be based on its combined effect on the sulfhydryl groups of proteins and the lipid component of the membrane. The strong stabilizing effect of DHQ on the membranes of isolated vacuoles may be associated not only with its antioxidant properties but also with changes in the membrane permeability affecting the ion channels.     

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.     

Two vacuole-mediated defense strategies in plants

As plants lack immune cells, each cell has to defend itself against invading pathogens. Plant cells have a large central vacuole that accumulates a variety of hydrolytic enzymes and antimicrobial compounds, raising the possibility that vacuoles play a role in plant defense. However, how plants use vacuoles to protect against invading pathogens is poorly understood. Recently, we characterized two vacuole-mediated defense strategies associated with programmed cell death (PCD). In one strategy, vacuolar processing enzyme (VPE) mediated the disruption of the vacuolar membrane, resulting in the release of vacuolar contents into the cytoplasm in response to viral infection. In the other strategy, proteasome-dependent fusion of the central vacuole with the plasma membrane caused the discharge of vacuolar antibacterial protease and cell death-promoting contents from the cell in response to bacterial infection. Intriguingly, both strategies relied on enzymes with caspase-like activities: the vacuolar membrane-collapse system required VPE, which has caspase-1-like activity and the membrane-fusion system required a proteasome that has caspase-3-like activity. Thus, plants may have evolved a cellular immune system that involves vacuolar membrane collapse to prevent the systemic spread of viral pathogens and membrane fusion to inhibit the proliferation of bacterial pathogens.Key words: plant-pathogen interaction, vacuole, hypersensitive cell death, caspase activity, vacuolar processing enzyme, proteasome     

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.