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.     

液泡膜转运蛋白在植物细胞代谢中的作用

液泡是植物细胞的一个多功能细胞器,其主要通过膜运输系统执行功能。液泡膜转运蛋白可以控制细胞内物质的储存和运输,参与细胞内的应答胁迫反应,隔离毒性离子,防止细胞质受害,调节Ca^2＋浓度和pH,维持细胞内环境的稳定。本文主要对液泡膜转运蛋白在营养储存、逆境胁迫、细胞内环境稳态中发挥的作用进行综述,以期为进一步阐释液泡复杂生理功能提供一些借鉴。     

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.     

Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.     

In vivo phosphorylation sites of barley tonoplast proteins identified by a phosphoproteomic approach

In plants the vacuolar functions are the cellular storage of soluble carbohydrates, organic acids, inorganic ions and toxic compounds. Transporters and channels located in the vacuolar membrane, the tonoplast, are modulated by PTMs to facilitate the optimal functioning of a large number of metabolic pathways. Here we present a phosphoproteomic approach for the identification of in vivo phosphorylation sites of tonoplast (vacuolar membrane) proteins. Highly purified tonoplast and tonoplast‐enriched microsomes were isolated from photosynthetically induced barley (Hordeum vulgare) mesophyll protoplasts. Phosphopeptides were enriched by strong cation exchange (SCX) chromatography followed either by IMAC or titanium dioxide (TiO₂) affinity chromatography and were subsequently analysed using LC‐ESI‐MS/MS. In total, 65 phosphopeptides of 27 known vacuolar membrane proteins were identified, including the two vacuolar proton pumps, aquaporins, CAX transporters, Na⁺/H⁺ antiporters as well as other known vacuolar transporters mediating the transfer of potassium, sugars, sulphate and malate. The present study provides a novel source to further analyse the regulation of tonoplast proteins by protein phosphorylations, especially as most of the identified phosphorylation sites are highly conserved between Hordeum vulgare (Hv) and Arabidopsis thaliana.     

An integrative view on vacuolar pH homeostasis in Arabidopsis thaliana: Combining mathematical modeling and experimentation

The acidification of plant vacuoles is of great importance for various physiological processes, as a multitude of secondary active transporters utilize the proton gradient established across the vacuolar membrane. Vacuolar-type H⁺-translocating ATPases and a pyrophosphatase are thought to enable vacuoles to accumulate protons against their electrochemical potential. However, recent studies pointed to the ATPase located at the trans-Golgi network/early endosome (TGN/EE) to contribute to vacuolar acidification in a manner not understood as of now. Here, we combined experimental data and computational modeling to test different hypotheses for vacuolar acidification mechanisms. For this, we analyzed different models with respect to their ability to describe existing experimental data. To better differentiate between alternative acidification mechanisms, new experimental data have been generated. By fitting the models to the experimental data, we were able to prioritize the hypothesis in which vesicular trafficking of Ca²⁺/H⁺-antiporters from the TGN/EE to the vacuolar membrane and the activity of ATP-dependent Ca²⁺-pumps at the tonoplast might explain the residual acidification observed in Arabidopsis mutants defective in vacuolar proton pump activity. The presented modeling approach provides an integrative perspective on vacuolar pH regulation in Arabidopsis and holds potential to guide further experimental work.     

Adaptation of plants to salt stress: the role of the ion transporters

Adaptation to high salinity is achieved by cellular ion homeostasis which involves regulation of toxic sodium ion (Na⁺) and Chloride ion (Cl⁻) uptake, preventing the transport of these ions to the aerial parts of the plants and vacuolar sequestration of these toxic ions. Ion transporters have long been known to play roles in maintaining ion homeostasis. Na⁺ enters the cell through various voltage dependent selective and non-selective ion channels. High Na⁺ concentration in the plasma membrane is balanced either by uptake of potassium ion (K⁺) by various potassium importing channels, by salt exclusion mechanism or by sequestration of Na⁺ in the vacuoles. Therefore, the role of high-affinity potassium transporter, the salt overly sensitive pathway, the most well-defined Na⁺ exclusion pathway that exports Na⁺ from cell into xylem and tonoplast localized cation transporters that compartmentalizes Na⁺ in vacuoles need to be studied in detail and applied to make the plant adaptable to saline soil. Knowledge on the regulation of expression of these transporters by the hormones, microRNAs and other non-coding RNAs can be utilized to manipulate the ion transport. Here, we reviewed paradigm of the ion transporters in salt stress signalling pathways from the recent and past studies aiding transformation of basic knowledge into biotechnological applications to generate engineered salt stress tolerant crops.

     

Physiological and molecular mechanisms of heavy metal accumulation in nonmycorrhizal versus mycorrhizal plants

Uptake, translocation, detoxification, and sequestration of heavy metals (HMs) are key processes in plants to deal with excess amounts of HM. Under natural conditions, plant roots often establish ecto‐ and/or arbuscular‐mycorrhizae with their fungal partners, thereby altering HM accumulation in host plants. This review considers the progress in understanding the physiological and molecular mechanisms involved in HM accumulation in nonmycorrhizal versus mycorrhizal plants. In nonmycorrhizal plants, HM ions in the cells can be detoxified with the aid of several chelators. Furthermore, HMs can be sequestered in cell walls, vacuoles, and the Golgi apparatus of plants. The uptake and translocation of HMs are mediated by members of ZIPs, NRAMPs, and HMAs, and HM detoxification and sequestration are mainly modulated by members of ABCs and MTPs in nonmycorrhizal plants. Mycorrhizal‐induced changes in HM accumulation in plants are mainly due to HM sequestration by fungal partners and improvements in the nutritional and antioxidative status of host plants. Furthermore, mycorrhizal fungi can trigger the differential expression of genes involved in HM accumulation in both partners. Understanding the molecular mechanisms that underlie HM accumulation in mycorrhizal plants is crucial for the utilization of fungi and their host plants to remediate HM‐contaminated soils.     

Phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don

 Transport of inorganic orthophosphate (Pi) across the tonoplast membrane was studied using intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus. Orthophosphate uptake was strongly stimulated in the presence of Mg-ATP and Mg-pyrophosphate and inhibited by bafilomycin and concanamycin which are potent inhibitors of the vacuolar H⁺-ATPase. These results indicated that the build-up of an electrochemical gradient by the H⁺ pumps was essential for the uptake of Pi. Potassium thiocyanate, which dissipates the membrane potential across the tonoplast, strongly inhibited the Mg-ATP-stimulated uptake of Pi, while only a weak inhibition was observed in the presence of NH₄Cl, which dissipates the pH gradient. These results indicate that, as observed for other anions like malate or chloride, the electrical component is the driving force of Pi uptake, whereas the ΔpH plays only a minor role. Possible competitive inhibitors of Pi, MoO²⁻ ₄, VO³⁻ ₄ and CrO²⁻ ₄ were tested. Among them, CrO²⁻ ₄ strongly inhibited Pi uptake into the vacuoles. Various inhibitors of anion transport were also tested. Only 4,4-diisothiocyanostilbene-2,2′-disulfonic acid strongly inhibited Pi uptake into the vacuoles. The function of the vacuolar Pi transporters for cytoplasmic Pi homeostasis is discussed. Received: 20 September 1999 / Accepted: 28 January 2000     

Turgor pressure changes trigger characteristic changes in the electrical conductance of the tonoplast and the plasmalemma of the marine alga Valonia utricularis

The giant marine alga Valonia utricularis is capable of regulating its turgor pressure in response to changes in the osmotic pressure of the sea water. The turgor pressure response comprises two phases, a fast, exponential phase arising exclusively from water shifting between the vacuole and the external medium (time constant about 10 min) and a second very slow, almost exponential phase adjusting (but not always) the turgor pressure near to the original value by release or uptake of KCl (time constant about 5 h). The changes in the vacuolar membrane potential as well as in the individual conductances of the tonoplast and plasmalemma accompanying turgor pressure regulation were measured by using the vacuolar perfusion assembly (with integrated microelectrodes, pressure transducers and pressure‐regulating valves) as described by Wang et al. (J. Membrane Biology 157, 311–321, 1997). Measurements on pressure‐clamped cells gave strong evidence that the turgor pressure, but not effects related to water flow (i.e. electro‐osmosis or streaming potential) or changes in the internal osmotic pressure and in the osmotic gradients, triggers the cascade of osmotic and electrical events recorded after disturbance of the osmotic equilibrium. The findings definitely exclude the existence of osmosensors as postulated for other plant cells and bacteria. There was also evidence that turgor pressure signals were primarily sensed by ion transporters in the vacuolar membrane because conductance changes were first recorded in the many‐folded tonoplast and then significantly delayed in the plasmalemma independent of the direction of the osmotic challenge. Consistently, turgor pressure up‐regulation (but not down‐regulation) could be inhibited reversibly by external addition of the K⁺ transport inhibitor Ba²⁺ and/or by the Cl^– transport inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). Extensive studies under iso‐, hyper‐ and hypo‐osmotic conditions revealed that K⁺ and Cl^– contribute predominantly to the plasmalemma conductance. Addition of 0.3 mm NaCN showed further that part of the K⁺ and Cl^– transporters depended on ATP. These transporters are apparently up‐regulated upon hyper‐osmotic, but not hypo‐osmotic challenge. These findings explain the strong increase of the K⁺ influx upon lowering turgor pressure and the less pronounced pressure‐dependence of the Cl^– influx of V. utricularis reported in the literature. The data derived from the blockage experiments under hypo‐osmotic conditions were also equally consistent with the experimental findings that the K⁺ efflux is solely passive and progressively increases with increasing turgor pressure due to an increase of the volumetric elastic modulus of the cell wall. However, despite unravelling some of the sequences and other components involved in turgor pressure regulation of V. utricularis the co‐ordination between the ion transporters in the tonoplast and plasmalemma remains unresolved because of the failure to block the tonoplast transporters by addition of Ba²⁺ and DIDS from the vacuolar side.     

Transport of primary metabolites across the plant vacuolar membrane

Mesophyll cells and most types of storage cells harbor large central vacuoles representing the main cellular store for sugars and other primary metabolites like carboxylic- or and amino acids. The general biochemical characteristics of sugar transport across the vacuolar membrane are already known since a couple of years but only recently the first tonoplast sugar carriers have been identified on the molecular level. A candidate sucrose carrier has been identified in a proteomic approach. In Arabidopsis, the tonoplast monosaccharide transporters (TMT) represent a small protein family comprising only three members, which reside in the vacuolar membrane. Two of three tmt genes are induced upon cold, drought or salt stress and tmt knock out mutants exhibit altered monosaccharide levels upon cold induction. These observations indicate that TMT proteins represent the first examples of tonoplast sugar carriers involved in the cellular response upon osmotic stress stimuli.     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

Role of the vacuole in the redox homeostasis of plant cells

The review focuses on the mechanisms employed by plant vacuoles for maintaining the redox homeostasis under generation of reactive oxygen species (ROS) promoted by various abiotic stressors. These mechanisms are based on functioning of diverse enzymes and transport systems of the tonoplast as well as on vacuole-specific redox reactions involving vacuolar antioxidants of enzymatic and non-enzymatic nature. The established antioxidant role of plant vacuoles provides a clear example of closely integrated activities of this organelle with the metabolism of other cell parts.     

Vacuoles and prevacuolar compartments

Plant vacuoles are complex and dynamic organelles. Important advances have been made in our understanding of the transporters present in the tonoplast and of the molecular interactions that allow targeting to vacuoles. Despite these advances, markers that permit vacuoles to be defined unambiguously have not yet been identified.     

The signaling lipid phosphatidylinositol‐3,5‐bisphosphate targets plant CLC‐a anion/H+ exchange activity

Phosphatidylinositol‐3,5‐bisphosphate (PI(3,5)P₂) is a low‐abundance signaling lipid associated with endo‐lysosomal and vacuolar membranes in eukaryotic cells. Recent studies on Arabidopsis indicated a critical role of PI(3,5)P₂ in vacuolar acidification and morphology during ABA‐induced stomatal closure, but the molecular targets in plant cells remained unknown. By using patch‐clamp recordings on Arabidopsis vacuoles, we show here that PI(3,5)P₂ does not affect the activity of vacuolar H⁺‐pyrophosphatase or vacuolar H⁺‐ATPase. Instead, PI(3,5)P₂ at low nanomolar concentrations inhibited an inwardly rectifying conductance, which appeared upon vacuolar acidification elicited by prolonged H⁺ pumping activity. We provide evidence that this novel conductance is mediated by chloride channel a (CLC‐a), a member of the anion/H⁺ exchanger family formerly implicated in stomatal movements in Arabidopsis. H⁺‐dependent currents were absent in clc‐a knock‐out vacuoles, and canonical CLC‐a‐dependent nitrate/H⁺ antiport was inhibited by low concentrations of PI(3,5)P₂. Finally, using the pH indicator probe BCECF, we show that CLC‐a inhibition contributes to vacuolar acidification. These data provide a mechanistic explanation for the essential role of PI(3,5)P₂ and advance our knowledge about the regulation of vacuolar ion transport.     

Malate and malate-channel antibodies inhibit electrogenic and ATP-dependent citrate transport across the tonoplast of citrus juice cells

Citrus juice cells accumulate high levels of citric acid in their vacuoles when compared to other organic ions including malate. Uptake of citrate into tonoplast vesicles from Citrus juice cells was investigated in the presence of malate, and after incubation with antibodies raised against the vacuolar malate-specific channel of Kalancho? diagremontiana leaves. Antibodies against the vacuolar malate channel immunoreacted with a protein of similar size in tonoplast extracts from three Citrus varieties differing in citric acid content. Malate channel antibodies inhibited both delta MicroH(+)-dependent and delta MicroH(+)-independent ATP-dependent citrate transport, indicating common domains in both transport systems and to the malate-specific channel of Kalancho? diagremontiana leaves. Malate strongly inhibited electrogenic citrate transport, whereas ATP-dependent citrate uptake was less affected. Kinetic analysis of citrate transport in the presence of malate confirmed the existence of two citrate transport mechanisms and indicated that both citrate and malate share a common transport channel across the tonoplast of Citrus juice cells.     

Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2

Plant copper P_1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu⁺ (K_m ∼1 or 0.5 μm, respectively) and similar affinity to Ag⁺ (K_m ∼2.5 μm). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu⁺ transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess.     

Biogenesis of the protein storage vacuole crystalloid

We identify new organelles associated with the vacuolar system in plant cells. These organelles are defined biochemically by their internal content of three integral membrane proteins: a chimeric reporter protein that moves there directly from the ER; a specific tonoplast intrinsic protein; and a novel receptor-like RING-H2 protein that traffics through the Golgi apparatus. Highly conserved homologues of the latter are expressed in animal cells. In a developmentally regulated manner, the organelles are taken up into vacuoles where, in seed protein storage vacuoles, they form a membrane-containing crystalloid. The uptake and preservation of the contents of these organelles in vacuoles represents a unique mechanism for compartmentalization of protein and lipid for storage.     

Novel families of vacuolar amino acid transporters

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed.