Sucrose transport at the tonoplast

Sucrose that leaked from maize scutellum slices upon transfer of slices from a hexose or hexitol solution to water or upon placing the slices in a buffered EDTA solution was considered to be cytoplasmic in origin; residual (after leakage) tissue sucrose was considered to be stored in the vacuoles. This paper presents a study of the movement of sucrose across the tonoplast between the vacuoles and the cytoplasmic compartment. It is concluded that; (a) sucrose transport into the vacuoles is directly linked to sucrose synthesis in such a way that free sucrose is not an intermediate in the coupled process, (b) cytoplasmic sucrose is not (cannot be?) stored, (c) sucrose transport out of the vacuoles is linked to the metabolic demand for sugar, and (d) the transport process removing sucrose from the vacuoles does not release free sucrose into the cytoplasm. The sucrose fluxes at the plasmalemma and at the tonoplast are calculated, and the transport processes at the two membranes are compared.     

Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts

Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Tonoplast-localized transporters catalyze the import and release of these molecules. The mechanisms determining the targeting of these transporters to the tonoplast are largely unknown. Using the paralogous Arabidopsis thaliana inositol transporters INT1 (tonoplast) and INT4 (plasma membrane), we performed domain swapping and mutational analyses and identified a C-terminal di-leucine motif responsible for the sorting of higher plant INT1-type transporters to the tonoplast in Arabidopsis mesophyll protoplasts. We demonstrate that this motif can reroute other proteins, such as INT4, SUCROSE TRANSPORTER2 (SUC2), or SWEET1, to the tonoplast and that the position of the motif relative to the transmembrane helix is critical. Rerouted INT4 is functionally active in the tonoplast and complements the growth phenotype of an int1 mutant. In Arabidopsis plants defective in the β-subunit of the AP-3 adaptor complex, INT1 is correctly localized to the tonoplast, while sorting of the vacuolar sucrose transporter SUC4 is blocked in cis-Golgi stacks. Moreover, we demonstrate that both INT1 and SUC4 trafficking to the tonoplast is sensitive to brefeldin A. Our data show that plants possess at least two different Golgi-dependent targeting mechanisms for newly synthesized transporters to the tonoplast.     

Calcium fluxes at plasmalemma and tonoplast

Abstract. An account is given of the characterization of calcium fluxes across plasmalemma and tonoplast membranes of root cortical cells, using compartmental analysis. Some of the assumptions associated with the method are discussed. Recent evidence regarding the concentration of free Ca²⁺ in plant cells, and the mechanisms driving active calcium transport across cell membranes, is reviewed. It is proposed that the evidence from whole cell studies and work at the molecular level is mutually supportive, and some speculation is ventured about the general pattern of calcium transport in higher plant cells.     

Cold acclimation induces changes in Arabidopsis tonoplast protein abundance and activity and alters phosphorylation of tonoplast monosaccharide transporters

Because they are immotile organisms, higher plants have developed efficient strategies for adaptation to temperature changes. During cold acclimation, plants accumulate specific types of solutes to enhance freezing tolerance. The vacuole is a major solute storage organelle, but until now the role of tonoplast proteins in cold acclimation has not been investigated. In a comparative tonoplast proteome analysis, we identified several membrane proteins with altered abundance upon cold acclimation. We found an increased protein abundance of the tonoplast pyrophosphatase and subunits of the vacuolar V-ATPase and a significantly increased V-ATPase activity. This was accompanied by increased vacuolar concentrations of dicarbonic acids and soluble sugars. Consistently, the abundance of the tonoplast dicarbonic acid transporter was also higher in cold-acclimatized plants. However, no change in the protein abundance of tonoplast monosaccharide transporters was detectable. However, a generally higher cold-induced phosphorylation of members of this sugar transporter sub-group was observed. Our results indicate that cold-induced solute accumulation in the vacuole is mediated by increased acidification of this organelle. Thus solute transport activity is either modulated by increased protein amounts or by modification of proteins via phosphorylation.     

Tonoplast-bound protein kinase phosphorylates tonoplast intrinsic protein

Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [³²P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [³²P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca²⁺-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases.     

Isolation of tonoplast vesicles from tobacco protoplasts

Vacuoles were isolated from protoplasts of Nicotiana glutinosa by the method of Mettler and Leonard (Plant Physiol 1979 64: 1114-1120) with minor modifications so that the number of intact protoplasts contaminating the vacuole preparation was reduced to less than 1% (by number). Isopycnic centrifugation of a [³H]choline-labeled, sonicated vacuole preparation on linear 5 to 40% sucrose gradients indicated that tonoplast vesicles equilibrated at a density of about 1.12 grams per cubic centimeter. When tonoplast vesicles were isolated on discontinuous sucrose density gradients substrate specific ATPase activity was not found to be associated with this membrane fraction. These results are discussed in terms of the energetics of ion transport through the tonoplast membrane.     

ATP-induced sucrose efflux from red-beet tonoplast vesicles

 Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H⁺-ATPase to bafilomycin-A and NO₃ ⁻. Inhibition of the H⁺-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H⁺-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism. Received: 12 October 1999 / Accepted: 18 November 1999     

Architectural remodeling of the tonoplast during fluid-phase endocytosis

During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute concentration.     

Vacuolar chloride regulation of an anion-selective tonoplast channel

Fluctuations in intravacuolar chloride concentrations affected the tonoplast inward (anion flux into the vacuole) currents of sugar beets (Beta vulgaris). Rising vacuolar chloride concentrations induced increases in the levels of nitrate, acetate and phosphate inward currents. These currents, evoked at physiological vacuolar potentials, showed a linear relationship with the concentration of vacuolar chloride between 6 and 100 mm. Single channel currents revealed that rises in vacuolar chloride increased the frequency and probability of channel openings at a given tonoplast potential by reducing the mean closed time of the anion channel. In addition, there was an increase in the gating charge for the channel and a decrease in the free-energy favoring the transition of the channel from the closed to the open state. Vacuolar chloride had a very different effect on malate currents. Increasing chloride concentrations resulted in decreased frequency and open probability of the channel openings, a decrease in the gating charge and an increase in the mean closed time of the channel. Our results support the role for vacuolar chloride concentrations regulating the influx of anions into the vacuole, in addition to osmoregulation. The activation of channel activity by chloride will provide a pathway for the storage of nutrients, such as nitrate and phosphate into the vacuole, while the reduction of the malate currents will allow the use of malate for mitochondrial oxidation and cytoplasmic pH control.This work was supported by the National Science and Engineering Research Council of Canada.     

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。     

Reconstitution of the tonoplast amino-acid carrier into liposomes

The tonoplast amino-acid transporter of barley (Hordeum vulgare L.) mesophyll cells was functionally reconstituted by incorporating solubilized tonoplast membranes, vacuoplast membranes or tonoplast-enriched microsomal vesicles into phosphatidylcholine liposomes. (i) Time-, concentration- and ATP-dependence of amino-acid uptake were similar to results with isolated vacuoles. Although the orientation of incorporation could not be controlled, the results indicate that the transporter functions as a uniport system which allows regulated equilibration by diffusion between the cytosolic and vacuolar amino-acid pools. (ii) The ATP-modulated amino-acid carrier was also successfully reconstituted from barley epidermal protoplasts and Valerianella or Tulipa vacuoplasts, indicating its general occurrence. (iii) Fractionation of solubilized tonoplasts by size-exclusion chromatography followed by reconstitution of the fractions for glutamine transport gave two activity peaks: the first eluted in the region of high-molecular-mass vesicles and the second at a size of 300 kDa for the Triton-protein micelle.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis This work was part of our research efforts within the Sonderforschungsbereich 176 of the University. We gratefully acknowledge experimental support by Marion Betz and valuable discussions with Professors U. Heber and U.-I. Flügge and Dr. Armin Gross (University of Würzburg) and Dr. E. Martinoia (ETH, Zürich, Switzerland).     

Studies on the tonoplast action potential ofNitella flexilis

Summary The origins of the two peaks of the action potential inNitella flexilis were analyzed by inserting two microelectrodes. one into the vacuole and the other into the cytoplasm. It was unequivocally demonstrated that the rapid first peak was generated at the plasmalemma and the slow second peak at the tonoplast. MnCl₂ applied in the external medium abolished the second, tonoplast, peak but not the first, plasmalemma, peak, MnCl₂ also inhibited the cessation of the cytoplasmic streaming accompanying the action potential. CaCl₂ added in MnCl₂-containing medium recovered generation of the tonoplast action potential and the streaming cessation. Since it has been established that the cessation of cytoplasmic streaming on membrane excitation is caused by an increase in cytoplasmic free Ca^2– (Williamson, R.E., Ashley, C.C., 1982.Nature (London) 296:647–651: Tominaga, Y., Shimmen, T., Tazawa, M., 1983,Protoplasma 116:75–77), it is suggested that the tonoplast action potential is also induced by an increase in cytoplasmic Ca²⁺ resulting from the plasmalemma excitation. When vacuolar Cl was replaced with SO ₄ ² by vacuolar perfusion, the polarity of the second, slow peak was reversed from vacuolar positive to vacuolar negative with respect to the cytoplasm, supporting the previous report that the tonoplast action potential is caused by increase in Cl permeability (Kikuyama, M., Tazawa, M., 1976.J. Membrane Biol.29:95–110).     

Enzymic and protein character of tonoplast from hippeastrum vacuoles

The membrane of anthocyanin containing Hippeatrum petal vacuoles was examined for protein and enzyme content after purification by equilibrium density centrifugation. Light scattering, protein, and a Mg²⁺-dependent nucleotide specific ATPase were associated with membrane having a density of 1.08 to 1.12 grams per cubic centimeter. A small amount of acid phosphatase was also present in this region of the gradient, but this activity peaked at about 1.12 grams per cubic centimeter. A component of yeast tonoplast, α-mannosidase, was not significantly present. UDP-glucose, anthocyanidin-3-O-glucosyltransferase, thought to be a cytosol enzyme in Hippeastrum, was absent from tonoplast of vacuoles isolated by osmotic shock in 0.2 molar K₂HPO₄ or 0.35 molar mannitol. Vacuolar acid phosphatase was insensitive to ethylenediaminetetraacetate but was 80% inhibited by 10 millimolar KF, while ATPase was inactivated by 2 millimolar ethylenediaminetetraacetate and only 50% inhibited by 10 millimolar KF. Five major and about 9 minor polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein on 5 to 30 and 6 to 16% gradient gels.     

Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots

A H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H⁺-PPase catalyzed the electrogenic transport of H⁺ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb⁺ = K⁺ > Cs⁺; but it was inhibited by Na⁺ or Li⁺. Maximum rates of transport required at least 20 millimolar K⁺ and the K_m for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K⁺-dependent PPase activity with an I₅₀ of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H⁺-ATPase (e.g. a disulfonic stilbene or NO₃⁻) had no effect on the PPase activity. Vanadate and azide were also ineffective. H⁺-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H⁺-PPase and the H⁺-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H⁺ pumping activity was strongly affected by Mg²⁺ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg²⁺, H⁺ pumping showed a K_maPP for PPi of 15 micromolar. The rate of H⁺ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H⁺-PPase.     

Evidence for an electrogenic adenosine-triphosphatase in Hevea tonoplast vesicles

The function of the Mg-dependent ATPase of Hevea tonoplast in active proton transport was investigated by using a purified tonoplast fraction containing tightly sealed vesicles. In the used experimental conditions, the uptake of [¹⁴C]triphenylmethyl-phosphonium ion ([¹⁴C]TPMP⁺) and [³H]tetraphenyl-phosphonium ion ([³H]TPP⁺) by the vesicles indicated a transmembrane potential difference, negative inside. In parallel, the uptake of [¹⁴C]methylamine into the vesicles monitored a transmembrane pH gradient, interior acid. The addition of 5 mM Mg-ATP markedly depolarized the membrane and increased the magnitude of trnasmembrane pH gradient. These ATP-driven events were substrate specific for Mg-ATP. They were strongly inhibited by ATPase inhibitors such as N, N′-dicyclohexylcar-bodiimide. They were completely eliminated by proton conductors such as carbonylcyanide-p-trifluoromethoxy-phenylhydrazone and 5-chloro-3-tert-butyl-2′-chloro-4-nitro-salicylanilide. They depended on the pH of the medium, the maximum being reached at about pH 7.0. These data provide in vitro evidence that the Mg-ATPase localized at tonoplast level is an electrogenic pump. They are consistent with the hypothesis that an electrogenic H⁺ pump is catalyzed by the tonoplast ATPase of higher plants.     

Electrophysiological properties of NtTPK1 expressed in yeast tonoplast

The patch clamp technique using enlarged yeast tonoplasts is an effective approach to characterizing the Nicotiana tabacum plant tonoplast K+ channel, NtTPK1. We report here that the NtTPK1-mediated currents comprise two phase components, both which were found to be highly selective for K+ over Na+ and Cl-.     

An abundant, highly conserved tonoplast protein in seeds

We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with M_r 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds.