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Enrico Martinoia - One of the best experts on this subject based on the ideXlab platform.
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the arabidopsis Tonoplast is almost devoid of glycoproteins with complex n glycans unlike the rat lysosomal membrane
Journal of Experimental Botany, 2016Co-Authors: Emanuela Pedrazzini, Enrico Martinoia, Andrea Caprera, Ilaria Fojadelli, Alessandra Stella, Alessandra Rocchetti, Barbara Bassin, Alessandro VitaleAbstract:The distribution of the N-glycoproteome in integral membrane proteins of the vacuolar membrane (Tonoplast) or the plasma membrane of Arabidopsis thaliana and, for further comparison, of the Rattus norvegicus lysosomal and plasma membranes, was analyzed. In silico analysis showed that potential N-glycosylation sites are much less frequent in Tonoplast proteins. Biochemical analysis of Arabidopsis subcellular fractions with the lectin concanavalin A, which recognizes mainly unmodified N-glycans, or with antiserum against Golgi-modified N-glycans confirmed the in silico results and showed that, unlike the plant plasma membrane, the Tonoplast is almost or totally devoid of N-glycoproteins with Golgi-modified glycans. Lysosomes share with vacuoles the hydrolytic functions and the position along the secretory pathway; however, our results indicate that their membranes had a divergent evolution. We propose that protection against the luminal hydrolases that are abundant in inner hydrolytic compartments, which seems to have been achieved in many lysosomal membrane proteins by extensive N-glycosylation of the luminal domains, has instead been obtained in the vast majority of Tonoplast proteins by limiting the length of such domains.
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cold acclimation induces changes in arabidopsis Tonoplast protein abundance and activity and alters phosphorylation of Tonoplast monosaccharide transporters
Plant Journal, 2012Co-Authors: Waltraud X Schulze, Thomas Schneider, Sabrina Starck, Enrico Martinoia, Oliver TrentmannAbstract: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.
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in vivo phosphorylation sites of barley Tonoplast proteins identified by a phosphoproteomic approach
Proteomics, 2009Co-Authors: Anne Endler, Sonja Reiland, Bertran Gerrits, Ulrike G Schmidt, Sacha Baginsky, Enrico MartinoiaAbstract: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 (TiO(2)) 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.
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identification of a vacuolar sucrose transporter in barley and arabidopsis mesophyll cells by a Tonoplast proteomic approach
Plant Physiology, 2006Co-Authors: Anne Endler, Thomas Schneider, Enrico Martinoia, Sacha Baginsky, Stefan Meyer, Silvia Schelbert, Winfriede Weschke, Shaun Peters, Felix Keller, Ulrike G SchmidtAbstract: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.
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proton pumps and anion transport in vitis vinifera the inorganic pyrophosphatase plays a predominant role in the energization of the Tonoplast
Plant Physiology and Biochemistry, 1998Co-Authors: Nancy Terrier, Enrico Martinoia, Christophe Deguilloux, Francoisxavier Sauvage, Charles RomieuAbstract:Abstract In order to gain insight into vacuolar changes that are associated with fruit specialization regarding the accumulation of organic acids, the properties of Tonoplast vesicles extracted from grape cell culture and ripe berries were compared. The two primary pumps, PPase (Pyrophosphatase) and V-ATPase (V-Adenosine Triphosphatase), which were found on both fruit and cell culture Tonoplast vesicles, exhibited classical behaviour for known inhibitors and activators. However, the 3-fold higher activity of the PPase in comparison to the V-ATPase was unexpected in a fruit exhibiting a vacuolar pH of 3.5. Surprisingly, the hydrolytic activities of both pumps were stimulated by temperatures up to 65 °C. Anion transport was studied by measuring simultaneously proton entry and membrane potential on the two types of Tonoplast vesicles. Maximal acidification rates could be observed upon simultaneous energization by ATP and PPi, and were abolished by imidodiphosphate and bafilomycin, while vanadate had little effect. Comparison of the sequence of transport rates observed with mineral and organic anions on fruit or cultured cell Tonoplast vesicles suggested that the fruit vacuole was more specialized in the transport of organic anions than the cultured cells. The transport of malate and tartrate was completely inhibited by 4,4′-diisothio-cyanato-stilbene-2,2′disulphonic acid while chloride transport was less affected. Km values for malate and tartrate transports were similar for vesicles extracted from cultured cell and fruits, around 14 mM. The transport rates of malate and tartrate were not additive, suggesting that a similar transporter was involved in the vacuolar uptake of the two major acids of grape berries.
Omar Pantoja - One of the best experts on this subject based on the ideXlab platform.
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quantitative proteomics of the Tonoplast reveals a role for glycolytic enzymes in salt tolerance
The Plant Cell, 2010Co-Authors: Bronwyn J. Barkla, Rosario Veraestrella, Marcela Hernandezcoronado, Omar PantojaAbstract:To examine the role of the Tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated Tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong Tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the Tonoplast from salt-treated plants. Moreover, Tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the Tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity.
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na h exchange in the halophyte mesembryanthemum crystallinum is associated with cellular sites of na storage
Functional Plant Biology, 2002Co-Authors: Bronwyn J. Barkla, Rosario Veraestrella, Jesus Camachoemiterio, Omar PantojaAbstract:The Tonoplast Na+/H+ exchanger is involved in sequestering Na+ in plant vacuoles, providing solutes for osmotic adjustment while avoiding cytoplasmic Na+ toxicity. As such it is assumed to be one of the key mechanisms involved in salt-tolerance in plants. In this study, we measured Tonoplast Na+/H+ exchange in roots and different leaf tissues of adult Mesembryanthemum crystallinum L. plants to determine if activity of the exchanger follows the gradient from roots to leaves previously observed for Na+ and pinitol accumulation. Na+/H+ exchange was absent from roots of control and NaCl-treated plants. In contrast, leaves showed constitutive Na+/H+ exchange that was enhanced by growth of the plants in NaCl. Highest activity was measured in the epidermal bladder cells in agreement with the highest concentrations of Na+ found in this tissue. Tonoplast H+-translocating ATPase activity was also greatest in this tissue, as were protein levels for myo-inositol-O-methyltransferase, a key enzyme in the pinitol biosynthesis pathway. The strong correlation between Na+/H+ exchange and Na+ accumulation confirms the role of this transporter in vacuolar sequestration of Na+ and plant salt tolerance.
Bronwyn J. Barkla - One of the best experts on this subject based on the ideXlab platform.
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quantitative proteomics of the Tonoplast reveals a role for glycolytic enzymes in salt tolerance
The Plant Cell, 2010Co-Authors: Bronwyn J. Barkla, Rosario Veraestrella, Marcela Hernandezcoronado, Omar PantojaAbstract:To examine the role of the Tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated Tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong Tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the Tonoplast from salt-treated plants. Moreover, Tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the Tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity.
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na h exchange in the halophyte mesembryanthemum crystallinum is associated with cellular sites of na storage
Functional Plant Biology, 2002Co-Authors: Bronwyn J. Barkla, Rosario Veraestrella, Jesus Camachoemiterio, Omar PantojaAbstract:The Tonoplast Na+/H+ exchanger is involved in sequestering Na+ in plant vacuoles, providing solutes for osmotic adjustment while avoiding cytoplasmic Na+ toxicity. As such it is assumed to be one of the key mechanisms involved in salt-tolerance in plants. In this study, we measured Tonoplast Na+/H+ exchange in roots and different leaf tissues of adult Mesembryanthemum crystallinum L. plants to determine if activity of the exchanger follows the gradient from roots to leaves previously observed for Na+ and pinitol accumulation. Na+/H+ exchange was absent from roots of control and NaCl-treated plants. In contrast, leaves showed constitutive Na+/H+ exchange that was enhanced by growth of the plants in NaCl. Highest activity was measured in the epidermal bladder cells in agreement with the highest concentrations of Na+ found in this tissue. Tonoplast H+-translocating ATPase activity was also greatest in this tissue, as were protein levels for myo-inositol-O-methyltransferase, a key enzyme in the pinitol biosynthesis pathway. The strong correlation between Na+/H+ exchange and Na+ accumulation confirms the role of this transporter in vacuolar sequestration of Na+ and plant salt tolerance.
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Expression of water channel proteins in mesembryanthemum crystallinum
Plant physiology, 2000Co-Authors: Hans-hubert Kirch, Christine B. Michalowski, Rosario Vera-estrella, Dortje Golldack, Françoise Quigley, Bronwyn J. Barkla, Hans J. BohnertAbstract:We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and Tonoplast, respectively. MIPs were most abundant in cells involved in bulk water flow and solute flux. The Tonoplast MIP-F was found in all cells, while signature cell types identified different PM-MIPs: MIP-A predominantly in phloem-associated cells, MIP-B in xylem parenchyma, and MIP-C in the epidermis and endodermis of immature roots. Membrane protein analysis confirmed MIP-F as Tonoplast located. MIP-A and MIP-B were found in Tonoplast fractions and also in fractions distinct from either the Tonoplast or PM. MIP-C was most abundant but not exclusive to PM fractions, where it is expected based on its sequence signature. We suggest that within the cell, MIPs are mobile, which is similar to aquaporins cycling through animal endosomes. MIP cycling and the differential regulation of these proteins observed under conditions of salt stress may be fundamental for the control of tissue water flux.
Teruo Shimmen - One of the best experts on this subject based on the ideXlab platform.
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role of ca2 on triggering Tonoplast action potential in intact nitella flexilis
Plant and Cell Physiology, 1997Co-Authors: Munehiro Kikuyama, Teruo ShimmenAbstract:Sensitive aequorin was microinjected into the cytoplasm of Nitella flexilis to study the role of Ca in the generation of the Tonoplast action potential. The temporal relation between the increase in cytoplasmic Ca and the Tonoplast action potential suggested that Tonoplast action potential is triggered by an increase in cytoplasmic Ca. This is also supported by the fact that Mn, extracellularly applied, inhibited both the increase in cytoplasmic Ca and the generation of the Tonoplast action potential.
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Analysis of temperature dependence of cytoplasmic streaming using Tonoplast-free cells of Characeae
Protoplasma, 1993Co-Authors: Teruo Shimmen, Shizuo YoshidaAbstract:The temperature dependence of cytoplasmic streaming in intact and Tonoplast-free cells ofNitellopsis obtusa was studied using a cryomicroscope. The streaming velocity decreases linearly with decrease in the temperature in well-buffered Tonoplast-free cells but non-linearly in some intact cells. These results suggest that low temperature causes a disturbance in the homeostasis of calcium and protons, which inhibit cytoplasmic streaming in intact cells.
Ed Etxeberria - One of the best experts on this subject based on the ideXlab platform.
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activity of membrane associated sucrose synthase is regulated by its phosphorylation status in cultured cells of sycamore acer pseudoplatanus
Physiologia Plantarum, 2004Co-Authors: Javier Pozuetaromero, Pedro Gonzalez, Diego Pozuetaromero, Ed EtxeberriaAbstract:Changes in sucrose synthase (SuSy) activity, protein level and degree of phosphorylation were investigated in plasmalemma and Tonoplast of sycamore cells cultured either in the presence of sucrose or after 24 h of starvation. SuSy activity was shown to be higher in the plasmalemma than in the Tonoplast of cells cultured in the presence of sucrose. In clear contrast, SuSy was shown to be more active in the Tonoplast than in the plasmalemma of starved cells. Western blot analyses on both membrane types did not show noticeable differences in SuSy protein levels under the two different regimes. However, phosphorylation state at the serine moieties of the enzyme was shown to be different in the presence or in the absence of sucrose. Plasmalemma-associated SuSy is not phosphorylated in the presence of sucrose, whereas Tonoplast-associated SuSy is phosphorylated under similar conditions. Starvation brought about a reverse in phosphorylation state of membrane-bound SuSy. Whereas plasmalemma-associated SuSy became phosphorylated, Tonoplast-associated SuSy was completely de-phosphorylated. Together, the data demonstrate that SuSy is simultaneously present in various cell membranes and also demonstrate a lack of direct relationship between membrane type location, and degree of phosphorylation, but substantiate the relevance of phosphorylation to enzymatic activity.
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vacuolar acidification in citrus fruit comparison between acid lime citrus aurantifolia and sweet lime citrus limmetioides juice cells
Journal of the American Society for Horticultural Science, 2002Co-Authors: Andreas Brune, Pedro Gonzalez, Lincoln Taiz, Mathias L Muller, Ed EtxeberriaAbstract:Vacuolar acidification was investigated in 'Palestine' sweet (Citrus limmetioides Tanaka) and 'Persian' acid lime ((Citrus aurantifolia (Christm.) Swingle) (vacuolar pHs of 5.0 and 2.1, respectively) using Tonoplast vesicles isolated from juice cells. The ATPase activity of Tonoplast-enriched vesicles from sweet limes was strongly inhibited by bafilomycin A1 and NO3 - , but was unaffected by vanadate. In contrast, the ATPase activity in acid lime membranes was only slightly inhibited by bafilomycin A1 and NO3 - and was strongly inhibited by high concentrations of vanadate. The vacuolar origin of the acid lime vesicles was confirmed by immunoblotting. After solubilization and partial purification of the two enzymes by gel filtration, their inhibitor profiles were largely unchanged. Based on equal ATPase activities, vesicles from sweet and acid limes were able to generate similar pH gradients. However, in Tonoplast vesicles from sweet limes, the maximum ∆pH was reached four times faster than in those from acid limes. Addition of ethylenediamine tetraacetic acid (EDTA) to chelate Mg +2 after the maximal ∆pH was attained resulted in collapse of the pH gradient in vesicles from sweet limes, whereas no change in ∆pH was observed in vesicles from acid limes, indicating a less H +
