Transport Properties of the Tomato Fruit Tonoplast - NCBI

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NANCY OLESKI2, PEIMAN MAHDAVI, GALEN PEISER3, AND ALAN B. ..... OLESKI NA, P MAHDAVI, AB BENNETT 1987 Transport properties ofthe tomato.
Plant Physiol. (1987) 84, 993-996 0032-0889/87/84/0993/04/$0 1.00/0

Transport Properties of the Tomato Fruit Tonoplast' I. IDENTIFICATION AND CHARACTERIZATION OF AN ANION-SENSITIVE H+-ATPase Received for publication November 18, 1986 and in revised form March 12, 1987

NANCY OLESKI2, PEIMAN MAHDAVI, GALEN PEISER3, AND ALAN B. BENNETr* Mann Laboratory, Department of Vegetable Crops, University of California, Davis, California 95616 ABSTRACT An anion-sensitive H+-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esclekntum) fruit. The H'-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by N03-, N,N'-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H'-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The K. for the H-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H'ATPase are very similar to those described for a number of plant cell tonoplast H'-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation.

The existence of a proton translocating ATPase on the tonoplast of many plant cells has been widely verified (9 and references therein). This H+-ATPase has been implicated in establishing an electrochemical potential gradient for H+ across the tonoplast. The gradient is believed to be utilized to drive H+/Ca2+ (5), H+/Na+ (3), and H+/sucrose (4) antiports present on the tonoplast. Tomato fruit tissue is quite acidic and thus may possess an unusually active H+ pump in the tonoplast. The tomato fruit vacuole is also the site of organic acid, sugar, and calcium accumulation, all of which contribute to quality characteristics of the mature fruit. In anticipation of experiments to assess accumulation of organic acids (6), sugars and calcium in the tomato fruit vacuole, we undertook a study to identify and characterize a H+-ATPase on the tonoplast of mature green tomato fruit.

MATERIALS AND METHODS Plant Material. Tomatoes (Lycopersicon esculentum L., cv Castlemart and Contessa) were grown in the greenhouse when possible or obtained commercially. Fruits harvested from the greenhouse at the mature green stage were used immediately after harvest. Fruits obtained commercially were not treated with ethylene prior to receipt. These fruit were kept at 20°C and used for membrane isolation within S d of harvest. Membranes iso'Supported by National Science Foundation grant DMB 84-04990. Plant Biology Laboratory, The Salk Institute for Biological Studies, P. 0. Box 85800, San Diego, CA 92138-9216. 3Present address: NPI, 417 Wakara Way, Salt Lake City, UT 84108. 993

2Present address:

lated from greenhouse grown or commercially purchased fruit were similar in the characteristics we examined. Membrane Preparation. Tomato microsomal membranes were prepared using a method similar to that for red beets (2). Approximately lOOg of tomato pericarp tissue (excluding the locular tissue) were chopped and added to 200 ml of homogenization buffer containing 250 mm sucrose, 0.1% BSA, 0.5% polyvinylpyrrolidine (PVP-40), 4 mM DTT, 3 mM EDTA, 70 mM Tris-Cl, and 20 mm Bis-Tris propane to a final pH of 8.5. The tissue was homogenized 1 min in an Osterizer blender and chilled for 20 min at 0°C. The pH of the homogenate was approximately 7.5. The homogenate was filtered through four layers of cheesecloth, and the filtrate centrifuged for 15 min at 13,000g in a Beckman SW28 rotor. The supernatant was then centrifuged for 30 min at 80,000g to pellet membranes. This microsomal membrane pellet was resuspended in 250 mm sucrose, 2 mm DTT, and 5 mm Tris/Mes (pH 7.0) to a final volume of 2 ml. The resuspended microsomes were then layered on to a continuous (15-45% w/w sucrose) or discontinuous (16, 26, 34, and 40% w/w sucrose) gradient containing 1 mM DTT and 5 mM Tris/Mes (pH 7.0). The sucrose gradients were centrifuged 2 h at 80,000g and fractionated. Discontinuous gradient fractions were collected and diluted 2-fold in 10 mM Tris/Mes (pH 7). Membranes were repelleted by centrifugation at 80,000g for 30 min and resuspended in 250 mm sucrose, 1 mM DTT, and 5 mM Tris/Mes (pH 7) to a final volume of 0.5 to 1.0 ml. ATPase Assays. ATPase activity was measured at 28°C for 45 min. The standard assay contained 5 mM Tris-ATP, 5 mM MgSO4, 50 mm KCI, 50 mm Tris/Mes (pH 7), and 5 to 10 ,g membrane protein in a volume of 0.5 ml. ATP concentration and pH were varied for some experiments as indicated in the text and figure legends. When KNO3 was used as an inhibitor, K2SO4 was added to the assays not receiving KNO3 in order to maintain a constant K+ concentration. Monovalent salts and inhibitors were added as indicated. Inorganic phosphate was determined by the method of Ames (1). H+ Transport. Formation of interior acid pH gradients was monitored as the quenching of fluorescence of the permeant amine dye, acridine orange. Vesicles (approximately 20 tg membrane protein), 3 mM Na2ATP or 0.1 mM sodium pyrophosphate, 50 mm KCI, 3 gM acridine orange and inhibitors were added as indicated to an assay buffer of 250 mm sucrose and 10 mM Tris/Mes (pH 7.0) to give a final volume of 1.5 ml. Fluorescence was measured at 28°C with a Perkin-Elmer 650-40s spectrofluorimeter at excitation and emission wavelengths of 472 and 525 nm, respectively. H+ transport was initiated by the addition of either 9 Al (with ATP) or 3 ul (with PPi) of 0.5 M MgSO4. Protein and Enzyme Assays. Membrane protein was measured by the method of Schaffner and Weissman (8). UDPase activity was assayed in 0.5 ml of 25 mm Hepes/Mes (pH 6.75), 1.5 mM UDP, 1.5 mm MnSO4, 0.05% Triton X-100 using 50 Ml of each gradient fraction. Each assay was incubated for 30 min at 28°C and Pi was determined by the method of Ames (1). Cyt c oxidase was assayed as previously described (2).

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RESULTS Continuous Density Gradients. Figure 1 shows the distribution ofvarious enzyme activities along a continuous sucrose gradient. As shown in the upper panel, ATPase activity measured in the absence of gramicidin peaks at a density of about 40% sucrose. ATPase activity is stimulated by gramicidin only in the lower density region of the gradient, suggesting that membrane vesicles in this region are tightly sealed. There is a large peak of nitratesensitive activity (middle panel) at 23% sucrose which is relatively free of azide and vanadate-sensitive ATPase activities. Vanadate sensitive ATPase activity (middle panel) peaks near 39% sucrose. Mitochondrial membranes (assayed by azide sensitive ATPase activity) are present between 32 and 40% sucrose and thus are not contaminants in the low density region ofthis gradient which is enriched in N03 -sensitive ATPase. The activities of two membrane marker enzymes, UDPase (Golgi) and Cyt c oxidase (mitochondria) are shown in the lower panel. UDPase shows a double peak of activity at 28 and 39% sucrose, while Cytochrome c oxidase activity peaks at 40% sucrose. Therefore, the lower density region of the gradient contains some Golgi membranes, but is essentially free of mitochondrial membranes. Based on these linear gradient results, discontinuous sucrose gradients were designed to collect a fraction enriched in nitrate-sensitive ATPase at the 16/26% sucrose interface.

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FIG. 1. Distribution of ATPase activities, Cyt c oxidase and UDPase associated with tomato fruit microsomal membranes. Upper panel, ATPase activity assayed in the presence of 5 mM Tris-ATP, 5 mM MgSO4, 50 mM KCI, and 0.1 mM Na-molybdate in the absence (0) or presence (0) of 2 uM gramicidin. Sucrose concentration (%, w/w) is also shown (0). Middle panel, N03-sensitive (A), vandate-sensitive (A), N3-sensitive (0), or gramicidin-stimulated (0) ATPase calculated as the difference in ATPase activity assayed in the absence or presence of 50 mM N03-, 50 gM vanadate, 1 mM N3-, or 2 uM gramicidin, respectively. Lower panel, Cyt c oxidase and UDPase activity assayed as in "Materials and Methods."

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FiG. 3. pH-dependence of H+-ATPase activity associated with low density (16/26% w/w sucrose interface) membranes from tomato fruit. A, ATPase activity assayed in the presence of 5 mM Tris-ATP, 5 mm MgSO4, 50 mM KCI, 2 uM grmicidin, and either 25 m K12504(0) or 50 mM KNO3 (0) at the indicated pH. Sodium molybdate was included at 0.1 mM in one treatment (0). N3O-sensitive ATPase (A) was calculated as the difference in activity assayed in presence of 25 mm K2504 or 50 mM KNO3. B, ATP-dependent H+ transport was measured as the quenching of acridine orange -fluorescence at the indicated pH values. Total fluorescence quench (0) or the initial rate of fluorescence quench (0) is plotted at each pH. In all cases pH was varied by adjusting the ratio of Tris/Mes but keeping a total buffer concentration of 25 mm.

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TOMATO FRUIT TONOPLAST H+-ATPase Table I. Effect of Monovalent Salts on Tonoplast Enriched H+-ATPase from Tomato Fruit ATPase activity was measured in triplicate at 28°C in the presence of 5 mM MgSO4, 5 mm Tris-ATP, and 50 mm monovalent salt. Gramicidin was present at 2 Mm in all assays. Approximately 10 gg protein was used per assay. Monovalent Salt in Specific Salt KCI Addition to MgSO4 Stimulation Activity Mmol Pi* mg % % protein* h' Control (MgSO4) 4.537 +KCI 8.855 95 100 +NaCl 9.023 99 104 +Choline-Cl 9.948 119 125 +LiCl 8.876 96 100 +RbCl 8.477 87 91 +CsCl 8.453 86 91 +KBr 7.636 72 68 +K-acetate 8.193 84 80 10.694 +KHCO3 136 143 +KI 5.861 29 31 +K2SO4 6.775 49 51 +KNO3 1.458 -68 +KSCN 0.313 -93 Table II. Effect ofInhibitors on Tonoplast Enriched H+-A TPase from Tomato Fruit ATPase activity was in measured at 28°C in the presence of 5 mm MgSO4, 5 mm Tris-ATP, 50 mm KCI, and inhibitors. The final pH was 7.0 and all assays except the control contained 2 gM Gramicidin. Approximately 10 ug protein was used per assay. % of Specific Inhibitor Gramicidin Activity MUmol Pi- mg protein-' *hControl 15.6 +Gramicidin (2 Mm) 25.1 100 +Na molybdate (1 mM) 26.0 104 +NaN3 (1 mM) 24.5 98 +Oligomycin (10 Mg/ml) 21.8 87 +KNO3 (50 mM) 4.6 18 +DCCD (100 gM) 4.0 16 +DES (100 uM) 3.6 14

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3 4 2 5 ATP Concentrotion (mM) FIG. 4. MgATP concentration dependence of ATPase activity associated with low density (16/26% w/w sucrose interface) membranes from tomato fruit. Upper panel, ATPase activity assayed in the presence of 50 mM KCI, 2 Mm gramicidin, and either 25 mM K2SO4 (0) or 50 mM KNO3 (A). ATP was added at the indicated concentration in the presence of MgSO4 which was consistently present in 2 mm excess of the ATP concentration. N03-sensitive ATPase (-) was calculated as the difference in activity assayed in the presence of either 25 mM K2SO4 or 50 mM KNO3. Lower panel, Hanes-Woolf plot of the N03 -sensitive ATPase activity shown in the upper panel.

(data not shown). In addition to ATP, PPi was also shown to be an effective substrate for H+-transport (Fig. 2). This PPidependent fluorescence quenching was insensitive to vanadate, but was partially inhibited by NO3. Under the assay conditions employed, the rates and extent of ATP- and PPi-dependent H+ transport were similar. Both ATP- and PPi-dependent fluorescence quenching were abolished in the presence ofthe ionophore

H' Transport. Membrane vesicles from the 16/26% sucrose interface were assayed for H+ transport using the fluorescent amine indicator of pH gradients, acridine orange. The ability of nigericin. this membrane preparation to catalyze ATP-dependent fluorespH Dependence. ATPase activity of the tomato tonoplast cent quenching of acridine orange (Fig. 2) indicates that the enriched vesicles was evaluated over a pH range of 5.5 to 8.5 in membrane vesicles are sealed, and competent to catalyze H+ the presence and absence of NO3 (Fig. 3A). In three separate transport. experiments the pH optimum for this ATPase varied slightly but Transport of H+ is insensitive to 50 AM vanadate but is a double peak of ATPase activity was consistently observed. The completely abolished by 50 mm NO;3. These results are similar lower pH optimum was consistently between pH 6.0 and 6.5 and to those described for other tonoplast H+-ATPases and indicate the higher pH optimum was consistently between pH 7.0 and that the NO3-sensitive ATPase is a H+ pump. The strong 7.5. The shape of this double pH optima was not affected by the inhibition of H+ transport by NO3 and lack of inhibition by presence of molybdate (Fig. 3A), suggesting that the acid pH vanadate indicates that contamination of this membrane prepa- optimum did not result from contaminating acid phosphatase ration by the plasma membrane H+-ATPase is negligible. Fluo- activity. In addition, ATPase activity was inhibited by NO3 over rescence quenching was not observed in the presence of the the entire pH range, indicating that this double pH optima is a general ATPase inhibitor DCCD4 or in the presence of DES feature of a single NO3-sensitive ATPase. When total fluorescence quench was used to determine H+ transport activity as a function of pH, a double pH optimum was observed (Fig. 3B). 'Abbreviations: DCCD, N,N'-dicyclohexylcarbodiimide; DES, dieth- However, the initial rate of fluorescence quench showed a single ylstilbestrol. pH optimum near pH 6.7. In four separate experiments the pH

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minimum varied slightly between pH 6.75 and 7.5 which accounts for the slight offset of the pH minimum seen when comparing ATPase activity and fluorescence quench (Fig. 3). Salt Stimulation. The H+-ATPase from tomato tonoplast enriched vesicles was characterized by assessing its response to monovalent salts (Table I). Chloride stimulates the ATPase activity, regardless of the accompanying cation. However, HCO3appears to be the most potent stimulator of the enzyme, while Br-, acetate, I-, and S04- are less effective than Cl-. Nitrate and SCN- are strong inhibitors of ATPase activity. Because of the unusual pH optima of this H+-ATPase the strong stimulation of activity by HCO3 may have resulted from a slight increase in the assay pH. Inhibitors. A variety of inhibitors were evaluated for their effect on tomato tonoplast H+-ATPase activity (Table II). Gramicidin stimulated ATPase activity indicating that these tonoplast vesicles are tightly sealed. Vanadate, azide, and molybdate had no effect on the tonoplast ATPase, indicating essentially no contamination by either the plasma membrane or the mitochondrial H+-ATPase, or by nonspecific acid phosphatase. Nitrate, DCCD, and DES strongly inhibited ATPase activity. MgATP Concentration Dependence. The activity of the tonoplast H+-ATPase was measured over a range of ATP:Mg concentrations. A velocity versus substrate concentration plot and a Hanes-Woolf plot are shown in Figure 4. The H+-ATPase demonstrated simple hyperbolic MgATP concentration dependence with a Km of approximately 0.8 mm.

DISCUSSION In this paper we have identified and characterized an anionsensitive H+-ATPase in membrane vesicles from mature green tomato fruit. Based on the membrane density of 1.1 1 g/cc and specific inhibition by NO3- we conclude that the H+-ATPase enriched in a 16/26% sucrose density gradient fraction is of tonoplast origin. Inhibition of ATPase activity in this fraction by NO3- is greater than 75%, suggesting low contamination by other ATP hydrolyzing enzymes. In most respects, the characteristics of this anion-sensitive H+ATPase from tomato fruit are similar to other tonoplast H+-

Plant Physiol. Vol. 84, 1987

ATPases recently characterized (9). One exception is the pH dependence of the tomato fruit H+-ATPase. In these experiments, a double pH optimum at acid (pH 6.0-6.5) and near neutral pH (pH 7.0-7.5) was consistently observed. Other tonoplast H+-ATPases have a single alkaline pH optimum (9). The pH dependence observed for the tomato fruit H+-ATPase may be related to the high acidity of this tissue. Identification of PPi as a substrate for H+-transport suggests that either PPi is also a substrate for the H+-ATPase, or that a H+-translocating pyrophosphatase (H+-PPase) is present on the tomato fruit tonoplast. Based on recent results (7, 10) we suspect that a H+-PPase also exists on tomato fruit tonoplast membranes. As previously demonstrated in oat root tonoplast vesicles (10), when assayed at low PPi and MgSO4 concentration (0.1 and 1 mM, respectively) H+ transport catalyzed by the H+-PPase is comparable in magnitude to H+ transport catalyzed by the H+ATPase. LITERATURE CITED 1. AMES BN 1966 Assay of inorganic phosphate, total phosphate, and phosphatases. Methods Enzymol 8: 115-118 2. BENNETr AB, SD O'NEILL, RM SPANSWICK 1984 H+-ATPase activity from storage tissue of Beta vulgaris. I. Identification and characterizations of an anion-sensitive H+-ATPase. Plant Physiol 74: 538-544 3. BLUMWALD E, RJ POOLE 1985 Na+/H+ antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris. Plant Physiol 78: 163-167 4. BRISKIN DP, WR THORNLEY, RE WYSE 1985 Membrane transport in isolated vesicles form sugarbeet taproot. Evidence for sucrose/H+-antiport. Plant

Physiol 78: 871-875 5. BUSH DR, H SzE 1986 Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells. Plant Physiol 80: 549555 6. OLESKI NA, P MAHDAVI, AB BENNETT 1987 Transport properties ofthe tomato fruit tonoplast. II. Citrate transport. Plant Physiol 84: 997-1000 7. REA PA, RJ POOLE 1986 Chromatographic resolution of H+-translocating pyrophosphatase from H+-translocating ATPase of higher plant tonoplast. Plant Physiol 81: 126-129 8. SCHAFFNER W, C WEISMANN 1973 A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56: 502-514 9. SZE H 1985 H+-translocating ATPases: advances using membrane vesicles. Annu Rev Plant Physiol 36: 175-208 10. WANG Y, RA LEIGH, KH KAESTNER, H SZE 1986 Electrogenic H+-pumping pyrophosphatase in tonoplast vesicles of oat roots. Plant Physiol 81: 497502