H+ Antiport System Driven by the Tonoplast Pyrophosphate-Dependent Proton Pump from Maize Roots

J. PlantPbysiol. Vol. 137. pp. 471-476(1991)

A Ca2 +/H+ Antiport System Driven by the Tonoplast Pyrophosphate-Dependent Proton Pump from Maize Roots ALAIN CHANSON

Institute of Plant Biology and Physiology of the University, Biology Building, 1015 Lausanne, Switzerland Received July 11, 1990· Accepted October 11, 1990

Summary Calcium uptake by tonoplast enriched membrane vesicles from maize (Zea mays L. cv. LG 11) primary roots was studied. A pH gradient, measured by the fluorescence quenching of quinacrine, was generated across sealed vesicles driven by the pyrophosphate-dependent proton pump. The fluorescence quenching was strongly inhibited by Ca2 +; moreover, when increasing Ca2 + concentrations were added to vesicles at steady-state, a concomitant decrease in the proton gradient was observed. Ca2 + uptake using 4SCa2 + was linear for 10 min when oxalate (10 mM) was present, while Ca2 +uptake was completely inhibited with proton ionophores (FCCP and monensin), indicating a Ca2 +/H+ antiport. Membranes were further fractionated using a linear sucrose density gradient (10-45 %) and were identified with marker enzymes. Ca2 + uptake co-migrated with the tonoplast pyrophosphate-dependent proton pumping, pyrophosphatase and ATPase activities: the Ca2 +/H+ antiport is consequently located at the tonoplast.

Key words: Calcium transport, H+ /Ca 2 +antiport, H+ ·PPase, tonoplast, root, Zea mays. Abbreviations and Symbols: BTP

=

bis-tris propane; DCCD

=

N,N'-dicyclohexylcarbodiimide; DES

= diethylstilbestrol; DIDS = 4,4'-diisothiocyanatostilbene-2,2'-disulfonate; FCCP = carbonylcyanide ptrifluoromethoxyphenylhydrazone; MDP = methylenediphosphonate; PPase = pyrophosphatase; 1 KS = 1,000 x g supernatant; 6 KS = 6,000 x g supernatant; 148 KP = 148,000 x g pellet. Introduction Calcium (Ca2 +) plays an important role in the regulation of growth and developmental processes in higher plant cells (Kauss, 1987). To avoid cytotoxic interactions, the free Ca2 + in the cytoplasm is maintained at a low level (0.1-1 I'M) against an electrochemical gradient favouring its passive influx. A transient increase in the cytoplasmic level in response to external stimuli is the mechanism by which Ca2 + exerts its function as second messenger (Kauss, 1987; Evans, 1988). Excess Ca2 + is pumped into the extracellular space or sequestered in intracellular organelles by the action of high affinity membrane-bound transporters (Kauss, 1987; Evans, 1988). Ca2 + is extruded across the plasmalemma by a primary ATP-dependent pump (Graf and Weiler, 1989; RasiCaldagno et al., 1989). A second ATP-driven pump is associated with the endoplasmic reticulum (Buckhout, 1984; Schumaker and Sze, 1985; Bush et aI., 1989), whereas a Ca2 +/ © 1991 by Gustav Fischer Verlag, Stuttgart

H+ antiport, depending on a protonmotive force, is located at the tonoplast (Schumaker and Sze, 1985; Blumwald and Poole, 1986; Joyce et aI., 1988). Mitochondria and plastids can accumulate Ca2 + from the cytosol, but it would appear unlikely that they play an important role in the regulation of Ca2 + concentration (Kauss, 1987; Evans, 1988). The localization of three Ca2 + transport systems in different membranes render their study very difficult (Hager and Hermsdorf, 1981; Zocchi, 1988). The Ca2 +/H+ antiport has been indirectly investigated using the tonoplast ATP-dependent proton pump to generate a transmembrane H +-electrochemical potential difference (Schumaker and Sze, 1985; Butcher and Evans, 1987a, b; Joyce et al., 1988), or directly by imposing pH gradients across tonoplast-enriched vesicles (Schumaker and Sze, 1985, 1986; Blumwald and Poole, 1986). A second H +-translocating activity is present at the tonoplast of higher plant cells (Chanson et aI., 1985; Chanson and

472

ALAm

CHANSON

Pilet, 1987, 1988). This electrogenic H +-pump is pyrophosphate (PPi)-dependent and in maize root tips its activity is higher than that of the H+-ATPase (Chanson and Pilet, 1987). The aim of the present study was to characterize the tonoplast Ca2+IH+ antiport activity of maize root tonoplast. The PPi-dependent proton pump, which is specifically associated with this membrane (Chanson, 1990), was used to avoid any interferences due to the ATP-dependent Ca2+_ pumps from the plasmalemma and the ER. The data given in this paper appear to be the first report of a pyrophosphate-driven H+ ICa 2 + antiport on the tonoplast of plant cells.

Materials and Methods Plant material Maize (Zea mays L. cv. LG 11, Association Suisse des Selectionneurs, Lausanne) root homogenates were prepared as previously described (Chanson and Pilet, 1987) and centrifuged 5 min at 1,000 x g (Beckman J2-21, JA-20 rotor) to sediment starch grains, unbroken cells, wall fragments and nuclei. The supernatant (1 KS) was collected and mixed with homogenization buffer (250 mM sucrose, 2 mM EDTA, 1 mM DTT, 0.1 % BSA, 50 mM Tris, pH adjusted to 7.8 with solid Mes) to a final volume of 21 mL. The diluted 1 KS fraction was added to the top of a non-linear sucrose gradient consisting of 5mL of 45% (w/w) (in gradient buffer: 0.5mM EDTA, 1 mM DTT, 2.5 mM Tris, adjusted to pH 7.5 with solid Mes), 5 mL 35 % and 5 mL of 10 % sucrose. After 3 h centrifugation at 80,000 x g (Kontron Centrikon T-2080, TST 28.38 rotor), the gradients were fractionated into 13 portions of 1.5 mL. Fractions 7 and 8, corresponding to the 10/35 % interface (Chanson and Pilet, 1988), were pooled and diluted to 10% sucrose with gradient buffer (sucrose concentrations were measured by refractometry). To remove EDTA, the membranes were sedimented (30 min at 148,000 x g, TST 28.17 rotor) and resuspended in 10 % sucrose in gradient buffer without EDTA. The different tubes were frozen in liquid nitrogen and stored at -70°C.

Linear sucrose gradients The 1KS fraction was centrifuged 20 min at 6,000 x g to sediment the majority of the mitochondria (Chanson and Pilet, 1987), and the supernatant (6 KS) was collected. The 6 KS fraction was sedimented (30min at 148,oooxg, TST 28.17 rotor) and the pellet (148KP) resuspended in 10% sucrose (in gradient buffer without EDTA). The 148KP fraction was added to the top of linear gradients consisting of a 2-mL layer of 45 % sucrose, 20 mL 10-45 % sucrose and a 1 mL overlay of 10% sucrose (in gradient buffer without EDTA). The gradients were centrifuged at 80,000 x g for 3 h (Kontron Centrikon T-2080, rotor TST 28.38) and fractionated into 16 portions of 1.5 mL each. All the fractions were frozen in liquid nitrogen and stored at -70°C.

Calcium transport assay Calcium transport was conducted by the membrane filtration technique (Buckhout, 1984) in a total volume of 0.5 mL containing 10 % sucrose, 25 mM BTP-Mes (PH 7.5), 50 mM KC1, 3 mM MgCh, 1 mM NaN3, 10 mM potassium oxalate (when present), 10 I'M CaCh plus 4sCaCh (32,000 cpm), in the presence or absence of 0.1 mM N~207. Disposable plastic tubes were used throughout to

minimize Ca2+ contamination (Butcher and Evans, 1987 a; Joyce et aI., 1988). The reaction was started with the addition of tonoplastenriched membranes (0.1 mL, 20 - 50 I'g protein, 10/35 % interface). At the end of the desired incubation time (0 to 30 min at 30 0e), the membranes were collected on 0.451'm filters (Gelman GN-6) and washed 3 times with 1 mL buffer solution (10 % sucrose, 25 mM BTP-Mes [pH 7.5], 50 mM KCl and 50 ~ CaCh). Filters were dissolved in 4mL Filter-Count™ (Packard Instrument) and the radioactivity determined in a Packard Tri-Carb Liquid Scintillation Spectrometer.

Quinacrine fluorescence quenching Membrane vesicles (200I'L, 40-100l'g protein), 200~ 10% sucrose (in gradient buffer without EDTA), the appropriate salt or inhibitors and quinacrine (5 ~ final concentration) were added to an assay buffer of BTP-Mes (PH 7.5) to a final volume of 0.6 mL (the final total concentration of BTP plus Mes was 25 mM). The initial rate of PPi-dependent fluorescence quenching was measured (%/min) at room temperature, as previously described (Chanson and Pilet, 1988). All experiments were repeated at least twice.

Enzyme assays and protein determination Pyrophosphate and ATP hydrolyzing activities were determined by detection of released Pi, as previously described (Chanson and Pilet, 1989). Protein determinations were carried out using the method of Bradford with BSA as a reference standard.

Chemicals 4SCa2+ (10-40mCi/mg Ca) was obtained from the Radiochemical Centre, Amersham, U.K. N~207 and La(N03h from Merck, Darmstadt, FRG. Monensin, Tris, Mes and BTP were obtained from Calbiochem-Behring and Na2ATP, imidodiphosphate, DES, BSA, verapamil, quinacrine, zeatin and bovine brain calmodulin (P 2277) from Sigma Chemical Company. All other chemicals were obtained from Fluka AG Buchs.

Results

Characterization 0/the Ca 2 +/H + antiport Light membrane fractions enriched in tonoplast were prepared from maize root homogenates using sucrose step gradients (10, 35, 45 %) (Chanson and Pilet, 1988). The membranes present at the 10/35 % interface were diluted to 10 % sucrose, pelleted and resuspended in 10 % sucrose in gradient buffer without EDTA. The effect of Ca2+ at different concentrations was tested on the PPi-dependent proton pumping activity (Fig. 1), measured by fluorescence quenching of quinacrine. Proton uptake in the vesicles was strongly decreased by Ca2+ and completely inhibited at 300 p.M. The rate of PPi hydrolysis was also inhibited by Ca2+, but at higher concentrations (Iso 80 p.M). The inhibition of proton pumping cannot be explained solely by a direct effect of Ca2+ on H +-PPase. Therefore, these results indirectly support the presence of a Ca2+1 H+ antiport at the tonoplast (Hager and Hermsdorf, 1981). The effect of added Ca2 + on the steady-state proton gradient, generated by the tonoplast H+-PPase, was determined (Fig. 2). With increasing Ca2 + concentrations added to tonoplast-enriched vesicles at steady-state, a concomitant de-

Pyrophosphate-dependent tonoplast Ca2+/H+ antiport 01

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avoid a direct inhibition of PPase activity (Fig. 1). Sodium azide (1 mM) was added to the incubation mixture to block the mitochondrial activity. In Fig.3, the time course of 45Ca2+ uptake by microsomal vesicles in the presence or absence of PPi (0.1 mM) and oxalate (10 mM) is shown. Oxalate was used as a calcium-trapping agent (Bush and Sze, 1986). In the absence of oxalate, the PPi-dependent 45Ca2+ accumulation reached a maximum after 20 min of incubation. When oxalate was present, a larger 45Ca2+ uptake was measured, linear during the first 10 min of incubation (Bush and Sze, 1986). In the absence of PPi, and independently of the presence or absence of oxalate, the accumulation of 45Ca2+ was very low. Several inhibitors, ionophores and growth substances were tested on the PPi-dependent 45Ca2+ uptake (Table 1) in the presence of oxalate (10 mM) and using a 10 min incubation period. All transport assays were carried out in the presence of azide (1 mM). The influx of Ca2+ in the tonoplast vesicles was completely prevented by the Ca2+ ionophore A23187. At high concentration (10 ~), this ionophore was not specific and also inhibited proton transport (Table 2). Monensin (an electroneutral H+ -Na+ ionophore) and CCCP (an electrogenic H+ ionophore), two substances which dissipate proton electrochemical gradients, completely inhibited the uptake of Ca2+. Valinomycine (10 p.M) (a K + ionophore) decreased the accumulation of Ca2+; this is perhaps due to a loss of ionophore specificity at high concentration. At 1 p.M, valinomycine had no effect on Ca2+ uptake. The uptake of Ca2+ was not inhibited by vanadate (100 p.M), a potent inhibitor of the ER Ca2+-ATPase (Bush et al., 1989). Several inhibitors of H +-PPase (Chanson and Pilet, 1988), MDP, imidodiphosphate, DCCD, DIDS and DES decreased the rate of Ca2+ uptake. DES (100 p.M) almost completely inhibited Ca2+ uptake and may act directly on the antiporter. Calmodulin slightly inhibited the PPi-dependent Ca2+ uptake, whereas it was ineffective on proton uptake.

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Fig. 2: Effect of Ca2+ concentrations (CaCh) on a steady-state proton gradient driven by the tonoplast PPi-dependent proton pump. Proton pumping was monitored as the decrease of quinacrine fluorescence. H20, MDP or CaCh (30/LL) were added after 6 min of proton pumping (steady-state). After 10 min, monensin (5/LM) was added.

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Table 1: Effect of different chemicals on PPi-dependent Ca2 + accumulation in sealed microsomal vesicles from maize roots. Calcium accumulation, measured after 10 min of incubation in the presence of oxalate (10 mM) and chemicals. Results are from two experiments using duplicates. Control: 12.6 nmol Ca2 + (mg protein) - I; Ethanol 1 %: 11.7 nmol Ca2 + (mg protein)-I. Each data represents the difference between Ca2 + uptake in the presence and absence of PPi

(O.lmM).

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100

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(10 JLM)

1.7 0.4 8.3 1.6 98.5 59.4

Ca + -transport effectors Calmodulin (2 !ill) Erythrosin B (2 JLM) Ruthenium red (10 JLM) La(N0 3)3 (10 JLM) Verapamil (200 !ill)

80.8 94.7 0.6 35.1 14.9

2

Growth substances AIA (10 !ill) ABA (10 !ill) Fusicoccin" (10 JLM) Zeatin· (10 !ill)

94.5 92.3 96.1 99.5

Lanthanum (a Ca2 + antagonist) and verapamil (a Ca2 +channel blocker) strongly inhibited uptake of Ca2 + and the PPi-dependent proton uptake (Table2). Ruthenium red strongly inhibited Ca2 + uptake, whereas the PPi-dependent proton uptake was only slightly inhibited by this substance (Table2). Finally, erythrosin B, an inhibitor of the ATP-dependent Ca2 + -pump from the plasmalemma (Graf and Weiler, 1989; Rasi-Caldagno et al., 1989), showed no effect on Ca2 + and proton uptakes.

Localization ofthe Ca] +/ H + antiport In maize roots, PPase activity can be used as a reliable tonoplast marker (Chanson, 1990). After linear sucrose gradient centrifugation of a maize root homogenate (Fig. 4), the PPase and PPi-dependent proton pumping activities were recovered at a density of 1,l1g/cm3 together with a peak of proteins corresponding to light membranes (tonoplast, ER and dictyosomes) (Chanson and Pilet, 1987, 1988; Chanson, 1990). The slightly different distributions of the two activi80

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Fig.4: Linear sucrose (10 to 45 %) gradient of the 148 KP fraction centrifuged for 3 h at 80,000 Xg. Protein (0) and percentage of sucrose (.) were analyzed with 50 and 20 JLL aliquots, respectively. PPase (6) and ATPase (A) activities were determined on 50 JLL aliquots, 45Ca2+ uptake (10 min incubation) (D) on 100 JLL aliquots and PPi-dependent proton transport (_) on 200 ILL aliquots.

Pyrophosphate-dependent tonoplast Ca2 +/H+ antiport ties might be due to the presence of some leaky vesicles. Distribution of the PPi-dependent 4SCa2 + uptake, measured by a membrane filtration technique (Buckhout, 1984), was identical to the PPi-dependent proton pumping activity, thus suggesting the presence of a Ca2 +/H+ antiport in the sealed tonoplast vesicles.

Discussion

In maize root cells, the PPi-driven Ca 2 + uptake is insensitive to vanadate and completely inhibited by protonophores and is therefore exclusively the result of a Ca2 +/H+ antiport. An ATP-driven accumulation of Ca2 + in purified tonoplast vesicles or isolated vacuoles from a number of tissues has been reported (Schumaker and Sze, 1985, 1987; Bush and Sze, 1986; Butcher and Evans, 1987 a, b; Joyce et aI., 1988; Malatialy et aI., 1988). Even if the ATP-dependent Ca2 +/H+ antiport was shown to be predominantly present on the tonoplast, some other membranes may possess such activity (Schumaker and Sze, 1985; Butcher and Evans, 1987b). In order to separate the secondary transport system from the primary H+ -pump, artificially imposed pH gradients were utilized to characterize the Ca2 +/H+ antiport independently of the proton-pumping ATPase (Schumaker and Sze, 1985, 1986; Blumwald and Poole, 1986). Most of the activity was associated with the tonoplast. However, a Ca2 +/ H + exchange system was shown to be present on several other different membranes from oat roots, using imposed pH gradients (Schumaker and Sze, 1986). Use of the tonoplast PPi-dependent proton pump to energize specifically the tonoplast vesicles in a mixture of membranes from different origins is a powerful method for several reasons. The H + -PPase of maize root cells is strictly localized on the tonoplast and only sealed tonoplast vesicles will induce a pH gradient. When ATP is used to energize the tonoplast vesicles, the plasmalemma and ER ATP-dependent Ca2 +-pumps will be active and interfere with the Ca2 +/H+ antiport, as reported for several materials (Hager and Hermsdorf, 1981; Zocchi, 1988). The only way to cope with this problem is to work with highly purified tonoplast fractions, which are very difficult to obtain when using maize root cells (Chanson and Pilet, 1987). The characteristics of maize root Ca 2 + /H+ antiport activity are similar to those previously described using other materials (Evans, 1988). Ca2 + uptake is not inhibited by vanadate (100 p,M), as reported for tonoplast fractions prepared from oat roots (Schumaker and Sze, 1985, 1986), cultured carrot cells (Bush and Sze, 1986), tomato fruits and red beet roots Goyce et al., 1988). The controversial result reported for tonoplast-enriched vesicles from spinach leaves might be explained by ER contaminations (Malatialy et aI., 1988). Vanadate is a potent inhibitor of the ER ATP-dependent Ca2 +-transport (Bush et al., 1989), whereas its effect on the ATP-dependent Ca2 +-pump from the plasmalemma is less clear (Graf and Weiler, 1989). The results of calmodulin effects on Ca2 + transport in higher plant cells are contradictory (Evans, 1988). In maize root vesicles, calmodulin slightly inhibited the PPi-dependent Ca2 + uptake, whereas it was ineffective on proton

475

uptake (Table2). A stimulatory effect of calmodulin on the Ca2 +/H+ antiport from tonoplast-enriched vesicles from spinach leaves has been reported (Malatialy et al., 1988). This could be explained by an ER contamination of the spinach fractions.

Conclusions The tonoplast Ca2 +/H+ antiport might be involved in the maintenance of Ca2 + cytoplasmic levels in maize root cells, even if the plasmalemma Ca2 +-pump is likely to be the longterm site for calcium efflux (Evans, 1988). Moreover, uptake of Ca2 + into the vacuole may be of particular significance in providing pools for rapid fluxes of Ca2 + in response to stimuli (Butcher and Evans, 1987 b). The recent report that inasitol1,4,5-triphosphate releases Ca2 + from tonoplast vesicles of oat roots (Schumaker and Sze, 1987) gives strong support to the idea that phosphatidylinositides may play a crucial role as regulators in higher plant cells. The regulatory mechanisms for the activity of the Ca2 +/ H+ antiport are not known, and such studies will help establish the physiological significance of the Ca2 +/H+ antiporter on the tonoplast. The inhibitory effect of Ca2 + on PPase activity may be essential in the regulation of Ca2 + entry into the vacuole. This problem will be the object of further investigation. The complete characterization of the maize root tonoplast Ca2 +/H+ antiport should be a conclusive step towards an understanding of its function. Acknowledgements I would like to thank Professor P. E. Pilet for valuable discussions during the course of this work and for reviewing the manuscript.

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