Copyright © Physiologia Plantarum 1999PHYSIOLOGIA PLANTARUM 106: 203–209. 1999ISSN 0031-9317Printed in Ireland—all rights reser6ed

A comparison of Zn, Mn, Cd, and Ca transport mechanisms in oat roottonoplast vesicles

Alonso Gonzalez, Victor Koren’kov and George J. Wagner*

Plant Physiology/Biochemistry/Molecular Biology Program, Room 200 L, THRI Bldg., Uni6ersity of Kentucky, Lexington, KY 40546-0236,USA*Corresponding author, e-mail: gwagner@ca.uky.edu

Received 13 October 1998; revised 4 March 1999

Oat root tonoplast vesicles were used to determine if tonoplast the order of Ca\Cd\Zn�Mn. The observed ion-concen-tration-dependent release of protons from sealed vesicles sug-transport of the divalent cations Zn and Mn occurs via angests that Zn and Mn, like Ca and Cd, can be antiported intoantiport mechanism, like that described for Ca and Cd. Also,the plant vacuole. In an effort to assess whether Ca and Cdinhibitors reported to affect Ca transport were tested for their

effects on Cd versus Ca transport and tonoplast ATPase use the same carrier, we tested the effects of verapamil,Do-Tea-Br, nifedipine, ruthenium red, and LaCl2 on 45Caactivity. The ability of Ca, Cd, Zn, and Mn to alter theversus 109Cd transport, and also on MgATPase activity. Theseproton gradient was monitored using both the fluorescentcompounds are shown to alter Ca transport in plants. Al-probe acridine orange and 14C-methylamine accumulation.

After the proton gradient was established in MgATP-ener- though some of the inhibitors had a negative effect on Mg-MAPase activity, the decrease in this activity did not accountgized vesicles, addition of Ca, Cd, and Zn to the reaction

restored the fluorescence of acridine orange, indicating dissi- for the decrease in Ca or Cd transport observed in any case.pation of the proton gradient. Fluorescence recovery was Particularly verapamil had a much greater effect on Calinearly correlated with metal concentration and followed the transport than Cd transport activity while not inhibiting

ATPase substantially. Data presented provide evidence for Znorder Ca\Cd]Zn. Addition of Mn did not restore thefluorescence of acridine orange. All four ions released 14C- and Mn antiport activity in oat root tonoplast and showmethylamine from MgATP-energized vesicles in an ion-con- differences in responses of Ca and Cd antiport activities tocentration-dependent manner, and with relative initial rates in several transport inhibitors.

It is now well established that tonoplast contains both anATP-dependent Ca pump and Ca/H antiport activities thattransport Ca from the cytosol to the vacuole (Bush 1995).The Km for the Ca/H antiporter has been variously mea-sured as 10–42 mM (Blumwald and Poole 1985, Schumakerand Sze 1986). Vacuolar transport of Ca has been exten-sively studied because of its role in Ca-mediated signaltransduction processes (Bush 1995). Antiport activities forNa/H, Cd/H, and Mg/H have also been described fortonoplast, these having apparent Km values of 7–26 mM,5.5 mM, and 2 mM, respectively (Blumwald and Poole 1985,Salt and Wagner 1993, Amalou et al. 1994). It has beensuggested that the Ca/H activity may also function in Mg/Htransport (Pfeiffer and Hager 1993). All are secondary iontransporters that are energized by the proton gradient acrossthe tonoplast. These activities are likely to play major roles

Introduction

The plant central vacuole is often assumed to be the compart-ment for containment of the bulk of most macronutrient,micronutrient, and pollutant ions in mature cells, includingthe ions studied here: Ca, Zn, Mn, and Cd. Indirect evidencefor this assumption comes from early ion efflux studies usingvarious plant tissues, studies that distinguished between slowefflux (vacuole) versus more rapid efflux (cytosol and cellwall) pools. Direct evidence for vacuolar ion compartmenta-tion has come more recently from vacuole/extravacuolelocalization studies in which isolated protoplast and vacuoleion contents were compared (Lin et al. 1977, Wink 1993,Brune et al. 1994). Other approaches have provided com-pelling evidence relating to vacuolar ion localization, includ-ing NMR spectroscopy studies for Mn (Quiquampoix et al.1993) and X-ray microanalysis studies for Zn and Cd(Vazquez et al. 1994, Salt et al. 1995).

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in ion homeostasis under physiological conditions. We re-cently found that Ni is not antiported in oat root tonoplastvesicles capable of antiportation of Ca and Cd (Gries andWagner 1998).

Although the catalog of antiporter activities at thetonoplast has increased in recent years, elucidation of thekinetic properties and ion specificity of the correspondingtransport protein(s) has lagged behind. This is due in part tothe low abundance of transport proteins, their membrane-bound nature, and the lack of plant mutants to separateactivities into specific entities. Perhaps the best biochemi-cally characterized tonoplast antiporter activities to date arethe Na/H and Ca/H transporters. Recently, the gene encod-ing a Ca/H activity was isolated from Arabidopsis (Hirschiet al. 1996).

Very relevant questions concerning the tonoplast antiportactivities for Ca and Cd are if related ions (Zn, Mn, etc.) aresimilarly antiported and whether all or some of these relatedions use the same or different transport proteins (Bush1995). These questions can be approached at the proteinlevel by comparing kinetics of transport of various ions, bysearching for differential responses of, e.g., Ca versus Cdtransport to inhibitors known to effect Ca transport. Otherapproaches are ion competition studies, comparative elec-trophysiological studies, in vitro studies of isolated trans-porters (if these were available), comparative studies usingmutants, or the gene complementation approach using, e.g.,yeast transport mutants.

Here, we compared the effects of Ca, Cd, Zn, and Mn onan established proton gradient using MgATP-energized oatroot tonoplast vesicles to examine the mechanisms of Znand Mn tonoplast transport. Also, in an attempt to differen-tiate between Ca/H and Cd/H activities, and to identifypossible probes for future isolation of transport proteins, weexamined the effects of several inhibitors commonly used toprobe Ca movement on Ca versus Cd transport.

Materials and methods

Plant material and preparation of sealed tonoplast-enrichedvesicles

Oat seeds (A6ena sati6a L.), purchased from Southern StatesCooperative, Lexington, KY, were germinated in the dark inan aerated solution of 0.5 mM CaSO4. Roots were harvestedafter 4 or 5 days. The tonoplast isolation procedure wasafter that of Randall and Sze with slight modifications (seeSalt and Wagner 1993). All steps were conducted at ice-bathtemperature and where used, DTT and PMSF were addedjust before use. The distal 4–5 cm of oat roots (150–200 g)were homogenized in a mortar and pestle in a mediumcontaining 250 mM mannitol, 6 mM EGTA, 0.1% (w/v)BSA, 0.1 mM PMSF, 50 mM HEPES-BTP (pH 7.4), 4 mMDTT, and 1% (w/v) insoluble PVP, using a medium-to-tissueratio of 2 ml g−1. The brei was strained through four layersof cheesecloth and insoluble material was extracted again.The combined filtrates were centrifuged for 5 min at 1000 gand then 10 min at 12000 g. Membranes in the supernatantwere pelleted by centrifugation at 60000 g for 30 min. The

pellets were gently resuspended with the tip of a paint brushin 1 ml of resuspension medium containing 250 mM manni-tol, 0.1 mM PMSF, 2.5 mM HEPES-BTP (pH 7.2), 1 mMDTT, pooled, and diluted to 30 ml with resuspensionmedium. The suspension was layered on top of 10 ml of adextran cushion containing 6% (w/v) dextran (Sigma, St.Louis, MO, USA; D-4751, average Mr=74 200), 2.5 mMHEPES-BTP at pH 7.2, and 250 mM mannitol. Gradientswere centrifuged at 70000 g for 2 h. The visible band at the0.6% dextran (w/v) interface was collected, diluted 10-fold inresuspension buffer, and this suspension was centrifuged at100000 g for 30 min. Pelleted membranes were resuspendedin resuspension buffer, divided into 100 ml aliquots andstored at −70°C until use.

Protein concentration determination

Membrane protein was determined using a modification ofthe method of Bensadoun and Weinstein as previouslydescribed (Salt and Wagner 1993). To 10 ml of vesicles, 30 mlof 1% (w/v) Na-deoxycholate was added, followed by incu-bation at 65°C for 5 min. One milliliter of Pierce BCAprotein assay reagent (Pierce Inc., Rockford, IL, USA) wasadded. After incubation at 65°C for 30 min, samples wereallowed to cool at room temperature for 15 min, then A562

was measured (BSA was used as a standard).

Effect of metals on the proton gradient: Acridine orangeassay

A proton gradient (acid inside) was generated via theMgATP-energized proton pump by adding tonoplast vesi-cles (20–30 mg protein ml−1) to a reaction mixture contain-ing 25 mM HEPES-BTP at pH 7.0, 175 mM mannitol, 28mM HCl (adjusted with BTP to pH 7.0), and 0.2 mMNaN3, 3 mM ATP, and 5 mM acridine orange. The protonpump was activated with MgSO4 (3 mM final concentration)and the formation of the pH gradient was followed spec-trofluorometrically by monitoring fluorescence quenching ofacridine orange (495 nm excitation, 540 nm emission) (Palm-gren 1991) at 20°C. After a steady state proton gradient wasachieved (4–5 min), the test metal was added and fluores-cence recovery rate was monitored using a chart recorder setat 1 mm s−1. The cuvette was constantly stirred.

Effect of metals on the proton gradient: [14C] methylamineuptake

A proton gradient (acid inside) was generated using theMg-stimulated proton pump by adding tonoplast vesicles(70–90 mg protein ml−1) to a solution containing 25 mMHEPES-BTP at pH 7.0, 250 mM mannitol, 28 mM HCladjusted with BTP to pH 7.0, and 0.2 mM NaN3, 3 mMATP and 20 mM [14C] methylamine (37 kBq ml−1). Theproton pump was activated with MgSO4 (3 mM final con-centration) and 90 ml aliquots were removed every 3 min tomonitor [14C] methylamine uptake using the direct vesiclefiltration assay, as described below. After a steady stateproton gradient was established (12 min), metal (Ca, Cd,

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Zn, or Mn) was added and aliquots were taken every minuteto measure [14C] methylamine retained by the vesicles. Alladditions were made in 2.5 mM HEPES-BTP at pH 7.0, andtubes were incubated at 22°C.

Proton-gradient-dependent Ca and Cd transport assays

Membrane vesicles (up to 90 mg protein ml−1) were incu-bated at 19°C for 8 min in reaction medium containing 25mM HEPES-BTP, pH 7.0; 250 mM mannitol, 28 mMHCl-BPT, pH 7.0, 3 mM ATP, and 2 mM NaN3. Tominimize variation between experiments, a volume of reac-tion medium with membranes equivalent to three assays (1.8ml) was made 75 mM with CaCl2 or CdCl2 and contained16.7 or 18.5 kBq ml−1 of 45Ca or 109Cd, respectively. Thisreaction mixture was divided into three aliquots (0.6 mleach), two received the inhibitor being tested, and the thirdwas used as a control. Inhibitors (100 mM final concentra-tion) were added from stocks in H2O (100% EtOH fornifedipine). After 10 min, MgSO4 was added (3 mM finalconcentration) to initiate formation of a proton gradient.Samples (90 ml) were removed from the medium every 3min, pipetted onto 0.45 mM pre-wet (reaction medium with-out ATP) filters (Millipore HATF type HA, MilliporeCorp., Bedford, MA, USA), and washed rapidly underreduced pressure (53 kPa) with 5 ml ice-cold wash medium.Wash medium was composed of 25 mM HEPES-BTP, pH7.0, 250 mM mannitol, and 28 mM HCl-BPT, pH 7.0, andincluded either 1 mM CaCl2 or CdCl2, as appropriate. Foreach inhibitor tested, one aliquot was taken before MgSO4

was added to the reaction mixture. The membrane bindingobserved was subtracted from all other measurements. Tri-ton X-100 (0.03%) was added after 18 min and activityremaining on the filter was assumed to represent metalbound to membranes. Negligible amounts (less than 0.1% oftotal metal retained by membrane vesicles) of 45Ca or 109Cdwere bound to the filters after Triton X-100 treatment.

Assay of ATP hydrolysis

Since the proton gradient was generated using the protonpump, it is important to determine the effects of the in-hibitors on its activity. MgATPase activity was assessed asthe rate of release of inorganic phosphate using the methodreported by Ames (1966). Vesicles were added to a mediumcontaining 25 mM HEPES-BTP, pH 7.0, 28 mM HCl-BPT,pH 7.0, 3 mM ATP, 0.2 mM NaN3, and the inhibitor beingtested (100 mM final concentration). After incubation for 15min in ice, MgSO4 was added to 3 mM and samples wereincubated at 28°C for 15 min. Reactions were stopped byimmersing tubes in ice.

Reagents

Chemicals, including [14C] methylamine, 337 MBq mmol−1,were purchased from Sigma (St. Louis, MO, USA). PierceBCA protein assay reagent was obtained from Pierce Chem-ical Co. (Rockford, IL, USA), 45Ca (411 MBq mg−1) wasobtained from ICN Radiochemicals (Irvine, CA, USA), and

109Cd (78 MBq mg−1) was obtained from DuPont-NewEngland Nuclear (Wilmington, DE, USA).

Results and discussion

Effects of metals on DpH: Acridine orange assay

Establishment of a proton gradient in vesicles (acid inside)by activation of the proton pump upon addition of MgATPresulted in maximum fluorescence quenching of acridineorange at about 4 min (time between points 1 and 2, insertto Fig. 1). The gradient was largely maintained for up to 20min (dashed line, point 2–3), attesting to the sealed natureof vesicles. Fluorescence was recovered when the pH gradi-ent was dissipated by addition of Zn, Cd, or Ca (trace frompoint 2–4). Addition of 1 mM NH4Cl or 0.03% (v/v) TritonX-100 resulted in rapid fluorescence recovery.

Fig. 1. Fluorescence recovery versus time in MgATP-energized oatroot tonoplast vesicles. Inset in Fig. 1 schematically shows: point1–2, acridine orange fluorescence quench from steady state uponaddition of MgCl2; point 2–3, maintenance of proton gradient;point 2–4, fluorescence recovery upon addition of ion. Fluorescencerecovery by various concentrations of Zn, Cd, or Ca: open circles,100 mM ; triangles, 75 mM ; closed squares, 50 mM, closed circles, 25mM ; and open squares, 10 mM ion. Values are the means (withstandard deviation) of three measurements.

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Fig. 2. Comparison of fluorescence recovery versus time after addi-tion of 75 mM Ca, Cd, or Zn. Values are the means (with standarddeviation) of three measurements.

concentration would be about 50 mM in an uncontaminatedsoil. The estimate (similarly calculated) for Cd in uncontam-inated soil is B0.4 mM (see Salt and Wagner 1993). Inearlier studies of Cd transport, non-linearity in transport(measured using the methylamine assay) only occurred be-tween 100 and 1 000 mM Cd (Salt and Wagner 1993).Qualitatively similar results to those shown in Figs. 1–3were obtained using a separate batch of vesicles, i.e., relativerates of fluorescence recovery were Ca\Cd]Zn (notshown). We conclude from acridine orange fluorescencequench assays that Zn, like Ca and Cd, causes dissipation ofthe pH gradient in a manner consistent with an antiportmechanism. Our observations indicate that transport of Znand Cd is similar, with the rate of Cd movement being onlysomewhat higher than that of Zn.

Mn is known to be accumulated in the plant vacuole(Quiquampoix et al. 1993). However, when Mn transportwas studied using the acridine orange assay, we found thataddition of 25 mM to 1 mM MnCl2 did not cause recoveryof fluorescence. In contrast, the methylamine assay (seebelow) showed exchange of protons upon addition of Mn,but only when relatively high concentrations of this ion wereused. Control experiments showed that Mn shifted neitherthe excitation nor emission wavelengths of acridine orangefluorescence, at pH 7 or 5. Addition of Cd to the assay inthe presence of 1 mM Mn resulted in a fluorescence recoveryresponse expected for Cd (not shown). Results with Mnsuggest that some uncharacterized interaction of this partic-ular ion with the acridine orange probe makes this assayproblematic for monitoring Mn transport. We note thatSchumaker and Sze (1986) reported that 50 mM Mn had noeffect on the proton gradient in K-nigericin-energized oatroot tonoplast vesicles, but in that study 50 mM Cd was alsofound to be without effect. Potential interferences associatedwith the use of the acridine orange assay have been dis-cussed by Palmgren (1991).

Addition of Ca, Cd, or Zn at various concentrations toenergized vesicles resulted in ion-concentration-dependentrecovery of fluorescence as shown in Fig. 1. The extent andrate of fluorescence recovery (real time measurements) wasdependent upon both the particular ion and ion concentra-tion. A single batch of vesicles was used for these measure-ments. These results are consistent with transport of Zn viaan antiport mechanism similar to that shown previously forCa and Cd (Schumaker and Sze 1986, Salt and Wagner1993). The initial rate of fluorescence recovery after Ca, Cd,and Zn addition is compared in Fig. 2 at 75 mM ionconcentration. The relative order of recovery rate was Ca\Cd]Zn. We previously found that the rate of Ca uptake inoat root tonoplast vesicles was greater than that of Cd (Saltand Wagner 1993). Results of Figs. 1 and 2 suggest thatDpH dependent vacuolar transport of Zn is nearly as effi-cient as that of Cd. While the Km for Zn has not yet beenmeasured (will require the use of 65Zn) proton exchangewith Zn and Cd monitored using acridine orange is similar.The Km for Cd/H of oat root tonoplast has been determinedto be 5.5 mM (Salt and Wagner 1993), lower than thatreported for Ca/H (Blumwald and Poole 1985, Schumakerand Sze 1986). In Fig. 3, the magnitude of fluorescencerecovery after 30 s is plotted against metal concentrationfrom 10 to 100 mM (25–100 mM for Cd). While the initialrates of proton efflux versus ion concentration were similarfor Zn and Cd, that for Ca was greater over the concentra-tion range tested. For all three ions, initial rates of recoverywere linear over a range of about 25–100 mM. This resultreflects upon the capacity of the pH gradient and iontransport mechanism(s) to accommodate even high concen-trations of all three ions. It has been estimated that plantsgrowing in an uncontaminated soil are exposed to Zn levelsof 25–190 mg g−1 soil with a mean of 50 mg g−1 soil(Hopps 1974). If one assumes a mean soil density of 1.3 gml−1 dry weight and that under neutral pH conditionsplants can access only about 5% of soil Zn, available Zn

Fig. 3. Comparison of fluorescence recovery rate (at 30 s) versusmetal concentration for Ca versus Cd versus Zn.

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Fig. 4. Proton/metal exchange in MgATP-energized tonoplast vesi-cles of oat roots monitored using 14C-methylamine. The relativeeffects of Zn versus Cd versus Ca were compared at 25 mM ion.Methylamine content of a control (no metal) assay was largelydissipated by addition of 0.03% (v/v) triton-X100 (addition atarrow, dashed line). Values are the means (with standard deviation)of three measurements.

Relative effects of various inhibitors on Ca and Cdtransport

Ca, Cd, and Zn (and perhaps also Mn) transport across thetonoplast are similar in apparent mechanism, but differ intheir ion concentration dependence. These differences mayreflect properties of a single carrier or indicate the presenceof more than one carrier for transporting these metals intothe vacuole sap. In an effort to distinguish between these

Fig. 5. Concentration-dependent proton/metal exchange in oat roottonoplast vesicles, determined using the methylamine assay. Dashedlines represent the response from 25 mM Ca. Data points formethylamine accumulation (open triangles) are the means (withstandard deviation) from ten separate experiments. Those for metaleffects are the means for three measurements.

Effects of metals on DpH: Methylamine assay

The use of 14C-methylamine and the direct vesicle filtra-tion assay is a well established method for quantitativelymonitoring the pH gradient across membrane vesicles. Asshown in Fig. 4, methylamine accumulation after additionof Mg to vesicles with ATP, reached a steady state levelafter about 12 min. Vesicles appeared to be well sealed asevidenced by maintenance of the steady state gradient inthe control, at least through 20 min and, as expected,addition of 0.03% (v/v) Triton X-100 dissipated the gradi-ent. Addition of Ca, Cd, or Zn (25 mM metal) resulted indissipation of the proton gradient. The relative effective-ness of these ions (Ca\Cd]Zn) was similar in order tothat observed using the fluorescence quench assay. Asshown previously (Salt and Wagner 1993), the rate andmagnitude of proton dissipation in response to Cd wasconcentration-dependent (Fig. 5). With Zn, dissipation wasalso observed, the magnitude being similar to that withCd at the same metal concentration. When Mn was testedin this assay, Mn-concentration-dependent proton gradientdissipation was observed (Fig. 5), but the concentration ofMn required to achieve the response of 25 mM Zn was250 mM Mn. These results suggest that Mn may be an-tiported into the vacuole, but with low efficiency relativeto Ca, Cd, or Zn. The phenomenon of Mn transport intothe vacuole may be highly important in terms of Mn toxi-city and tolerance in plants growing in acid soils. In thiscircumstance, tissue Mn can reach in excess of 4 mg g−1

dry weight (Horst 1988). In all experiments described inFig. 5, addition of 0.03% (v/v) Triton X-100 to controlassays at about 20 min resulted in dissipation of the pro-ton gradient (not shown).

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Fig. 6. Effects of various ion transport inhibitors on 45Ca versus109Cd transport versus ATPase activity in MgATP-energized oatroot tonoplast vesicles. Inhibitor concentrations were 100 mM andion concentrations were 75 mM. La effects were monitored using theacridine orange assay. Verapamil effects were measured using bothMgATP-energized and K-loaded, nigericin-activated vesicles.

nium red, a compound that was found to prevent the rise incytosol Ca under anoxic conditions in maize (Subbaiah etal. 1994), and reduce Ca uptake in reconstituted oat roottonoplast vesicles (Schumaker and Sze 1990), inhibited Caand Cd uptake by 37 and 14%, respectively. Do Tea Brinhibited Ca and Cd transport by 38 and 19%, respectively.Amiloride, as expected, had little effect on Ca or Cd trans-port. LaCl3 (tested using the acridine orange assay) inhibitedboth Ca and Cd transport. This ion is reported to reduce Cauptake in reconstituted oat root tonoplast vesicles (Schu-maker and Sze 1990) and Ca release in beet root tonoplastvesicles (Calver and Sanders 1994).

The effects of inhibitors on Ca and Cd transport observedhere using MgATP-energized vesicles could be due to inhibi-tion of the proton pump (ATPase activity or other aspectsof the pump) or the antiport carrier(s). We monitoredATPase activity in the presence and absence of the variousinhibitors and found inhibition levels to be considerablybelow the levels of inhibition of Ca and Cd transportactivities, particularly in the cases of verapamil and ruthe-nium red (Fig. 6). Regarding possible inhibition of othercomponents of the proton pump (e.g., proton translocation),we observed with the acridine orange assay that verapamiland nifedipine reduced the level of fluorescence quench(proton gradient) by about the same levels observed inATPase assays (data not shown).

Variable results with inhibitors and in ion competitionexperiments is often observed in various studies reported inthe literature. This variability may be due to differences inthe source of vesicles or assay conditions and is difficult toexplain because the modes of action of inhibitors are notprecisely known. Also, interactions of inhibitors and ionsmay be at several points critical to a transport process(transporter iself, the membrane involved, other componentsin a particular assay system). We have taken care in thisstudy to compare transport of two ions using one tissue, oneisolation/assay system, and a single membrane batch (con-trol versus inhibited) in an effort to minimize variability.Data obtained are considered to be comparative in thiscontext. From inhibitor studies, we conclude that suitableanalogs of verapamil or ruthenium red may be candidatesfor labeling, differentiating, and possibly isolating Ca, Cd,Zn, Mn membrane transporting proteins from oat roots.Experiments to this end are underway.

In summary, we have observed DpH-dependent transportof Zn in tonoplast vesicles from oat roots. This activity issimilar in character and apparent magnitude to Cd trans-port and is different in magnitude and kinetics from Ca/Hactivity. Since the occurrence of substantial Cd is generallyrestricted to soils polluted through human activities fromthe beginning of the industrial revolution, but Zn is om-nipresent at relatively high levels, Cd/H transport may occurvia the antiporter evolved for transport of Zn (that evi-denced here). Support was also obtained for the occurrenceof Mn/H transport (methylamine assay), but the magnitudeof this activity was 7- to 10-fold less than that of Cd/H orZn/H. Responses of Ca/H versus Cd/H exchange to vera-pamil and ruthenium red differed substantially in the magni-tude of inhibition observed. While further study is needed todetermine if one or more distinctly different antiporter

two possibilities, we monitored 45Ca or 109Cd transport intoMgATP-energized vesicles in the presence and absence ofvarious known inhibitors of Ca transport, using the directvesicle filtration assay. To minimize variability, Ca and Cdtransport in the presence and absence of each inhibitor wereassayed under identical conditions, using a single batch oftonoplast membranes. Control (no inhibitor) and inhibitor-containing assays were run simultaneously. Since some in-hibitors might affect the proton pump and thus the protongradient, their effects on vesicle ATPase activity was alsoassessed. Results (Fig. 6) are presented for each inhibitortested, as percent of control (no inhibitor present). Resultswere similar in two separate experiments. The inhibitorstested are all reported to effect Ca transport. Some havebeen identified as Ca channel blockers (verapamil, nifedip-ine, dodecyltriethylammonium bromide [Do Tea Br], La)(Pfeiffer 1995). Amiloride is shown to interact with theNa/H antiporter of beet root tonoplast (Barkla et al. 1990)and Mg/H antiporter activity in latex vesicles from He6ea(Amalou et al. 1994).

Different inhibitors (all at 100 mM concentration) showeddifferent effects on Ca versus Cd transport (monitored using75 mM ion). Verapamil decreased Ca and Cd uptake by 45and 19%, respectively. When verapamil inhibition of Ca andCd transport was monitored in K-loaded, nigericin-ener-gized vesicles (see Salt and Wagner 1993 for methods),inhibition of transport was 73 and 32%, respectively. Thus,under conditions where the proton pump is not involved(artificially-generated gradient), Ca transport is also muchmore inhibited by verapamil than is Cd transport. Ruthe-

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proteins exist for tonoplast transport of Ca versus Cd versusother related ions, our results suggest some tools for ap-proaching the testing of this question, one that is highlyrelevant to ion homeostasis and future efforts to manipulateion storage and sequestration in plants.

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