Barkla trasnport ion.pdf

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Annu.Rev. Plant Physiol. Plant Mol. Biol. 1996. 47:159–84Copyright© 1996by AnnualReviewsInc. All rightsreserved

PHYSIOLOGY OF ION TRANSPORTACROSSTHE TONOPLAST OFHIGHER PLANTS

Bronwyn J.Barkla andOmar Pantoja

Departamento de Biología Molecular de Plantas,Instituto de Biotecnología, UNAM,Cuernavaca,Morelos, México, 62271

KEY WORDS: V-ATPase,V-PPase,channels, cotransporters,vacuole

ABSTRACT

Thevacuoleof plant cellsplaysanimportant rolein thehomeostasis of thecell.It is involved in the regulation of cytoplasmic pH, sequestration of toxic ionsandxenobiotics, regulation of cell turgor, storage of amino acids, sugars andC02 in the form of malate, andpossibly asa sourcefor elevating cytoplasmiccalcium. All these activities aredrivenby twoprimaryactive transport mecha-nisms present in the vacuolar membrane (tonoplast). These two mechanismsemploy high-energy metabolites to pump protonsinto thevacuole,establishingaprotonelectrochemical potential that mediatesthetransport of adiverserangeof solutes. Within the past few years, great advances at the molecular andfunctional levels have beenmadeon thecharacterization andidentifi cation ofthesemechanisms.Theaim of this review is to summarize thesestudiesin thecontext of thephysiology of theplant cell.

CONTENTSINTRODUCTION..................................................................................................................... 160PROTON TRANSLOCATING PUMPS.................................................................................. 160

Vacuolar H+-ATPase.......................................................................................................... 160Vacuolar Pyrophosphatase................................................................................................. 165

ION COTRANSPORTERS....................................................................................................... 169Na+/H+ Antiport .................................................................................................................. 169Ca2+/H+ Antiport................................................................................................................. 170

ION CHANNELS...................................................................................................................... 171

Slow-Activating Channels ................................................................................................... 171Fast-Activating Channels.................................................................................................... 172Ca2+-Selective Channels..................................................................................................... 173Malate-SelectiveChannels.................................................................................................. 177Tonoplast IntrinsicProtein ................................................................................................. 178

CONCLUDING REMARKS.................................................................................................... 179

INTRODUCTION

Ion transportacrossthe tonoplastplays a central role in the control of cellhomeostasisandosmoregulation,andhasled toinvestigations ofthe tonoplasttransportmechanismsinvolved in thesekey cellularprocesses.Primarytrans-port activitiesrequiringhigh-energymetabolitesfor their operationhavebeencharacterizedat the functional and molecularlevels. In contrast,secondaryactivetransporters,aswell aspassivetransporters,haveonly beendefinedatthefunctionallevel,with theexceptionof thetonoplastintrinsicprotein.Thesestudieshaveled to our understanding of therole of thevacuolein thephysiol-ogy of plant cells. Evidencehas beenpresentedfor the importanceof thetonoplasttransportersin sodium accumulationand salt tolerance,vacuolarCa2+ release andsignaltransduction,andfor thestorage of CO2 in the formofmalate.For theseprocesses,passiveand/orsecondaryactivetransportershavebeenimplicated,whoseactivitiesaremaintained by thedriving force providedby theprimaryactive transporters.

PROTON TRANSLOCATING PUMPS

VacuolarH+-ATPase

It is not surprisingthatthe firstplantvacuolar transportprotein(s)to beclonedbelong to the vacuolartype H+-ATPase(V-ATPase).Theseprimary activetransportersrepresenta ubiquitousclassof protonpumpsthat arefound on avariety of cellular organellesincluding lysosomes,endosomes,secretoryandstoragevesicles,and protein sorting organelles,as well as on the vacuolarmembrane(tonoplast)of higherplants,fungi, andyeast(44, 65, 112). Becausethey werefirst characterizedon the tonoplast,they weredesignatedvacuolartype despitethe misleadingimplication.V-ATPaseshavealsobeenfound asfunctioningenzymeson the plasmamembraneof specializedvertebratecellssuch as osteoblasts and neutrophilsand epithelial cells of the kidney andbladder(50, 79, 123). The V-ATPasesfunction in biological energyconver-sion, generatinga proton-motive-force(PMF) by hydrolyzing ATP and thusproviding the driving force for a wide rangeof secondaryactiveandpassivetransportprocesses.Theseenzymesalsofunction in cellular pH homeostasisaswell asin acidification of theinterior of severalorganellesin thecaseof an

160 BARKLA & PANTOJA

endomembrane distribution, or in acidificationof a localized extracellularcompartmentin thecaseof the plasmamembraneV-ATPase (84).

The enzymeresemblesthe F0F1-ATPases(F-ATPases)of mitochondria,chloroplasts,andeubacteriain their multimericstructure,lackof a phosphory-lated intermediate,and insensitivity to vanadate(84). An evolutionary rela-tionship betweenF-type and V-type ATPasesis indicatedby sequenceho-mologiesof someof the subunits(68, 69, 83, 122). However,V-ATPasesrepresentauniqueclassof H+-ATPasesbecausetheyhaveaneutralratherthanan alkaline pH optimum and becausethey show a distinct inhibitor profile.They are insensitive to azideand more sensitive than the F-ATPasesto N-ethylmaleimide (NEM),nitrate,and bafilomycin A1 (114).

SUBUNIT COMPOSITION The V-ATPasesare large protein complexescom-posedof 7 to 10 different subunits that appearto copurify with the enzyme,dependingonthespecies,with anativemolecularweightbetween400and650kDa (113).However,in plantsonly themajorsubunitshavebeencloned,andmuchof theworkonthestructureandfunctionof theV-ATPasehasbeencarriedoutusingyeast as a modelsystem.

Thesubunits thatmakeup the functionalenzymecanbegroupedinto twocategoriesbasedon their associationwith either the peripheral,hydrophiliccatalyticsector(V1) or the integral,hydrophobicmembranesector(V0). TheV1 sectoris composedof five or six soluble proteins.Among these arethetwoubiquitous major subunits, A and B, presentin threecopiesper functioningenzyme.The 70-kDacatalyticnucleotide-binding subunitA and the 60-kDaregulatorysubunit B were initially cloned in carrot (122) and Neurosporacrassa (26), respectively,with the plant sequencefor the B subunit fromArabidopsis thaliana clonedsoonafter (69). Othersubunitsof the V1 sectorthathavebeenclonedaresubunitsC andE (81). Althoughproteinsof similarsizehavecopurifiedwith theplantV-ATPase(46,89,113), thecorrespondingplant sequenceshaveyet to beobtained.Recently,two additional subunits ofthe V1 sectorhavebeenclonedin yeast(subunitF) and in yeastandbovine(subunitD) (81,82).SubunitF, a14-kDapolypeptidenecessaryfor theassem-bly andactivity of theV-ATPase(81), is thoughtto play a role in themodula-tion of enzymeactivity. In Saccharomycescerevisiae,thesubunitis releasedwith the V1 sectoruponcold inactivation of the enzyme,a responsethat hasonly beendemonstratedfor the solublesubunits of the V-ATPaseenzymes(81). Subunitsof similar size have beenshown to be associatedwith theV-ATPaseholoenzyme from plants including mung beanandbarley(46,113).In oat,a subunitof 13 kDa was alsoidentified;however, itwas concludedthatthis polypeptidewasassociatedwith theV0 sector(63).SubunitD wasidenti-fied asa proteinthatalsocopurifiedwith thecold-inactivatedV1 sectorfrombovine chromaffin granules.The geneencodesa protein with a calculated

TONOPLAST ION TRANSPORT 161

molecularmassof 28 kDa. Comparisonof relatedsequencesresultedin theidentification of two identicalpartial cDNAclonesfrom A. thalianaexhibitingapproximately50% identity with the internalnucleotide sequence for thebovine subunitD, which suggeststhat thesefragments areprobablecandidatesfor the gene encoding the A. thaliana subunit D (82). V-ATPase-relatedsubunitsof this sizehavepreviouslybeenidentifiedin Kalanchoëdaigremon-tiana (27),Mesembryanthemumcrystallinum(28),andredbeet(89);however,theassociationof thesepolypeptideswith theV1 sectorof theV-ATPasewasnotdemonstrated.

TheV0 sectorof theV-ATPaseis composedof the16-kDaproteolipid andoneto four othersubunits.Theproteolipid, presentin six copiesperfunction-ing enzyme,is thought to function in the conductionof protonsacrossthemembrane.First clonedin mammals(68), this subunit hasalsobeenclonedinK. daigremontiana (DM Bartholomew,M Bettey, FM Dewey, JAC Smith,personalcommunication),M. crystallinum (MS Tsiantis, DM Bartholomew,JAC Smith, submitted for publication),andoat (64). The 16-kDaproteolipidmay alsoact to direct the assemblyof the functionalenzymeby servingasatemplatefor theassemblyof the remainingV0 subunits andthereafterfor theassemblyof the catalytic subunits (111). Asecond 16-kDapolypeptide(M16),distinct from the proteolipid,hasbeenassociatedwith the V0 sectorfrom S.cerevisiae(111). The geneencodesa highly chargedhydrophilic protein ofpredictedmolecularmass13kDathatcopurifieswith theV-ATPase;however,becauseit is not releaseduponcold inactivation of theenzyme,it is thereforeclassified asmembrane associated. Another of theV0 subunitsis Ac45,clonedfrom bovinebrain tissueandshownto encodea putativeglycosylatedmem-braneproteinof 51 kDa (109).Parryet al (89) identifieda V-ATPasesubunitof 52 kDa in Betavulgaris; however,this proteinappearedto be associatedwith theV1 sectorratherthanwith theV0 sectorbecauseit wasreleasedfromthemembranefollowing cold inactivation. Ac115is alsoa glycosylatedputa-tive membraneproteinassociatedwith theV-ATPase(109)andis thoughttoplay a role in the targetingof the enzymeto the appropriateendomembranelocation (70), althoughit may not participatedirectly in the mechanismofaction of the V-ATPase(111). A subunit of this size has beenpositivelyidentified in themembranesectorof the tonoplastV-ATPasefrom Beta(89),anda 115-kDapolypeptidewasresolvedfrom FPLC-purifiedV-ATPase frombarley(46). However,thepurified oatV-ATPasedoesnot appearto containasubunitof this size (113).

ISOFORMS Isoformsof theV-ATPasesubunits aresuggestedon thebasisofseverallinesof evidence.Thefirst is thevariationin subunitcompositionamongandwithin species.The secondis the presenceof multigenefamilies for thedifferentsubunits.Plantshavebeenshownto haveisoformsfor severalof the


differentsubunitsof theV-ATPase.Oathasbeenshownto have a familyof atleastfour genesencodingthe16-kDaproteolipidsubunit (64). Isoformsof theA subunitof theV-ATPasealsoseemto bepresentin plants.In carrot,genomicfragmentsrepresentingthreedifferentgenesfor theA subunithavebeenclonedandsequenced(51),andin tobaccouptofourgenesencodingthe70-kDasubunithavebeendetected(79). Isoformshavebeenshownfor the B subunitof theV-ATPaseas well. In barley, two different clonesfor the B subunit wereidentified (15). The presenceof multigenefamilies could suggestthat whilesomegenesfor theV-ATPasearepresentashousekeepinggenes,othersmaybeundertissueor cell-specificcontrolmechanisms or mayrespondto specificdevelopmentalorenvironmentalcues,whichalloweachsubunittobeamplifiedor suppressedas required.

REVERSIBILIT Y OF THE V-ATPASE One of the propertiesof the V-ATPasefundamentalto our understandingof its role in thephysiology of theplantcellis its couplingratio (molesof H+ transportedper molesof ATP hydrolyzed).Thecouplingratioandreversibility of theplantV-ATPasehasbeendetermined(41). Davieset al (41) demonstratedthat the coupling ratio of the V-ATPasewasnotafixedparameterbutonethatdependednotonlyonthepHatbothsidesof the tonoplastbut alsoon the absolutepH differenceacrossthemembrane.Thus,in conditionssimilar to thosefoundin vivo with vacuolarpH (pHv) = 4.8andcytoplasmic pH(pHc) = 7.6,acouplingratioof 3wascalculated.DeviationsfromthisvaluewereobservedwhenthepHwasmademoreacidicateitherside.Thevalueof thecouplingratio decreasedto 2 whenthepH differenceacrossthetonoplastwassetat3.68pH units(pHv = 4.32andpHc = 8.0)anddecreasedfurther to 1.75by increasingthepH differenceoneadditionalpH unit (pHv =3.26).The reversibilityof the V-ATPase was demonstratedunderthe presenceof ADP andPi, with a gradientof 2.8pH unitsacrossthetonoplastof redbeet(41).Undertheseconditionsandemployingthepatch-clamptechniquetowholevacuoles,an inward-directedandbafilomycin A1–inhibitablecurrentwasre-cordedbetween±90 mV. Possibleinterferencefrom the vacuolarpyrophos-phatase(V-PPase)waspreventedby employing K+-freesolutions.Theresultspresentedby Daviesetal (41)demonstratedthepartialuncouplingof thepumpthroughchangesin pH at both sidesof the tonoplastandhelp to explainthecapacity of theV-ATPase in establishing differentvacuolar pH values.

REGULATION OF THE V-ATPASE In plantcellsaccumulating NaCl, thedrivingforcefor secondaryactiveNa+ transportinto thevacuoleisprovidedbythePMFgenerated across thetonoplastby the two H+ pumps, the V-ATPaseandV-PPase.This suggeststhat the V-ATPasemay play a fundamentalrole inenergizingNa+/H+ antiportactivity incellsaccumulating significant quantitiesof NaCl.


Severalgroupshavestudiedtheregulationof V-ATPaseactivity andlevelsof expressionof subunits for the enzymeduring growth of the halophyteM.crystallinum in NaCl. Measurementsof both V-ATPaseH+-transportactivityandATP hydrolytic activity weretwofold higherin tonoplastvesiclesisolatedfrom the leavesof salt-treatedplants(200 mM NaCl) when comparedwithmeasurementsof activity in controlplantsof the sameage(13). Bremberger etal (27) andRockelet al (99) alsoreportedincreasesin hydrolytic activity forthe V-ATPasefrom leavesof salt-treatedM. crystallinum plants basedoncomparisonswith controlplants.At day23 of salt treatment,the increasewashighestin plantstreatedwith 400 mM NaCl and was seento increasewithrespectto NaCl-treatmentconcentration(99). Evidencehas beenpresentedcorrelatingthis increasein V-ATPaseactivity in salt-treatedM. crystallinumwith alterationsin theamountandsubunitcomposition of theenzyme(28,94,99). Two polypeptidesof molecularmass31 kDa and27 kDa wereshowntobe inducedwith salt stressin preparationsof the purified V-ATPaseenzymefrom M. crystallinum (28, 94). However,thoughthesepolypeptidescross-re-actedwith a K. daigremontiana antibodyagainstthe V-ATPaseholoenzyme,and were immunoprecipitatedwith the holoenzymeusing antiserumagainsttheV-ATPaseA subunitfrom M. crystallinum(94), thepossibility that thesesubunits may represent degradation products of higher molecular masssubunitscannotbe ruled out. Changesin composition of the V-ATPasehavealsobeenimplied on thebasisof studiesof freeze-fracturecarbonreplicasoftonoplast,revealingthe enlargementof intramembranousparticlesputativelyassociatedwith the V0 sectorof the V-ATPaseand leading the authorstosuggest anincrease inparticlesizewith progressivesalttreatmentof theplants(99).

V-ATPaseactivity hasalsobeenstudiedin several speciesfollowing short-termexposureof rootsto NaCl (74, 78) andin salt-adaptedcell lines (79, 96,124).Matsumoto & Chung (74)reportedadoublingof theH+-transport rateofthe V-ATPasefollowing a3-day treatment ofbarley roots with 200 mM NaCl.Theprotein synthesis inhibitors, cycloheximideandantimycin D, inhibitedthesalt-inducedincreasein V-ATPaseactivity, suggesting that exposureof thebarley roots to NaCl inducedthe synthesisof V-ATPasesubunits,thus ac-countingfor the increasedtransportactivity (74). Nakamuraet al (78), whomeasuredV-ATPasehydrolytic activity in salt-treatedmungbeanrootsdem-onstrateda 1.4- and1.3-fold increasein V-ATPaseactivity following 3- and12-h exposureto 100 mM NaCl, respectively.This rapid induction led theauthorsto suggestposttranslationalmodifications oftheV-ATPaseratherthaninductionof proteinsynthesis,in contrastwith theresultspresentedfor barleyroots. Enhanced V-ATPase hydrolytic activity has alsobeen observed inNaCl-adaptedcellsof Acer pseudoplatanus(124). Nitrate-sensitive V-ATPasespecificactivity wastwofold higherin microsomal membranesisolatedfrom


salt-growncells (80 mM NaCl) at thebeginningof thestationaryphasewhencomparedwith activities measuredin the unadaptedcell line at the samegrowthstage.In tobaccocell suspensionculturesReuvenietal (97)studiedthehydrolytic andH+-transportactivitiesof theV-ATPasein unadaptedvs NaCl-adaptedcells grown in the presenceof 428 mM NaCl. To overcomediffer-encesin purity of tonoplastfractions,Reuveniet al normalizedresultsto thelevel of expressionof the70-kDacatalyticsubunit. Usingpolyclonalantibod-ies againstthe red beet70-kDasubunit, they determinedthat the amountofimmunodetectedproteinin the tonoplastfraction from NaCl-adaptedtobaccocells was fourfold less than in unadaptedcells. Consequently, the relativeH+-transportcapacityand ATPhydrolytic activity, perunit of 70-kDasubunit,from NaCl adaptedcells was threeto four times greaterthan that obtainedfrom unadaptedcells.However,to obtain50%inhibition of transportactivityin the NaCl-adaptedcells a greaterconcentration(1.6 times)wasrequiredof70-kDaantibody serumproteinthanwasrequiredfor 50%inhibition of trans-port activity in the unadaptedcells (97), suggesting that the antibodyhad areducedaffinity to the70-kDasubunitin thesetobaccocells.Therefore,nor-malization of the transportactivities to the amountof 70-kDa polypeptideusing this antibody would lead to possiblemisinterpretationof the results.Beforeconclusions canbe drawn,it will be necessaryto reexaminethe nor-malizationprocedure.Regulation by NaCl of mRNA levels for the 70-kDaV-ATPasehasalsobeenstudiedin tobaccocells(80).ThemRNA levelsfromunadaptedcells andadaptedcells grown continually in the presenceof NaCl(428 mM) showedno significantdifference.However,the amountof immu-nodetected70-kDa polypeptidewas fourfold less in the adaptedcells (97).Whenadaptedcells grown in the absenceof NaCl wereexposedto NaCl for24 h,the levelsof the70-kDa polypeptide mRNAincreased 2.3timesover thecontrol levels, but only when treatedcells were in the midlinear stageofgrowth (80). This wasattributed to the enhancedability of the adaptedcells,upon reexposureto salt, to regulate mechanisms necessary forgrowthin salinemedium.

VacuolarPyrophosphatase

TheV-PPasebelongsto a fourth categoryof primary ion translocasesdistinctfrom the F-, P-, andV-type H+-ATPases(101). Like the V-ATPase, theV-PPasecatalyzeselectrogenictonoplastH+ translocation. However,unlikethe V-ATPase,the V-PPasederivesenergyfrom the hydrolysis of PPi andappearsto bepresentonly in plantsandphototrophicbacteria(seesectiononRelationship to OtherPPases).Sincethe isolationandsequencingof the firstcDNA clone for the 64-kDa to 67-kDa substrate-binding subunit from A.thaliana (101), work on the structureand function of the V-PPasehasad-vancedrapidly. cDNA sequencesfor the substrate-binding subunitof the


enzymehavenowbeenclonedin Hordeumvulgare(115)andB. vulgaris(61).In addition,a partialN-terminalaminoacidsequencehasbeenobtainedfromVignaradiata (67). In B. vulgaris,cDNA sequenceanalysisandSouthernandNorthern blotanalysisindicatedmultiple genes encoding thissubunit,andtwodifferent cDNA cloneswereobtained(61). GenomicSouthernanalysisof A.thaliana indicatedthepresenceof only a singlecopyof thegene(101).Fromthe plant sequencescloned to date, it appearsthat the V-PPasecatalyticsubunitis highly conserved.The barleysequenceshowed86% homology attheaminoacid level to thededucedsequencefrom A. thaliana (115),andthetwo B. vulgaris sequencesexhibited 89% identity with eachother and 89%identitywith the correspondingpolypeptide fromA. thaliana (61).

Evidencefor a catalyticfunctionof the64–67kDa V-PPasesubunitanditsparticipationin substratebinding hasbeenimpliedby thekinetics ofinhibitionand labeling by the sulfhydryl reagent[14C]-NEM (95). Inhibition studiesdemonstrated that NEM needed only to modify, via alkylation, a singlecysteineresidue,presumedto be theMg2+ + PPi-bindingsite, to irreversiblyinactivatethe V-PPase(95). To identify the implicatedcysteine,the 64–67kDa subunitwas specifically labeled with [14C]-NEM, purified, and sub-sequentlydigestedwith V8 protease.From the generatedprotein fragmentsonly a single labeledbandof 14 kDa wasobtained(121).Themicrosequenceof this bandalignedto the carboxy-terminalsegmentof the deducedaminoacid sequencefor the 64–67kDa subunit.Within this region only a singlecysteine(Cys634) was conservedamongthe known plant sequences,whichsuggestedthat alkylation of this specificresiduewasresponsible for enzymeinactivation. Moreover,the location of Cys634, on hydrophilic loop X, indi-catedthat this loop was orientatedto the cytoplasmic face of the tonoplast(121).Theseresultsindirectly implicatedthe 64–67kDa subunitasthe cata-lytic subunitof the enzyme;however,the heterologousexpressionof the A.thaliana V-PPasesubunit in S. cerevisiaeclearly establishedits function inbothPPi hydrolysisandH+ transport(59).

HETEROLOGOUS EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF THE V-

PPASE The heterologouslyexpressed64–67 kDa subunit showedidenticalcharacteristicsto the nativeplantV-PPasewith respectto PPi hydrolysis, H+

translocation,K+ andCa2+ regulation,andinhibitor sensitivity (59),presentingunequivocalevidencethatthecatalyticfunctionsof thenativeV-PPasecanbedirectly explainedby thefunctioning of the64–67kDa subunit.Theseresultsinvalidated the proposalthat putative subunitsof 21 and 20 kDa may benecessaryfor theH+ translocation propertiesof theenzymeasobservedfor thereconstituted64–67kDa subunit(29).

The demonstratedfacility of heterologousexpressionof the V-PPaseinyeast,togetherwith theevidencethat theexpressedenzymefunctionedasthe


native plant enzyme,openedthe way for site-directedmutagenesis studiesaimedat understandingthe structureandfunction of the V-PPase.Kim et al(60) individually mutatedthe 9 Cys residuesconservedamongthe knownplantV-PPase sequencesto determinetherole of these aminoacidsin enzymeinactivationby NEM and substrate binding. MutationswereCys toSer,allow-ing for chargeconservation,or Cys to Ala, maintaining the size of the sidegroup (60). With the exceptionof Cys634, the previously identified NEMreactiveresidue(121), the other eight mutations had no effect on enzymeactivity. Mutationsin Cys634 to eitherSeror Ala resultedin anNEM-insensi-tive enzyme(60). The Cys634 NEM-insensitive mutantwas still capableofbothPPihydrolysisandH+ translocation. This indicatedthatthesitefor NEMinhibition is not the substratebinding site as previouslypostulated (95) andsuggestedthat the inhibitory action of NEM and relatedcompoundson theV-PPaseis dueto a conformational changein the substratebinding site (60).Indicationsof a possible substratebinding site havecomefrom site-directedmutagenesisstudiesandanalysisof alignmentsfor all the sequencedsolublePPases(39) with the A. thaliana and B. vulgaris sequences(61). Althoughdemonstrating litt le overall sequencehomology, a singlemotif of acidic andbasic residues,D or E(X)7KXE, appearsto be conservedbetweenthe V-PPasesand amongthe soluble PPases(61) and may participatein substratebinding.

RELATIONSHIP TO OTHERPPASES Although exhibiting no apparent relation-ship to thesoluble PPases, theV-PPase doesappear to share common featureswith thePPisynthasefromthepurple,nonsulfurbacteriumRhodospirillum (95).It hasbeenproposedthattheseenergyconserving/transducingPPasesbelongtothe samecategoryof ion-translocase(120). Both showedthe sameinhibitorsensitivity profile to the pyrophosphate analogs 1,1-diphosphonates,with ami-nomethylenediphosphonatein particular being a highly specific and potentcompetitive inhibitor of both enzymes(14, 120). In addition,both enzymesappearto be madeup of a single immunologically cross-reactivesubunitofsimilar molecularweight, andSouthernanalysisof Rhodospirillum genomicDNA also demonstratedsequencesimilarities to the Arabidopsis and Betasequences (95).

ROLE OF THE V-PPASE Severalrolesfor theV-PPasein plantcellshavebeensuggested(95). The enzymemay function asan energyconservationsystemthroughtheestablishmentof a pH gradientacrossthetonoplastthat is utilizedto energizesecondaryactivetransport,andit mayalsofunctionasamechanismfor the regulationof cytosolic pH. The role of theV-PPaseasa K+ pumpingmechanisminto thevacuolehasalsobeendefined(42),andtheimplicationsforthis enzyme inturgorregulationhave been postulated (95).


REGULATION OF THEV-PPASE Evidencefor differentisoformsof theV-PPasemaybeanindicationof differentialregulationof thisenzymeby developmentalor tissue-specificcues or of enzyme regulationby environmentalfactors.Althoughprovidingnecessaryinsight into thephysiological importanceof thePPasein thetonoplastof plantcells,few studieshaveconcentratedonthis fieldof research.In B. vulgaris,Northernanalysisof a wide rangeof tissuesfromdifferent developmentalstagesusing sequence-specificprobesfor the twoV-PPaseisoformsindicatedthatsteadystatelevelsof expressionof theisoformswerethesame(61). However,astheauthorssuggested,morecomprehensivestudiesemploying techniqueswith highersensitivity andresolutionarerequiredto determinedifferentialexpressionof thesetwo Betaisoforms.

Severalstudieshavefocusedon the regulationof the V-PPaseactivity bygrowth in NaCl. In contrastwith the generalsodium-induced increase inV-ATPaseactivity, thereappearsto be a decreasein V-PPaseactivity withexposureto NaCl (28, 74, 78). The V-PPasehydrolytic activity in tonoplast-enrichedmicrosomesfrom barleyrootstreatedwith 200mM NaCl for 3 dayswas only50% ofthe activityseen in thecontrolroots(74). InM. crystallinum,theactivity of theV-PPasein thetonoplastfrom salt-treatedplants(400mM)waslower thanin thenontreatedplantsat all stagesof treatment,andin bothcontrol andsalt-treatedplantsthe V-PPaseactivity decreasedwith plant age(28). Mung beanV-PPaseactivity was severelyinhibited when mung rootswereexposedto NaCl (100 mM) (78). Moreover,this inhibition of enzymeactivity wasalsoobservedon isolatedtonoplastvesiclesupontheaddition ofNaCl to the reactionmedium.From theseresults,it was concludedthat theinhibitory effectsof Na+ weredirectly on theV-PPase,which accountsfor thedecreasein activity following exposureto NaCl andaccumulationof this ionby theplant(78).

Evidencefor exceptionsto this Na+-induceddecreasein V-PPaseactivityhas beenpresented(38, 124). In NaCl-adaptedcells of A. pseudoplatanus,both theV-ATPaseandV-PPaseactivitieswerehigherthanin theunadaptedcell line, with theV-PPaseactivity increasingby 100%in thecellsadaptedto80 mM NaCl (124).Unadaptedcellsof Daucuscarotagrown in thepresenceof 50 mM NaCl for 10 daysalsoshoweda doubling of V-PPase activity whencomparedwith theactivity of thecontrolcells(38).Thediscrepancyin resultsbetweenstudiesmaybedueto differencesin theNa+ concentrations employedor maysimplybedue tospecies differences.

Regulation of the V-PPaseby environmentalfactorsother thanNaCl hasalsobeeninvestigated.Conditionsof anoxiahavebeendemonstratedto resultin anincreasedV-PPasespecificactivity of up to 75-fold in tonoplastvesiclesisolatedfrom rice seedlingsgrown in the presenceof nitrogen(31). V-PPaselevelsdecreasedto control valuesfollowing the returnof theseedlingsto air.This anoxic-inducedincreasein V-PPasespecific activity was mirrored by


increasesin transcriptand proteinlevels(31).During anoxia,levelsof cellularATP were severelyreducedwhile levels of PPi showedno variation (95).Therefore,upregulationof theV-PPaseduringanoxiawould function to con-servelevelsof ATP andmaintaintonoplastenergizationandcytoplasmicpHhomeostasis, essentialrequirementsfor survival of the plantsunderoxygenstress (31,95).

Regulation of V-PPaseactivity would alsobeessentialunderconditionsofchilling stresswhere,concomitantwith reducedlevelsof cellularATP, thereiscold-inactivation of the V-ATPase(95). Rice seedlingsexposedto tempera-tures of 10°C for severaldays showedincreasesin both V-PPase-specificactivity (20-fold increase) and amountof immunoreactive enzyme (31).

ION COTRANSPORTERS

Na+/H+ Antiport

The presenceof a tonoplastNa+/H+ antiporter,or exchanger,involved invacuolarNa+ sequestrationhasbeenwell documentedin severalplantspecies(25, 48, 52, 108,andreviewedin 10). Secondaryactivetransportof Na+ intothe vacuolevia the tonoplast Na+/H+ antiporterwould be energizedby thePMF generatedby the activity of the V-ATPaseand/or the V-PPase.As afundamentalmechanismin salt tolerance,anactiveantiportwould functiontosequesterNa+ into thevacuole,which resultsin avoidance ofcytoplasmicNa+

toxicity and maintenanceof a high cytoplasmicK+/Na+ ratio. In parallel,vacuolarNa+ would serveasanosmoticumnecessaryfor cellularH2O homeo-stasis.Evidencefor aroleof thetonoplastNa+/H+ antiport insalttolerancehasbeenprovidedfrom severalstudiesdemonstrating induction of antiportactiv-ity uponexposureto NaCl. In sugarbeetcell suspensions, increasesin consti-tutiveantiportactivity wereobservedupon growth of cells inNaCl (25) andinbarley roots; althoughNa+/H+ antiport activity was not detectedin plantsgrown in theabsenceof NaCl, antiportactivity wasrapidly inducedin plantstreatedwith NaCl (48). Furtherevidencewasprovidedby comparisonof twoPlantago speciesshowing markeddifferencesin their sensitivity to NaCl.Studies revealedthat tonoplastNa+/H+ antiportactivity couldbedetected onlyin tonoplastvesiclesof the salt-tolerantP. maritima but not in the salt-sensi-tive P. media(108).

More recently,evidencefor a Na+/H+ antiport hasbeenpresentedin thehalophyteM. crystallinum(13).Whenplantsof M. crystallinumwereexposedto salt for severalweeks,measurementsof bulk tissueconcentrationsof NaClwereashigh as1.0 M, andin the largeepidermalbladdercells thatcovertheshootsurface,Na+ concentrationswereshownto exceedthis value(1). Thedemonstratedefficiency of Na+ sequestration in this plant presentsit as a


modelsystemwith which to studythemechanismemployedfor vacuolarNa+

accumulation. Electroneutral Na+/H+ antiport activi ty was detected intonoplastvesiclesisolatedfrom leavesof both control and salt-treated(200mM) M. crystallinumplants(13) and indicatesconstitutive levelsof expres-sion and activity of this protein in agreementwith resultspresentedfor cellsuspensionculturesof B. vulgaris (25). Initial ratesof Na+/H+ exchangewere2.1 times higher from vesiclesof salt-treatedplantscomparedwith vesiclesfrom control plants andindicated aspecific induction of antiport activityfollowing salt treatment.As demonstratedfor the Na+/H+ antiport from B.vulgaris (23), the M. crystallinum antiport was inhibited by amiloride (13).TheNaCl-inducedincreasein Na+/H+ antiportactivity in M. crystallinumwasclosely correlatedwith an increasein V-ATPaseactivity in the salt-treatedplantsandimplicatedtheV-ATPasein energizingvacuolarNa+ accumulationvia theNa+/H+ antiport.

Although biochemicalstudiesaimedat the identification of the tonoplastNa+/H+ antiport have been carried out (10, 12)and a170-kDa tonoplastpolypeptide hasbeenassociatedwith the antiporter (11), no recentadvanceshave been madein this field.

Ca2+/H+ Antiport

The accumulationof vacuolarCa2+ in plants is now well recognizedto beexplainedby the activity of a tonoplastCa2+/H+ antiport(24, 104,105),andpreliminaryresultson thereconstitution of this exchangerhavebeenreported(107). The stoichiometry of the Ca2+/H+ antiport hasbeensuggestedto beequalto 3 andto bestimulatedby vacuolarpositive potentials, leadingto thethermodynamically possible accumulationof Ca2+ inside the vacuoles(19).PharmacologicalstudieshavedemonstratedthattheCa2+/H+ antiportis inhib-ited by severalagentsknownto affect otherCa2+ transportersincludingruthe-nium red,verapamil,La3+, Cd2+, andotherdivalentcations(33, 34). In viewof thesereportsit is interestingto bring into contexttheresultsconcerningthepresenceof a Cd2+/H+ antiport in the tonoplast of oat roots (100). Salt &Wagner(100)demonstratedtheuptakeof Cd2+ by tonoplast-enrichedvesiclesfrom oatrootsenergizedeitherby ATP or by K+/nigericin. Initial ratesof Cd2+

accumulationshowedsaturationkineticswith a Km = 5.5µM anda Vmax= 14nmolmg protein-1 min-1. Theseresults led the authorsto suggestadetoxificat-ing role for the Cd2+/H+ antiport by which Cd2+ is removedfrom the cyto-plasmto preventits potentialtoxic reactionwith -SH-containingcompounds(116).However,it is possible thattheactivity of theCd2+/H+ antiportmaybethe sameas thatfor the Ca2+/H+ antiport, as indicatedby the results ofChanson,who demonstratedthe inhibition of the Ca2+/H+ antiport by Cd2+

(34). This view was also suggestedby Salt & Wagner,who recordedtheactivity of a Ca2+/H+ antiportin theoat tonoplast vesicles(100).


ION CHANNELS

The third categoryof ion transporterspresentin the tonoplastcorrespondstothe ion channelsthatmediatethemovementof ionsdown their electrochemi-cal potentialgradient.

Slow-ActivatingChannels

The slow-activating (SV) channelswere the first channelsdescribedin thetonoplastof plantcells (53) andarepresentin all speciesstudied.Thedistin-guishing characteristicsof theSV channelsareaslowactivationtimeconstant,a markedoutwardrectificationat positive tonoplastpotentials1, activationbycytoplasmic Ca2+ (Ca2+

c) at levelshigherthan10-6 M, anda low selectivityfor monovalentcations.However,becausethesechannelsaremainly openatnonphysiological positivetonoplastpotentials, their role in thephysiology ofplantcells is not clear,although attempts havebeenmadeto assigna functionto thesechannels.Lowering pHv from 7.2 to 5.5 had no effect on the SVchannelactivity from onionguardcells (7). However,acidicpHv reducedtheSV activity from Vicia faba guardcells without affectingthe singlechannelconductance(103).DecreasingpHc alsoresultedin a diminishedactivity of V.faba vacuolarSV channelswith a pKd of 6.8, with no changein the single-channel conductance(103).

In order to gain more information on the role of tonoplastSV channels,their regulationby othercytoplasmic factorshasbeenaddressed.The regula-tion of SV channelsby calmodulin (CaM) hasbeendemonstrated(118) andappearsto be dependentuponCa2+

c. Although additionof CaM in the pres-enceof 0.1 mM Ca2+

c did not increasethevacuolarcurrentsof V. fabaguardcells (103), CaM stimulated the SV currentsfrom storageprotein vacuolesfrom barleyaleurone(18),but only at Ca2+

c below10-5 M. This indicatesthatSV channelsmay be regulatedby the associationCa2+-CaM at Ca2+

c below10-5 M, a mechanismthat may be overriddenby Ca2+

c above10-5 M. TheCaM antagonists [N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide] (W7),trifluoperazine,andcalmidazolium directly inhibited the SV channelsby af-fectingtheirgatingmechanism without changingthechannelconductance(18,103). In vacuolesfrom barleyaleuronea partial recoveryof the SV currentswasobserveduponthe additionof 3.5 µM CaM in the presenceof W7 (18),leadingthe authorsto conclude thatthestimulationof vacuolarSV channels isthroughtonoplast-associated CaMactivatedby Ca2+

c.


1The directionof thecurrentsandthe tonoplastvoltageareaccording to Bertl etal (17).

Changesin thelevelsof cytoplasmic chloride(Cl-c) havealsobeenreportedto regulateSV channelactivity. In vacuolesfrom sugarbeet cell cultures,decreasinglevelsof Cl-c reducedtheactivity of theSV channels,with a linearrelationshipbetweenwholevacuolecurrentandCl-c from 10 to 100mM (86).Studiesat the single-channellevel demonstratedthat the effect of decreasingCl-c wasa reductionin the meanopentime andthe possible induction of anadditionalclosedstate,with no effecton thesingle-channelconductance(86).Similar resultswerereportedfor theSV channelsof V. fabaguardcells(103),althoughtheseauthorsconcludedthat the reductionin the magnitude of thecurrentswasdueto blockingof theSV channelsby gluconate.If this conclu-sion were correct,the reductionin the SV channelcurrentsseenwith otheranionsemployedassubstitutesfor Cl- (86) would alsohaveto be dueto theblockage ofthe SV channels.

Regulation of theredbeettaprootvacuolarSV channelsby Mg2+c hasalso

beenreported(43). The induction of SV-like currentswasobservedin vacu-oles isolated in a Mg2+-free medium by including 0.4 mM Mg2+ on thecytoplasmic side.When thevacuoleswereisolatedin amediumcontaining0.4mM Mg2+, raisingtheconcentrationof Mg2+ to 2.4 mM resultedin a furtherincreasein magnitudeof the SV currents,an effect that wasgreatestafter20min (43). However, theseresultsdiffer from early reportswhere only theactivity of FV channelswasrecordedat low Ca2+

c in thepresenceof 1–2mMMg2+

c (54,66, 87).Why theSV channelswere stimulatedby Mg2+c under the

conditionsreportedby Davies& Sanders(43) is not clear,but it is possiblethat isolation of thevacuolesin a Mg2+-freeor Mg2+-low mediummaybeofrelevance.

Fast-Activating Channels

The fast-activating(FV) channelsin plant vacuoleswere first identified byHedrich & Neher(54). Thesechannelsare active at physiological levels ofCa2+

c, arevoltageindependent,andhavea selectivity of 6:1 betweenK+ andCl-. Similar characteristicshavebeenreportedfor the vacuolesof sugarbeetcell cultures(87), V. faba guardcells (117) andred beetstoragetissue(43).The FV channelsfrom beet were as selectivefor K+ as for Na+, with apermeabilityratio Pc+/PCl- of 7. A higherselectivity wasreportedfor the FVchannelsfrom guardcellswith a permeability sequenceK+ > Rb+ > NH4

+ >>Cs+ ≈ Na+ ≈ Li+ (117).The FV channelsfrom guardcell vacuoleswerealsostimulatedby small increasesin Ca2+

c. Recordingsof FV channelactivity atthe whole vacuoleandsingle channellevel suggestthat thesechannelsmayfunction inthereleaseanduptakeof K+ during cellular osmoregulation, andinthe particularcaseof guardcells during the openingandclosing of stomata(117). Insight into the role of theFV channelshasbeenobtainedby studyingtheregulationof thesechannelsby cytoplasmic factors.Davies& Sanders(43)


havereportedthestimulationof thevacuolarconductancein redbeetthroughFV channelsby cytoplasmic ATP or ATPγS, in theabsenceof eitherCa2+

c orMg2+

c, indicatingthiseffectwasindependentof proteinphosphorylation.ATPwasalsoreportedto stimulate the movementof argininethroughthesechan-nels (43). Regulation of the FV channelsby pH, independentof ATP, wassuggestedby thestimulationof the FV channelsby increasing pHc from 7.3 to8.0 (43). However,inhibition of FV channelsby increasingpHc wasreportedfor vacuolesof V. faba guardcells (117), suggesting that regulationof FVchannelsby pHC maybe tissueor speciesspecific.Furtherstimulation of redbeet FV channelswas observedwith Mg2+

c between0.4 and 2.4 mM inconditionsof low Ca2+

c (43). Regulationof red beetFV channelsby ATP,however,must be takenwith carein view of the resultsthat were obtainedundermore physiologicalconditions. Davies& Sandersshowedthatwith pHVsetbetween5.5 and4.3, stimulation of the inward FV channelsby ATP wasprevented(43).Thus,regulationof theFV channelsby physiological vacuolarpH may suppressthe effectsof cytoplasmic ATP. In spite of this, the rolesascribedto the FV channelsare convincing (43). It is proposedthat thesechannelsserve as the shunt conductancefor the V-ATPase, allowingthereleaseof positive chargesfrom the vacuolarinterior facilitating the estab-lishmentof a ∆pH. The FV channelscould alsofunction asa mechanismforthe releaseof K+

v into thecytoplasmunderconditionsof low K+ availabilityandcould thusplay an important role in K+

c homeostasis(43). An additionalrole for the FV channelswas proposedby Ward & Schroeder(117) whosuggestedthe involvement of thesechannelsin tonoplastdepolarization(seenext section).

Ca2+-SelectiveChannelsIt is now well establishedthat Ca2+

c playsan important role in stimulus-re-sponsecouplingin plants.Someof thestimulus-responsecouplingprocessesin which changesin Ca2+

c havebeenimplicatedare thoseinducedby plantgrowth regulators(76), touch and cold (62), as well as in the regulationofprotein kinases(20) and ion channels(54, 102). Becauseof the size of thevacuoleandthe concentrationof Ca2+ within, it is arguedthat this organelleservesasthe sourcefor the increasesin Ca2+

c (58). Two classesof tonoplastCa2+-selectivechannelshavebeendescribedto date:voltage-dependentchan-nels andagonist-activatedchannels.

VOLTAGE-DEPENDENT Ca2+ CHANNELS Thefirst evidencefor theexistenceofCa2+-selectivechannelsin plantswasreportedin the tonoplastof sugarbeetemployingBa2+asthechargecarrier(88).Underbi-ionicconditionswith Ba2+

cand K+

v as the chargecarriers,Pantojaet al (88) reportedthe activationofputativeoutwardCa2+ channelswith a permeability ratio of 5 to 7.Thesecurrents,observedat thewholevacuoleandisolatedpatchlevel,showed

P /PBa K2 ++


a conductanceof 40 pSwith 100 mM Ba2+c, andwereinhibitedby La3+ and

verapamil,two Ca2+ channelblockersidentified in animal cells. A furthercharacteristicof thesecurrentswas the saturationobservedwith increasingBa2+

c with adissociationconstant(Kd) = 16mM (88).Similarchannelactivitywasobtained withvacuolesof redbeetandtobacco.Undercomparableexperi-mentalconditions,Pingetal (90) recordedanoutwarddirectedchannelwith aconductanceof 30 pSwith 50 mM Ba2+

v, which alsocarriedSr2+. Employingexperimentalconditionsamenablefor the detectionof inward Ca2+ channels,Pinget al failed to recordanychannelactivity (90).Similar outwardCa2+-se-lectivechannelshavebeenreportedfor thetonoplastof guardcellsfromV. faba(103,117).EmployingsymmetricalCaCl2, SV-likecurrentswererecordedwithwholevacuolesandisolatedchannels.Thesecurrentsreversedneartheequilib-rium potential for Ca2+ with a calculatedpermeability ratio of 5;single-channelrecordingsshoweda conductanceof 16 pS for thesechannelswith 50 mMCa2+

v (117).Voltage-dependent inward Ca2+ channels have been described for the

tonoplastof red beet taproot (57, 58) and V. faba guard cells (6). Thesechannelsareproposedto functionasareleasemechanismfor Ca2+. Employingisolatedinside-outtonoplastpatcheswith 50 mM K+

c and 5 mM Ca2+v as

charge carriers, researchers found thatnegativepotentialsstimulated the activ-ity of inwardchannelswith a conductanceof 12 pSin redbeet (57,58) and 27pSin V. faba (6), which showedsaturationat 5 mM Ca2+

c. Positive potentialsinducedtheopeningof outwardchannelswith a conductanceof between120and 200 pS (6, 57). Analysis of thesecurrent-voltagerelationships showsaresemblance to theoutward rectificationof the SV channels.The inwardchannels showed a selectivity towards Ca2+ with a permeabil i ty ratio

in therangeof 6 to 20,with theuppervaluecorrespondingto theredbeetchannels.IncreasingCa2+

v between5 and20 mM shiftedthe activationpotential ofthecurrentstowardless-negativepotentialsandcausedaconcomi-tant increasein the open-channelprobability in both species(6, 57). Regula-tion of the Ca2+-releasechannelswas effectedby pHv. Underphysiologicallevelsof pHV, a markeddecreasein theopenprobabilityof thechannelswasobserved withrespect tothatrecorded at pH 7.3(6, 57). Acharacteristic ofthered beettonoplastCa2+ channelswasa “noticeablerundown” in the channelactivity atphysiologicalpHV (58).BothCa2+ channelswereinhibitedby Gd3+

with a half-maximalinhibition (K1/2) of 10–20µM (6, 57). In addition, redbeetchannelswereinsensitive to Ca2+

c, thealkaloidryanodine,inositol 1,4,5-triphosphate(IP3), or heparinbut wereinhibitedby Zn3+ (57), whereastheV.faba channelswere reversibly blocked by the dihydropyridine, nifedipine,with a K1/2 of 77µM (6).

Allen & Sanders(6) alsoreportedthepresence ofa secondtypeof voltage-dependentCa2+ releasechannelin the tonoplastof guardcells.This channel

P /PCa K2 ++

P /PCa K2 ++


hadasinglechannelconductanceof 14pS,apermeability ratio of 4,andwasfoundonly in 8% of thepatchesanalyzed. Thevoltagedependence ofthe openprobability was similar to the 27 pS channel.The presenceof twoCa2+-releasechannelsin the tonoplastof guardcellswassuggestedto conferthecapacityto respondto a varietyof stimuli knownto controlthephysiologyof the guardcells (6).

Permeabilityof the redbeetinwardvacuolarCa2+ channeltowardK+ wasdemonstratedby Johanneset al (58). Using K+ as the only chargecarrier,alinear current-voltagerelationshipwasobtained.Upon elevatingCa2+

v a de-creasein the single-channelcurrentat negativepotentials with half-maximalinhibition at 0.3mM Ca2+

v wasobserved(58).This resultsuggeststhatCa2+v

may be blocking the channels atnegative tonoplastpotentialsand thus causingan outward rectification similar to that observedfor the SV channels(seesectiononSlow-ActivatingChannels).This inhibition by Ca2+

v resemblesthatobservedfor the inwardrectificationcausedby Mg2+

c in cardiacK+ channels(72, 73). Johanneset al (58) also reportedthat Ca2+

v was requiredfor thegatingof the inward Ca2+ channels.At nonphysiological levelsof Ca2+

v (30µM), channelopeningswereslowandrecordedonly atverynegativetonoplastpotentials,with increasedCa2+

v causinga shift in the activation potentialtowardpositive potentialsaswell asa fasteractivation(58). However,obser-vations on the effects of Ca2+

v on the single-channelcurrent andgatingmechanismof the Ca2+ channels—withK+ asthe chargecarrier—wereonlyobtainedat thesingle-channellevel, makingdirectcomparisonwith thevacuo-lar SV channels difficult.

Theresultsreportedfor vacuolarCa2+ channelsleadusto suggestthat mostif not all ofthese activitiescould correspond tothefunctioningof SV channelsin thetonoplast.Evidencefor this is bestdemonstratedby theresultsof Ward& Schroeder(117)andSchulz-Lessdorf& Hedrich(103).Ward& Schroederhaveproposeda modelby which Ca2+-inducedCa2+ releasefrom thevacuolemayoccurthroughthesynchronizedactivity of FV andSV channels(117).Inthis model,increasesin Ca2+

c above10-6 M would activateFV channelsandstimulate the releaseof K+ from the vacuole,following its electrochemicalpotential, andcauseadepolarization ofthetonoplastmembranepotential. Thisdepolarization,togetherwith the increasein Ca2+

c, would in turn activatetheSV channelsand thus causethe releaseof Ca2+ into the cytoplasm. Thevalidity of this proposalrestson the postulatedcapacityof SV channelstorelease Ca2+. For these channelsto beinvolvedin stimulus-response coupling,a tight regulatorymechanismmust exist to preventuncontrolledreleaseofCa2+ to the cytoplasm. One suchmechanismcould be vacuolarpH. AcidicvacuolarpH has beenshown to down-regulatethe channel-mediatedCa2+

release(6, 58). This togetherwith thehigh concentrationratio K+/Ca2+ in thevacuole(around20:1) and the low permeability ratio of the Ca2+

P /PCa K2 ++

P /PCa K2 ++


channelswould reducethe potentially constantreleaseof Ca2+ into thecyto-plasm.

Although theactivity of theCa2+ channelsin the inwarddirectionwasnotdetectedby severalgroups (88, 90), it is possible that the channelswereinactivated or inhibitedby theexperimental conditionsemployed. Pantojaetal(88) did not observeoutwardor inwardcurrentsemploying symmetricalBa2+

solutions, which leadsto the conclusionof a possible inhibition of outwardcurrentsby Ba2+

v and inhibition of inward currentsby Ba2+c. On the other

hand,undersimilar experimentalconditions to thoseemployedby Johannesetal (57, 58)and Allen& Sanders(7), Pingetal (90)alsofailed to recordinwardCa2+ currents,employing Ca2+-gluconatein the cytoplasm, a condition thathas been demonstrated to inactivate SVchannels(85,103).

LIGAND-GATED Ca2+ CHANNELS IP3-gatedchannels Although stimulus-re-sponsecouplingcanbe mediateddirectly by changesin Ca2+

c, this couplingmay require the participation of intermediate signalingmoleculesincludingCaM, IP3, or cyclic ADP ribose(cADPR).Thepresenceof endogenousCaM(8) and IP3 (55) hasbeenreportedin plant cells. However,the presenceofcADPRin plantshasnot beendemonstrated.CaM hasbeenshownto regulatethe activity of Ca2+-ATPasesfrom the plasmamembrane(45, 92, 98) andendoplasmicreticulum(9,49a),aswell astheion channelsin thetonoplast(18,103,118). IP3 on theotherhand,hasbeenshownto causestomatalclosing, aresponsethoughtto be mediatedby increasinglevelsof Ca2+

c (22, 49). Thesourcefor this increasein Ca2+

c hasbeensuggestedto be intracellularpoolsincluding the endoplasmic reticulumand/orthe vacuole(49). Evidencefor apossibleroleof IP3 in therelease ofvacuolarCa2+ hasbeenpresented(30, 91,106).Employing purifiedtonoplastvesiclesfrom redbeet(30),oatroots(106),andisolatedvacuolesfromAcer(91),researchersshowedreleaseof 45Ca2+upontheapplication of IP3. Applying thepatch-clamptechniqueto isolatedvacuolesof red beet,researchershavealsoshownIP3 to directly activateCa2+-releasechannels(2, 3). Attempts by severalinvestigatorsto repeattheseresultshavefailed,whichthrowsinto questiontheexistenceof suchaCa2+

c-releasemecha-nism(35).However,Allen & Sanders(5) recentlyshowedthatadditionof 1µMIP3 to isolatedvacuolesfrom redbeetrootsexposedto 1 mM Ca2+

c and200µM Zn2+—conditions thatshould inhibit SV andFV channels—stimulatedthemagnitudeof theCa2+inwardcurrents.Thereversalpotentialof theIP3-inducedcurrentsindicateda permeabilityratioPC of 200for thesechannels.Interestingly,preplasmolysis of the vacuolesat high osmoticpressures(851mOsmol) was requiredto observethe stimulation of Ca2+ currentsby IP3.Furtherstimulationof thesecurrentswasobservedwhenvacuoles—underthewhole-vacuoleconfiguration—were exposedto hypoosmotic solutions (5).Thus,analmosttwofold increasein themagnitudeof theIP3-inducedcurrents

P /PCa K2 ++


wasrecordedwhenadifferenceof 250mOsmol kg-1 wasestablishedacrossthetonoplast(5).TheIP3-inducedcurrentswereindependentof thelevelsof Ca2+

cbetween0.1 µM and1 mM, andthesingle-channelconductancewasvariablebetween11 and182 pS.Although it wasconcludedthat hyperosmoticstresswasa prerequisiteto recordIP3-inducedcurrents(5), Pinget al (90) failed toobservethe sameresponseemploying similar hyperosmotic conditions forvacuoleisolation. Fromthemanyreportsthathavefailed to detecttheactivityof IP3-inducedCa2+ currents(35) and the particularconditionsnecessarytorecord these channels(3, 5), itis clear thatmore exhaustivework is required.

Cyclic ADP ribose-gatedCa2+ channels The presenceof a secondmecha-nism, independentof IP3, for the releaseof Ca2+ from intracellular sources hasbeen reported in animal cells and is suggested to be associated with theryanodine receptor(16, 47).Reportsby Galioneet al (47) andMészároset al(77)suggestedthattheryanodinereceptorsmaybeunderthecontrolof cADPR.However,it appearsthatcADPRdoesnothaveanyeffectonsomeanimalcells,and direct demonstration that cADPR levelschangein responseto certain stimuliis still lacking(16).Stimulation of Ca2+ releasefrom vacuolesby cADPRhasrecentlybeendemonstratedin plants(4). Addition of cADPR to a red beetmicrosometonoplast-rich preparation,previouslyloadedwith 45Ca2+, inducedthe releaseof 15% of the total 45Ca2+ uptake.This activity was inhibited byrutheniumred,aninhibitorof theryanodinereceptor.Ryanodinealsostimulatedthereleaseof 45Ca2+ andpreventedtheeffectof cADPR,if previouslyadded,indicatingthat thesetwo ligandsbind to a commonreceptoron thetonoplast.Employing isolatedvacuolesandthepatch-clamptechnique,researchersfoundthat addition of cADPR inducedan increase inthe magnitude of inwardinstantaneous currents with a Km of 20–25 nM and a permeabili ty ratio

between9 to 27. Specificity for cADPR wasdemonstratedby theinsensitivity to thenoncyclicanalog,adenosine5′-diphosphoribose. Similartofindingsfrom reportsonanimalcells(47),additionof IP3 increasedthemagni-tude of the control inward currents,and a further rise was inducedwith theaddition ofcADPR(4), indicating that theeffectsof cADPRwereindependentof theIP3-inducedCa2+ release.Althoughthis reportsuggeststhepresenceofasecondsignalingpathwayinvolvedin vacuolarCa2+release,reservationsmustbeconsideredbeforecADPRis acceptedasa plantsecondmessenger.First, itisnecessarytodemonstratethatcADPRandtheenzymeinvolvedin itssynthesisarepresentin plants,andsecond,it is importanttodemonstratethatcytoplasmiclevelsof cADPR are affectedby a specific externalsignal.

Malate-SelectiveChannels

The organicanionmalateis accumulatedin the vacuoleof mostplant cells.The role of malatein the physiologyof plant cells is manifold: asa storage

P /PCa K2 ++


form of CO2, asa chargebalance,andasanosmolyte involvedin themainte-nanceof cell turgor.The transportof malateinto thevacuoleis important forthe regulation of cytoplasmic pH and thecontrol of cellular metabolism,particularly in plantsshowingCrassulaceanacid metabolism(CAM), wherelargefluxes ofmalate occurduring theday/night cycle.Previously, ithadbeenconsideredthat themechanismmediating theaccumulation of malateinto thevacuolewas a carrier (85, 119), and its reconstitution from barley and K.daigremontianavacuoleshasbeenreportedby Martinoiaet al (71) andRata-jczacket al (93), respectively.In both cases,two polypeptidesof molecularmass20–30kDa wereassociatedwith thereconstitutedactivity.

More direct evidenceon the natureof the malate transporterhas beenobtainedby employing thepatch-clamptechnique.UsingCa2+

c below10-7 Mto eliminateSV channelactivity, andemploying potassium malateas themainelectrolyte,the activity of voltage-activated and inward rectifying channelsselectiveto malatein the tonoplastof the CAM plant Graptopetalumpara-guayense(55) and sugarbeet cellcultures (86)has been demonstrated.In bothcasespHc was7.5, indicating thatthedivalentform of theanionis thespeciesmoving throughthechannels.Studiesat thesingle-channellevelwith vacuolesfrom G. paraguayensedemonstratedthat the channelopenprobability wasslightly voltagedependentwith a rundownobservedwithin 10–15min (56).Characterizationat thewhole vacuolelevel in redbeetcell suspensions dem-onstratedthe presenceof slow-activatinginward malatecurrents(87). Theselectivityof theslow-activatingchannelswas6 to 10 timeshigherfor malateoverK+ (87). Malate-selectivechannelshavealsobeenfound in thevacuolesof theCAM plantK. daigremontiana (O Pantoja& JAC Smith,manuscriptinpreparation)andin vacuolesof A. thaliana (32). The whole vacuolecurrentsfrom K. daigremontianaandA. thalianashowedsimilarkinetics to those fromsugarbeet with aclearinward rectification. Varying thelevelsof Ca2+

c hadnoeffect on the malatecurrentsfrom the two species;however,inhibition bycytoplasmic acidification within the physiological rangeof pH 7.0–6.5wasobservedin K. daigremontiana. This independentelectrophysiological evi-dencedemonstrated thattheplausiblemechanismfor vacuolarmalateaccumu-lation is ananionchannelthathastheparticularpropertyof beingselectivetoan organic ion, rather than to inorganic ions. From thesereportson malatechannels,thediversity ofspeciesthathavebeenemployed, andtheimportanceof this organicanionin plant cell physiology, it is temptingto speculatethatplantvacuolar malatechannelsmay be ubiquitous.

Tonoplast Intrinsic Protein

The tonoplastof plant cells alsocontainsa channelselectivelypermeabletowater, the tonoplastintrinsic protein (TIP). Thoroughreviews on this andrelatedmembrane intrinsic proteins (MIP)have appearedrecently(36,37,96).


Therole of γTIP asa waterchannelhasbeendemonstratedthroughheterolo-gousexpressionof the protein in Xenopuslaevis oocytes.Oocytesinjectedwith γTIP mRNA showedrapid swelling and burst within 6 min followingexposureto hypoosmotic solutions. Uninjected or water-injectedoocytesswelled very slowly upon the sametreatment(75). From theseresults,theauthorscalculatedthatγTIP causeda six- toeightfoldincrease in oocytewaterpermeability,similar to resultsobtainedwith CHIP28, a well-characterizedwaterchannelin humanerythrocytes(90a).γTIP, on the otherhand,did notaffecttheoocyte’s glyceroluptakerateor resultin theappearanceof additionalionic currentswhen assayedin Bart’s medium. Herewesuggestthepossibilitythatundertheexperimental conditionsemployed(75),thepotentialpermeabil-ity of γTIP to particularions mayhavebeenoverlooked.For this, two condi-tions needto be considered.The first is that during heterologousexpressionstudiesthe proteinwill insert in the Xenopuslaevisoocytemembranein thesameorientation inwhich it is foundin thetonoplast, i.e.with its vacuolarsidefacing the extracellularsideof the oocyte,and the secondis that γTIP is aninwardrectifying anionchannel,for example,themalatechannel.If thesetwoconditionsare met, the activity of γTIP as a malatechannelwould only berecordedif thisanionwerepresentin theinteriorof theoocyte,aconditionthathasnotbeentested(75).Therefore,it wouldbeinterestingto assayundertheseconditionstheactivity of theheterologousexpressedγTIP. To preventpoten-tial effectsof malateon theoocyte,theemploymentof macropatchescouldbea better approach toassistin resolvingthis concern.

CONCLUDING REMARKS

In the next few yearsit will be important to addressthe molecularcharac-terizationof ion cotransportersandchannelsat the tonoplast in order to ad-vancethe understanding of the role of the vacuolein thephysiology of plantcells.Furtherstudiesaimedat unravelingtheregulationof thetransportersbybiotic andabiotic factorsshouldalsobeemphasized.While researchover thepastfew yearshasenlightenedour understanding of tonoplastion transport,questionsthat remainunansweredinclude:Are the SV channelsresponsiblefor thereleaseof vacuolarCa2+?Is cADPRpresentin plants?Is it regulatedbyexternalstimuli? Is therea Ca2+-ATPasein the tonoplast?Are channelstheonly mechanismby which malateis accumulated?How is malatereleasedfrom thevacuole?Whatis themechanisminvolvedin thetransportof Cl- intothe vacuole?Does the Ca2+/H+ antiport participatein the sequestrationofheavy metals?


ACKNOWLEDGMENTS

We thankall the colleagueswho helpedus by makingavailablereprintsandpreprintsof their work and by respondingso quickly. We also thank Dr.FedericoSánchezfor his encouragementto undertakethis task.We wish todedicate thisto our parents.

Any Annual Reviewchapter, aswell asany arti clecited in an Annual Reviewchapter,may bepurchased fromthe Annual ReviewsPreprints and Reprints service.

1-800-347-8007; 415-259-5017; email: [email protected]

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