1040-2519/97/0601-0399$08.00

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MAURELAQUAPORINSAnnu.Rev. Plant Physiol. Plant Mol. Biol. 1997. 48:399–429Copyright© 1997by AnnualReviewsInc. All rightsreserved

AQUAPORINSAND WATERPERMEABILITY OF PLANTMEMBRANES

Christophe MaurelInstitut desSciences Végétales, CNRS, Avenue de la Terrasse, F-91198 GIF-SUR-YVETTE Cedex, France

KEY WORDS: hydraulic conductivity, lipid bilayer,osmosis,turgor, waterchannel

ABSTRACT

Themechanismsof plantmembranewaterpermeability haveremainedelusiveuntil the recent discovery in bothvacuolarandplasmamembranesof aclassofwater channel proteinsnamedaquaporins. Similar to their animal counterparts,plant aquaporins havesix membrane-spanning domainsandbelong to theMIPsuperfamily of transmembranechannel proteins.Their very high efficiency andselectivity in transportingwater moleculeshavebeenmostly characterizedusingheterologous expression in Xenopus oocytes. However, techniques set up tomeasuretheosmotic waterpermeability of plant membranessuch astranscellu-lar osmosis, pressureprobemeasurements, orstopped-flow spectrophotometryarenow being used to analyze thefunction of plantaquaporins in their nativemembranes.Multiple mechanisms,at the transcriptional and posttranslationallevels,control theexpression and activity of the numerousaquaporin isoformsfoundin plants. Thesestudiessuggestageneral rolefor aquaporinsin regulatingtransmembrane water transport during the growth, development, and stressresponsesof plants. Future research wil l investigate the integrated function ofaquaporins in long-distancewater transport andcellular osmoregulation.

This review is dedicated to Professor Jean Guern on the occasion of hisretirement.

CONTENTSINTRODUCTION..................................................................................................................... 400BACKGROUND....................................................................................................................... 401MEMBRANE WATERPERMEABILITY MEASUREMENTS............................................ 402

Diffusional WaterPermeabil ity........................................................................................... 402Osmotic WaterPermeabil ity ............................................................................................... 403A WideRange ofMeasuredPermeabil ities......................................................................... 404

EMERGENCE OFWATER CHANNELSIN PLANT PHYSIOLOGY................................. 404TheRedBlood Cell Paradigm............................................................................................. 404Early Evidencefor Plant WaterChannels .......................................................................... 405Molecular Identification of Plant Aquaporins..................................................................... 407

MOLECULAR FEATURESOF WATERCHANNELS......................................................... 408MIP Homologs,Aquaporins, and Other Water-TransportProteins................................... 408AquaporinsAre Expressed in thePlant Vacuolar and PlasmaMembranes....................... 409Molecular Structure ofthe Aquaporin WaterChannel....................................................... 410

FUNCTIONAL STUDIESOF PLANTWATERCHANNELS.............................................. 411Heterologous Expression of Plant Aquaporins in XenopusOocytes.................................. 411Contributionof Aquaporinsto Membrane Water Transport.............................................. 412TransportSelectivity............................................................................................................ 414Mechanismsof Water Permeation ...................................................................................... 415

THE REGULATION OFAQUAPORINACTIVITY .............................................................. 415GeneExpression.................................................................................................................. 416Cell Localization.................................................................................................................. 417Posttranslational Modifications.......................................................................................... 417Other Regulatory Mechanisms............................................................................................ 418

THE INTEGRATED FUNCTION OF AQUAPORINSIN PLANTS..................................... 418Transcellular and Long-DistanceWater Transport............................................................ 419Cell Volumeand Osmoregulation....................................................................................... 421

PERSPECTIVES....................................................................................................................... 422

INTRODUCTION

Becauseof their lack of mobility in anoftenchallengingenvironment,plantsdependon a supply of water for their growth and development and havetotightly control water balance.Numerousstudieshaveaddressedthe overallphysiological andbiophysicalmechanismsof plantwaterrelations (7,14,104)andpointedto a variety of physiologicalprocesses,from long-distancewatertransportto singlecell expansionandosmoregulation, that requirethe trans-portof wateracrosscellularmembranes(7, 104).However,themechanismsofplant membranewater permeability have remainedelusive until the recentdiscoveryin animalsandplantsof a classof water-transportproteinsnamedaquaporins(3, 11). Thesewater channelproteinsfacilitate the passiveex-changeof wateracrossmembranes,andtheir discoveryin plantsemphasizedthe limitation that transmembranewater transportmay exerton a numberofphysiological processes(12). Their discoveryalsosuggestedthe existenceofyet to bediscoveredregulatoryprocessesthatmight becritical to plantwaterrelations.

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Thefirst aim of this reviewis to bridgethegapbetweenthenewmolecularinsightsmadepossible by the discoveryof aquaporinsanda wealthof infor-mationreportedover the yearsby plant physiologistsandbiophysicists. Thesignificanceof aquaporinsfor our understanding of plant waterrelationsandthe experimental and theoretical perspectives that they open are also dis-cussed.

BACKGROUND

In plants,transmembranewatermovementsareprimarily determinedby hy-drostaticand osmotic pressuregradients,respectivelynoted∆P and ∆π (14,105).

The buildup of a hydrostaticpressureor turgor is madepossible by themechanicalresistanceof thecell wall whoserelationto cell volume is charac-terized bythe elasticmodulus(ε) (105).

Theosmoticdriving force∆ψosmcan be expressedas

∆ψosm= σ∆π = σRT∆C. 1.

It is determinedby the soluteconcentrationgradient(∆C) but alsoby thesolute reflection coefficient (σ) for the membrane(R is the universalgasconstant,T is absolutetemperature)(22). This σ coefficient quantifiesthemembraneselectivity for the soluteanddeterminesits osmotic efficiency. Inmostcases,thesolutecannotsignificantly permeatethemembraneor at mostat a rateseveralordersof magnitudelessthanwater,andσ is maximal andequal to unity. However,σ may take lower valuesand is null (no osmoticeffect)whenthe solutepermeatesthe membraneas efficientlyas water (22).

The basic flow equation(Equation2) that governstransmembranewatertransporthasbeenderivedfrom thetheoryof irreversiblethermodynamics(14,53, 105). It dictatesthat, if only onesoluteis considered,the net transmem-branevolumeflow Jv is proportionalto the motive force (∆P-σ∆π), and thesurfacearea (A) and thehydraulicconductivity(Lp) of the membrane.

Jv = Lp A (∆P-σ∆π). 2.

This equationemphasizesthe major role played,besidesσ and ε, by Lp,which describestheintrinsic permeability of themembraneto water.Lp canbeconvertedinto Pf, the osmoticwaterpermeability or filtration coefficientac-cordingto

Pf = LpRT / Vw, 3.

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whereR andT havetheir usual meaning andVw is the partial molar volume ofwater (22). Examining the biophysical,molecular,andphysiological signifi-canceof theseparameters,Pf andLp, is theobjectof this review.Reviewsthatcover all aspectsof plantwater relationscan be foundelsewhere (7, 104).

MEMBRANE WATER PERMEABILITY MEASUREMENTS

While Pf describesoverall water movementin responseto hydrostatic orosmoticpressuregradients,the diffusional permeability coefficient (Pd) de-scribesthe unidirectionalflux (diffusion) of watermoleculesthat still occursacross the membranewithout any drivingforce(22).Although of lesserphysi-ologicalsignificance,this parametergivesanestimateof themembranewatertransportcapacityand,whencomparedwith Pf, providesvaluableinformationaboutthe biophysical mechanismsof water permeation(see below).

Diffusional WaterPermeability

All earlymeasurementsof plantcell andtissuediffusionalwaterpermeabilitywere performedusing heavy(HDO) or tritiated (HTO) water (28, 40, 101).However,the rapiddiffusional waterexchangeratesand,aboveall, unstirredlayer effectsrenderdiffusional water permeability(Pd) measurementsusingthesetracerflow techniques very difficult (14, 22,105).Unstirredlayereffectswere especiallyprominent when Pd was determinedin multicellular plantsystemsor in cells with high waterpermeability; mostof the reportedvalues(101)maywell bespurious. Recently,Henzler& Steudle(34)measuredthePdof Chara internodalcells takingadvantageof theosmotic (solute-like)effectsof HDO andfollowing its diffusion by meansof a pressureprobe(seebelow).Protonnuclearmagneticresonance(1H-NMR) providesapproachesto circum-vent the extracellularunstirredlayer artifactsand permitsprobing of watercompartmentsin intact plant tissues(77, 100, 110, 135a).Membranewaterpermeabilitycanbe derivedfrom saturationtransfer(77) or morecommonlyfrom relaxationtimes of intracellular water protons.For this, the 1H-NMRsignalof the watermoleculesresidingin the extracellularspaceor diffusingfrom theintracellular spaceis doped by anextracellular paramagnetic ion suchas Mn2+. However,the Pd valuesdeducedfrom thesemeasurementsgreatlydependon assumptions aboutthe penetrationand compartmentation charac-teristicsof theparamagneticagentinto theplantcell or tissue(100,110,135a)and can stillbe dominatedby intracellularunstirredlayers(110).

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OsmoticWaterPermeability

Theoverallexchangeof waterinducedosmotically in intact tissuesor organscanprovideindicationson cell waterpermeability(25, 32). More direct esti-matesof osmoticwater permeability(Pf) in single plant cells were first ob-tainedby the plasmometric method(reviewedin 102). A preliminary plas-molysisseparatestheplantprotoplastfrom any interactionwith thecell wall,andPf valuesarederivedfrom therateof protoplastswelling observeduponasubsequenthypotonictreatment.This techniquehasbeencriticized(51, 137)mostly becauseof its low resolutiondue to the restricteddiffusion of theosmoticumand/orunstirredlayers in the plant cell walls. Thesedrawbackshaverecentlybeenavoidedthroughthe direct micromanipulation of isolatedprotoplasts(91).

Thegiantinternodalcellsof theCharaceaeprovideconvenientmaterialsforstudyingmembranetransportand allowed in particularPf measurementsbymeansof transcellular osmosis (51).For this, thetwo endsof acell arebroughtinto contactwith externalmediaof differentosmolarities.This createsa tran-scellular water flow from the less to the most concentratedcompartment,whoserate indicatesthe cell Pf. Despitepitfalls due to intracellularosmoticpolarization (15, 51), this methods provides Pf measurements with littlechangein cell volumeandturgor. It alsoallows an easydistinction betweenendo- (inward) andexo- (outward) osmotic water movements.

In contrastwith transcellular osmosis, thepressureprobetechniquecanbeapplied to giant algal cells but also to normal-sizedhigher plant cells (re-viewedin 103,136).An oil-filled microcapillary is insertedinto thecell anditscouplingto a mechanicaldeviceallowsto primarily measureor clampthecellturgor pressure.Water movementis generatedupon imposition of a suddenosmotic or hydrostatic pressuregradientand can be followed through theassociatedturgorpressurerelaxation.Alternatively, volumerelaxationscanbefollowedunderpressure-clampconditions(135).Thesetypesof measurementsallow thesuccessivedetermination of thecell volumetricelasticmodulus, thecell hydraulic conductivity, and the membranesolutereflectioncoefficients,which togetherdefine the plant cell water relation(103, 136). Anothergreatpotential of the pressureprobe isthat it allows in situ measurementsonindividual cellsunderosmotic or hydrostaticconditions(108,112).However,owing to technical difficulties, this techniquehas only been appliedto arestrictednumberof higherplantcell types.Two otherdrawbackshamperitsapplicationto the direct analysisof membranewater transport.First, the re-spectivecontributions of the membranesand cell-to-cell connections(plas-modesmata)canhardlybedistinguishedin theoverallcell Lp. Second,because

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the probeis insertedinto the vacuole,the cell Lp integratesthe resistanceinseriesof the tonoplastand the plasmamembrane.This restrictionappliestonearlyall techniquesdescribedaboveandhaslong beenrecognizedasa maindifficulty in interpretingwater transportmeasurementsin plant cells (14, 28,124). In Chara, these difficulties could be circumventedby the selectivedisruptionof the tonoplast(58) or by a three-compartmentanalysisof turgorrelaxationkinetics(124,125).

More recently,optical methods(reviewedin 117) have beenapplied tomeasuringthePf of purifiedplantmembranevesicles(81,96).Themixingof avesiclesuspensionwith ananisoosmotic solution is accomplishedin <5 msina stopped-flowapparatus.The subsequentosmotic adjustmentof the sealedvesiclevolume canbefollowed, for instance,from the simultaneous changeinscatteredlight intensity. A Pf valuecanbederivedfrom boththetimecourseofvesiclevolumeadjustmentandthevesiclesize,determinedby anindependenttechniquesuch as electronmicroscopy.

A Wide Range of Measured Permeabilities

Early estimates(14, 101) of plant membranewaterpermeability rangedovermorethanthreeordersof magnitude.Becauseof methodologicaluncertainties,this variability couldhardlybe interpretedat that time (14). Sincethen,novelmethodshave been developedand their reliabilityassessed. Forinstance,consistentPf valuesof Characellscanbemeasuredusingeithertranscellularosmosis or a pressure probe (Table 1). The latter technique indicated thatLp valuesof different plant cell typesrangefrom 2 × 10−8 to 10−5 m • s−1 •MPa−1 (i.e.Pf ranging from 3× 10−4 to 10−1 cm •s−1) (Table 1;105).Stoppedflow measurementsalso indicatedthat membranesubfractionsisolatedfromthe sametobaccocells can display strikingly different water permeabilities,from 6 × 10−4 to 6 × 10−2 cm • s−1 (Table1; C Maurel,F Tacnet,J Güclü,JGuern& P Ripoche,unpublishedmanuscript). Thus,thewide rangeof waterpermeabilitiesmeasuredin plantcellsappearsto reflecta genuinepropertyoftheir membranes.The reportedvaluesalsocorrespondto the rangeof perme-abilitiesdeterminedfor animalcell membranes(Table1; 22,116).

EMERGENCEOF WATER CHANNELSIN PLANTPHYSIOLOGY

The Red BloodCell Paradigm

The lipid phaseof membraneswaslong consideredthe major pathfor waterexchangein living cells. Transportalong this path is basedon the solubil-

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ity/diffusionof individual watermoleculesinto thephospholipid bilayerandischaracterizedby equalPf andPd anda high Arrheniusactivationenergy(Ea =14–16kcal • mol−1) (22,30).Thedeterminationof aPf/Pd greaterthanunity intheerythrocyteandotheranimalcell membraneshasbeenthefirst experimen-tal evidencefor transmembraneaqueouspores thatwould mediate, astheoreti-cally predicted,anapparentpositiveinteractionduringosmoticwaterflow (59,83). The low Ea (<5 kcal • mol−1) of this processin erythrocytesfurthersuggestedthe existenceof a bulk flow of wateracrossmembranepores(66,116). That the red blood cell water channelsmight containa proteinaceouscomponentwasinferredfrom their reversibleblockadeby mercurysulfhydrylreagents (66).

Early Evidencefor Plant Water Channels

Thepossible existenceof aqueousporesin plantmembraneswasdiscussedinthe early 1960sby Ray (92) andDainty (14). To investigatethis possibility,Gutknecht(28) determinedthe Pf/Pd ratio of Valonia cells. Its valueclosetounity and the low measuredvalueof Pf of thesecells (2.4 × 10−4 cm • s−1;Table 1) suggestedthe absence of aqueousmembrane pores. Probablybecauseof the well-recognizedartifactscausedby unstirredlayersin suchan experi-ment (14), this was, until recently (34), the only report of Pf/Pd in a plantmembrane(Table 1).

Thedependenceof watertransporton temperaturewasalsocharacterizedinseveralplant systems(15, 26, 40). The derivedEa values,higherthanthe Eafor freewaterdiffusion or waterdiffusion in deadtissues,wereinterpretedtomeanthat plant membranes,and more specificallyaqueouspores(15), maycreatean impediment to water flow. However,Ea has not beenuseduntilrecentlyto distinguish betweenlipid- andchannel-mediatedwatertransports.Thesearchfor inhibitorsof watertransportin plantsled to theeventualuseofmercuryderivatives(26, 40). Although thesecompoundswere suggestedtodirectlyalterthepermeability of plantmembranes(40),theywereusedin mostcases as general metabolic inhibitors (26).

Actually, it wasgenerallyassumedthat, if present,aqueousporesin plantmembranesshouldallow thesimultaneousflow of waterandsolutes.This ledto thesearch ofpossible frictional interactionsbetweensoluteandwaterin themembranethat yield σ valuesinferior to unity. Now that thevery high trans-port selectivity of aquaporinshasbeendemonstrated(seebelow), it appearsthat the absenceof such friction (σ = 1) (29) is not evidenceagainstthepresenceof aqueouspores.In contrast,Dainty & Ginzburg(16) and others(107)noticed—andthis argumentstill remainsvalid (106)—thattheσ values

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Table 1 Waterpermeabil itiesof cellsandmembranes from plants andanimals

Species Tissue SystemaPermeabil ity(10–4 cm • s–1)b Method Reference

Materials andmeasurement techniques

Characorallina

Internode Cell Pf: 260 ± 72Pf: 243 ± 15

Pressure probeTranscellularosmosis

34121

Elodeanuttalli i

Leaf Cell Pd >300 1H-NMR 110

Elodeadensa

Leaf lowerepidermis

Cell Pf: 19.0 ± 3.1(P<4bar)

Pf: 7.5± 2.7(P>4bar)

Pressure probe 109

Pisum sativum Epicotylepidermis

Cell Pf: 3–30 Pressure probe 13

Epicotylcortex

Cell Pf: 50–1200 Pressure probe 13

Cell, plasma membrane andendomembranes

Charaaustralis

Internode Cell

PM

Pf: 97± 17

Pf: 94± 13

Transcellularosmosis

Transcellularosmosis

58

Alliumcepa Bulb innerepidermis

CellVacuole

Pf: 6–8Pf: 40–540

DeplasmolysisDeplasmolysis

113

Nicotianatabacum

Cellsuspension

PM ves.TPves.

Pf: 6.2± 0.4Pf: 659 ± 83

Stopped-flowStopped-flow

c

Apple Fruitparenchyma

Vacuole Pd: 24.4 1H-NMR 100

Liri odendrontulipifera

Leaf Chloroplastenvelope

Pd: 9 ± 2 1H-NMR 77

Animalcell membranes

Hog Stomach PM ves. Pf: 2.8± 0.3 Stopped-flow 116

Toad Bladder Apicalmembraneves.

Pf: 3.9± 0.4(– vasopressin)

Pf: 450(+ vasopressin)

Stopped-flow

Stopped-flow

116

Human Erythrocyte PM ves. Pf: 230 ± 30 Stopped-flow 71

Pf/Pd of plantmembranes

Valoniaventricosa

— Cell Pf: 2.4± 0.3

Pd: 2.4 ± 0.2

Transcellularosmosis

Tracerflow

28

Characorallina

Internode Cell Pf: 260 ± 72Pd: 7.7 ± 3

Pressure probePressure probe

34

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for organicmoleculesin Characeaecells areso low that this mustreflect thepresence of water-filled pores.

Nevertheless,the characterizationof membranepermeability in Characeaecells progressivelyled to the idea that solute transport,but also most of theosmoticflow of water,occursacrossmembraneporesin thesecells (55, 121,122).For instance,Wayne& Tazawa (121,122), priorto the molecularidenti-fication of animalandplant aquaporins,presentedmostof the now classicalargumentsfavoring membranewaterchannelsandclearly demonstratedtheirmajorcontributionto osmotic water transport.

Surprisingly, this view remainedmarginal in plant physiology. High Pfvalues(>10−2 cm • s−1) hadbeenmeasuredin severalhigherplant cell typesandmight havesuggestedwater-channel-mediatedtransport.Yet the prevail-ing idea was that the reportedrangeof Pf could be accountedfor by waterdiffusion acrossplant lipid membranesof distinct composition (102). Thisview can indeedbe supportedby studieswith artificial membranesthat re-portedPf valuesrangingfrom 2 × 10−5 to 1 × 10−2 cm • s−1 (21, 22, 61) butwas scarcely investigated (27, 96)usingplantlipid membranes.

MolecularIdentificationof PlantAquaporins

The abundance ofaquaporinsin plantsis undoubtedly themainreason that ledto their redundantidentification by biochemicalandmolecular biological ap-proaches,well beforetheir functionwasclearlyidentified.Thehighhydropho-bicity of theseintrinsic membraneproteinsalso facilitatedtheir biochemicalpurification. The α-TonoplastIntrinsic Protein (α-TIP), for instance,repre-sents2% of the total extractableproteinsof beancotyledonsand could beeasilypurified by Triton X-114 extractionandpartitioning into thedetergent-rich phase(46).Surprisingly,theepitopescarriedby aquaporin-likeproteinsinplantmembranesaresoprominentthatseveralpolyclonalantibodiesraisedtototal purified plasma membranes or tonoplastswerefoundto mostly reactwiththeseproteins(52, 70). A largeclassof Arabidopsis plasmamembraneaqua-porins was thus isolatedusingsuchantibodies,by immuno-selectionfrom amammalian COS cell expressionsystem(52). Severalaquaporinssuch asγ-TIP are also someof themostabundantlyexpressedsequence tags(ESTs) inArabidopsisand were repeatedlyidentified in systematic cDNA sequencing

aPM, Plasma membrane;TP,tonoplast;ves.,vesicles.bPd: Diff usionalwaterpermeabil ity coefficient; Pf: osmotic waterpermeabil ity coefficient. When

not providedby the authors, Pf values werederived, for comparison with otherPf values,from theindicatedLp values according to Equation 3, with T = 293 K. Pf andPd valuesare indicated± SD.

cC Maurel, F Tacnet, J Güclü, J Guern& P Ripoche,unpublishedmanuscript

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programs(35). Finally, the modulation of aquaporingeneexpressionin re-sponseto variousphysiological stimuli hasalsoled to their identification byproceduressuchasthe differential screeningof cDNA libraries(49, 86, 126,127).γ-TIP andRD28 werethusidentified asgenesup-regulatedby, respec-tively, gibberellicacid (GA3) anddrying treatments(86, 127).More recently,the duplicatedAsn-Pro-Ala(NPA) motifs conservedin the aquaporinaminoacidsequences(seebelow)haveprovideda uniqueregionfor thePCRampli-ficationof putativeaquaporingenes.

ThearchetypalanimalwaterchannelCHIP28,subsequently renamedaqua-porin-1 (AQP-1),wasalsofirst identifiedby biochemicalmeansin theeryth-rocyte membrane(reviewedin 116). The work of Prestonet al (87), whohypothesizedfrom the expressionpatternof CHIP28/AQP-1that it mightcorrespondto oneof thelong soughtafterwaterchannelsof theredbloodcellandkidney epithelia,hasprovedto be a major breakthroughfor animalandplantbiologists (11). Evidencethatplantaquaporinsfacilitatethetransportofwateracrossmembranescomesfrom theobservationthattheseproteins,simi-lar to AQP-1, inducea specific and mercury-sensitiveincreasein the Pf ofXenopusoocytemembranes(75). The two othercriteria that functionally de-fine waterchannels,i.e. a low Ea anda Pf/Pd > 1, havebeendemonstratedforAQP-1 (71, 87). This and the reconstitution of functional waterchannelsinproteoliposomescontainingthe purifiedprotein(115,130,131) have providedunequivocalevidencethatAQP-1,andby extensionits functionalanimalandplanthomologs, have an intrinsic water transportactivity.

MOLECULAR FEATURES OF WATER CHANNELS

The aquaporinsdefinea functional classof water-transportproteinsthat be-long to the larger Major Intrinsic Protein (MIP) family of transmembranechannels(3). This ancientfamily was namedafter its archetype,the MIP ofmammalian lensfiber, and includesmembers in awide variety of livingorganisms(93). At present,aquaporinshave beenidentified in vertebrates,higher plants,bacteria, and insects(8, 63, 75,87).

MIP Homologs,Aquaporins andOtherWater-TransportProteins

Sequencesavailablein thegenedatabanksindicatethat the largeMIP familyof membranechannelsis evenmoreextendedin plantsthanin animals(93).For instance,23 MIP-like uniqueESTshavebeenfound in Arabidopsis (AZweig & MJ Chrispeels,personalcommunication) while only 7 MIP-like

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sequenceshavebeenidentified in humans(118).However,mostof the plantMIP proteinsandcDNAs remainto befunctionallycharacterized.While theypotentially representnovel aquaporins,they may alsoencodetransmembranechannelsfor othersubstrates.ThebacterialMIP homolog, GlpF, for instance,facilitatesthe diffusion of small polyhydric alcoholsbut is not permeabletowater (33, 75, 76). The function of mammalian MIP, as an ion or a waterchannel,remainscontroversial(54, 118).The watertransportactivity of soy-beannodulin-26remainsto bedetermined,but thepurified proteinwasshownto formlarge conductanceion channelsin artificial membranes(123).

Protein-mediated transmembranetransportof water is not necessarilydueto aquaporins.Becausethey containa hydratedpath,all membranetransportproteinshavea finite permeability to water.It wasfound,for instance,that inmost ion channelsthe transportof one ion is coupledto that of 5–10 watermolecules,which correspondto the numberof water moleculesfillin g theconstrictionof the pore (4, 22). Higher water transportactivities havebeenassignedto animalmembraneproteinssuchas the cystic fibrosis transmem-braneconductanceregulator(CFTR), glucosetransporters,or inorganic ioncotransporters(reviewedin 132). In plants,the tonoplastof Chara internodalcells contains achannel that allows the coupling of 25moleculesof waterwitha singleK+ ion (39). The inhibition of water transportin Chara cells by K+

channelblockers(122) providesevidencethat eithernonclassical aquaporinsor other proteinscan significantly contributeto the Pf of plant membranes.Nevertheless,with a unit permeability of 3–12cm3 • s−1 (115,130,131), thewater transportefficiency of aquaporinsis at least20 timesgreaterthanthat ofany otherknown protein.

Aquaporins Are Expressedin the Plant VacuolarandPlasmaMembranes

Thesequencerelationshipbetweenall plant MIP-like cDNAs (126) indicatesthat the encodedproteinsfall into threesequencesubclasses.The first twoclassescontain,respectively,the TonoplastIntrinsic Proteins(TIP) and thePlasmamembraneIntrinsic Proteins(PIP) that have been localized in thetonoplastand in the plasmamembrane.This was shownby severalgroupsusing immunocytochemistry(36, 46, 48) and protein immunodetectioninisolatedorganelles(36) or purified membranefractions(18, 48, 52, 68). Thethird class comprisesnodulin-26, which is expressedin the peribacteroidmembraneof root symbiotic nodules(23). However,the localizationof nodulin-26 homologues innonlegumesremainsto be determined.

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MolecularStructureof theAquaporin Water Channel

All of theknownaquaporincDNAs typically encode25–30kDa polypeptides(116, 118). Although the overall identity amongMIP homologsmight fallunder25%, their aminoacid sequencescontainvery well-conservedresiduesincludinga SGxHxNPAVTmotif (NPA box) (Figure1; 93).Sequenceanaly-ses also suggestthat the MIP gene family aroseafter an early intragenicduplication. Thustheamino- andcarboxy-terminal halvesof theproteinssharesequencehomology, each possessa NPA box,andeach is orientedin oppositedirectionsin the membrane(Figure 1;93).

Theconservedhydropathyprofilesdeducedfrom MIP-like cDNAs suggesta typical structure,with six putativemembrane-spanningsegmentsandcyto-plasmicamino- and carboxy-terminaldomains(Figure 1; 93). Experimentalevidencefor this topological modelmostlycomesfrom theanalysisof vecto-rial proteolysis at defined epitopesintroducedin recombinantAQP-1 (89).Phosphorylationand glycosylation studieswith other animal and plant MIPhomologues areconsistent withsucha model(6, 74,79).

Figure 1 Proposedstructureof plantaquaporinsbasedontopologicalstudiesonplantandanimalMIP homologs (seetext). The most conservedresiduesin the MIP family areencircled.The twoNPA boxesareshaded. Phosphorylation sitesidentifi ed in α-TIP aresquared off. Stars indicatepositionsfor cysteine residuesthatconfer mercurysensitivity onplantaquaporins.

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The low electrophoreticmobility of nativeplant aquaporins(68) andtheirpropensityto aggregatein vitro suggestedthattheymayoccurasoligomersinplantmembranes(17,18, 46, 52,68). This view is consistentwith sizeexclu-sionchromatographyandhydrodynamic analysesof nativeAQP-1 thatdeter-mineda stoichiometry of 4 subunitsper oligomer (99). Recently,X-ray andelectron diffractionof two-dimensional crystals ofAQP-1yieldedhigh resolu-tion projection maps.Thesemaps depict a structural motif of 6–7 nm indiameterwith acentraldepressionandfour trapezoidsubunits,eachdisplaying6–7 putative transmembranesegments(43,80, 119).

Although aquaporinsassemblein homotetramericor evenmoreorganizedstructures, radiation inactivationanalyses indicateda functional size of30kDafor animal water channels(114). In addition, the coexpressionin Xenopusoocytesof wild type AQP-1 monomers with inactive permeability mutantsindicatedno dominantnegativeeffectsof the latter (88, 97, 133).Thesedatasuggestedthat in animals,and probably in plants as well, eachaquaporinmonomerformsa functionallyindependentpore (118).

Mercury derivativeshave beenextensivelyusedto probe the molecularstructureof water channels.They are thought to block thesechannelsviaoxidationof cysteineresidue(s)proximal to theaqueousporeandsubsequentocclusionof thelatterby thelargemercuryion. Themercury-sensitivesitesofAQP-1andvasopressin-regulatedAQP-2havebeen identified at,respectively,Cys189 andCys181, eachin thethird extracytoplasmicloopnextto thesecondNPA box (6, 88,133).Thefunctionalimportanceof this andof thesymmetri-cal loop carryingtheotherNPA box (seeFigure1) wasalsosuggestedby theconverseobservationthat animalandplant mercury-resistantproteinscanbesensitizedto the inhibitor by introduction of targetcysteineresiduesin eitherof thesedomains(18, 47, 98). However, the cysteineresiduesthat confermercurysensitivity to plant γ-TIP and δ-TIP sit in the third transmembranesegment,on the hydrophilic sideof a putativeamphiphilic α-helix (17). Thispointsto another domainthatpossiblylinestheaqueoustransmembranepore.

FUNCTIONAL STUDIES OFPLANT WATER CHANNELS

Heterologous Expression of PlantAquaporins in XenopusOocytes

Thecapacityof Xenopusoocytesto translateintracellularlyinjectedmRNAs,their largesize,andthe relatively low Pf (≤1 × 10−3 cm • s−1) of their nativemembranemakethesecellsconvenientfor thefunctionalexpressionof water-transportproteins(134).Oocytesareclassicallyincubatedin isoosmotic con-

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ditionsfor few daysaftermRNA injectionto allow exogenousproteinexpres-sion.Theinitial rateof cell swelling observeduponsubsequenttransferof theoocytesinto a hypoosmotic solution indicatestheir membranePf (134).Simi-lar to animalAQP-1 (87), severalplantMIP homologsof eitherthetonoplastor theplasmamembrane(18,52,75)werefoundto inducea2-20-foldincreasein thePf of theoocytemembraneandwere thusidentifiedasnovelaquaporins(Table 2).Oocyte expressionalso providespowerful approachesto studyaquaporintopology (89), oligomer assembly(97), posttranslationalregulation(60, 74),transportselectivity(75), and structure-functionrelationships(47).

Contributionof Aquaporinsto Membrane Water Transport

Becausemoststudieson plantaquaporinshavebeenperformedafterheterolo-gousexpressionof proteinsin oocytes,severalgroupsarenow concentratingon thestudyof waterchannelsin plantmembranes(34,81,122)(C Maurel,FTacnet,J Güclü,J Guern& P Ripoche,unpublishedmanuscript). In all theseexperiments,aquaporinfunction wasprobedthroughmercuryinhibition. Be-causesomeplant andanimalaquaporinsareresistantto mercuryaction(18,31), this approachonly indicates theminimal contribution of waterchannelstothe total watertransportcapacityof themembrane.Nevertheless,1 mM mer-curic chloride (HgCl2) can reduceby more than 80% the Pf of tonoplastvesiclespurified from tobaccosuspensioncells(C Maurel,F Tacnet,J Güclü,J Guern & P Ripoche, unpublished manuscript). Thehigh intrinsic waterpermeabilityof thesemembranes(Pf = 6 × 10−2 cm • s−1) andthe low Ea oftransport(∼2.5kcal • mole−1) provideadditional evidencethatwatertransportin thesevesiclesoccurspredominantly throughmembranechannels.In con-trast,plasmamembranevesiclesisolatedfrom thesametobaccocellsexhibiteda low Pf (6 × 10−4 cm • s−1) anda highEa (∼14 kcal • mole−1), bothindicativeof a low densityof functionalaquaporins,if any. The membranesof Charaand Nitellopsiscells are also highly permeableto water,and mercurialsre-ducedtheirPf by upto 70 and30%,respectively(34,122).This inhibition wasassociatedwith either a decreasein Pf/Pd (34) or a twofold increasein Ea(122),bothindicatingtheblockadeof pore-mediatedtransport. Although thesestudiesindicatethatthehigh waterpermeabilityof certainplantmembranesismainly accountedfor by waterchannelactivities,they provideno molecularidentification of thecorrespondingchannelproteins.To addressthis question,Kaldenhoff et al (48) have used transgenicArabidopsisthat constitutivelyexpressedantisensePIP1baquaporintranscriptsanddisplayedan associated40–80%reductionin PIP1aandPIP1bmRNA levels(R Kaldenhoff,personalcommunication). The osmoticswelling of isolatedprotoplasts was three-to

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Table 2 Cell localizationandexpressionof plantaquaporins

ExpressionName Species Aquaporin

functionaLocalizationb Organ-specific Stimulus-

induced

TonoplastIntrinsic Protein (TIP) subfamily

α-TIP Phaseolusvulgaris

74c TP(36, 46)c Seedsandseedlings(46, 78)c

γ-TIP Arabidopsisthaliana

75 TP(37) Roots& shoots(elong. zone)—flowers (37, 65)

GA3 (86)

δ-TIP Arabidopsisthaliana

17 TP(17) Shoots >roots(vasc. tissues)—flowers (17)

pRB7 Nicotianatabacum

82 n.d. Roots (meristem,immaturecentralcylinder)(128)

Root-knotnematode in-fection(82)

PlasmamembraneIntrinsicProtein(PIP)subfamily

PIP1a,PIP1c

Arabidopsisthaliana

52 PM (52) Roots (central cylin-der>cortex)—leaves(vasc. tis-sues>mesophyll )(52; AR Schäff-ner, personalcommunication)

PIP1b(AthH2)

Arabidopsisthaliana

48, 52 PM (48, 52) Roots& shoots(differentiatingandelong. tissues,guardcells) (48,52)

Bluelight,ABA, GA3(48–50)

PIP2a,PIP2b

Arabidopsisthaliana

52 PM (52) Roots& shoots (52)

RD28(PIP2)

Arabidopsisthaliana

18 PM (18) All organsexceptseeds (18)

Desiccation(up-regula-tion) (127)

MIPA Mesembryan-themumcrys-tall inum

126 n.d. Leaves(vasc.tis-sues,meristem)—roots (epiderm.,develop. xylem)(126)

Saltstress(down-regu-lation) (126)

MIPB Mesembryan-themumcrys-tall inum

126 n.d. Root tip (126)

aAquaporin function wasdetermined by oocyte expressionin all cases,exceptin Reference 48wheretransgenic Arabidopsis expressinganantisensegene construct was used.

bCell localization: TP,tonoplast;PM, plasma membrane;n.d., not determined.cNumber(s)indicate(s)the correspondingreference(s).

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fourfold slowerin transgenic protoplasts thanin controls,suggestingthat PIP1aquaporinssignificantly contributeto watertransportin thesecell membranes(48).

Transport Selectivity

Well beforethemolecularidentification of aquaporins,indirectevidencesug-gesteda reasonablyhigh selectivity for animalandplant waterchannels(55,66). For instance,the water permeability of Chara cell membranescan bemodulatedwithout changein electricalconductance,suggesting theexistenceof two distinct paths,for waterandions,respectively(55). More recently,theselectivityof plantaquaporinshasbeendirectly supportedby theobservationthat neitherγ-TIP nor α-TIP hasdetectableeffectson glycerol andion trans-port in oocytes(74, 75). In particular,currentmeasurementsduring osmosisdid not indicateanydetectablepassiveion permeability or anysolvent-dragofionsin conditionswherethewaterchannelswereknownto operate, indicatingtheeventualpassageof at most1 ion for every2 × 105 watermolecules(75).This view hasrecently been challengedby the findingthatwhenstimulated byforskolin in theoocytemembrane,animalAQP-1exhibits a high permeabilityto cations(129).

Using the pressureprobetechnique,Henzler& Steudle(34) investigatedtheselectivity ofplant waterchannelsdirectly in themembraneof Characells.Mercury wasfound to reducethe membranepermeabilityto waterbut not tosolutessuchas dimethyl formamide,ethanol,or acetone.This indicatesthatthe major transportpath for thesesolutesis not throughwater channelsbutrather through the lipid bilayer. Using a theoreticaltreatmentof membraneorganizationasa composite structurewith arraysof waterchannelsandotherswith lipid phase,Henzler& Steudle(34) further calculatedreflectioncoeffi-cientsfor thesesolutesalongthewaterchannelpath.Thesecoefficientswereless thanunity andwereinterpretedto meanthatthesemolecules canpermeatethe channelpore, thoughat a very slow rate, and developstrong frictionalinteractionswith watermolecules.Recentinvestigationsusingoocyteexpres-sion have also revealedbasalpermeabilities of animal aquaporinsto smallsolutessuchasurea,ethyleneglycol, andglycerolbut not to largermolecules(2, 20,42).α-TIP andγ-TIP displayeda selectivity in theseassayshigherthanthatof their animalcounterparts (1).It remainsto bedeterminedwhetherplantcells possessother aquaporinsthat, similar to animal AQP-3, discriminatepoorly betweenwater and small neutral solutes (20, 42). Nevertheless,ourcurrentknowledgeof plant aquaporinselectivity, andtheir assumedcapacity

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to excludeprotons(75, 131), points to the remarkablecapacityof thesepro-teinsin improving thesemipermeablecharacteristicsof plantmembranes.

Mechanismsof Water Permeation

The molecularbases ofwater channel selectivity are currentlyunknown(118).Thesechannelshavebeenassumedto form narrow poresof 0.3–0.4nm indiameterthat would excludeany moleculelarger thanwater (55, 66). Watermoleculeswould flow one by one in the constriction of the pore, and thenumberof moleculesin a singlefile would determinethePf/Pd ratio, typicallygreaterthanunity in waterchannels(22,59,83).Theexclusionof small ions,and protonsin particular(75, 131), further suggeststhat size exclusion, butalsoothermechanismssuchaselectricalfiltering, determineaquaporinselec-tivity. Thecomparativestructure/function analysisof aquaporinswith distinctselectivity profiles, suchas plant TIPs and animal AQP-3, will be of greatinterestto addressthese issues.

Wateris thoughtto flow acrossthe waterchannelporein eitherdirection,down itspotential gradient.However, early reports (quoted in 57)mentionedadifferencein the ratesof plasmolysis anddeplasmolysis of plant protoplasts.This might well beexplainedby a solute sweep-awayor otherunstirred-layereffectsduringosmosis (14).However,morepreciseanalysesof hydrostatic orosmoticwatermovements in Nitella suggestthatthesecell membranesat leastpossessan intrinsic capacity torectify water flow,by a factor of 1.1to 2.7 (15,51,57,108,121).It remainsto bedeterminedwhetherthis polarity results,asfirst proposed,from a waterflow–induceddehydrationof the lipid membrane(15) or, in view of the most recentstudies,from intrinsic featuresof waterpermeabilityin membranechannelsor from their differentialexpressionand/or regulationin subcellularmembranedomains(see below).

THE REGULATION OFAQUAPORINACTIVIT Y

Although not fully understood, thelarge variability of water permeabilityvaluesreportedin differentplantcell types(Table1) suggeststhe importanceof speciesspecific,developmental, and physiological factorsin determiningwater transportin plant membranes.In Chara, the Lp of internodalcellsdependsontheirdistancefrom theapexandonthevegetativeandreproductivestatusof theplant (120).Lp canalsobemodulatedby theexternalsolute(15,56) or CO2 (120)concentration.In addition,in higherplantsnumerousphysi-ological processesmight involve the modulation of transmembranewatertransport.For instance,the Lp of roots varies in responseto a variety of

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environmental factorsincluding diurnal cycling,drought andsalinity, lowtemperature,and nutrient and O2 deficiency(5, 10, 85, 104, 135). Phyto-chrome(Pfr) (9)andauxin(64) were shownto increasethedeplasmolysis rateof epidermalcellsof TaraxacumandAllium,respectively,while abscisicacid(ABA) down-regulateswater transportin carrot disks(26).

Changesin membranelipid composition and fluidity areknown to occurduringplantdevelopment(111)or in response tospecific physiologicalcondi-tions (10, 72). Thesechangesmight provideplant cells with distinct baselinemembranewater permeabilities(61, 96). However, water channelproteinsappearas the most powerful meansto mediaterapid and large amplitudechanges inthe water transportcapacityof membranes.

GeneExpression

A detaileddescriptionof plant cell typesandtissuesknown to expressaqua-porinsis presentedin Table2. Althoughaquaporinexpressioncanbefoundinmost tissues,the distribution of eachindividual aquaporinisoform is undertight developmentalandphysiological control andsuggestsspecificrolesforeachof theseproteins.For instance,severalaquaporins(Arabidopsisδ-TIP,tobaccopRB7)arepreferentiallyexpressedin theparenchymacellsof vascu-lar tissues andarepossibly involved inlong-distancewatertransport(17,128),whereas otherisoforms(ArabidopsisPIP1b/AthH2,γ-TIP) have patternsasso-ciatedwith elongatinganddifferentiating cells (48, 65). Detailedanalysesofseed-specificexpressionof α-TIP indicated that developmental controlofplantaquaporinscanbeexertedat thelevelsof genetranscription, translation,or subcellular routing of protein (reviewedin 73). Table 2 also showsthataquaporinexpressioncanbe controlledby varioushormonal(ABA, GA3) orenvironmentalfactors (blue light, waterstress, pathogen infection) (48–50,82,86,126,127).Of particularinterestis the up- ordown-regulationof aquaporingenesfrom Arabidopsis,ice plant, and sunflower in responseto droughtorsalinity thatsupportsa role for theseproteinsin regulatingoverallplantwaterbalanceunderstressconditions(126,127) (X Sarda,D Tousch& T Lamaze,personalcommunication). All thesefactors can interact in providing eachaquaporinwith a physiologically relevantpatternof expression.The γ-TIPgene,for instance,is preferentiallyexpressedin elongatingtissuesor afterstimulation by GA3 (65, 86). The TobRB7 promotercontainstwo distinctregulatorycis-actingelementsthat direct root-specificexpression,undernor-maldevelopmentalconditions orafterroot-knotnematodeinfection(82, 128).

Although morework is neededto integratethespatialand temporalexpres-sionof all plantaquaporinisoforms, thevarietyof theseisoformsandexpres-

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sion patternsmight well reflect the needfor distinct cell typesandtissuestoaccommodatevarious hydric requirementsin specific physiological condi-tions.

Cell Localization

It is well knownthatwaterreabsorptionin themammaliankidneyis regulatedby vasopressinvia the rapid (<5 min) insertion and removal,by a vesicleshuttlemechanism,of AQP-2 proteinsin the apicalmembraneof collectingduct epithelial cells (reviewedin 54). Although similardynamicregulation hasnotbeen reported for plantaquaporins,their very strict subcellular localizationpointsto a critical role in thefunctionalspecialization of membranecompart-mentsor evendomains.For instance,γ-TIP and α-TIP can coresidein thesameseedparenchymaor root tip cells,but areconfinedto, respectively,theproteinstoragevacuoleor the nascentvegetativevacuole(38, 84), two func-tionally distinct vacuolarcompartments(84). In Arabidopsismesophyllcells,PIP1aquaporinsaccumulatein highly convolutedinvaginationsof theplasmamembranecalledplasmalemmasomes(94). This might indicatelocal regula-tion in water permeabilityfor membranedomainsin closecontactwith thetonoplast.

Posttranslational Modifications

α-TIP canbephosphorylatedin beanseedsby a tonoplast-boundcalcium-de-pendentprotein kinase(45). Putativeaquaporinsin the spinachleaf plasmamembranearealsophosphorylated,in responseto changesin apoplastic waterpotential (44). The protein kinase(s)and protein phosphatase(s)that targetplant aquaporinsmay thus provide an efficient link betweenwater transportand thesignalingcascades involved in plantcell osmoregulation (44,73).

The functionalsignificanceof α-TIP phosphorylation was investigatedinXenopusoocytes(74). For this, a setof site-specificmutantsof α-TIP wherethreeputative phosphorylationsitesweredisrupted,individually or in combi-nation,was usedfor in vitro andin vivo phosphorylationby animalcAMP-de-pendentproteinkinase(PKA). Parallelfunctionalassayof thesemutantpro-teinsshowedthat direct phosphorylation of α-TIP at thesethreesitesstimu-latesby 100–150%its waterchannelactivity. From theseandsimilar resultsobtainedon animalAQP-2(60), it hasbeenproposedthataquaporinphospho-rylation can provide a mechanismto control in situ openingand closing ofwater channels(60, 73, 74). Alternatively, this might representa signal forsubcellularproteintransport,assuspectedin thespecificcaseof AQP-2regu-lationby vasopressin(62).

AQUAPORINS 417

Otherposttranslationalmodificationsoccurin aquaporins,suchasthepro-teolytic maturation from a largerprecursorof a 23-kDaTIP in pumpkinseeds(41). Theypossibly representnovelmechanismsfor regulatingaquaporinex-pressionand activity.

OtherRegulatory Mechanisms

Theendo- andexo-osmoticLp measured inCharacellsby transcellularosmo-sis canbedifferentially inhibitedby, respectively,pCMBS or theK+ channelblockernonyltriethylammonium (122).This suggeststhe presenceof distincttypesand/orregulationsof waterchannelsat the two polesof thecell, whichmay explain the observedcell polarity for water transport.In particular,thespecificsensitivity of endoosmotic waterflow to cytochalasinsB andE pointsto a role for theactincytoskeletonin regulatingtheactivity or localizationofsomeof these water-transportproteins(121).

The Lp of giant-celledalgaecanalsobe modulatedin responseto hydro-staticor osmotic pressuregradients(15, 56, 107,108,137).For instance,theintracellularandextracellularosmotic pressurescan inducea linear increaseby up to 100% and 30%, respectively,in the hydraulic resistanceof Nitellaflexilis membranes(56). A pressure-dependentmembranebehaviorhasalsobeenobservedin the leaf cells of the higherplant Elodea(Table1; 109),butwasfound in neitherTradescantianor Mesembryanthemum(seereferencesin105). Turgor- and concentration-dependentwater permeability might be ofprime importanceto plant cell waterrelations,but its molecularmechanismsremain unknown. This phenomenonseemsto be relatedto the polarity ofwatertransportobservedin certaincells,becausepolarity wasstrongerathightonicity (57), andsimilar mechanismsinvolving lipid membraneextensionorfolding, compression, or dehydrationhavebeenproposedto explain the twophenomena(reviewedin 103).Noneof theclonedplantor animalaquaporinshas beenreportedto be directly gatedby hydrostaticor osmotic pressuregradients.

THE INTEGRATED FUNCTIONOFAQUAPORINSINPLANTS

Studiesin animalsclearlyindicatea role for aquaporinsin renalfunctionsandmoregenerallyin epithelial fluidabsorption/secretion.In contrast,thefunctionof aquaporinsin red blood cells or as putative osmoreceptorsin the brainremainsunclear (54). The physiological and developmentalprocessesthatinvolve aquaporinfunction in plants can beinferred from the expression

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patternof theseproteins,but this providesno direct indication on their basiccellularfunction(s).For instance, water-stressregulatedaquaporinsmightcon-tribute to the toleranceof plants to drought or salinity, but it is not clearwhethertheyareinvolvedin adjusting theoverallLp of theplantto its growthcapacity(126), locally facilitate water mobilization toward critical cells andorgansduring drought (12), or help in preadaptingdessicatedtissuesto asuddenrehydrationstress after drought(75).

TranscellularandLong-DistanceWater Transport

In plants,most of the long-distancelongitudinal transportoccurs throughphloemandxylem vesselsthatposeno or poorly resistantmembranebarriersto waterflow. In contrast,the intensewaterflow that radially traversesrootsandleavesis mediatedthroughnonvascularliving tissues(7). The respectiverolesof the apoplasmic (acrosscell walls) andcell-to-cell pathsfollowed bywaterin thesetissuesarestill a matterof debateandmay well dependon thephysiological conditions,theorganor theplant speciesconsidered(Figure2;7, 104). Although it has been interpretedthat cell-to-cell water transportmostly occursby thesymplasmicpath(acrossplasmodesmata) (Figure2; 85,135), it hasnot beenexperimentallypossible to distinguish its contribution

Figure 2 Putative functions ofaquaporins inplantcells.

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from thatof the transcellular path (acrossmembranes). Aquaporins,in particu-lar thoseat theplasmamembrane(PIPs),mightplayamajorrole in controllingthelatter(12).However,cell typeswhere transmembrane waterflow might belimit ing remainto beclearly identified.In roots,theCasparianbandof endo-dermal cell walls is thought to createan impediment to radial water flowtowardthesteleandthevasculartissues(85,104).Thismeansthateitherin theroot cortexor in the endodermis,apoplasticwaterflow hasto enterthesym-plasm.Theexpressionof PIP1aquaporinshigherin theroot endodermisthanin the root cortex of Arabidopsis (A Schäffner,personalcommunication)ratherfits with the latter possibility. The xylem parenchymacells arealsoasite of high aquaporinexpression(Table 2) and surely play a key role incontrollingosmoticandhydrostaticwater movements.

Although integratedmodelsof watertransportin rootshavebeenproposed(85,104),adirectanswerto thesequestions willrequiretheprecisequantifica-tion of membranePf in the different root cell types.The combination of thecell androot pressureprobes,togetherwith theuseof mechanicallymodifiedrootsbring a first hint at thesequestions(5, 24,104).Theuseof plantsalteredin theexpressionor activity of root aquaporinswill behelpful to complementthese approaches.

Theblockadeof aquaporinsby mercuryhasrecentlybeentakenasa basisto investigatethe overall role of cellular membranesin long-distancewatertransport(10, 32, 69). Maggio & Joly (69) measuredpressure-inducedsapflows in excisedtomatorootsandfoundthattreatingtheserootswith 0.5mMHgCl2 induceda 57%decreasein their hydrostatic Lp, without anychangeinglobal K+ transport.Carvajal et al (10) demonstratedan effect of a lowerHgCl2 concentration(50 µM) on sapexudationin excisedwheatroots.Al -thoughtheir measurementsof osmotic Lp in rootsaremorequestionable thanthe hydrostaticLp measurementsperformedby others(69, 103), their resultssuggestthat the down-regulation of mercury-sensitivewater channelsmightaccountfor the decreasein root Lp observedupon nitrogen or phosphorusdeprivation.A simultaneousreduction inplasmamembranefluidity points to acomplementaryrole for lipids in regulatingmembranepermeability (10). Theosmoticextensionandshrinkageof peeledsunflowerhypocotylsegmentswasalso sloweddown after mercury treatment(32). Although mercury-inducedchangein Lp hasbeenidentified in thesethreeapproaches,it remainsunclearwhetheraquaporinswerethetargetof the inhibitor. Mercurymaytargetothermembranetransportproteinsor actasa metabolic poison, thusgreatly alteringlocal waterpotentialgradients. However,mercury inhibition couldbereversedin all casesby reducing agents,suggestingthat the blocker did not alterintratissueorganization.

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Cell Volume andOsmoregulation

The abundanceof aquaporinsin the tonoplastof different cell types(46, 67,70) andthe few reportson watertransportin vacuoles(58, 100,113,125; CMaurel, F Tacnet,J Güclü, J Guern& P Ripoche,unpublished manuscript),bothsuggestthatmostplantcellsexhibit a high tonoplastwaterpermeability.This mightprovidethecells witha reduced vacuolarresistanceto transcellularwaterflow (12).Theobservationthatin tobaccosuspensioncellsandin onionepidermalcells the tonoplastcanbe up to 100-fold morepermeableto waterthan theplasma membrane (Table 1)suggestsasecondrole for watertransportat the tonoplast(75). In manyplantcells,thevacuole(s)occupiesmostof theinterior, andthe cytoplasmis restrictedto a thin layer betweenthe tonoplastand the plasmamembrane(Figure 2). Nonlimiting water transportat thetonoplastmight allow plant cells to efficiently use their vacuolarspacetobuffer osmotic fluctuations occurring in the cytoplasm(75). This would, inparticular,avoid thecollapseor theswelling of the latter, in caseof a suddenosmoticchallengeoriginating from the extracellularspace.The clusteringofArabidopsisplasmamembraneaquaporins,in plasmalemmasomesthat pro-trude deepinto the vacuole(94), might also contributeto optimizing waterexchangebetweenthevacuoleandtheapoplast,with minor osmotic perturba-tion in the cytoplasm. All together,thesestudiesstronglysuggestan originalosmoregulatoryfunction of aquaporinsin plant cells directly relatedto thetypical compartmentation of these cells.

The role of cell Lp (watersupply) in controlling extensiongrowth is stillunclear(7, 104). A baselinewater permeabilityat the plasmamembraneofindividual elongating cells could be a priori sufficient,evenundera reducedwaterpotentialgradient,to accountfor theoverall flow of waterthatmediatestheir relatively slow expansion.Suchhydraulicresistancemight alsoexplainthedisequilibria in waterpotentialobservedin growingtissues(7).However,ahighLp hasbeenreportedfor someelongatingcellsandmightbenecessaryfora cell-to-cell supplyof water, from xylem to growing tissues(13, 104). Ac-cordingly, the plasmamembraneaquaporinPIP1bis preferentiallyexpressedin the elongatingcells of Arabidopsisroot and hypocotyl (48). The parallelexpressionof γ-TIP in thesecells (65)might reflect a criticalrole for tonoplastaquaporins during vegetative vacuole differentiation and expansion. Thiswould permit, in particular,water uptakeinto the vacuoleor transvacuolarwater flow inquasi-isoosmotic conditions.

Aquaporinsaresurely involved in someotheraspectsof plant cell turgorandvolume regulation:for instance,there-imbibition of dessicatedplantcellsthat canoccurduring seedgermination(73, 74) or afterexposureto drought.

AQUAPORINS 421

The observationthat pollen grain hydrationin Brassicais regulatedthroughprotein synthesisin the stigma and that this control possibly relatesto theself-incompatibility mechanisms (95) might alsosuggesta key role for aqua-porins in plant reproduction.Finally, plant infection by root knot nematodesleadsto theswelling of two adjacentroot cellsthatserveasnutrientreservoirsandfeedingsite for the parasites.The trans-induction of aquaporinpRB7 bythe pathogenin thesecells providesa striking exampleof the role that aqua-porinsmay playin adjustingmembrane Pf to cell size andwater demand(82).

In conclusion,aquaporinsseemto fulfill two main functionsin plants,inindividual cell osmoregulation,on theonehand,andin thecontrolof trans-cel-lular and-tissuewatertransport,on theotherhand.Thesetwo functionsappearto be intrinsically relatedto the dual presenceof theseproteins,respectively,on thetonoplastand the plasmamembrane(Figure 2).

PERSPECTIVES

Thediscoveryof aquaporinsin plantsandotherorganisms providesa molecu-lar basis for the passivepermeability of membranesto water. Beyond itsimmediate implications in molecularbiophysics, this discoveryhasraisedanovel emphasison the notion of hydraulicconductivity of plant membranes,cells, and tissuesandopensnew perspectivesto understandthe role and thecomplexityof this parameter.Two majorpoints havereceivedstrongmolecu-lar supportfrom themostrecent workon plantaquaporins.First, the hydraulicconductivity of membranesappearsto be crucially determinedat the spatiallevel in the plant. This control is exerteddown to the level of subcellularcompartmentsand maybeeven membranedomainsand determinesfundamen-tal featuresof cell organizationandpolarity. In particular,water transportinandthroughthe plant cell is intrinsically relatedto its compartmentationintovacuoleandcytoplasm. Thespatialcontrolof membranehydraulicconductiv-ity also contributesto determining water exchangebetweenadjacentcells.This is especiallyimportant because,due to plant tissueorganization,anysinglecell type hasto copewith specificandlocal waterpotentialgradients.Finally, aquaporinslikely participate in the complex integration of watermovementin the whole plant.For instance,the radial flow of water in rootsthat involvesdistinct parallelpathsconvergingtoward the vasculartissueofthesteleillustratestheexpectedcomplexity of this integration andcannow beaddressedin molecularterms.The secondidea that emergesis that a largevariety of regulatorymechanisms cannow be envisagedthat cancontrol theintensity of transmembranewater flow and its relative contribution to totalwatertransport.Thesemechanismsallow a dynamicadjustmentof membrane

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propertiesandappearto be integratedin mostdevelopmentalprograms,fromseed toflower, and ina varietyof physiological responsesto biotic and abioticstimuli. The discoveryof aquaporinshasdirectedour attentionto the impor-tanceof watertransportregulationin mostof theseprocesses.Conversely,theneedfor such an extendedspatial and temporalcontrol of water transportthroughplant membranesmay well providea basisto explain the wealthofaquaporinisoformsandassociatedregulatorymechanismsthat arebeingdis-covered.

Thedescriptionof asyetunidentified plantaquaporinsandtheirexpressionpatternswill surelyrevealnewphysiological situationsthat involve aquaporinfunction.Nevertheless,numerousmolecularprobesarealreadyavailablethatallow accurateaquaporinmRNA andproteinquantificationand localizationinplantcells.Thesetools, coupledto accuratewaterpermeabilitymeasurementsby means of the cell pressure probe or stopped-flow spectrophotometry,shouldenablethedevelopmentof novelcell biologicalapproachesfor investi-gating the variety of physiological and developmentalregulationsthat havealreadybeen identified in algal and higher plant cells. The most excitingdevelopmentsopenedby aquaporinsdiscoverymight yet be expectedin thefield of genetics.In humanbeings,individuals mutatedin the AQP-1 andAQP-2 geneshaveprovidedvaluableinsightsinto the physiological signifi-canceof theseaquaporins(19,71, 90). In plants,reversegeneticsusingantis-enseaquaporintransgenesseemsto beequallypromising (48) (R Kaldenhoff,personalcommunication).Thedevelopmentof othergeneticalstrategies,suchastransposonor T-DNA insertionmutagenesis, shouldallow a moreaccuratetargetingof aquaporingenes andfunctions.

In conclusion,aquaporins provide amolecular basisto thepassivetransportof wateracrossplant membranes.However,it remainscrucial to dissectthecomplementarymechanismsthat determinethedriving forcesresponsibleforwatertransportandbalanceat thedifferentorganizationlevelsof theplant.Weare just at the beginningof understanding the entire significanceof plantaquaporins,but thecharacterizationof theseproteinshasalreadyinseminatedoriginal physiological and theoreticalapproachesto the studyof plant waterrelations(10, 69,106).

ACKNOWLEDGMENTS

I thankJ Dainty, MJ Chrispeels,J Guern,andP Ripochefor discussions; MJChrispeels,R Kaldenhoff, X Sarda,and AR Schäffnerfor communicatingunpublished results; and H Barbier-Brygoo, MJ Chrispeels, D Geelen,JGuern, CLurin, and P Ripochefor critical reading ofthe manuscript.

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