Evidence for Symplastic Involvement in the Radial Movement of Calcium in Onion Roots

EvidenceforSymplasticInvolvementintheRadialMovementofCalciuminOnionRoots1

EwaCholewa2andCarolA.Peterson*

DepartmentofBiology,UniversityofWaterloo,Waterloo,Ontario,Canada,N2LG1(C.A.)

ThepathwayofCa21movementfromthesoilsolutionintothesteleoftherootisnotknownwithcertaintydespiteaconsiderablebodyofliteratureonthesubject.Doesthisioncrossanintact,matureexodermisandendodermis?Ifso,isitsmovementthroughtheselayersprimarilyapoplasticorsymplastic?Thesequestionswereaddressedusingonion(Alliumcepa)adventitiousrootslackinglaterals.RadioactiveCa21appliedtotheroottipwasnottransportedtotheremainderoftheplant,indicatingthatthisioncannotbesuppliedtotheshootthroughthisregionwheretheexodermisandendodermisareimmature.Amorematurezone,inwhichtheendodermalCasparianbandwaspresent,delivered2.67nmolofCa21mmÿ1treatedrootlengthdÿ1tothetranspirationstream,demonstratingthattheionhadmovedthroughanintactendodermis.Fartherfromtheroottip,athirdzoneinwhichCasparianbandswerepresentintheexodermisaswellastheendodermisdelivered0.87nmolCa21mmÿ1rootlengthdÿ1tothetranspirationstream,http://wendang.chazidian.compartmentalelutionanalysesindicatedthatCa21hadnotdiffusedthroughtheCasparianbandsoftheexodermis,andinhibitorstudiesusingLa31andvanadate(VO43ÿ)pointedtoamajorinvolvementofthesymplastintheradialtransportofCa21throughtheendodermis.Itwasconcludedthatinonionroots,theradialmovementofCa21throughtheexodermisandendodermisisprimarilysymplastic.

ThepathwaywherebyCa21movesradiallyfromthesoilsolutiontothetrachearyelementsofaroothaslongbeenasubjectofdebate(forreviews,seeClarkson,1991;White,2001;WhiteandBroadley,2003).Toreachtherootstele(thelocationofthetrachearyelements),anionmustpassthroughtheepidermis,exodermis(ifpresent),centralcortex,andendodermis.Becauseintypicalrootsthewallsoftheepidermisandcentralcortexcellsarepermeabletoions,thecurrentdebateconcernsthepassageofionsthroughtheendodermisandexodermis.

OneideaisthattheCa21deliveredtothexylemmaymovepredominantlyorentirelyapoplastically.Thishasbeenconsideredbyavarietyofauthors(White,1998;McLaughlinandWimmer,1999;Whiteetal.,2000;White,2001,WhiteandBroadley,2003).Insomestudies,theamountofCa21translocatedwasposi-tivelycorrelatedwiththetranspirationrate,aswould

¨,beexpectedifitsmovementwereapoplastic(Hylmo

1953;BellandBiddulph,1963;LazeroffandPitman,1966).ThelackofselectivitybetweenCa21,Ba21,andSr21uptakebyrootshasalsobeencitedasevidenceforthepresenceofanapoplasticradialpathwayforCa21(BowenandDymond,1956;White,2001).Theargu-ThisworkwassupportedbyaDiscoveryGrantfromtheNaturalSciencesandEngineeringResearchCouncilofCanadatoC.A.P.2

Presentaddress:BIOSAgriculture,2111LakeshoreRoad,P.O.Box187,Ste-Anne-de-Bellevue,QC,Canada,H9X3V9.

*Correspondingauthor;e-mailcpeterso@uwaterloo.ca;fax519–746–0614.

Article,publicationdate,http://wendang.chazidian.com/cgi/doi/10.1104/pp.103.035287.

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mentagainstthisviewisthatCasparianbandsoftheendodermisandexodermishaveprovedimpermeabletoseveralions(deRufzdeLavison,1910;Baker,1971;RobardsandRobb,1972;Peterson,1987;Petersonetal.,1993)includingCa21(SinghandJacobson,1977,Kuhn

¨cklingetal.,2002)withinthelimitsofetal.,2000,Bu

thetestsemployed.

AsecondideaisthattheCasparianbandsintheendodermisandexodermisarecircumventedbyasymplasticstep.Inthiscase,theoverallradialmovementofCa21tothestelewouldshowfeaturesofsymplastictransport.Insupportofthisidea,therearesomereportsthatCa21movementtotheshootisnotcorrelatedwiththespeedofthetranspirationstream(Atkinsonetal.,1992;JarvisandHouse,1970).DrewandBiddulph(1971)foundthatthepassageofCa21intothetranspirationstreamofbean(Phaseolusvulgaris)wasreducedbycoldtemperaturesandseveralmetabolicinhibitors.Also,whenthebathingsolutioncontainedarelativelylowconcentrationofCa21,theionwasconcentratedinthetranspirationstream.InacorrelativestudyofCa21movementintothesteleofmarrow(Cucurbitapepo)roots,Harrison-MurrayandClarkson(1973)foundthattheentryofCa21intothestelewasinverselycorrelatedwithsuberinlamelladevelopmentintheendodermis.TheirinterpretationoftheresultswasthatCa21movedthroughtheendodermissymplastically,enteringthecytoplasmbymeansofCa21channelslocatedinthemembranesliningtheoutertangentialwallsoftheendodermalcells,andexitingthecytoplasmbymeansofCa21-ATPaseslocatedonthemembranesliningtheinnertangentialwallsofthesecells.Suberinlamellaewouldinterferewiththesechangesof

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CholewaandPeterson

compartment,inhibitingCa21movementtothestele.(Forafulldiscussion,seeClarkson,1991.)Thecounter-argumenttoapathinvolvingasymplasticstep(s)isthataradial owofCa21wouldmakeitdif cultforthecytoplasmtomaintainitsnormallowconcentra-tionoftheion(FlowersandYeo,1992).However,Clarkson(1984)pointedoutthatappropriatecytosolicconcentrationscouldbemaintainedbyrapid uxesthroughtheendodermalcells.Afterreviewingtheevidenceonthesubject,WhiteandBroadley(2003)state‘‘AlthoughtherelativecontributionsoftheapoplasticandsymplasticpathwaystothedeliveryofCatothexylemareunknown,itislikelythatafunctionalseparationofapoplasticCa21 uxes(fortransfertotheshoot)andsymplasticCa21 uxes(forcellsignaling)wouldenabletheroottoful llthedemandoftheshootforCawithoutcompromisingintracellular[Ca21]cytsignals.’’

Athirdidea,onethatreconcilestheopposingviewsonthepathway(apoplasticversussymplastic)ofradialCa21movementthroughtheendodermis,isthatentryintothesteleoccursneartheroottipwheretheendodermisisimmature(FlowersandYeo,1992).AdditionalpointsofentryinmorematurezoneswouldbethroughdiscontinuitiesintheCasparianbandsassociatedwiththegrowthoflateralrootprimordia(Ferguson,1979;PetersonandMoon1993,WhiteandBroadley,2003).

Inthisstudy,thepassageofCa21fromthesoilsolutionintothetranspirationstreamofonion(Alliumcepa)rootswasstudiedwithaviewtoansweringtwoquestions:(1)Doesthisionmoveradiallythroughanintactexodermisandendodermis,oristheionprovidedtotheplantbytheroottip(aregiondevoidofCasparianbands)?(2)Iftheiondoespassthroughanintactexodermisand/orendodermis,isitspath-waypredominantlyapoplasticorsymplastic?Young,adventitiousrootssproutedfrombulbswerechosenforthisstudybecausetheserootslacklaterals,theformationandemergenceofwhichleadstodisconti-nuitiesintheCasparianbandsoftheendodermisandexodermis,respectively(Esau,1940;PetersonandMoon,1993).Thus,allareasexceptfortheroottiphaveacompleteCasparianbandintheendodermis,andtheexodermalCasparianbandisalsocompleteinolderrootregions.Toanswerthe rstquestion,45Ca21wasappliedtodiscretezonesofrootsandtheamountsmovedtothexylemwereestimatedfromradioactivityinallpartsoftheplantsproximaltothetreatedareas.Toanswerthesecondquestion,differentmethodsprovedappropriatefortheexodermisandendoder-mis.Fortheformer,thepermeabilityoftheCasparianbandtoCa21wastestedbycompartmentalelution,atechniquethatcanbeusedtomeasurethequantitiesofionsinvariouscompartmentsofplantcells(Cram,1968;Jeschke1982;Peterson1987;Maclonetal.,1990).ThepermeabilityoftheexodermalCasparianbandstoCa21wastestedbymeasuringtheamountoftheionelutedfromthecellwallcompartmentsof‘‘intact’’segmentswithsealedendsand‘‘dissected’’segments

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thathadbeenlongitudinallybisectedandhadtheirstelesremoved.IfexodermalCasparianbandsdonotrestricttheapoplasticdiffusionofCa21,theamountselutedfromthecellwallsinintactanddissectedsegmentsshouldbethesame.If,however,thesebandsdorestrictapoplasticdiffusion,theamountofCa21elutedfromthecellwallcompartmentintheintactsegmentswillbesubstantiallysmallerthanthatfromthedissectedones.Fortheendodermis,theeffectsofLa31(aninhibitorofCa21channels)andVO43ÿ(aninhibitorofCa21-ATPases)onthemovementofCa21tothesteleweretestedinasystemwhereexudatewascollectedfromexcisedroots.Iftheradialmovementisentirelythroughtheapoplast,theinhibitorswillhavenoeffectoniondeliverytothestele.However,ifmovementisproceedingwithasymplasticstep(whichnecessitatestheionmovingintoandoutoftheendodermalcellsthroughtheirmembranes),theamountofCa21inthesteleshouldbesubstantiallyreduced.ThiswillbetrueevenifmembraneeffectsotherthaninhibitionofCa21channelsandCa21-ATPasesareelicitedbytheinhibitors.

RESULTS

SitesofCa21UptakeandAxialTransportAlongOnionRoots

Adventitiousrootssproutedfromonionbulbsde-velopeduniformlywhengrowninthefavorableconditionsprovided.Suchrootswerewithoutlaterals,eitheremergedorintheformofprimordia.Therootsweredividedintothreeanatomicalzonesofinterestwithreferencetothematurationoftheendodermis,earlymetaxylemvessels,andexodermis(Fig.1).Thetipzonewastheapical5mmoftherootwherefunctionalxylemandCasparianbandswereabsent.Theyoungzone,15to40mmfromthetip,hadfunctionalxylemandCasparianbandsintheendo-dermis.Theoldzone,whichbegan100mmfromtheroottipandextendedtotherootbase,possessedfunctionalxylem,andCasparianbandsinallcellsandsuberinlamellaeinmostcellsofboththeendodermisandexodermis.Thus,passagecellswerepresentinbothofthelatterlayers.Fortheexperiments,thetiporthecentralregionsoftheyoungandoldzonesweresealedintoatreatmentchamber(Fig.2).

When1mM45Ca21wasappliedtothetipzone,nomeasurableradioactivitywasdetectedintherestoftheplant(TableI).Duringthe24htreatmenttime,theroottipshadgrownanaverageof3mm,whichresultedina nallengthof8mmbeingtreated.Insomecases,Casparianbandshadformedintheendo-dermisintheproximalpartofthetreatedzone,buttheearlymetaxylemwasalwaysimmature.Otherwise,theanatomyoftherelevantstructuresdidnotchangeduringtreatment.Whentheyoungzonewastreated,radioactivitywasdetectedintheplantpartsproximalbutnotdistaltothetreatedsite.Theamountof45Ca21extractedfromtheplantwasequivalenttoadelivery

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RadialMovementofCalciuminOnionRoots

atripleexponentialfunction(Fig.4)withaveragehalf-timesrangingfrom47to66s,5.6to7.5min,and102to210mincorrespondingtowall,cytoplasm,andvacuole,respectively(althoughtheidentityofthelastcompartmentisnotknownwithanycertainty;TableIIelutionat228C).TheamountofCa21inthewallcompartmentoftheintactsegments(2.0mmolgÿ1segmentfreshweight[fw])wassubstantiallysmallerthanthatinthedissectedsegments(8.8mmolgÿ1segmentfw),indicatingthattheexodermalCasparianbandsarenotfreelypermeabletotheion(TableII,Fig.3).Identi cationofthecellwallcompartment,criticalforthisstudy,wasvalidatedbyresultsofelutionsat48C.Heretheef uxfromthewallcompartmentwasunaffected(averagehalf-timeof63s),butthatfromthenext(cytoplasmic)compartmentcouldnotbedistin-guishedfromtheslowest(vacuolar)compartment(TableII).Thehalf-timeofelutionfromthelattercombinedcompartmentwasnotsigni cantlydifferentfromthatofthevacuoleat228Cineitherwholeordissectedsegments(TableII).

EffectofInhibitorsonCa21TransporttotheStele

Figure1.Medianlongitudinalsectionofanonionroot.Majordevelopmentallandmarksareindicated,aswellaszonesofinterestinthisstudy.Notdrawntoscale.AtoC,Crosssectionsofonionrootstainedwithberberine-anilineblueandviewedwithUVlight.A,Sectiontakenfromtheoldzoneoftheroot(150mmfromtip).Casparianbandsandsuberinlamellae(asterisks)arepresentintheexodermisandCasparianbands(arrowheads)http://wendang.chazidian.comtemetaxylemvesselsarematureandligni ed.B,Sectiontakenfromyoungzoneoftheroot(30mmfromroottip).Casparianbands(arrowheads)arepresentinendodermisonly.Earlymetaxylemvesselsaremature.C,Sectiontakenfromtipzoneoftheroot(5mmfromtip).NoCasparianbandsweredetected;xylemvesselsareimmature.Bars¼500m

m.

Intheyoungzoneoftheroot,treatmentwithinhibitorssubstantiallyreducedthe uxofCa21tothestele,asmeasuredbytheamountoftheionappearingintherootexudate.Additionof1mMLa31tothetreatmentchamber(Fig.5)resultedin73%lessCa21intherootexudatecomparedtothecontrol.Similarly,priortreatmentofthecellsinthestelewithVO43ÿfedintothetranspirationstreamresultedina73%inhibitionofCa21translocation(TableIII).Neitherinhibitoraffectedthevolumesofexudatesproducedbytheroots(Table

III).

tothetranspirationstreamof2.67nmolmmÿ1treatedrootlength(TableI).Treatmentoftheoldzoneresultedintransportof0.87nmolmmÿ1treatedrootlength(TableI).Aswasthecasefortheyoungzone,45Ca21movementfromtheoldzonewasonlyinanupwarddirection(i.e.towardtheshoot).Nopositivecorrela-tionbetweentheamountoftheiontransportedandtranspirationratewasevident(TableI).

ImpermeabilityofExodermalCasparianBandstoCa21

Thedatafromthecompartmentalelutionof45Ca21frombothintactanddissectedsegments(Fig.3) tted

Figure2.Sectionaldiagramoftheapparatususedtoapply45Ca21todiscretezonesofonionroot.Anentireonionwithitsnumerousadventitiousroots(onlytwoofwhichareshown)wasplacedinalargetraysothattheleavesprojectedintotheatmosphere.Onerootwassealedintoamulti-chamberedplexiglassbox.Flanking(f)andbordering(b)chamberswere lledwith1mMCaSO4,andthetreatmentchamber(t)withradioactiveCa21(seetext).Thecoverandsidesofthelargetraywerelinedwithwetpapertowels,andtheuntreatedrootswerecoveredwithmoistvermiculite(notshown).

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CholewaandPeterson

TableI.Translocationof45Ca21fromdevelopmentallydifferentzonesofonionroots,andtranspirationrates

DistancefromRootTipmm

Transpirationgwaterlostduring24h

CaTranslocationnmolmmÿ1treated

rootlength

0–8(tip)

15–30(young)[100(old)13.565.88.760.918.164.30.0

2.6760.74*0.8760.36*

Thetotallengthofroottip(8mm)attheendofexperimentwasconsideredincalculations.*,statisticallysigni cantdifferencesinthetwovaluesanalyzed(n¼4,6SD,Student’sttest,P¼0.06).

DISCUSSION

Onionprovedtobeanidealspeciesfortestingthepermeabilityoftheendo-andexodermistoCa21astherootsdidnotdeveloplateralsand,therefore,theCasparianbandsoftheselayers,onceformed,re-mainedintact.Moreover,theserootsdidnotdeveloproothairs,facilitatingacleardistinctionofthewallcompartmentincompartmentalelutionexperiments.

SitesofCa21UptakeandAxialTransportAlongOnionRoots

When45Ca21wasappliedtotheroottip,theonlylocationintherootnotinvestedwithatleastonecompleteCasparianband,nomeasurableamountwastransportedawayinthetranspirationstream.Perhapsthisisnotsurprisingasthiszonelackedmaturexylemvessels.Thegeneralapoplasticimpermeabilityofthe

¨ttgeandWeigl,1962;tipnotedinotherstudies(Lu

Rasmussen,1968;Wierzbicka,1986;EnstoneandPeterson,1992;CholewaandPeterson,2001)alsoappliestoCa21ionssincetherewasnodetectablemovementthroughparenchymatothepointwherethexylemelementswerefunctional.EnstoneandPeterson(1992)postulatedthattheapoplasticimper-meabilityoftherootapexmaybeaconsequenceofanabsenceofsuf cientlylargeintermicro brillarchan-nelsinthewallsofthecellstoallowtracermovement.Inviewofthefactthattheendodermismaturesclosertothetipthanthexylem(PetersonandLefcourt,1990),onewouldexpectthattheroottipwouldnotsupplyCa21tothetranspirationstream.Onthecontrary,Huangetal.(1993)foundthatCa21transportcouldoccurfromthetipzoneinwheat(Triticumaestivum).However,itisunknownwhetherornotsomeofthetrachearyelementswerematureinthetreatedzone.Inyoungzonesofonionroots(between15and40mmfromtheroottip),wheretheendodermisandearlymetaxylemvesselsweremature(Fig.1),radio-activeCa21movedtotheshoot.Infact,thelargestamountwastransportedfromthiszone(TableI).Inthisregion,nosuberinlamellaeareasyetdevelopedinendodermalcells(BarnabasandPeterson,1992)sothatallcellscomprisingthislayerwouldbeavailableforimportofCa21intothesymplastandexportagainonthestelesideoftheCasparianbands.Robardsetal.

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(1973)correlatedtheanatomyofbarley(Hordeumvulgare)rootswithCa21(using85Srasatracer)trans-porttothesteleandfoundthatthezoneofmaximumuptakewas20mmfromtheroottip.Asinonion,thisisinthezoneinbarleywheretheendodermisismaturebutsuberinlamellaehavenotyetformed.ThemaximumamountofCa21thatenteredthesteleinbarleywas0.13nmolmmÿ1treatedrootlength(calculatedfromRobardsetal.,1973,Fig.29;whereamaximumof260pmolofCa21mmÿ3rootwasreported,andassuming0.4mmrootradiusfromAlonietal.,1998,Fig.13).Thisismuchlessthantheaveragevalueof2.7nmolmmÿ1inonion.Clearly,movementthroughanintactendodermisinoniondidnotresultinlessiondeliverytothestelethaninbarleyrootswherelateralrootprimordiawerepresumablyde-veloping.

Inthemostmature(old)zoneoftheroot(greaterthan100mmfromtheroottip),Casparianbandswerepresentintheendodermisaswellasintheexodermis(Fig.1).DespitethepresenceoftwosetsofCasparianbandsontheradialpath,thiszonewascapableoftransferringCa21tothestele(TableI).Thisiondid,therefore,passthroughanintact,matureexo-andendodermis.Thelessef cientradialtransportintheoldzone(comparedtothatoftheyoungzone)couldbeaconsequenceofthepresenceofsuberinlamellaeinboththeendo-andexodermis,assimilarresultswerepreviouslyobtainedwithmarrowandbarley(Harrison-MurrayandClarkson,1973;Robardsetal.,1973;FergusonandClarkson,1976).

FromtheabovecomparisonofCa21transportthroughvariousrootzonesitisclearthat,inthisstudy,theiondoesnotenterthesteleapoplasticallyattheroottipwhereCasparianbandsarenotyetformed,norapoplasticallyinolderregionsthrough

discontinuities

Figure3.Crosssectionsofonionrootsusedforcompartmentalelution.ShadingindicateshypotheticalareasfromwhichCa21couldbeelutedfromthewalls.A,Sectionfromawholesegmentwithwax-cappedends.Elutionoccursthroughtheepidermis.Lefthalfofroot,assumingtheexodermalCasparianbandispermeabletoCa21;righthalfofroot,assumingtheexodermalCasparianbandisimpermeabletoCa21.B,Sectionfromadissectedsegment,i.e.onethathadbeenbisectedandhaditssteleremoved.Elutionoccursthroughtheepidermisandcortex.Forthediagram,itisassumedthatCa21isnotelutedfromtheanticlinalwallsoftheexodermis.

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RadialMovementofCalciuminOnionRoots

intheCasparianbandsoftheendodermisandexo-dermisbroughtaboutbylateralrootproduction.Rather,Ca21movedthroughanintactendodermisandexodermisonitswaytothestele.

Inallzonestested,therewasaconsistentlackofCa21transporttowardtheroottip,supportingthegeneralviewthatCa21isimmobileinthephloemandisnotcirculatedwithinplants(Marschner,1995).Mostpreviousreportsagreewiththeexclusivebasipetaltranslocationofthision,e.g.inonion(Maclon,1975)andmaize(Zeamays,MarschnerandRichter,1973).Butinonestudy(Huangetal.,1993),Ca21transporttowardthetipofwheatrootsanditsredistributiontoothernontreatedseminalrootswasdetected.

PathwayofCa21MovementThroughtheExodermis

Figure4.Graphedresultsfromonereplicateofatypicalelutionexperiment(wholesegments).BeginningwithgraphA,thetimesoftheelutionstepsontheabscissa(linearscale)areplottedagainstalog(ordinate)scaleofthedpmfromthe45Ca21thatwouldhavebeenpresentinthesegmentsatthetimesindicated.Thelineofbest twascalculatedforthelinearpartofthecurve.TheamountofCa21indicatedbytheinterceptofthelineontheordinateattime0(indicatingtheamountoftheioninthemostslowly-elutingcompartment)wassubtractedfromallhighervaluestoobtainthesetofdataplottedinB.Again,alineofbest ttothelinearportionofthecurvewasobtained,andthevalueattheordinate(theamountoftheioninthesecondmostslowly-elutingcompartment)wassubtractedfromtheremainingvaluestoproducedataforgraphC.IngraphC,thestraightlineofbest tincludesthepointontheordinate,indicatingthatthisisthefastest-elutingcompartmentoftheroot.Thehalf-timesofelutionfromthethreecompartmentsareobtainedfromtheslopesofthestraightlines.TheelutionofCa21fromonionrootsegmentswasresolvedintothreecompartments:A,vacuole(orotherslowlyelutingcompartment),B,cytoplasm,C,cellwall.

ThetechniqueofcompartmentalelutionwasusedtodiscernthepathofCa21asitmovedthroughthematureexodermis,alayerwithCasparianbandsneartherootsurface.Withthistechnique,itisimperativetoidentifythecompartmentofinterest(i.e.thewall)correctly.Inthisstudy,when45Ca21waselutedfromeitherintactordissectedsegments,threecompart-mentsweredistinguished.Thefastestelutingofthesewasidenti edasthecellwallbasedonacomparisonofitshalf-time(about1min)withliteraturedata(DrewandBiddulph,1971;Maclon,1975;Rygiewiczetal.,1984;Peterson,1987;Rauser,1987;Whiteetal.,1992;DiTomasoetal.,1993;Devienneetal.,1994;Kronzuckeretal.,1995a,1995b)anditslackofchangeduringelutionatcoldtemperature.Asurface lm,whichwouldhaveelutedmorerapidlythanthewallcompartment,wasnotevident.PerhapstheCa21insucha lmhadassociatedwiththewall.Thesecondfastestelutingcompartmentcouldbeunequivocallyidenti edasthecytoplasmbasedonitshalf-timeofelutionandextremesensitivitytocoldtemperature.Thethirdcompartmentmayhavebeenthevacuoleorsomeotherveryslowlyelutingcompartment.Thewallcompartmentwasidenti edwithcertainty,butitincludedboththewaterfreespaceandtheDonnanfreespace.MostoftheCa21(99.6%)inthiscompart-mentwasintheDonnanfreespace(calculationsnotshown).

Havingidenti edandcharacterizedthewallcom-partment,itwaspossibletotestthehypothesispresentedintheintroduction,namelythatasmalleramountofCa21elutedfromthewallsoftheintactsegmentswithsealedendscomparedtotheamountelutedfromthedissectedsegmentswouldindicatethattheCasparianbandisnotfreelypermeabletoCa21.(ItisnecessarytoassumethattheDonnanfreespaceoftheepidermalandouterexodermalwallsisthesameasthatofthewallsofthecentralcortex.)Theseresultswere,infact,obtainedinexperimentsrunat228Cand48C(TableII).TheamountsofCa21inthewallcompartmentsbasedonweight(asreportedinTableII)canbecorrectedforsamplevolumesbyconsideringthatthelengthoftheintact

segments

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