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Factors affecting absorption and transportofpotassium inmaizeroots

./ v. IN08201.720

W.G.Keltjens

W.G.Keltjens

Factors affecting absorption andtransport of potassium in maize roots

Proefschrift terverkrijgingvandegraadvan doctorindelandbouwwetenschappen, opgezagvanderectormagnificus, dr.H.C.vanderPlas, hoogleraarindeorganischescheikunde, inhetopenbaarteverdedigen opwoensdag26april1978 desnamiddagstevieruurindeaula vandeLandbouwhogeschoolteWageningen

Centre for Agricultural Wageningen - 1978

Publishing

and

Documentation

Abstract

Keltjens,W.G.(1978)Factorsaffectingabsorptionandtransportofpotas?«™ ono ^ J ° 0 t S - A g r i ° - R e s - R e P- ( V e " Llandbouwk.Onderz.)876, ISBN902200662X,(viii)+ ,03p.,60figs, 11tables,5diagrams,145 rets,Eng.andDutchsummaries. Also:Doctoralthesis,Wageningen. Beforeshort-termabsorptionexperiments,excised rootsofyoungmaize plantsweredifferently treatedtoalterpropertieslikemembranepermeabilityandconcentrationsoforganicandinorganiccompounds.Subsequently influxandeffluxofpotassium ionswereestimatedduring theinitialandthesteady-statephase. Inotherexperiments,absorptionandtranslocationofK +wereestimated simultaneouslym excisedrootsofyoungplantsand indecapitatedroots ofmaizeplants5weeksold. Absorption,accumulationandupwardtransportofpotassiuminmaize rootswerecloselylinked.Freshlyabsorbedpotassiumwasaccumulated »nH ,,t 7A iW l t h t i m e ' i n t e r ™ l saltconcentration,osmoticpressure andupwardxylemtransport (exudation)steadily increase.Duringthe wereIquatand T'fT* °f ^ ^ ^ t r a n s P ° « °** toaerialparts. n 0 t accum 1 » « ,-™ A- I ? > ate inmaizeroots.FreshlyabsorbedK + int n r r f f ? 1 t r a n S p ° r t e d u P w a r d s ° rexchangedwithK*alreadypresent beforPT,J;LC! a n t h e "tem P° ra rilyaccumulated inrootcellvacuoles oelorebeingtransported longitudinally. l o c a t i o n ^ 0 " ! r i E 6 '6 X C i S e d r 0 ° t S ' S a l t deficiency,absorption,translocation,accumulation,potassium ions,bleeding sap,composition.

Thisthesisw i n a l s o b e p u b U s h e d a g A g r i c u U u r a l ^ ^ ^

R e p o r t s 876>

©Centre forAgriculturalPublishingandDocumentation,Wageningen,.978. microfilmoranyother a means r w?^ dU ^ d ° rP u b l i s h e d " anyform,byprint,photoprint, =>witnoutwrittenpermissionfromthepublishers.

Stellingen

1.Hetbestuderenvandeionenopnamemetbehulpvanafgekniptewortels,geheelondergedompeldindeopnamevloeistof,isalleenjuistbijgebruikmakingvanisotopenen gedurendeeenperiodevanmaximaal6tot8uur,ongeachtdezoutstatusvanhetwortelmateriaal. Ditproefschrift.

2.Wortelpotentialen,gemetenopeenwijzezoalsbeschrevendoorHelmy,zijnnoch anatomisch,nochelektrocheraischteinterpreteren. A.K.Helmy,M.Tschapek,N.PeinemanandE.A.Ferreiro.PlantandSoil35: 549-553 (1971). 3.Snelhedenbiedenvoordelenbovendekumulatieveweergavevandeionenopnamedoor plantewortels. Ditproefschrift.

4.Eenkonklusiealszou,naeentoedieningvanDNP,eenafnameindehoeveelheidopgenomenzoutentijdensdeinitielealssteady-statefaseeropduidendatdeopname tijdensdesteady-statefasedirektafhankelijkisvanengestuurdwordtdoordeomvang vandeapparentfreespace (AFS)isonjuist. U.IgheandS.Pettersson.Physiol.Plant.30:24-29 (1974).

5.Vanwegehetsuspensieeffekt,zoalsweergegevendoorSchuffelenenLoosjes (1946), zijninternepHmetingeninplantecellen,uitgevoerdmetbehulpvaneenmicropHelektrode,onjuist. A.C.SchuffelenandR.Loosjes (1946).Koninkl.Akad.Wetenschap.,Proc.Ser.A, 49,80-86. 6.Dekritiekophetdoenvanplantevoedingsonderzoek zondergrondisongegrond.

7.Dekwaliteit(b.v.NO.-gehalte)vanophydrocultuur (teeltzonderaarde)gekweekte groentegewassendientextranauwlettendindegatengehoudenteworden.

8.Deionenbalans,gebaseerdopresultatenverkregenuitlangeduurbemestingsproeven, isgeengoedemaatterkarakteriseringvanopnamemechanismenvanzoutendoordeplant.

9.Metingrijpendewijzigingenindevochthuishoudingvande droogtegevoelige Nederlandse zandgronden,doormiddelvankunstmatigeberegening enprofielverbetering, dientbijhetopstellenvandelegendavanbodemkaartendiedienenalsbasisvoorde bodemgeschiktheidwaarderingraeerrekening teworden_gehouden. 10.Teneindedetemperatuurenmineralenhuishoudingvanhetwortelmediumvandeplant betertekunnenregulerenenoptimaliseren,zalbij eenverdere intensivering vande Nederlandseglastuinbouwdeteeltzonderaardeeensnelleuitbreidingondergaan. 11.Ooktenaanzienvanhetbeheervanlandschapsparken geldtdevoorde hoog-produktieve landbouwgeldendeleuze: 'Stilstand is achteruitgang'. 12.Bijeenjuridischeveroordeling totdeontzeggingvande rijbevoegdheid dientbij devaststellingvandetermijnvanontzegging rekening gehoudentewordenmet deburgerlijkestaatvandeveroordeelde.

Stellingenbehorendbijhetproefschriftvanir.W.G.Keltjens, getiteld 'Factorsaffectingabsorptionandtransportofpotassium inmaize roots' Wagenmgen,26april1978.

Woord vooraf

Allereerstwilikalienbedankendiebijgedragenhebbenaandetotstandkomingvan ditproefschrift. Helaasheeftwijlenprof.dr.A.C.Schuffelendedefinitieye afrondingvandit onderzoeknietmogenmeemaken.Aanzijninspirerendvoorbeeld endeinteressantediscussiesdieikmethemhebmogenvoeren,zalikaltijddierbareherinneringenbewaren. HooggeleerdeVervelde,hooggeachtepromotor,zeererkentelijkbenikuvoorde bereidheid omalsprcmotor tewillenoptreden.Zeerveeldankbenikuverschuldigd voor dewijzewaaropumijtijdensdeafrondingvanmijnonderzoekhebtbegeleid.Vooraluw waardevolleadviezenbijdeinterpretatievandeproefresultaten enuwkritische zinbij hetredigerenvandeteksthebbeneengroteinvloedgehadbijdetotstandkomingvandit proefschrift. • NaasteenwoordvandankaanallemedewerkersvanhetLaboratoriumvanLandbouwschelkundewil ikenkelemetnamenoemen. -JaapNelemans,jouwinzetenbelangstellingwasaltijdenormgroot.Dank zijjoukon ikaltijdbeschikkenovervoldoendemaiswortels;jouwanalytischehulpheb ikbijzonder opprijsgesteld. -Voordeanalyses inniet-gelabeldegewasmonstersdankikAdaHoogendijk enhaar medewerkers. -PietJansen,veeldankvoorjouwaandeel indeomslagvanditproefschrift. -Datdeadministratieheelwatmans is,bleekooknuweer;hetveletypewerkwerdmet veelinzetenbereidwilligheid doorKarindeKatenDinyEleveldverzorgd.DeheerMatser wist ineenzeerbeperkte tijdeengrootaantal figurentetekenen. Demedewerkingvanhet ITALindevormvaneensnelleenaccurate leveringvan radioaktieve isotopenvoorkwamongewenstevertraging,waarvoordankaandeheerDignum. Veeldankbenikverschuldigd aandemedewerkersvanhetPudoc,metnamedeheren Aalpol,vandenHeuvelenRigg. Marijke,albenjijpas tijdensde laatsteaktetentoneleverschenen,tochheb jij achterdecoulissenveelvoormijbetekend.

Curriculum vitae

Deauteurwerdop11juli1944teGrubbenvorst geboren.Nahetbehalenvanhet diplomaHBS-BaanhetR.K.LyceumteVenrayin1963,werdindatzelfde jaareenaanvang gemaaktmetdestudieaandeLandbouwhogeschool teWageningen. Nahetafleggenvanhetkandidaatsexamenwerdvanapriltotnovember1968de praktijktijddoorgebrachtaandePurdueUniversity,Indiana,USA.Injanuari 1971behaaldedeauteurhetdoctoraalexamenindebodemkundeenbemestingsleermetalskeuzevakkendealgemenebodemkundeenbemestingsleer,deregionalebodemkunde,delandbouwplantenteeltendeplantenfysiologie. Per1februarivandatjaartradhijalswetenschappelijk medewerkerindienstvan devakgroepbodemkundeenbemestingsleervandeLandbouwhogeschool enwerdeenaanvang gemaaktmethetpromotieonderzoek.Binnendezevakgroepgeefthijonderwijsenverricht hijonderzoekvooralophetgebiedvandeplantevoeding.

Contents

List of

abbreviations

1 Introduction

1

2 Literature

2

2.1 2.2 2.3 2.3.1 2.3.2

Absorptionofionsintheplantroot(cells),apassiveoractiveprocess? 2 Activeiontransportandenergy;theroleofadenosinetriphosphatase (ATPase)3 Radialandlongitudinalsaltandwatertransportinroots 4 Radialtransport Longitudinaltransport

4 5

3 Materials and methods 3.1 Growth 3.2 Experimentaltechnique 3.2.1 Uptakeexperiments 3.2.2 Transportexperiments 3.3 Chemicalanalysis 3.4 Presentationofresultsandstatisticalanalysis

6 6 6 6 8 9 9

4 Preliminary experiments 4.1 4.2 4.3

11

Short-termK/Rbsubstitutionexperimentswithexcisedroots SubstitutionofRbforKinlong-termexperimentswithintactplants Conclusions

5 Potassium uptake in excised roots 5.1 Timecourseofionabsorption 5.2 Factorsaffecting ionuptake 5.2.1 EffectofpHvalueoftheabsorptionsolution 5.2.2 EffectofthecalciumstatusandCasupplytotheroot 5.2.3 EffectoftheinternalK statusoftheroot 5.2.4 Effectofatreatmentwithvariousinorganicandorganicsaltsolutions 5.2.5 Surface-activechemicalsandionuptake 5.2.6 Metabolicinhibitorsandpotassiumuptake 5.2.7 Effectofglucosesupplytotherootsandofdurationoflightingplants 5.2.8 Temperatureoftheabsorptionsolutionandionuptake

11 13 15 17 17 19 19 27 32 34 41 47 50 56

6 Potassium transport through excised roots 6.1 Transportandaccumulationwithtime 6.2 EffectofKstatusoftheroot 6.3 Effectoftheanion 6.4 TransportandaccumulationofKasaffectedbytheexternalK concentration 6.5 Effectofglucose,CNandDNPonabsorptionandxylemtransportof potassium 6.6 EffectofpHoftheexternal mediumonpHofexudate

->9 60 63 69 73 77 81

7 Discussion 7.1 Accumulationofpotassiumintheroot 7.1.1 Phasesinthetimecourseoftheuptake 7.1.2 Uptakekinetics 7.1.2.1 Initialuptakeandapparent freespace 7.1.2.2 Steady-stateofionuptake 7.2 Transportmechanismofsaltinroots 7.3 Modelofsaltandwatertransport

83 83 83 85 85 86 90

91

Summary

94

Samenvatting

•96

References

98

List ofabbreviations

indiffusibleorrestrainedanion apparentfreespace adenosine triphosphate adenosine triphosphatase carbonylcyanidem-chlorophenylhydrazone citrate cyanide Donnanfreespace 2.4-dinitrophenol drymatter fumarate indiffusibleorrestrainedundissociatedweakacid chloride KionwithCIascounterion v K ionwithNO.ascounterion nitrate K ionwithSO,counterion sulphate 4 LSD leastsignificant difference M malate Mo malonate S.C. selectivitycoefficient Succ succinate WFS waterfreespace WSC watersolublecarbohydrates A AFS ATP ATPase CCCP Ci CN DFS DNP EMFum HA

Subscripts c i o v X

s

cytoplasm internal outerorexternal vacuole,exceptinJ ,wherevmeansvolume xylem saltorsolute

Fluxes

*oc CO

cv vc

inwardplasmalemmafluxorfluxbetweenoutersolutionandcytoplasm outwardplasmalemmafluxorfluxbetweencytoplasmandoutersolution inwardtonoplastfluxorfluxbetweencytoplasmandvacuole outwardtonoplastfluxorfluxbetweenvacuoleandcytoplasm

Inthetextionicspeciesareusuallyrepresentedbytheirchemicalsymbols,omitting chargesigns,e.g.KinsteadofK + .

1 Introduction

A fundamentalproblemofplantgrowthishowinorganic ionsenterrootcellsand thenmove throughtherootanduptotheshoot.Thefirststep,ionabsorption,has beenthetopicofmanystudiesbyagreatnumberofplantphysiologists (e.g.Epstein, 1955; Hodges,1973;Lundegardh&Burstrom,1933;Lycklama,1963;Pitman, 1975;WynJones, 1975).Inmostexperiments dealingwiththeuptakeoraccumulationofsaltsintheplant root,attentionisfocusedonprocessesofiontransportatacellular level,shortdistanceiontransport.Mechanisms regulatingionfluxesatplasmalemmaandtonoplast, theouterandinnermembraneofthecytoplasmrespectively,havebeenstudied extensively. Thedistributionofionsbetweenthetwocellcompartmentscytoplasmandvacuoleis oftenusedtoevaluatetheionabsorptionmechanismasawhole.Inordertostudydifferentaspectsoftheionabsorptionmechanism,theexternalmedium (e.g.saltconcentration,pH,temperature,CL-tension),aswellasfeaturesoftheroot (permeabilityand structureofmembranes,internal saltstatus,energy level)arevariedduringtheuptakeexperiments. Astheplantrootconsistsofdifferent tissues (epidermis,cortex,endodermis, stele),eachwithitsowncharacteristic cells,iontransportinplantrootswillnotbe restrictedtoaccumulationofsaltsinthecellcompartmentscytoplasmandvacuole.It alsoincludesthesymplasmaticiontransport fromcelltocellandsubsequently theupwardxylemtransporttoaerialparts.Therefore,themechanismofionabsorptionorion transportinplantrootswillbecomplexanddynamic. Asaconsequence,ionuptakestudies,frequently carriedoutwithunicellularorganisms,suchas Sitella andChora (MacRobbie,1973;Spanswick&Williams,1964;Vredenberg, 1971;Barber&Shieh, 1972),provideapoorrepresentationofthemineral relations ofplantrootsorcomplete intactplants. Absorptionandradialdisplacementofionsbytherootareonlythefirststepsin salt transportintheplant.Furtherstepsarethelongitudinalupwardxylemtransport, transportofsalts fromtherootsandsupplytoaerialpartsoftheplant.Both steps havemostlybeeninvestigated separately. Investigatorshaveeitherbeenengagedinabsorptionexperimentswithexcised rootsorinexudationexperimentswith decapitated root systems (Anderson,1975a;Ariszetal., 1951;Klepper,1967;Meiri, 1973).Forthis reason,itseemedrelevanttoinvestigatebothprocessessidebysideandtofindout whetheruptakewas relatedtoupwardsalttransportandwhetherbothprocessesare regulatedbyidenticalmechanisms. Inthisstudy,Kabsorptionwas studiedwithexcisedlowsaltmaizerootstoinvestigateandidentifythebehaviourofmaizerootsinrelationtoKabsorptionandKaccumulationprocesses.Subsequently,absorptionandexudationwasstudiedtoanalysesimultaneouslytheshort-distanceandlong-distance transportprocessesofKinthemaize root,andtocorrelateuptakeandupwardtransportofsalts. 1

2 Literature

Thischapterisashortreviewofliteraturedealingwithanumberofselect aspects ofionabsorptionandtransportintheplantroot(cell).SectionsOfChapter 5and6will refertorelevant literature inmoredetail.Comprehensive reviews onionuptake andion transporthavebeenpresentedbyBowling (1976),Brouwer (1965),Hodges (1973),Fried& Shapiro (1961),Higinbotham (1973),MacRobbie (1971),Pitman (1977). 2.1ABSORPTIONOFIONSINTHEPLANTROOT(CELLS),A PASSIVEORACTIVE PROCESS? According toHodges (1973),Luttge (1973),Nobel (1970),active iontransport isthe movementofionsagainsttheirelectrochemical gradient,whereas transportwillbepassive ifjlonsaremovingdowntheelectrochemical gradient.Possiblephysicaldriving forces in saltoriontransportare: 1.concentrationgradientsofthesaltsand ions involved, 2.electricalgradients,sincethemovingparticles carryanelectrical chargeandplant cellsshowarestingelectricalpotentialofabout 100mVormore,the interiorbeing negative (Clarkson,1974;Higinbotham etal., 1961;Pitmanetal., 1970). Hence,thepassiveoractivenatureofsalttransport canbedeterminedbyinvestigatingwhether'theionfluxesinvolvedobeytheNemst criterion,theGoldmanequation, ortheUssing-Teorellcriterion (Baker&Hall, 1975;Bowling, 1976;Nobel,1970),describingpassive iondistribution,orwhether theydeviate fromthese laws.Theoriginof theelectricalpotentialacrossmembranesisprobably aresult ofthreeprocesses,diffusion,absorptionbyfixedchargesandactiveelectrogenic transport.A diffusionpotentialcanarisefromadifference inmobility ofionsinamembraneorbydifferences in therelativepermeability ofamembranetovarious ions.Fixedcharges aredue todissociationoforganicmoleculesorcomplexesbeingheldwithin thecellenvelope.Electrogenictransport isanactivetransport inwhichanetchargeistransferred across a membraneattheexpenseofmetabolicenergy. • * Membranepotentials thereforeariseasthesumofthesethreeprocesses.Theelectrochemicalmembranepotential isbuiltupbyanenergy-independentcomponent,thediffusionpotential,aswellasbyanenergy-dependentpart,anelectrogenic component.This meansthattermslikeenergy-dependentandenergy-independentsalttransportarenot identicaltoactiveandpassive salttransport,respectively. . Comprehensive studiesoftheelectrochemical status6fions inplant tissues have beencarriedoutbyanumberofinvestigators.Higinbotham etal. (1967)found,inroots of Pisum sativum, thatallanions (Ci;S0 4 ,N 0 3 andH ^ ) weretransported andaccumulatedactively,thatisagainsttheelectrochemicalpotential gradient.Transport and accumulationofcationsprovedtobemorevariableandunclear.According to

Higinbothametal. (1967),thereisnoevidence foractive accumulationofCaandMg. ThebehaviourofNaandKisrathercomplex.According toEtherton (1963)sodium isactivelyextrudedbyrootcells,whereasothers (Shepherd&Bowling,1973)believe that rootsofsomeplantspeciesaccumulateNaactively,dependentontheexternalandinternalsodiumconcentrationoftherootcell. Thenatureofpotassium transport inplantcellsseemstobeevenmorevariable. Measurements ofactiveaccumulation (Pierce&Higinbotham, 1970),passive equilibrium (Higinbotham etal.,1967)andactiveextrusion (Etherton,1963)ofK inplantrootshave beenreportedinevidence.Jeschke (1970b),ontheotherhand,foundevidence forthe existence ofaK/Napump inwhichactiveeffluxofNaislinkedwithactiveKinflux. Altogether,onehastobecarefulwithstatements aboutactiveorpassivemovementsofions,because thenatureofthetransportprocessdependsonplantspecies,salt andenergy statusoftheroot,whiledataofelectricalmembranepotentials,measured in rootcellsofhigherplants,shouldbeinterpretedwithcaution. 2.2ACTIVE IONTRANSPORTANDENERGY;THEROLEOFADENOSINETRIPHOSPHATASE (ATPase) Respirationandphotosynthesis aregenerallyconsidered tobethemajorsourcesof metabolicenergy thatdrivesactive ionfluxes inplantcells.According toLtittge (1975), theenergy supply isnotspecific.Adenosine triphosphate (ATP)seemstodrive ion transport,irrespectiveofthenatureoftheATP-providingpartialreactionofenergy metabolism (e.g.oxidativephosphorylation,non-cyclicphotophosphorylation,cyclic photophosphorylation orevenglycolysis).As active iontransportmustbe directly coupledtoanenergy-releasing reactionandATP istheenergysourceforiontransport inroots,attemptshavebeenmadetofindoutwhethermembranesofplantcellspossess ATPaseactivityjust likeanimalcells.Hall (1969)proved thepresenceofATPase in plantcellmembranes.Kylin&Gee (1970)andLeonard&Hodges (1973)showed ion-stimulatedATPase activityinisolatedmembranesofoatrootsandinleavesofthemangrove. Moreover,Fischeretal.(1970)haveshownthatthecomponentoftheATPase,activated byKorRb,ishighly correlatedwithKandRbabsorptionbyrootsoffourplantspecies. AlthoughsomeevidenceforaconnextionbetweenATPaseactivityandsaltuptakehascome frompreviouswork,uptillnowstrictproofislackingofthepresenceofan ion-specific ATPaseactivity inplantcellmembranesor,ifpresent,ofalinkbetweenthisenzyme activity andactiveuptakeofrelatedcationsoranions. According toBowling (1976),active iontransportmaybebroughtaboutdirectlyby theATPaseactingasacarrier,butactive iontransportmayalsobebroughtaboutby carriersystemswhichareonlyindirectlyconnected totheATPase.Themembrane ATPasewouldhavenodirecttransportrole,butwould actonlybyproviding energy for active iontransport.ThislackofspecificityofATPase fordirectcation transport suggeststhattheATPaseisnotprimarilyacarrierofionsacross themembrane,but its mainroleistomakeenergyavailabletospecificcarriersystemsbyhydrolysis ofATP (Bowling, 1976).

2.3RADIALANDLONGITUDINALSALTANDWATERTRANSPORTINROOTS 2.3.1 Radial

transport

Besidesabsorption,radialtransportofionsthroughthedifferentroot tissues needstobeconsidered.Theassumption,madebyHouse&Findlay (1966)andSlatyer (1967) fortheosmoticflowofrootpressureexudation,thatonlyasinglemembrane system existswithintheroot,wouldseemanoversimplification.Amodifiedandimproved model,introducedbyCurran&Mcintosh (1962)andGinsburg&Ginzburg (1970)isbased onexistenceoftwomembranesinseries. Inpenetratingtherootcentripetally,theionspasstwotissues,theepidermisand thecortex.Althoughtheepidermisisonlyonelayerofcells,itcanfulfilanimportant roleintransportprocesses:inolderrootssuberizationoftheepidermis cells often leadstoformationofanidentifiableexodermisandalimitationofthetransportof waterandsalts.Thecortexoccupiesabout90*»oftherootvolumeinmaizeplants (Anderson,1975b).Therearetwoparallelpathways formovementofsaltandwateracross thecortex,oneistheextracellularspaceorapoplasmandthesecondisthesymplasm,the continuationofthecytoplasmofonecelltothenextbywayoftheplasmodesmata. Saltandwater,presentintheapoplasm,havenotyetpassedabiologicalmembrane; bothareabletomovefreelywithintheapoplasm inwardsasfarastheendodermis,but alsooutwardstotheoutersolution.Diffusionandmassflowwillbethedrivingforces, modifiedbyfactors limitingtransportratesofsaltsandwaterwithintheapoplasm. Aftertheapoplasm,saltsandwaterhavetopasstheplasmalemmabeforebeing takenup inthiscellularcytoplasmicstream.Thisplasmalemmaflux isheldtobethe rateoc limiting factorforcorticaltransport.Inthesymplasm,theplasmodesmatal transfer willbetherate-controllingstepforthesymplasmaticpartoftransport (Tyree, 1970). AccordingtotheworkofArisz (1956),thesymplastictransportissufficiently rapid toaccountforthemajority(upto90?«)ofcorticalsalttransport.Forwatermovement, ontheotherhand,itislikelythattheapoplasm,becauseofits highhydraulicconductivity,isthepreferentialpathwayacrossthecortex,ratherthanthesymplasm (Anderson,1975b). TheaspectofionexchangehasbeentreatedclearlybyHodges&Vaadia (1964). Transportofsalts,presentinthesymplasm,isnotsimpleandstraightforward,butis accompaniedbyatwo-wayexchangeofsymplasticandvacuolarsalt,expressedbythe saltfluxescvand^ . Dependentonthesalt statusoftherootcellsandtherateof symplasticsalttransport,thisexchangemechanismcanbepredominantorinsignificant. Thefirstrealbarrierinsaltand.watertransportwillbetheendodermis.The Casparianbandswillblockapoplasmatictransport.Soluteandwaterhavetoenterthe cytoplasmoftheendodermiscellsinordertopassthismonolayeralongsymplasmic pathways. Thesteleformsthenexttissueintherootcross-section.Beforejoiningthe xylemflow,bothsaltandwaterhavetopassthexylemparenchyma.Thewayandthe natureofthisstelartransportarestillobscure.Initially,theworkofCrafts&

Broyer (1938)assumedanoxygendeficiencywithinthestele,inducingaleakinessofthe parenchyma cellsforions. Ionscouldthenleakoutoftheparenchymacellsandbe transported fromonecelltothenextultimately intothexylemvessels.However,more recentworkdisprovesasituationofanaerobiosisandpassivetransportwithinthe stele.Respirationmeasurements (Halletal., 1971)andfeaturesofadualmechanismin isothermsforlong-distanceiontransport (Lauchli&Epstein,1971;Lauchli, 1972)prove a symplasticsalttransportintheparenchymacellsofthestele.Contrarytothis mechanism,Baker (1973)providesevidencethatthefinalpassage intothexylemvessels wouldbemorepassive. 2.3.2 Longitudinal

transport

Subsequenttothetransfer intothexylemvessels,waterandsaltsaretransported longitudinally;inthisway,aerialpartsoftheplantaresuppliedwithnutrientsand water.Theanatomicstructureofthepathwaysusedforthisvertical transportiscrucial. Longtubular cellswithperforated transversewalls (tracheids)formcontinuous tubes withintheplantthroughtherootandthestemupintotheleaves.Protoplastsofthese cells,activeduringtheearlyphasesoftheirformationdieaftercelldifferentiation; consequentlythefunctional transportchannelsaredeadandbelongtotheapoplasmic pathwaysofsaltandwatertransport.Inthisway,vertical transportofwaterand soluteswithinthexylemvesselsispassive. AccordingtoAnderson (1975a),ahydraulicandanosmoticcomponentareinvolvedin longitudinalwater flow.Transportofwatertotheaerialpartsoftheplantswill dependonrateoftranspirationbytheleaves (hydrauliccomponent)andsaltabsorption bytheroots (osmoticcomponent),upwardsalttransport,simultaneouswiththewater flow,isassumedtobethesumofaconvective (massflow)anddiffusive flow.Thus longitudinal transportofwaterandsaltswithinthexylemvesselsispassive.

3 Materials and methods

Thischapterdescribesstandard conditionsofplantgrowthandexperimental techniques.Modificationsinthesestandard conditions arementionedinChapters4,5and6. 3.1 GROWTH Foruptakeexperiments,seedsofmaize (Zea mays L.,cv. CIV2'Prior')weresown intraysfilledwithcoarsegravelandmoistenedwithdemineralizedwater.After germination,28traysweretransferredonacontainer,filledwith120litersofa CaSO,solutionof \ mmoll - 1 .Thesolutionwasmixedandaeratedbyanelectricpump (Slangen,1971).RootsoftheplantsgrownonthisCaS0 4solution (lowsalt roots) wereused10-15daysaftergerminationforthedifferentuptake experimentsandalso forTransportExperiments35,36,37and41.Forallothertransportexperiments, seedsofthesamemaizevarietyweregerminatedinquartzsandandmoistenedwith demineralizedwater.Oneweek aftergermination,seedlingswere transferredtoacontinuouslyaeratednutrientsolutionwithacompositionasshowninTable1.Oncea weekthenutrientsolutionwascompletely replenished;eachdaypHwas adjusted back to5.0withaHNO solution1mol1~.Fourweeksaftergerminationallplantswere transferredfromthecompletenutrientsolutiontoajmmol1 CaCH.PO) solution. Oneweek latertransportmeasurementswereperformedwiththeexcised rootsystemsof thesemaizeplants (exudationexperiments). Thelowandhighsaltplantsweregrowninaglasshousethroughout theyear. DuringOctober-April,anilluminanceof20000lxandaminimum temperatureof20 C wasguaranteedbyartificial lighting (HPLlamps)andheating,respectively. During summer,temperaturesoccasionallyreached30-35°C.Asaresultofdiurnaland seasonalfluctuationsintemperatureandlightintensity,plantsofsuccessive experimentsweresometimesdifferent. 3.2EXPERIMENTAL TECHNIQUE 3.2.1

Uptake experiments

Thefollowing typesofmeasurementswereperformedonplantsorplantorgans: 1.Influxmeasurements (atracermethod).Rootswereexcisedandrinsedindemineralized water.Dependentontheaimoftheexperiment,rootswereusedfortheinflux experiment directlyorafterfurthertreatment.Afterblottingtoremoveexcesswater,portionsof 10gfreshrootwereplacedincheesecloth 'teabags' (Epsteinetal., 1963)inavolume of500mlaeratedexperimental labelledsolutionsattheappropriate concentrationand

Table1.Compositionofthenutrientsolutionsinmmol1 .Traceelements (mg1 ): 0.5B,0.5Mn,0.05 Zn,0.02Cu,0.01 Mo,0.4FeasFe-EDTAand0.4FeasFeSO,. K NO, H2P04. SSO,

2.5 0.5

£Ca

^Mg

5.0 2.0

containinginaddition0.05mmol1 CaCl.. Inpreliminaryuptakeexperiments (Chapter 4), with

Rbwasusedeithersimultaneously

Kasatracerforpotassium (doublelabelling),orasatracerforRbinflux

measurements.Inuptakeandtransportexperiments,describedinChapters5and6, Rb, oC.

or

CI,

oo

Sand Na (RadiochemicalCentre,Amersham)wereusedasradioactivetracersfor

K,CI,SandNa,respectively.Themolaractivityatthebeginningoftheexperiment wasabout333MBqmol monovalentcationoranion.Thetemperatureofthesolution duringtheexperimentwasmaintainedbetween20and23°C.Allinfluxexperimentswere carriedoutatleastinduplicate.Whennecessary,pHwasadjustedbymeansofacid orbase.Experimentaltimewas 1-10h.Therateofioninfluxwasmeasuredeitherby: -depletion:atappropriatetimeintervals(t=0,15,...,600min),10mlaliquots ofthewell-mixedandlabelledexperimentalsolutionswerepipettedintocounting tubes.Attheendoftheabsorptionperiod,depletionoftheambientsolutionwas calculatedfromradioactivityofthedifferentsamples,orby -accumulation:theabsorptionperiodwasterminatedbydesorptionofexchangeablebound ions.Theteabag,containingtheroottissue,wasdroppedintoavolumeofabout 200mlofacold (4°C)unlabelledsolutioncontaining 10mmol 1~ KC1and 0.05mmol1

CaCl..Thistreatmentwasrepeatedfourtimesinsuccessivefresh

aliquotsofidenticalsolutions.Thefivedesorptionperiodstook3 x 5 , 15and 30min,respectively.Finally,thetissuewasrinsedtwicewithwaterfora totalrinsingtimeoffiveminutes.Afterthesetreatments,thefreshmaterialwas driedat70°Cfor24handweighed.Afterdigestionofthedryroottissue, ambientsolutionandthedigestedsampleswereanalysedbyliquidscintillation counting. 2.Influx/effluxmeasurements (atracermethod).Effluxwasmeasuredalwayssimultaneously withtheinflux.For12h,twoportionsIandIIofexcisedrootswereallowedto accumulateionsfromalabelledoranidenticalunlabelledsaltsolution,respectively. Subsequently,afterwashingtherootsfor10sindemineralizedwater,theportionsI andIIweretransferredeithertofreshidenticalunlabelledorlabelledexperimental solutionsandeffluxandinfluxweremeasuredduringthenext4-10h.Atappropriate times,10-mlaliquotswerepipettedoutoftheexperimentalsolutions.Arelease or depletionoflabelbytherootswasmeasuredbycountingofradioactivityofthesamples. 3.Netuptakemeasurements (continuoustitrationmethod).Netuptake (influx-efflux)of potassiumwasmeasuredbycontinuoustitration (Breteler,1973).Withanautomatic titrationequipment (Radiometer,Copenhagen)incombinationwithanion-specificK electrode (Philips),concentrationofK intheabsorptionsolutionwaskeptconstant for4h.Portionsof10goffreshlyexcisedlowsaltrootmaterialwereputin500ml

ofabsorptionsolutionatappropriateconcentrationofK.Thetemperatureofthe absorptionsolutionwasmaintainedat20°C.Onarecordersheet,theamountoftitrationsolution,apotassiumsaltsolution,thatwasusedtokeepKconstantwas recorded.Netuptakewascalculatedastheproductoftitrationrateandconcentration ofthetitrationsolution. 4.Saltaccumulationbyintactplants(long-termexperiments).Theuptaketechniques, aswellastheplantgrowthconditions,employedinpreliminarylongtermK-Rbuptake experimentswithintactplantsweredifferentandwillbediscussedinChapter4.

3.2.2 Transport

experiments

Thefollowingtypesofmeasurementswereperformedonplantsorplantorgans: 1.Vascularinfluxandeffluxbyexcisedlowsaltroots.Longitudinalxylemtransportofpotassiumwasmeasuredinexcisedrootsoflow-saltgypsumplants.Asshownin Diagram1,10excisedrootswerefixedwithparaffininaplasticcup.Onlythecutend oftheexcisedrootswasplacedintheuppercompartment (I);therootitselfwas immersedinthelowercompartment (II).FlowoftheuppersolutiontoCompartmentIIwas preventedbytheparaffin.ThevolumesofCompartmentsIandIIwere25and500ml, respectively.Bothcompartmentswerefilledwitha1mmol1 KC1solution;onlySolution IIwasaeratedcontinuouslyduringthefluxexperiments.Thetemperatureofbothsolutions was20-22°C.InvasculareffluxexperimentsonlythelowerSolutionIIwaslabelledwith Rb.Atappropriatetimes,samplesweretakenfrombothcompartments.Aftermeasurement ofradioactivity,potassiuminfluxandvasculareffluxoffreshlyabsorbedKwerecalculated.ForK(total)transport,measurementsof [K(total)] werecarriedoutinthe compartmentIsamples.Invascularinfluxexperiments,labellingwasthereverse. Samplesfrombothcompartmentswereanalysedandinfluxbythecutendoftheexcised rootswascalculated.Alltransportexperimentswereintriplicate. 2.Exudationexperimentswithcompleterootsystemswithcutstump.Forexudation experimentstopsofplantswereremovedbycuttingabout5cmabovethestembase. Therootswererinsedindemineralizedwaterandeachplantwasplacedin61of aeratedabsorptionsolutionat20-22°C.Arubbertubewasfastenedtothestump.

Diagram1.Experimentalarrangement formeasurementof vascularinfluxandeffluxofKinexcised low-saltmaize roots.Excisedrootswerefixedwithparaffinintheplasticuppercup (Compartment I),whilerootsthemselveswere immersedinthelowerCompartment II.Bothcompartments arefilledwithsolutionsofequalcomposition,withor withoutlabelling.

Exudateswerecollectedperiodically fromtherubbertubeswithapipetteandstored in adeep-freezer.At theendoftheexperimentweightandcompositionofallexudateswere measured. Ioninfluxandsubsequent transport offreshlyabsorbedsaltsweremeasured bydepletionandbyanalysis ofthebleeding sap.Insamplesofabsorptionsolutionand exudates,collected atappropriate timeintervals,radioactivitywasmeasured. For K(total)transport alsoK(total)concentration intheexudateswasanalysed. InExperiments39and40,absorption,accumulationandtransport isothermsofdifferent ionswere measuredwithoutradionuclides.Afterexperiment,rootswererinsed threetimesfor5 mineachindemineralizedwateranddriedfor24hat 70 C.Subsequently,cationsand anionswereanalysed intheexudates andindriedrootasdescribed inSection3.3. Totalsaltuptakewasassumedtobethesumofsaltaccumulationplussalttransport. Exudationexperimentswereatleast intriplicate;exudationwasmostly for24h. 3.3 CHEMICALANALYSIS Radioactivity insamplesofabsorptionsolutionsandexudateswerecountedwithout anyfurther treatment.Analysisoftherootsampleswasdoneafteradigestionofthe driedrootmaterial inconcentrated sulphuricacidandhydrogenperoxide.Counting of radioactivity (g-radiation)waswithanautomaticNuclearChicagoMark Iliquid Q/-

/O

00

/^

Scintillationcounter.For Kb, K, Naand Ca,Cerencovradiationwasmeasured. Tomeasure CIand S,scintillationsolutionwas added,e.g. amixture of 1,4-dioxane (800ml), 2-ethoxy-ethanol (160ml),naphthalene (48g)and 2,5-diphenyl-oxazole (9.6g). Ofthissolution,9mlwasmixedwith 1mlsample.As aresultofdouble labelling in Experiments 1and2,sampleswerecountedtwice.Immediately afterfinishing the influx experiment,thesum K+ Rbwasmeasured.After Kdecay (onemonth), Rbwas counted. Inorganicconstituents innon-radioactivesamplesofrootmaterial,absorption solutions andexudatesweremeasuredbythemethodofSlangen&Hoogendijk (1970). Potassium concentrations inradioactive sampleswereanalysedwithan ion-specific K-electrode (Philips). Inplantmaterial,Rbwasestimatedbyatomic absorption spectrophotometry.Organicconstituents likecarboxylatesandwater-soluble carbohydratesweremeasuredbythemethodofBreteler&Wittich (1973). 3.4PRESENTATIONOFRESULTSANDSTATISTICALANALYSIS Dataonuptakeandtranslocationexperimentsaremostly calculated onarate basisandpresented infiguresortables.Further: -Extremelyhighabsorptionratesduringtheinitialphaseofmostexperimentshad tobeleftout. -Meanratesofuptakeandtranslocationareplottedatthemidpointofeach measurementperiod.The lengthofthedifferentmeasurementperiodsisindicated in mostfiguresbyverticalblocks.Measurementperiodsareequalforthe different curvesinonefigureaswellasforcurvesincombinedfigures (A,B, . . . ) . -Duringperiodsoffastchangeinrates,forexample initially,drawing thecurve

rate

time

Diagram 2.Graphical representation of the rate of ion absorption versus time.Measurement periods are indicated by vertical blocks. • midpoints of the different measurement periods.

through themidpoints of the measurement periods would be wrong, since areas inside and outside theblocks have tobe equal (Diagram2 ) . The mathematical method to calculate the rate ofuptake and translocation as a differential of relevant incremental curves is not feasible in thiswork,because of the limitednumber of dataper experiment. In some experiments,results are checked for statistical significance by the Student's t test (Snedecor & Cochran, 1967). In tables,data that differ significantly from the control data aremarked with a single (P= 0.05) or a double (P= 0.01) asterisk.To check statistical significance of data presented graphically, least significant differences (LSD) (Snedecor & Cochran, 1967) are placed above the curves. These LSDvalues (P= 0.05) are only calculated for influx, efflux, transport during the steady-state phase of the experiment or, ifnot reached, for the last measuremental period.

10

4 Preliminary experiments

Becauseofthefastdecayof K (2\=12h ) ,themorestableisotope Rb 42 (T, =18d)isfrequentlyusedasaphysiologicalsubstitutefor Kinpotassiumabsorptionandtransportexperiments.Althoughliteratureonthissubjectisquiteextensive (Marschner&Schimansky,1971;Mesbahuletal.,1971;Schimansky,1970),thereisno clearindicationthatapotassium-rubidiumsubstitutionisfullyjustifiedunderallcircumstances.BecauseofthenegativeresultsofJeschke (1970a)andWest&Pitman (1967), afewexploratoryshort-termKinfluxexperimentsweredonetoinvestigate: K- RbsubstitionwithexcisedmaizerootslowandhighinsaltandatdifferentK concentrationsoftheabsorptionsolution; -KandRbinfluxbyexcisedhighandlowsaltmaizeroots; -theK/Rbselectivityinuptakeduringalong-termexperimentwithintactmaizeplants. 4.1 SHORT-TERMK/RbSUBSTITUTIONEXPERIMENTSWITHEXCISEDROOTS

Experiment 1: 86 Rb as a tracer for potassium influx measurements. Influx from a 0.1 mmol I KCl solution in excised low salt roots, estimated from depletion. Figure 1showsthatafter15-30min(initialphase),theratesofpotassiuminflux, measuredwith

Kand Rbdonotdiffersignificantly.Obviously,theselowsaltmaize

rootsdonotdiscriminatebetween4 2 Kand Rbasatracerforpotassium,atleastata potassiumconcentrationof0.1mmoll - intheabsorptionsolution.

mmolKkg^DMh"1 200

LSD=3,83

4h

Fig. 1.Experiment 1.InfluxofKinto excisedlow-saltmaizerootsfroman absorptionsolutionwithKClatsubstanceconcentration0.1mmoll - 1 , estimatedwith4 2 K (x)and86Rb (•) astracersforK.

11

Toinvestigatetheeffectoftheexternalpotassiumconcentration,thenextexperimentwasstarted.

Experiment 2: 86Rb as a tracer for potassium influx measurements with excised low salt roots. Influx from KCl absorption solutions 0.1, 0.5, 1.0, 5.0 and 10.0rnmol I , for 4 h, estimated from accumulation. Bothinfluxisothermsofthelowsaltrootmaterial (Fig.2A)showagoodagreement inthelowerconcentrationrangeoftheabsorptionisotherm.AtKClconcentrationsof 5.0mmol1 _1andmore,86 Rbgivessignificantly lowerKinfluxthandoes

Ktracer.

InFigure2B,bothisothermsarepresentedformaizerootsrichinK.Rootsofplants, grown8dbeforetheinfluxexperimentonacompletenutrientsolution (Table1)were investigated.ExceptwiththehighestexternalconcentrationofKCl (10mmol1 ) , preloadingoftherootswithpotassiumdepressedpotassiuminflux,measuredwiththe tracers4 2 Kand86 Rb.Thisequalinhibitionofboth4 2 K and86 Rbinfluxwithanincreased internalcellularconcentrationindicatesanidenticalbehaviourofKandRbintheplant cell,atleastundertheseconditions.

mmolKkg"'DMh"1 100n

80-

60

40-

20

0V 30-i

20-

10'

0-lV-

0.1

12

0.5

1.0

5.0 10.0 mmolKI"

Fig.2.Experiment2.InfluxofKinexcisedmaizerootsatdifferentexternal concentrationsofKCl.A.Low-saltroots. B.Roots loadedwithK.InfluxofKwas estimatedwithboth4 2 K (x)and86 Rb (•) astracers.

Experiment 3: Influx of K and Rb in excised roots low and high in salt from a. 0.1 mmol 1 KCl and RbCl solution, respectively. K and Rb influx were both measured by depletion with the tracers K and Rb, respectively. The time courses oftheinflux forKandRbindicate, that -forroots richinK,also theRbinfluxwasreduced significantly (Fig. 3A,B); -forlowsalt roots,therateofRbabsorption dropped significantly after about 2-3h, whereasK influx reached steady-state after about 1h (Fig. 3 A ) . Under equal experimental conditions,potassium absorption froma0.1mmol 1 solutionofKClismuch higher thantheRbuptake fromaRbCl solutionofequal concentration. However,theresults inExperiment 2suggest thatatextremely highK/Rbratioin ftfi the absorption solution ( Rbonlyasa tracer),maize root doesnotdiscriminate betweenKandRb. Obviously,theK/Rbmolar selectivity coefficient approaches unity only at high substance ratiosofKtoRbintheabsorption solution. 4.2 SUBSTITUTIONOFRbFORKINLONG-TERM EXPERIMENTS WITH INTACT PLANTS

Selectivity andtheroleofRbasaphysiological substitute forpotassiumwas investigated inlong-term absorption experiments with intact plants.After germination, 5 maize plants were placedon500mlofawell aerated nutrient solutionwhich,in addition toKandRb(aschlorides) contained thefollowing salts inmmol 1 :

mmolkg-'DMh"1 200-

LSD=3,38

80i

40-

—i

Ah

Fig. 3.Experiment3.A.InfluxofK (x)andRb (A)toexcisedlow-salt maizerootsfrom0.1 mmoll - 'solutionsofKClandRbCl,labelledwith ^ 2 Kand86 Rb,respectively.B.Influx ofRbtoexcisedK-preloadedmaize rootsfrom0.1 mmoll - 'RbClabsorptionsolution,labelledwith 86 Rb.

13

1 Ca(NCL)„,0.5Mg(NO),1NaHPO, andFeandtrace elements (Table 1 ) .Inthese experiments,theconcentrationso fKandR bandtheir ratio were varied. Fresh nutrientsolutions were supplied daily. After 12d,theexperiments were terminated. Theplants were harvested,androotsandshoots separated. Plant materialw a sdried, weighed and, after digestion, KandRbwere estimatedb yflame photometry andatomic absorption spectrophotometry, respectively.T h eselectivity coefficient, K/Rb

S.C.

K/Rb

K/Rb

intheplant intheabsorption solution

was used tocharacterise theselectivity orpreferenceo fthemaize plant forKandRb at varying ratiosintheexternal solution. Thus values greater than 1.0would indicate a preference forK,whereas values smaller than 1.0would indicate thereverse. Experiment 4: Selectivity

in uptake of K and Rb (accumulation)

for 12 days on a complete nutrient a. K and Rb at respective 0.0, 2.0 mmol I

concentrations

(K-substitution

b. K and Rb at respective 2.0, 2.0 mmol I

solution

maize

of 2.0, 0; 1.5, 0.5; 1.0, 1.0; 0.5, of 2.0, 0; 2.0, 0.5; 2.0, 1.0; 2.0,

series).

mmol/5 plants x 2.4-

2.0'

1.6

1.2-

0.8-

0.4

o-Jo: 0.0mmolK[-1 2.0mmolR bI"1 K/Rb Fig. 4.Experiment 4.AccumulationofK (x)andRb(o)inintact maize plants (samplesof 5 plants), grown for12doncomplete nutrient solutions with different K andRbconcentrations (K-substitution series). 14

1.5;

series).

concentrations

(Rb-addition

by intact

with:

1.5;

plants

AccumulationcurvesforKandRbinFigure4showthatasubstitutionofRbforK decreasedaccumulationofKandsimultaneouslyenhancedaccumulationofRb.Withinthe rangeK/Rb2.0/0.0-1.0/1.0,thereductioninaccumulationofKwascompensatedbyan equalincreaseinRbaccumulation.Thesum [K+Rb] accumulatedbytheplantsisalmost constantandtheselectivitycoefficientdoesnotsignificantlydifferfromunity.At substanceratiosof0.5/1.5orless,plantgrowthwasinhibited (Table 2),andselectivitywaschanged.AsS.C. , withinthisrangebecamegreaterthanunitytheplant prefersK;thereductionindrymatterproductionatK/RbratiosRb>K>Na>Liispreferred,andastheelec-

Table2.Drymatter (DM)productionfrom5plantsandselectivity coefficientofKover Rb(S.C.K/Rb)i nintactmaizeplants,grownfor 12donaseriesofnutrientsolutions. DMproductionand S.C.^/gj,werestatistically tested,withDMofthezero-rubidium treatmentandS.C.K/Rb= 1ascontrols,respectively.Experiment4. Rb- addition series

K-substitut ionseries e(mmol1 K

Rb

2.0 0.0 1.5 0.5 1.0 1.0 0.5 1.5 0.0 2.0

')

DM

S-C

-K/Rb

(g/5plants) 1.85 1.92 1.62 1.45* 0.91**

0.97 0.95 1.23**

c(mmol1

')

DM

K

Rb

(g/5plants)

2.0 2.0 2.0 2.0 2.0

0.0 0.5 1.0 1.5 2.0

1.68 2.01 1.70 1.82 1.59

S

- C- K/Rb

1.02 0.98 0.95 0.99

15

mmol/5 plants x 1A

2.0H

1.6

1.2-

0.8-

0.4-

2.0 mmol KI' 2.0 mmol RbI' S C

- -K/Rb

Fig.5.Experiment4.AccumulationofK (x)andRb (o)inintactmaizeplants (samples of5plants),grownfor12doncompletenutrientsolutionswithconstantKbutincreasingRbconcentrations (Rb-addition series).

triefieldstrengthofthenegativesiteincreases,thespecificityofionbinding progressivelyshiftsathighelectricfieldstrengthtothesequenceLi>Na>K>Rb> Cs.Thus,ashiftinselectivitywithanincreaseinelectricalfield,inducedbyan increaseinexternalionconcentrationisinagreementwithpredictionsofHodges (1973),

16

5 Potassium uptake in excised roots

5.1TIMECOURSEOFIONABSORPTION

Intheliterature,biphasicabsorption-timecurveshavebeenpresentedbymany investigators.Afterarapiduptakeofsaltsduringthefirstperiod,theinitialphase, therateofabsorptionbecomesconstantduringasubsequentphaseofsteady-stateuptake. However,potassiumabsorptionmeasuredbycontinuoustitration(Section3.2) showed,inadditiontothesetwophases,alsoatransitionphase.Itseemstobeastep betweenthephaseofinitialandthephaseofsteadystateuptake. Suchanuptake-timecurve,preciselyandcontinuouslyrecorded,wasobtainedin Experiment5.

Experiment 5: Rate of potassium absorption in excised low salt maize roots during absorption for 90 min. Absorption from a solution with KCl at concentration 5 rnmolI was measured continuously by titration. Figure6showsthreephasesduringthe1.5hoursofabsorption.Justasreported byHelleretal. (1973)andLiittge&Pallaghy (1972),afterafirstshortperiodof rapiduptake,therateofpotassiumuptakewasextremelylowduringasecondphaseof about15min.Duringthethirdphase,uptakebecamelinearwithtime. Thequestionariseswhetherthissecondphaseinthethreephasicpatternof

90min

Fig.6.Experiment5.Rateofpotassiumabsorptionintoexcisedlow-saltmaizeroots duringa90-minabsorptionperiod.AbsorptionsolutionwithKClatsubstanceconcentrationrnmol 5 1 _ I ;absorptionofKwas measuredbytitration.

17

potassiumuptakeisarealtransitionalphasebetweenaprocessofinitialuptakeand asubsequentprocessofsteady-stateuptake.Atransitioncouldimplythatatthe beginningofabsorptiononlytheinitialuptakeorfillingofapparentfreespace (AFS) exists,whereasaftercompletionofthisprocess,thesteady-statephasestartsafter about15min(lagphase).Inthethreephasicabsorption-timecurve,thesecondphase canthenbeexplainedasatransitionbetweenthetwoprocessesofionuptake.

Experiment 6: Potassium absorption in excised maize roots low in salt from an absorption solution with KCl at concentration 1 mmol I . Absorption was calculated after 0, 5, IS, SO, 60, 120 and 240 min by depletion and accumulation. Rubidium-86 was used as a tracer for potassium. Attheendofabsorption,therootsweretreatedinanunlabelledsolutionofKCl Rfi

—1

atconcentration10mmol1 for1htoremovethepotassium ( Rb)fromtheAFS (Fig.7).Thefractionretainedbytheroot-tissuewascalledFractionBandequalsthe potassiumaccumulatedintheroot.Afterabout 1h,thefillingoftheAFSseemedto becompleted andsubsequentKabsorptiononlyaddstoFractionB. Fromthebeginningoftheabsorptionperiod,theprocessofaccumulationperhapsexists andconsequentlytheinitialuptakeissuperimposedonanaccumulation.Figure8also provesthattherateofpotassiumaccumulationissimilartotherateofKabsorption foundinExperiment5bytitration.Aphaseofrapidaccumulationduringthefirst15-20 minwasfollowedbyashortperiodoflittleornoaccumulationandathirdstageof steady-stateuptake. Thusuptakeofpotassiumshowsathree-phasicabsorption-timecurveandnotatwophasicpattern.Ionaccumulationstartsimmediatelyafterthebeginningofabsorption

mmolKkg-1DM 120T

•AA-B

Fig.7.Experiment6.Timecurves ofabsorption (A),accumulation (B)andpotassiumpresentinthe apparentfreespace (A-B)in excised low-saltmaizeroots.AbsorptionandaccumulationofK froma 1mmol l -1KClabsorption solutionwerecalculated fromdepletionandaccumulation,respectively,with 86 Rb.

mmolKkg"1DMh""1 24

20H

16'

12H

m 4h

Fig. 8.Experiment6.RateofaccumulationofKinexcisedlow-saltmaize rootsover4h.Absorptionsolution withKC1 1mmoll-'.Accumulationof Kwascalculated fromaccumulationof 86 Rb.

andproceedssimultaneouslywithinitialuptake.Thethree-phasicmodelisshownnot onlyinthedepletioncurve,butalsoincurvesofaccumulation. Thissecondphaseinthethreephasicmodelisnotatransitionalstagebetweeninitial andsteady-stateuptake.

5.2 FACTORSAFFECTING IONUPTAKE

S.2.1 Effect of pH value of the absorption

solution

SoilpHaffectsgrowthandionuptakeofplantsmainlyindirectlybyachangein nutrientavailability,microbialactivityorsoilstructure.However,thepatternsof saltuptakebyplantsgrowingonanutrientsolutiondependonpHtoo.Suchadirect effectofpHontheabsorptionofsaltshasbeendemonstratedfordifferentcations andanions (Jacobsonetal.,1957;Lycklama,1963;Rainsetal.,1964;Tromp,1962). AsmentionedbyRainsetal.(1964)thispHeffect,consistingofareductionincation uptakewithdecreasingexternalpH,canbecausedeitherbyinjurytotherootcellsor bycompetitionbetweencations. ThereisevidencethatH +maycauseageneralderangementof,ordamageto,the ionabsorptionmechanism.OnetypeofinjuryatlowpHcouldbedenaturationof proteins,nucleicacids,phospholipidsandotherpolymersinvolvedinmembranestructureandfunctions.Asecondcouldbereductionincalciumuptakebytheplantatlow pH (Arnonetal.,1942;Pala,1975).Calciumdeficiencyinplantcellsresultsin disintegrationofcellwalls,lossofintegrityofcellularmembranesandconsequently inachangedpermeabilityofcellularmembranesforelectrolytes (Albrecht,1968; Waisel,1962).Sohydrogenioninteractswithcalcium. Accordingtothecompetitionmechanism,pHwillnotaffectcellularwallsand

19

membranes;achangeinpermeabilityorenhancedleakageofsaltswillnotoccur.At lowpHoftheexternalsolution,therateofabsorptionofpotassiumorcationsin generalwillonlybereducedbycompetitionbetweenH andthesubstratecationsfor availablecarriersites. EffectsofpHwerestudiedwithexcisedlowsaltrootsduringshort-termabsorption.SpecialattentionwaspaidtotheeffectofexternalpHoninitialionuptakeand onsubsequentsimultaneousinfluxandeffluxofsaltsintheroot.

Experiment 7: Potassium influx

in excised maize roots low -in salt for 10 h, at —1 absorption

a low (2.0), medium (5.0) and high pH (7.6) of the 1.0 mmolK I QQ

solution.

K influx was estimated from depletion

with

Rb.

Theinfluxdataofpotassium (Fig.9)confirmthedataofJacobsonetal. (1960). Insteady-state,theinfluxofpotassiumatapHof2.0wassignificantlylessthanat pH5,whileafurtherincreaseinexternalpHfrom5.0to7.6doesnotalterpotassium -2 influxfurther.Onlyatrelativelyhighhydrogenionconcentrations (10 mmol1 or

-1

more),thisionisinvolvedincationabsorption.Atlowerconcentrations,concentration ofH issolowthatanyfurtherdecreasedoesnotaffectpotassiuminflux.ToinvestigatewhetherthispHeffectisaninjuryoracompetitioneffect,asimultaneous influx-effluxexperimentwassetup.IfpHisactivebyinjuryofthewallsand membranesofrootcells,alowpHoftheexternalsolutionwouldprobablyincreaseefflux (leakage)ofcytoplasmicorvacuolarpotassium.

Experiment 8: Simultaneous influx

and efflux

of potassium in excised low salt maize Off

roots. Flux measurements for 10 h by the standard method with Kb as tracer. The -1, 7.6, 5.5, and 2.0 pH of absorption solutions (1 mmolK I ) were

mmolKkg"1DMh"1

LSD=5.23

9 20

h

Fig.9.Experiment7.Influxofpotassiumin excisedlow-saltmaizerootsover10hfrom a1.0mmol1~1Kabsorptionsolution,ata lowpH(2.0;A ) ,mediumpH(5.0;x)andhigh pH (7.6; o ) .

Figure10confirmsthatsubstancefluxofKinsteady-statewashalvedatpH2. However,influxcurvesinFigure10Aincombinationwithcorrespondingeffluxcurvesin Figure10BshowthattheinhibitioninpotassiuminfluxatpH2wasnotaresultofan enhancedeffluxoffreshlyabsorbedpotassium.Allthreeeffluxcurvesdonotdiffer significantlyover10h.EvenanextremelylowpHoftheexperimentalsolution (pH2) didnotchangeordestroyrootcellmembranestosuchextentthatsaltsalreadypresent intheserootcellswouldleakoutimmediatelyafterthemaizerootshadbeentransferredtothisextremelyacidmedium.Soduringtheseshort-termexperiments (10h ) ,pH effectsarenotduetomembraneinjury.Probablythehydrogenionisonlyactiveby competition.EspeciallyathighH (1mmol1 andhigher), theconcentrationsof hydrogenionandsubstratecationarealmostequalandcationcompetitionisprobable. Effectsofinjury,directorindirect,probablybuilduponlyonthelongterm. DataofExperiment7(Table3)showthatvaluesforKaccumulationintheAFS duringtheinitialphaseofionabsorption,obtainedbygraphicalanalysis,werehigher athighpHof7.6,thanwiththemediumandlowpHoftheexternalsolution. ThisaspecthasbeeninvestigatedmoreextensivelytocheckwhetherpHmight regulatesteady-stateKuptakebyachangeininitialuptake.

^ -1 -1 mmolKkg DMh

12 LSD=1,18

4-

x

x u —

o

A 1 ~

-A—=;

LSD=0,80

0\

9 h

Fig. 10.Experiment8.Potassiumfluxesinexcisedlow-saltmaizerootsover 10hfroma 1.0mmolK1"'absorptionsolutionatalow pH (2.0;A),medium (5.5;x)andhighpH (7.6;o), A.Influx.B.Efflux.

21

Table3.EffectofthepHoftheexternalmediumonKaccumulated initially intheAFS andonKinfluxduringthesubsequentsteady-statephase.Statistically testedwithpH 5.0ascontrol.Experiment7. pH

2.0 5.0 7.6

Kaccumulated inAFS

Steady-stateKinflux

(mmolkg

(mmolkg"'DMh~')

109.8 111.4 129.6*

DM)

1 .41* 5.62 6.57

Experiment 9: Initial uptake of K and Cl in excised roots low in salt from a 2 mmolI-1 KCl absorption solution with a low (2.8) and high pH (6.5). Influx of K and Cl were measured by depletion, using Rb and CI as tracers. Before the absorption experiment, intact plants were grown for 48 h on a h mmol I CaSO solution with a low and high pH of 2.8 and 6.5, respectively. CumulativecurvesfortheinitialKandClabsorption,presentedinFigure 11, provethat -atlowpHoftheexternal solution,potassium accumulationintheAFSisonly about 403,ofthatathighpH(Fig. 11C); -atlowpH,initialaccumulationofcationsisalmostequaltothatofanions;this contrastswiththeratioathighexternalpH(Fig. 11A.B); -accumulationofClisalmostequalatlowandhighpHoftheexternal solution (Fig. 11D). Thesefindingssuggestthattheinitialabsorptionofcationsandanionsisadual process,e.g.aprocessofpuremassflowofsolventplus solutes intothewater free space (WFS)andasecondprocessofsaltfluxandaccumulation intoaDonnan free space (DFS),controlledbyphysicochemicalforces.AccordingtoBriggsetal. (1961) andNobel (1970),fixedcarboxylgroupsatcellwallsurfacesorrestrained indiffusible anions,suchasdissociatedorganicacidsoraminoacidswithinthecytoplasm,are responsibleforthesephysico-chemical forcesandthesubsequent accumulationofcations anddepletionofanionswithinthisDFS.Ifso,thesecondcomponentoftheinitial cationabsorptionmechanismwillbedependentonpH,whileanionabsorptionduringthe initialphasewillberestrictedtothemassflowcomponentandthuswillbealmost independentofpH. Theregioncontainingthechargedsites,suchascarboxylgroupsinthecellwall, isfrequently referredtoastheDonnanphase.Atequilibrium,aDonnandistributionof oppositelychargedions,electrostatically attractedtotheimmobilecharges,occurs betweentheDonnanphaseandtheadjacentaqueousone,asdescribedbyNobel (1970)and byBolt&Bruggenwert (1976)foradsorptionofsaltsontoclayminerals.Accordingto Nobel (1970),Donnanphasesalsooccurincytoplasm,whereimmobilechargesareoften duetoproteinsandorganicacids.Theseorganiccompoundsarefixedinthesensethatthey cannotdiffuseacrosseithertheplasmalemmaorthetonoplast. Theconcentrationofthesecarboxylgroups,proteinsororganicacidsinplant cell compartmentswillnotaloneberesponsibleforthetotalnegativechargewithinthe Donnanphase,butalsothedegreeofdissociationoftheseorganiccompounds.Asboth

22

® , mmol kg"1 DM

® 1 mmol kg 'DM

12(H

yj)

mmolKkg"DM

1

mmolCIkg DM

/OM

120-

80-

°/°

40-

Fig. 11.Experiment9.Initialabsorptionof K (o,»)andCI(A,A)inexcised low-saltmaize rootsfroma2mmoll-'KC1absorptionsolutionoflowpH (2.8)andhighpH (6.5),indicatedwithclosedandopensymbols,respectively.

/>*" 'A-

1 I 00 30 60 min

0 30 60 min

pK (Kisdissociationconstant)oftheorganiccompoundandpHoftheDonnanspace willdeterminethedegreeofdissociation,thewholeDonnansystemwillbecharacterized bytheconcentrationoftheimmobileorrestrainedorganiccompounds [HA],theexternal concentrationofinorganicsalt,pHandpX. TocheckwhetherdatagatheredinExperiment9resembleaDonnandistribution, anidealDonnanequilibriumwascalculatedforconcentrationsofthehypothetical non-diffusibleorganiccompoundHAandoftheexternalinorganicsalt (KC1);pHofthe externalmediumandpXofthehypotheticalnon-diffusiblecompoundswerevaried (Diagram3). Underequilibriumconditions

[ClT!

[A"

IK£J

IHTJ

(D

(electricalneutrality)and

tcr] [CIT]

[K!]

" [¥]

[H^

~

(2)

[H+]

1 o o (equalionratios;Briggs,1961).FromEquations1and2,

23

Diagram3.TheschemeofanidealDonnanmodel;anexternalandinternalcompartment, separatedbyamembraneorcellwall,indiffusibleanions(A-)andanunivalentcation andanion,e.g.KC1. Concentrationsatequilibrium sideo(externalsolution)

sidei

[K + ]= a=1mmoll"1

[A']

(internalsolution)

0

+

[H ]=b

[HA]

0

[ci~]= (a+b)= o

a =[A]+[HA]=100mmoll"1

[Ht]

volume«oo

[KT] 1

[ciT] volume indefinite phaseboundary (membrane,cellwall)

[H + ]

[a]

be

[ClT]

[HT

[Ht] 1

and

I

[Ht] [K*]

(3)

[H+] o

a [Ht]

For a, b and a, see Diagram 3. Substitution of Equation 3 in 1 gives: ,+i2 «[Ht] bo + [A~][Ht] By d e f i n i t i o n : [A"] [Ht] =

1HAJ—

[A~]

[Ht]

a - [A~]

or

24

,+l2 [Ht

(43

[A~

Ka

(5)

[H*]+ FromEquations 4and5:

K l 3 (f

[H+]2(f + *)- [Ht] (to-**)+ l )+

Kbo - 0

Thistrinomial equationfor [H.] hasbeensolved forfixedvaluesof a and [K ](100 -1

_i

1

ramol1 and 1mmol1 ,respectively).pXvaluesofthehypothetical acidandpHvalues oftheexternalsolutionwerevaried. DatainFigure 12showthatloweringthepHoftheexternalrootmedium from7to 2resultsinafastdecreaseof [A~]atbothpXvaluesoftheweakacidHA.AtapHof 2, [A]isreducedtoalmost zero.Consequently,anaccumulationofcationswithinthe DFS,asshownathighpH,doesnotoccuratthislowpH (Fig.13).Concentrationcurves fortheanionwithintheDFSshowthereverse;onlyatexternalpHvalues 3

1 li iral

Fig. 26.Experiment 16.Uptakeofpotassium (•) and chloride (gl)inexcisedrootsofmaizeplantsafter treatmentofintactplantsfor96honsolutionsof Ca(N03>2,CaCl2andCaSC>45mmol1~'.Absorptionsolutionwasasolution 1mmoll - 1 KC1.UptakeofK andCIbyrootstreatedwithCaS04 takenasreference (100).

Kconcentrationsbelow1mmol1'(System 1),potassiuminfluxwasmeasuredalsoata higherconcentrationoftheabsorptionsolution (System2 ) . Experiment 17: Potassium influx in excised maize roots after a treatment as in Experiment 15. Influx of K from a 20 mmol l~ KCl solution was measured over 4 h by accumulation with ov86 Rbr DataofKuptake (Table 5), showthat: -Athighexternalsaltconcentrations,influxofKwasmarkedlyaffectedbytheuptake rateofthecounterion.AnenrichmentwithCI (Table 6 ) , depressedinfluxofCI,and reducedinfluxofK. -AtreatmentwithCa(N0) aidnotenhanceinfluxofKathighexternalsaltconcentrations.Thisisincontrasttotheobservationswith lowsaltconcentrationsinthe absorptionsolution. Experiments 15-17showthatcontrarytolowexternalsaltconcentrations,high externalconcentrationstendtomakecationuptakesignificantlydependentontheuptake ofanion.ThisconfirmstheideaofEpsteinandHagen (1952)andHiatt (1968)ofthe existenceofatleastadualisothermofcationuptake;ananion-independentonefor lowexternalconcentrationsandananion-dependentoneforhighexternal concentrations ofK.Theexistenceofamultiphasemodeloftheuptakeisotherm,aspostulatedby Nissen (1973),canbeneitherdisproved,norconfirmedbytheseexperiments. Intheprecedingexperiments,theanioneffectmaybemixedupwithbothacarboxylateandaprotein/aminoacideffect.Therefore,inthenextexperimentsnotonlymore, butalsopurecarboxylateandnitrogeneffectsareintroduced. Experiment 18: Influx of potassium in excised maize roots after a treatment of the intact gypsum plants for different times on a 5 mmol l" solution of CaSO^, Ca(NOs)2, (NH4)2S04 or urea. Influx of K from a 1 mmol I solution of KCl was measured over 4 h by depletion with Rb.

38

Table5.RelativeuptakeofKinexcisedmaize roots after treatmentofintact plants for96hondifferent enrichment solutions.Absorptionmediumisa20mmol l - 'KC1 solution.Absorption timeis4h;CaSO, treatmentascontrol.Experiment17. Enrichment solution

RelativeKuptake

substance (mmol1 ) CaCl, S Ca(N0O, 5 CaSO/2 5 4

70 98 100 _ _

-

Table6.Content CI, Na,SO,andN inmaize roots after treatmentofthe intact plants for96hondifferent enrichment solutions.Experiment17. Enrichment solution

Content (mmolkg DM)

substance (mmol1')

CI

NO3

S0.4

Norg

CaCl, 5 CaNOa), 5 CaSO 2 5 "4

382 17 48 702 54 405 62 1230 82 7 104 813

Table 7.RelativeKuptakeinexcised maize roots after treatmentofiftact plantsfor 96hondifferent enrichment solutions.Absorption medium is1mmol1 KC1 solution. Absorption timeis4h. CaS0 4 treatmentascontrol.Experiment 18. Enrichment solution

Relative K uptake

substance (mmol1 ) CaSO, 5 Ca(N0 3 ), 5

0™4)K5 Urea

5

100 1°*

g 85

•

Table7presents potassiumuptake forroots treatedondifferent containing nitrogen solutionswithaCaS0 A solutionascontrol.Ifthestimulating effectofC a ( N 0 3 ) 2 ,as showninExperiments 15and 16,isapure nitrogen effect,andactsbyenhancementof internal cellularproteinoramino acid content, treatmentwithNcompounds suchas M A andurea should stimulate influxofK.However, influx datainTable7disprove this assumption. Afteratreatmentoftheplantswith N H 4orurea, influxofKwassignificantly less thanwith CaSO,ofCa(N0 3 ) 2 . Thepositive effectofC a ( N 0 3 ) 2 cannotbeanitrogen effectingeneralbutacarboxylate effect inducedbyaconsiderable assimilationofNO3 (reduction),orelseapositive effectofnitrogenisexceededbyanegative effectof m . . Figure27demonstrates theeffectoftheN 0 3 andtheN H ,treatmentwith different timesoftreatmentorincubation.TheNO3andNH A curves differ almost immediately after thebeginningoftreatment,while after about 150hnofurther divergence takesplace. A further resolutionofthedifferent effectsoninfluxofKwasattempted m the next experiment. 39

10-

40

80

120

160

200

240 h

Fig. 27.Experiment 18.Potassium absorption intoexcised rootsof maizeplantsover4hafteratreatmentofintactplantsfordifferent times (0-220h)onsolutionsof Ca(N0 3 ) 2 (x)and (NH4)2S04 (o) 5mmol1~1.Absorptionsolutionwas asolution 1mmol l -1KC1.

Experiment 19: Potassium influx in excised maize roots after treating the intact gypsum plants for 96 h on solutions with substance concentration of glutamic acid 10, aspartic acid 10, succinic acid 10, CaSO^5 and CaiflO^g 5 mmol l~ . Ihe first three solutions were adjusted to pH 5.5 with NaOH; to other solutions, NaClwas added to obtain equal concentrations of Na. Influx of K in excised roots was measured as in Experiment 18. Theresults (Table8)indicatethatsuccinatedidnotsignificantlyalter influx ofKcomparedwiththeCaSO,ascontrol.Similarly,Breteler (1975)andDijkshoorn (1973) foundnopositiveorevennegativeeffectsofsuccinateonuptakeofNH,orKbyexcised maizerootsgrownonaCaSO,medium.Thisexperimentconfirmsthelackofeffectof succinatewithlowsaltroots.Thereasonforthisineffectivenessmaybethehigher internalcontentofcarbohydratesintheserootsthaninhighsaltroots.Treatment withaminoacidsgivesasimilarincreaseinKinfluxtoatreatmentwithaCa(N0j) 2 solution.Thus,unliketreatmentswithsuccinate,NH,orurea,incubationwithamino acidsstimulatedinflux.Thisleadstothefollowingconclusions: 1.TheabsenceofanysuccinateeffectoninfluxofKprovesthepositive Ca(N0 3 ) 2 effectnottobetheresultofanincreasedcarboxylatecontent. 2.Sincetreatmentwithoneoftheaminoacidsasparticacidorglutamicacidaffected subsequentKinfluxpositively,itisprobablethat -aminoacidsandproteinsaffecttheuptakecapacityoftherootsdirectly; -nitratestimulatesinfluxofKbyenhancedproductionofaminoacidsorproteins. 3.ThenegativeeffectofNH^orureaoninfluxofKcanbeexplainedbytheroleof NH 4asinhibitoronthesubsequentinfluxofK(cationcompetition).Thisinhibitionmust exceedthepositiveeffectofastimulatedaminoacidorproteinproductionassuggested

40

Table8.RelativeKuptakeinexcisedmaizerootsaftertreatmentoftheintactplants for96hondifferent solutions.Solutionswithaminoacidsandorganicacidswere broughttopH5.5withNaOH;toCaSO^andCaCNO^^solutionsequivalentamountsofsodiumwereaddedasNaCl.Absorptionsolutionisa 1.0mmoll -1KC1solution(CaSO, treatmentascontrol). Treatmentsolution substance

(mmol1

glut,acid aspar.acid succ.acid CaSO^ Ca(N0^)?

10 10 10 5 5

RelativeKuptake

_1

) 116*

101 100 119*

before. 4.Thefact thatthepositiveeffectofNO. wasfoundonlyatlowexternalconcentrationsofKsuggests thatthisinfluenceisactiveonlyatlowexternal concentrations (System1). 5.2.5

Surface-active

chemicals and ion uptake

AsreportedbyKuiper (1967)andNewman&Kramer (1966),thepermeabilityof cellularmembranescanbemodifiedbysurface-activeorganicchemicals.Accordingto theirfindings,permeabilityofcellularmembranesincreasedforsolventaswellas forsolutesaftertreatmentofplanttissuewithcertainconcentrationsofthesechemicals. Sincethepermeabilityofamembranegovernsuptakeofsingleionsandrelativeuptake ofions (ionselectivity),itsometimesmaybeusefultoalterthepermeability features ofamembrane (permeability coefficientP)toincreasefluxofwaterandsaltswithin thetissue.AsdemonstratedbyKuiper (1967),organiccompoundswithahydrocarbon chain,carbonamidesofdecenylsuccinic acid,acetylatedcompoundsandcertainfluorinated compounds increasetheelectrolytepermeabilityofplantrootssignificantly.However anincreaseinpermeabilityofplantrootsbytheseorganicchemicals,measuredbyan enhancedelectrolyte effluxorleakage,doesnotnecessarily stimulateinwardfluxof salts.Achangedpermeabilityofrootsystemstowaterandsaltswillprobablyresult inapositivenetinward saltfluxifthechemicaldoesnotseriously injuretissue, andonlyaltersthestructureorconfigurationofthemembrane.AccordingtoAriens& Simonis (1976),thiscanbecausedby -changesinpermeabilityofpores (size,polarity) -changesinpermeabilityofthelipiddouble-layer.Incorporationofthemoleculeof theorganicchemicalintothelipid layerofthemembraneordissolutionofhydrophobic orhydrophilicpartsofthelipiddouble layercanprobablyalterthepermeabilityof themembraneforpolarandapolarcompounds. Thebehaviourofoneoftheeffectiveacetylatedcompoundsglyceryl triacetate (Kuiper,1967)willbeexamined.Tocheckwhetherthissurface-activechemicalaffects influxoreffluxofthedifferentionsandwhetheritalterstheselectivityofthe membranefordifferentionstoo,anumberoffluxexperimentsweresetup. 41

Experiment 20: Potassium influx in excised maize roots low in salt after a treatment of the excised roots for 12 h in CaCl? solutions with concentrations of 0, 10~6, 10~4, 10~3, 10~2 or 10 mol l~ glyceryl triacetate (triacetin). Influx of K from a 1 mmol l~ KCl solution was measured for 6 h by depletion and accumulation with fib. WithCaCl 2andtriacetinfor12h,therewasasignificanteffectoftheconcentration ofthesurface-activechemicalontheincreaseinelectricalconductivityofthe -4 incubationsolution (Fig. 28).Atconcentrationsoftriacetingreaterthan10 mol1 , —2

-1

— 1

conductivityincreasedmarkedly,butatconcentrationsgreaterthan10 mol1 ,there wasnofurtherincreaseinconductivity. InfluxofKdecreasedafter increasingconcentrationoftriacetin (Fig.29).EspeciallyKaccumulatedanalogouslytoconductivity oftheincubationsolution (Fig.28).Atconcentrations exceeding 10~ mol1 ,triacetin reducedaccumulationofKdrasticallydowntonearlynilataconcentrationof10 mol 1 .InfluxofKcalculatedfromdepletiondroppedwith increasingconcentrationof triacetin,butmaintainedasignificantlyhighervaluethanindicatedbyaccumulation. Figure29correspondswiththetimecoursesofinfluxpresentedinFigure 30,which indicatesthatonlythecontrolandtreatmentat10~ mol1~ hadanormal influx,but higherconcentrationsoftriacetindiffered.Afterrapid initial influxofK,absorption declinedveryfastandevenreversed,sothatafterincubationoftheexcisedrootsin triacetinsolutionswithconcentrationsexceeding 10 mol1 ,rootswere 'filledup 1 withpotassiumduringtheinitialphaseofthesubsequentKabsorptionexperiment. However,duringthenextphaseofabsorption,nonetaccumulationbutaneteffluxor ionexcretionOccurred.Obviously,afteranincubationinconcentrated triacetinsolutions, membranepermeabilitychangedinsuchawaythatsaltcouldnotaccumulate.After

_2

A conductivity

|Ln. 200H

160-

12OH

80H 40

0i410-

42

10"

10-3

10'-2 10" triacetin mol l"

Fig. 28.Experiment20.Increasein conductivity (Ayfl)oftheincubation solutionafter 12hincubationofexcised low-saltmaizeroots.Incubation solutionsweresolutions0.5mmol1"' CaCl2withdifferenttriacetinconcentrations.

triacetin mol1"

Fig.29.Experiment20.Potassium absorptioninexcisedlow-saltmaize rootsafterincubationfor 12hin asolution0.5mmol1~'CaCl,with differentconcentrationsoftriacetin.AbsorptionsolutionhadKC1at substanceconcentration 1mmol1~1. Absorptionwascalculatedfromdepletion (x)andaccumulation (+).•

mmolKkg"1DMh"1 28i A

Fig.30.Experiment20.Potassium influxinexcisedlow-saltmaize rootsfor6h.Beforetheabsorptionexperiment,intactmaize plantsweretreatedonaCaCl2 solution0.5mmoll -1withtriacetinatsubstanceconcentrations 0 (x), 10"5 (A), 10-4 (o),io-3 (A), 10 - 2 (•) and I0~! (+)mol1 . AbsorptionsolutionhadKC1at1 mmol 1 " .

43

incubationintriacetin,membranepermeability allowedsaltsalreadypresentinthe roottoleakoutmucheasierandfaster;anenhanced influxofKaftertreatmentwith thesurface-activechemicalisoutofthequestion. Theeffectofincubationtimewas investigatedinthenextexperiment. Experiment 21: Potassium influx in excised maize roots low in salt after a —2 —1 treatment of the excised roots in a 10 mol I triacetin solution for 03 1, 3, 5 —2 and 12 h. Influx of Kwas measured also with untreated roots, but with 10 mol l~ triacetin in the absorption solution. Influx of K was measured from a 1 mmol I KCl solution over 6 h by depletion and accumulation. Figure31confirmstheeffectofthischemicalonthepermeabilityoftherootcell membranes.Aftertreatmentfor1h,electricalconductivityoftheincubationsolution increasedsignificantlyoverthecontrolina0.05mmol1~ CaSO,solution.Thecurves alsodemonstratedthatduring12htherootcellmembranesbecamemoreandmorepermeable tosalts. Increasingincubationtimecausedpotassium influxtofall (Fig.32),asshownboth bydepletionandaccumulation.Obviously,withincreasingtimeoftriacetintreatment, rootcellmembranesbecomemorepermeable;however,thisincreaseinpermeability facilitateseffluxorexcretionofsaltsbutnotinfluxofsalts.Consequently,thenet uptakeoraccumulationofsaltsbytherootslowsdownwithincreasing timeoftreatment ofrootsina10 mol1 triacetinsolution. Contrarytothispicture,theabsorptionofKbyrootswithouttriacetintreatment, -2 -i butwith10 mol1 triacetinaddedduringinfluxcausedasignificantincrease.

A conductivity U-TL

200

2 4 6 8 10 12h Fig.31.Experiment 21.Changeinconductivity (Auft)ofincubation solutionsbyexcised low-saltmaizerootsduringanincubationperiodof12h.Incubation solutionshadCaCl, at0.5mmol1 with (o)andwithout (x)triacetinat10 - 2mol1 _ 1 .

44

AccumulationofKwasroughlydoubled (Fig.32).Probablythesurface-activechemicalis directlyinvolvedinuptakebyanincreasedinflux,adecreasedeffluxorboth. Tocheckwhetherthistriacetinintheabsorptionsolutionisoperativebyinflux orefflux,thenextexperimentwassetup.

Experiment 22: Potassium influx and efflux in excised low salt roots over 4 h. —1 —2 Experimental solutions were 1 mmol I KCl solutions with and without 10 mol —1

I

fiR

triacetin.

Influx and efflux were estimated by the standard method with

Rh.

Theresultsofthisexperimentconfirmthesuggestion,thattheglyceryltriacetate isactivebybothfluxes.Anenhancedinfluxtogetherwithadiminishedeffluxfinally stimulatedKabsorption (Table 9).Thisexperimentalsoconfirmedthatsurface-active chemicalaffectsnetabsorptionofKpositivelyonlyifthechemicalispresentinthe absorptionsolution.Perhapsthesurface-activechemicaltriacetinisactivebyadirect couplingofpotassiumandtriacetinmoleculetoacomplexpermeatingmorereadily throughthemembranethanKalone,orperhapsitisbuiltintothelipiddouble-layerof therootcellmembranes.Washingoftherootsaftertriacetintreatmentandbeforeflux measurementcanremovetheorganicmoleculesfromthemembraneandinhibitthepositive triacetineffectorevenenhancethepermeabilityorleakageofthecellmembrane. Sincepermeabilityofcellularmembranesdiffersfordifferentions,theeffect ofatriacetintreatmentwasinvestigatedalsoinrelationtoionselectivity.If

mmolKkg"'DM 120-.

100-

80-

60

40-

20

0

2

4

R

H

iu

12h

Fi

g. 32.Experiment21.Potassiumabsorptioninexcisedlow-saltmaizerootsafterincubationfordifferenttimesinasolutionof 10"2moll"1 triacetin.Absorptionduringthe subsequent6hfromsolutions 1mmoll -1 KClwasestimated fromaccumulation (+)anddepletion (x).Solidcirclesattheendofthedottedlinesrepresentabsorptiondataof rootsnottreatedwithtriacetin,butwithtriacetinat 10~zmoll"1 intheabsorption solution 1mmol1"'KCl.

45

Table 9. Steady-state influx, efflux and net absorption of K in excised maize roots low in s a l t for 4 h. Absorption medium i s a 1 mmol l - ' KC1 solution without (control) and with 10"^ tnol l - ' t r i a c e t i n . Experiment 22. Steady-stateKfluxes (mmolkg DMh )

Treatment

influx

efflux

19.19 30.29**

control triacetin10 mol1

3.11 0.48**

netabsorption 16.08 29.81**

triacetinaltersthepermeabilityoftherootcellmembranes,thechangesmaydiffer fordifferentions. TheeffectoftriacetinonthemembranefluxoftheionsK,Ca,NaandCIwasstudied inthenextexperiment. Experiment 23: Influx and efflux

of K, Ca, Na and CI in excised low salt were 1 mmol l'1 solutions

roots for 4 h. Absorption solutions —P

NaClwith and without 10 86

method with

4S

Rb,

mol I PP

Ca,

Na and

maize

of KCl, kCaCl„ and

—7

triacetin.

Flux was measured by the standard

^R

CI as tracers for K, Ca, Na and Cl,

respectively.

InFigure33,theinfluxandeffluxdatarelativetocontrols (withouttriacetin) arepresentedforthefourions.Theeffectoftriacetinonionfluxwastotally differentforthefourionicspecies.Onlytheinfluxofpotassiumwasstimulatedby triacetin,andinfluxofCa,NaandClwasinhibited.Alsotheeffectoftriacetinon effluxwastotallydifferent.Again,triacetinaffectseffluxofpotassiumnegatively, whereaseffluxofotherionswasenhancedbythisorganicchemical.Consequentlyaddition ofthissurface-activechemicaltotheabsorptionsolutionstimulatednetuptakeofKand inhibitednetuptakeofCa,NaandCl.

relative fluxes 150 7

LSD=176 LSD=13,6

i ^

will beenhanced.The latterwillresultinafasterreleasefrom thevacuole ofunlabelled potassium,whichwillthenbetransported togetherwiththenon-exchangedpartofthe freshlyabsorbed labelledpotassiumtothexylemvessels andsubsequently tothecutend oftheexcisedroots.Figure49Eillustrates thatthemajority ofthepotassiumtransportedupwards isunlabelledpotassium,stored inthevacuolebefore the absorption experiment. 2.Aftertransferofhigh-saltrootstoalow-salt (K)mediumthetotalxylem transportof potassiumexceedstheabsorptionoffreshpotassiumby theroot.Consequently,netaccumulationrate (Fig.49F)turnsnegative,while theaccumulationrateoffreshly absorbed potassium ismaintainedwithpositivevalues. Symplastic labelledpotassiumcouldbe isotopically dilutedbyexchangewithunlabelledKalreadypresent inthecorticalcellvacuoles.Thisnon-physiological process could leadtomisinterpretations oftransport andexchange ifbasedonnothingbutlabel. Onecouldeasilyoverestimateorunderestimate exchangeandtransport rates $ , and R (Diagram4 ) . According tothisandtheprevious experiment,thetime (At)betweenthebeginning of theexperimentandthefirstreleaseofpotassiumfromthexylemvesselsatthecutendof theexcisedrootsdependsontheinternalcellularpotassium statusoftheroots.This timelagAtisalsoimportant ininterpretationofabsorptionorinflux experiments (Chapter 5). Criticismthatexudationmayinterferewithuptakedataderived frompartitionofsaltsbetweenexcisedrootmaterialandabathing solutionwillbe justified iftimeof absorptionexceedsAt.Absorbed saltsmaythenbepartly excreted again into theabsorptionmediumbytheexcisedxylemvessels,that areinopenconnexionwiththe externalmedium.Consequently,absorptiondatameasured fromdepletionor accumulation willbeunderestimated.Abetterunderstanding aboutthevalueofAtisthendesirable. Experiment35indicatesveryclearlythatexcised low-saltroots,asused inthe uptakeexperiments (Chapter5)donotstartupwardxylem transportbeforethe6thto 8thhour.Onlyafterloadingofthelow-salt rootsfor24h ina1.0or 10.0mmol l - 1 KC1solution,anupwardxylemfluxofpotassiumcanbemeasured immediately after cutting offtheaerialpartoftheplant.However,Figure49CandE indicates thatthemainpart oftheupwardpotassiumfluxthroughthexylemconsists ofafractionofunlabelledpotassiumaccumulated inthecells (vacuole)during loading,andxylem transport of freshly absorbed labelledpotassiumdoesnotreallygetunderwayuntilabout4-6 hafterstarting theexperiment.Soalthough thereisavasculareffluxofpotassium fromthe high-potassium roots,thiseffluxconsistsofunlabelledKduring thefirst 4-6 h.Consequently,theuse ofexcisedrootmaterial,oflowandhigh-salt status,willbejustified inuptakeexperimentsofmaximal6handwiththeuseofisotopes.Beyondthisperiod,vascular efflux 66

offreshlyabsorbedpotassiumwillbetoogreatandabsorptionmeasurementsbythe depletionorbytheaccumulationmethodwillbeincorrect (toosmall). Inafewfurtherexperiments,specialattentionwaspaidtothevascularefflux (upward xylemtransport)andvascular influx,bothinthepotassiumabsorption.Upwardxylemflux, bothoflowandhighsaltexcisedroots,wasblockedbysealingthecutendoftheroot withparaffin. Experiment 36: Potassium absorption (accumulation) in excised young maize roots with and without sealing the cut end of the roots with paraffin. Before the absorption experiment intact plants low in salt were grown for 24 h on solutions of CaSO. 0.05 -1 -1 -1 mmol I (control), or KCl 1 mmol I (K-loading). Absorption from a 1 mmol I KCl solution was measured from depletion and accumulation, for 10 h with Rb as tracer. Theresults (Fig.50)showthat: -sealedlowsaltroots (control)accumulatesignificantly lesspotassium thanthe unsealedones; -thereisnodifferenceinpotassiumaccumulationbetweensealedandunsealed excised rootsafter loadingtherootswithpotassium. Blockageofthevascular fluxofpotassiumandwaterwouldenhanceKaccumulation, especiallywiththehigh-saltroots (highvascularefflux).Yet,theexperimentproves exactlythereverse.Probably,arelativelyhighvascularinfluxofpotassiumcanbe responsibleforapparentextraKaccumulationbythe 'openroots' (Diagram 5). Thatthis apparentKaccumulationoccursonlyinlow-saltrootmaterialsupports thepostulate, sinceinfluxofpotassiumbythe'open'orcutxylemvesselsandthesubsequentradialsalt transportbackwardtothestelarandcorticalcells,lowinK,willbemuchhigherin K-starved thaninK-loadedrootmaterial. Experiment 37: Vascular influx and efflux of potassium in excised low-salt maize roots, measured by the standard method, for 10 h. The 1 mmol I KCl absorption Of*

solutions were labelled with

Rb as a tracer for K.

Initially,therewasafastvascular influxofpotassiumbythe'open'xylemvessels (Fig. 51).However,afterabout30mintheamountoflabelledK,enteringbythiscutend, startedtodecline.After3h,allpotassiumthatenteredtherootbythiswayhadbeen 'pumpedout'andfromthenonanetvasculareffluxofpotassiumstarted.Thispotassium hadbeenabsorbedbytherootcells (epidermisandcortex)andsubsequently transported radiallyandlongitudinallytothecutend.Althoughanetvascularinfluxexistedtemporarily,thestimulatedKaccumulationwithexcised 'open'roots,asfoundinExperiment 36,cannotbeexplainedfullybythisphenomenonofvascularinflux.Fromthedata,itis hardtosaywhethersealingofthecutendblocksorinhibitsspecialprocesses relatedto saltandwateraccumulationortransportinexcised low-saltroots,orthereverse,that theseprocessesareenhancedbyan'open'cutend. Enhancedosmoticpressureofvacuolarsapduringsteady-state saltaccumulationmight increasethewaterabsorptionandwatercontentoftherootcellscontinuously,becauseno 67

theoutersolutionthroughapoplasmandsymplasmintothexylemstream,beinglessmixed orexchangedwithapoolofpotassiumalreadypresentinthecorticalcellvacuoles. PlantssuppliedwithK.SO,clearlyreachsteady-stateatmuchlowerAc orAnthan plantssuppliedwithKNO.orKCl,butthereasonisstillobscure.AccordingtoCooil (1974),sulphateseemsnottoaccumulateinthecellsandtobetransportedtothexylem slowly,suggestinglimitedpenetrationattheplasmalerama.Consequently,transportofK exceedsthatofSO,andendogenousanionsarerequiredtobalanceexcesscationtransport fromK,SO.. 24 Therefore,inthenextexperiment,absorptionandtransportratesweremeasuredof bothcationsandanionsbyplantssuppliedwithKC1andK.SO,.Exudateswereanalysedfor organicanionsandinorganiccationsandanions.Osmoticpressureofexternalsolutions (n)andexudates (n.)wasmeasuredtofindAn. O

1

Experiment 38: Absorption and xylem transport

of potassium and its oounterion

decapitated

root systems of maize plants 5 weeks old. Experimental solutions

10 mrnol I

solutions

of KCl and %KpSO..

tracers for K, Cl and SO. respectively

Rubidium-86,

Cl and

in were

S were used as

during this 24 h experiment

Thisexperimentconfirmsearlierresults.Oncesteady-stateisreached,about12h afterthebeginningoftheexperiment,almostequivalentamountsofKandClareabsorbed

mmolkg DMh

25K 2 S0 4

\ 20-

s,

! * > •

\

10-

\

• ^v

b0-

70

^

A

py^-ii—=

v ^ r

s

—4— 16

20

=

24h

Fig.52.Experiment 38.Absorptionandupward xylemtransportofpotassium (A,A)andits counterions (o,»)over24hindecapitated rootsystemsofmaizeplants,from10mmol l -1 solutionsofKClandJ^SO^.Solidand opensymbolsrepresentabsorptionandtransport,respectively.

andtranslocatedtothecutendoftherootsystem (Fig.52).WithSO ascounterion absorptionandtransportofKaremuchlessthanwiththeKC1treatment,butalsothe amountofabsorbedandtransportedanion(SO.)ismuchlessthanthatofthepositiveion K .Asaresult,adifferenceinchargehastobecompensatedtomaintainelectrical neutralitywithintheroottissue. Table11presentsdataofthecompositionoftheexudatescollectedfromrootsystems, suppliedwithKC1andjKSO.,eachatexternalequivalentconcentrationsof0.5and20.0 -1 mmol1 .Exudateswerecollected18-24hafterstartingtheexperiment (steady-state phase).Atbothexternalconcentrations,KandCIintheKC1exudatesarealmostequally represented;consequently,theorganicanionconcentrationintheseexudatesisminimal. TheK„S0,exudateischaracterizedbyanexcessofKoverSO,.Thisexcessinpositive chargeiscompensatedbyorganicanions,mainlyofmalicacid (about851).Theseanions movetogetherwithSO,inthexylemstreamandneutralizethexylemsapelectrically. Measurementsoftheosmoticpressureoftheexudatesconfirmthebigdifferencebetween KC1andK2SO exudates,collectedinsteady-state.ThelowosmoticpressureoftheK 2 S0 4 exudates,combinedwiththerelativelyhighcontentoforganicanions,provesthatat loworhighexternalsaltconcentrationsthecounterionSO^retardstheupwardpotassium/ watertransportsignificantly. TransportisothermsofKwithCIorSO,ascounterionweredeterminedinthenext experiment. Table 11.Compositionofexudates,collected frommaizeroots 18-24hafterdecapitating and transferring theplantstoabsorptionsolutionsofKC1and lK2S04,eachatsubstance concentrations of0.5and 20mmol1-1.Fum=fumarate,Succ=succinate,Mo=malonate, 0=oxalate,M=malate,Ci=citrate. Componentsinexudates

o (KC1)

Iorganic (mmol1

(mmol 1 titratable)

Fum Succ Mo 0 M Ci Ecarboxylates

O (JK 2 S0 4 )

(mmol1 )

')

0.5

20

0.5

20

0.01 0.19 0.05 0.16 1 .53 0.75 2.73

0.09 0.14 1 .16 0.06 0.30 0.40 2.15

0.20 0.41 0.00 0.00 4.89 0.30 5.81

0.00 1.01 1.53 0.08 14.42 0.00 16.77

26.50 25.00

45.24 42.00

13.25

24.25

1.50

3.24

2.12 11.12

3.62 20.63

50.25 1.00 49.25

82.50 37.00 45.50

21.00 1.00 20.00

39.25 25.00 14.25

IIinorganic (mmol1 _1 ) K CI

so 4 (cations-inorg.anions) IIIosmoticequivalent (mmol1~') exudate(n^) ext.medium (n) An= (n.-It)° l

o

71

Experiment 39: Upward xylem transport of potassium in decapitated root systems of maize plants 5 weeks old. Rate of K transport was measured during steady-state phase, 18-24 h after transferring the root systems to solutions containing 0.1, 0.5, 1.0, 5.0 or 10.0 mmolKCl or hKJSO. I . Radioactive tracers were omitted. Transportisothermssignificantlydeviatedfromuptakeisothermsontwoimportant points (Fig. 53): 1.Quitedifferentfromabsorptionisothermswithexcisedroots (Fig.15),thetransport isothermsdidnotshowadualpattern,eitherwithCIorwithSO,ascounterion.Increasingtheexternalconcentrationofpotasssiumraisedtherateofstationaryupward Ktransportcontinuouslyandgradually,insteadofstepwise (twoormoresteps)asfound foruptakeisothermsbyNissen (1973). 2.Whereasabsorptionofthecationwasinsensitivetotheanion,suppliedwithitin solutionatequivalentconcentrationsbelow1mmol1~ ,thepresenttransportisotherms provethattheupwardKtransportwasaniondependentoverthewholerangefromlowto highexternalconcentrationsofsalt. Bothobservationsindicatethatthekineticsofxylemtransportdiffersfromuptake kinetics.Thisphenomenonneedsexplanation,becauseinsteady-stateabsorptionand subsequentxylemtransportarenotonlyinterdependentbutevenhavevaluesclosetogether. Consequently,undertheseconditionsabsorptionandtransportshouldshowsimilarkinetics. ThelackofthedualcharacterofthetransportisothermsupportsthesuggestionbyPitman (1970)andPitmanetal. (1968)thatthedualabsorptionisotherm (System1and2)maybe duetoartefactsofexcisedlow-saltroots.Intheseexperiments,xylemtransportofpotassiumhasbeenmeasuredinsteady-state,16-24hafterstartingtheexperiment.Onthe otherhand,mostabsorptionoraccumulationexperimentsareduringthefirst4-6h,mainly

mmolKkg-1DMh"1 100

80-

60

40

20

0-4:>, none fr. ^- e.xii-hntdurine 'long-term'experiments dualcharacter.Thisconfirms theidea (Section 6.3) thatduring B v *. cw,th ^i_•- A,„I character BecauseKisothermswith absorptionandaccumulation isothermslosetheirdualcharacter,ce u tt,P K oneandthe transport S0 4 andClascounterionhadaboutthesameshapeastne% i t r a t e 73

mmolkg DM 2000n

1600

12004

800

400^ 8

o\ 800-

400H

o\

_i

0.1

0.5

1.0

5.0 10.0 mmolKNOoI

Fig.54.Experiment40.Absorption(I), transport inxylem (II)andaccumulation (III)over24hofpotassium (x) andNO3(o)indecapitated rootsystems ofmaizeplants5weeksold.Absorption solutionshaddifferent concentrations ofKNO3.

isothermsforK c h l o r i d e andK s u l .t g havebeenpresentedalreadyinFigure53,the accumulationandabsorptionisothermsforthesetwocombinationsareomittedhere. Figure55AshowstheKconcentrationsinthedifferentKNO,exudatesagainsttime. Alltheseconcentrationcurvesshowasimilarpattern.Thepotassiumconcentrationofthe xylemsapincreasedcontinuouslyforabout 16handthenbecamealmostconstant. The concentrationwashigherwithhigherexternalconcentration.Overthe16hours,aconcentrationgradienthadbuiltup;consequently,theosmoticequivalentdifferenceAn,the exudationrate J^ andKtransportinxylemJ Racceleratedsimultaneously.Figure55B illustratesthatafterabout 12-16htherateofxylemtransportofpotassiumwasstationary,butwashigherwithhigherexternalKconcentration. AsshowninEquation6,theexudationrate J dependsonthedifferenceinosmotic equivalentbetweentheexternalmediumandthexylemexudate.Althoughosmoticpressure wasnotmeasuredinthisexperiment,osmoticpressureofxylemsapandexternalmediumin similarexperimentswasmeasuredbyMinting (1977).ValuesprovethatforKN0 3andKC1 treatmentstheosmoticequivalentdoesnotsignificantlydifferfrom n.o, where nis thesumofthecationandanionvalencesofthecompletelydissociatedsaltand aisthe concentrationofsolutes (mmoll"').Thiscanbetrueforthechemicalcompositionof theseexudates (Table 11).Bycontrast,theK,,S04exudatecontains,apart fromanorganic potassiumandsulphateions,aconsiderableamountofdissociatedorganicanionsasa compensationfortheexcessofcations (K).Therefore,forthisexudate n.o /n,but

74

mmolKkg1exudate LSD=5,35

40

mmolKkg"1DMh"1 LSD=14,39

24h

Fig. 55.Experiment 40.Decapitated root systems keptfor24hinsolutionswith substance concentrationsofKN0 30(x),0.1(0),0.5(A),1.0 (+), 5.0 (•)and10.0 (A)mmol I"1.A.Concentrations ofKinexudates.B.Upward transportofpotassium inxylem.

n-2 n , where cv isthepotassium concentrationintheexudate.

nresented

Tnepotassium concentrations observedintheexudatesinsteady-sate r p r e ^ t e d fordifferent externalKconcentrations and threedifferentanions inFigure 56A. following features shouldbenoted:

,s i e n i _

-Thepotassium concentrationsoftheKC1andKNO3exudateswerealmos e o u a l » d sign ficantlyhigher than thoseoftheK ^ exudates.Thiswasvalidforallexternal concentrations. h -a100-fold increaseinexternalK concentrationresultedm les inK exudate concentration.The concentrationratio [ K e x u d a t ? ]'l

twofold increase

^ ^ ^ ^ ^ riiJ

^

(1

^ ^

concentrationsisextremely high (130-260),whereas thisratioisre forhigh externalKconcentrations. externalsalt concentrations Tnesedifferencesina c c ^ a t i o n ratiooflow^ * *+-««. ^ resultinanoteworthyphenomenonofexudationasmeasuredby P ^^ _ (Fig. 56B).insteady-state,theosmoticeouivaen ferenc^^ ^ ^ ^ cantly affectedbytheexternal concentration,at than 0.1-10m.01!-.Secondly,ifK.SO,iss^plie to™ ^ « reach forroots suppliedwithKC1orKN0 3 . Thxs- a n s that e P * o £t h e e x t e r n a l steady-state,characterizedbyacertainAHthat isalmost in ^ 75

mmolKkg"exudate

30-

20

10-

o-*V ATT(mmolI"1) 60n x 40 20-

o-. © kgkg"1DMh"1 2A-i

1.6 Fig. 56.Experiment 40.Exudation experiment with decapitated root systems transferred to absorption solutions with different concentrations ofKNO3 (x),KC1 (o) and 5K2SO4 (A).A. Substance concentration ofK in exudate in steady-state. B. Osmotic equivalent difference (An) in steady-state. C.Exudation rate