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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<j> 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
saltfluxes<|>cvand^ . 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/Rbratios<1provesthatRb
cannotcompletelytakeovertheroleofpotassiumwithintheplant.Halfthepotassium
canbesubstitutedwithoutgrowthreductionordeviationoftheS.C...,,fromunity.
IncreasingadditionsofRbtoaconstantKconcentrationof2.0mmol 1 decreased
accumulationofKandincreasedtheaccumulationofRb (Fig.5).However,forallsubstanceratios,theS.C..,,,didnotdiffersignificantlyfromunity;alsotheproduction
ofmaizedrymatterwasnotsignificantlyaffectedbyadditionalRbinthenutrientsolutionuptoaratioof1(Table 2).ThisRbadditionseriesalsoprovedthatRbtook
oversomefunction(s)ofpotassiuminthemaizeplant.WithRbsubstitutionoraddition
downtoaratioj1,themaizeplantdidnotdiscriminatebetweenKandRbinitsuptakefunctions,norshowanydepressionindrymatterproductionfor12days.
4.3CONCLUSIONS
Theuseof Rbasatracerfor Kinuptakeexperimentswithexcisedrootsof
maizeisjustifiable.Eveninlong-termexperiments,Rbsubstitutedforuptohalfthe
Kinthenutrientsolution.Astheconcentrationof RbinKabsorptionsolutionswas
alwayslow(onlytracers),theratiowashigh.Resultsofinfluxexperimentswithexcisedroots,anddoublelabellingwith K+ Rbconfirmedthis.Onlyathighexternal
—i
ftfi
potassiumconcentrations (5mmol1 orhigher)wasuseof Rbrisky.
AccordingtoHodges (1973),selectiveiontransportbyplantsisalmostsureto
dependonelectricfieldstrenghtoftheion-bindingsitesandonshiftsintheelectric
fieldstrengthofthesites.Whentheelectricfieldstrengthofthenegativesiteis
weak,theionabsorptionsequenceCs>Rb>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, [CI.]
differsfrom zero,butthis isduetotheexternal [CI]increase inthemodel.For
o
relativelyhigh pKvaluesofHA,datagathered inExperiment 9agreewith theoretical
calculationsasshowninFigure 14.AtpXbetween4andS,thecalculated[A ]and
[JL]willbenegligibleatpH 2andhighathighpH.ThiscationadsorptionathighpH
andtheabsenceofanionaccumulationatanypHareinagreementwiththeexperimental
dataofExperiment9.At lowpH,bothcationandanionabsorptionresultfrommass flow
ofsolventandsolute.AthighexternalpH,supplementary cationabsorptionoradsorptioncomes intoplay,regulatedbyphysico-chemicalforces.
ThequestionwhetherthispH-dependent initialcationabsorptionregulates the
subsequentstationarypotassiuminfluxremainsunanswered.
o-M^
2pH
°
Fig. 12.Diagram3.Theoreticalrelationbetweendegreeof dissociation
(expressed aspif)of thehypothetical
indiffusibleacidHA andpHofthe
external solution.Calculations from
aDonnandistribution.
25
mmoll"
1001
pH
Fig. 13.Diagram
ternalsubstance
potassiumandchl
externalpHand2
sociationofthe
HA.Calculations
tribution.x, pK
lines,K;solid1
3.Theoreticalinconcentrationsof
orideatdifferent
degreesofdisindiffusibleacid
fromaDonnandis3;o, pK5;broken
ines,CI.
[A"]mmolf
PK(HA)
Fig. 14.Diagram3.Theoretical
relationbetweendegreeofdissociationand pKofthehypotheticalindiffusibleacidHAata
lowpH (3.0;A ) , medium (5.0;o)
andhighpH (7.0;x)oftheexternalsolution.Calculations
fromaDonnandistribution.
Remarks
AlthoughdataoninitialabsorptionofKandCI,gatheredinExperiment9,support
theexistenceofaDonnanphaseinplantcells,
1.TheassumptionofthepresenceofonlyoneacidHAintheDonnanphasewillbean
over-simplification.Dataofseveralinvestigatorsprovetheexistenceofdifferent
membrane-boundorcytoplasmicacidcompounds,eachwithitsownspecificpXvalue(s).
Particularly thismixtureofHAcompounds,coveringabroadrangeofpKvalues ensures
thepresenceofnegativeindiffusibleorrestrainedanions (A~)~overawiderangeof
externalpH.However,thetotalnegativeindiffusibleorrestrainedchargewill
dependonboththeamountandqualityofthedifferentacids (HA)andtheexternalpH
(Fig. 14).
2.Calculationsof[A~], [K+.],[ClT]and[HtjarebasedonanidealDonnandistribution
26
ofbothcationsandanionsinplantcellsandequilibrium conditionsofthisDonnan
distribution.WhileacceptingtheexistenceofaDonnanphaseinplantcells,itstill
keepsunclearwhetherreal 'equilibrium'willbereachedunderdifferentexternal
conditions,forinstanceofpH.
3.Inprevious calculationstheexternalpHwas introducedasanindependentvariable
intheDonnanmodel,whilethecompositionoftheDonnanphase itselfwillbedependent
onoradaptedtotheexternalpH.Howeverastronglybufferedandhigh-electrolyteHA
phaseisnoteasilyadaptedtoanunbuffered low-electrolyteabsorptionsolution.
4.AtlowpHoftheexternalmedium,calculatedpHoftheDonnanphaseislowtoo.
Acceptingabuffering capacityofaplantcell,thiscanindicatethat
-extremely lowpHoftheDonnanphasewillbereachedonlyatequilibriumand;
-equilibriumisnotreachedintheshortterm;
-reachingequilibrium,inthelongterm,resultsinextremely lowpHoftheDonnanphase.
Thiscansupportthelong-termeffectofanextremely lowexternalpHontheionuptake
andgrowthofplantsingeneral.
5.2.2 Effect of the calcium status and Ca supply to the root
Inadditiontothenutritionalroleofcalciumforplants (Albrecht, 1968),this
cationisinvolvedinthemaintenanceoftheintegrityandstructureofcellwallsand
membranes (Steveninck, 1965;Waisel,1962).AccordingtoMarschner&GUnther [1964),
rootsofbarley,starvingforcalcium,showaclearreductioninpotassiumuptake.
Electronmicrographsofthesecalcium-starved roottissuesshowedcellswithabroken
tonoplast,amixtureofcytoplasmicandvacuolarsap,andconsequently lossofstructure
ofthecellprotoplasm.ThisisinfullagreementwithfindingsofMarinos (1962).
Inaddition,thepresenceofcalciumintheabsorptionsolutionwasfoundto
promotetheuptakeofotherions,aphenomenonwidelyconfirmedbyHooymans (1964) and
Rainsetal. (1964). Thiseffectofcalciumsupplytorootsduringabsorptionexperiments
isnotwellunderstood.Severalalternativeexplanationsforthestimulationofpotassium
absorptionaddtothisconfusion.They includeblockingoforinterferingwiththeuptake
ofcationssuchasHorLi(Jacobsonetal.1960)orincreasingthediffusionofKacross
theoutercellmembrane (Waisel,1962),oraroleofaribonucleoproteincomplexcontainingfreeSHgroups (Tanada, 1962).
Toinvestigatehowcalciumisinvolvedinthepotassiumabsorptionbymaizeroots,
experimentswere carriedoutatdifferentexternalconcentrationsofcalciumandwith
rootsdifferentincalciumstatus.
Experiment 10: Potassium influx in excised low salt maize roots from absorption
solutions
with KCl at concentrations
0.10, 0.25, 0.60, 1.0, 1.5, 2.5, 5.0, 10.0
and 40.0 rnmol I1 with and without calcium (h mmolCaCl^ I ) . Absorption of K
was measured over 4 h by accumulation with
Rb.
Theresultsofthisexperiment (Fig. 15)demonstratethatpotassium influxes
differently affectedatthevariousexternalconcentrationsofKby0.5mmol1 Cain
27
mmolKkg-1DMh"1
AOT
30H
20-
10
o-H0.1
-i
0.25
1.0
i_
2.5
10
40
mmolKI"1
Fig.15.Experiment 10.Influx
ofpotassiumintoexcisedlowsaltmaizerootswith (•) and
without (o)CaCl2atsubstance
concentration0.5mmoll - 'in
theabsorption solution.SteadystateinfluxofKismeasured
for4h.
theabsorptionsolution.Bothinfluxisothermsdonotsignificantlydifferforexternal
concentrationsofKupto2.5mmol1~.AthigherconcentrationsofK,additionof
0.5mmol1 CaCl-totheabsorptionsolutioninhibitedinfluxofKsignificantly
(P=0.05).Thisinhibitionathighsubstrateconcentrationssuggeststhatcalciumcomes
intoplayintheconcentrationrangeoftheisothermthatEpstein (1966)andCram&
Laties (1971)calledthelow-affinityisothermorSystem2.Inviewofthepostulations
byTorii&Laties (1966a),calciummustaffectthepotassiuminfluxatthesecondmembrane,
thetonoplast.
Sinceacalciumconcentrationof0.5mmol1 didnotsignificantlyaffectthe
potassiuminfluxatanexternalpotassiumconcentrationof1.0mmol1 ,inthenext
experimenttheeffectofthepresenceofCaontheeffluxofKwascheckedtoo.
Experiment 11: Potassium influx and efflux in excised maize roots low in salt from
a 1 mmol I KCl solution with and without 0.OS mmol I • CaCl
Fluxes were
2'
Of*
measured by the standard method for 4 h with Kb.
Dataofbothinfluxandefflux (Table4;Fig.16)confirmtheabsenceofanyeffect
ofcalciumoninfluxofK;buteffluxofKwassignificantly (P=0.05)enhancedinthe
absenceofCaintheexperimentalsolution.InspiteofthissignificanteffectofCa
ontheeffluxofK,therateofnetabsorptionofKwasnotaffectedsignificantlyby
Ca.EitherCainhibitedeffluxoreffluxwasstimulatedundercalcium-freeconditions.
Thelattermayindicatethatrootcellsneedaconstantcalciumsupplytomaintainan
optimumfunctionofcellularmembranesandwalls.AdditionofCatoallabsorption
solutionsprovednecessary,asrecommendedinmostoftheliteratureonionabsorption
byplanttissues.Calciumcanbeoperativeinionabsorptionbyastimulatedinfluxas
28
mmolKkg"'DMh
LSD=3,94
Fig.16.Experiment 11.Potassiumfluxesinexcisedlowsaltmaizerootsover4hwith
(•) andwithout (o)CaCl2at
substanceconcentration0.5
mmoll -1 inthe1mmolI"1KC1
absorptionsolution.A.Influx.
B.Efflux.
- 1 , absorption
Table 4. Effect of the presence or absence of calcium (0.5 mmol 1 ) in ,the
solution on influx and efflux of potassium in excised low-salt roots. Absorption solution
with 1 mmol l - 1 KC1. Experiment 11.
Ca - treatment
+ Ca
- Ca
Steady-state K fluxes (mmol kg
DM h )
influx
efflux
16.3
13.9
2.06
2.38*
well as by an i n h i b i t i o n i n efflux.
In how far stimulation of K influx depends on the external concentration of Ca was
tested.
Experiment 12: The effect of the external calcium concentration on the potassium
influx in excised low salt roots. Influx of K was calculated for 4 h from both
depletion and accumulation. Calcium concentrations in the 1 mmol I KCl absorption solutions were 0.0, 0.025, 0.05, 0.25, 0.5, 2.5 and 5.0 mmolCaCl2 I .
The calcium effects are shown in Figure 17 r e l a t i v e to control without calcium.
S t a t i s t i c a l analysis proves t h a t calcium concentrations of 0.5, 2.5 and S.O mmol 1
enhanced influx s i g n i f i c a n t l y (P = 0.05), for influx calculated from depletion and from
accumulation. Contrary t o Hooymans (1964), a t external Ca concentrations < 1 mmol 1 ,
29
thesegypsumrootsdidnotshowanysignificanteffect.Althoughallcalciumtreatments
showapositiveeffectontheKinfluxinFigure 17,thehighestpositiveeffectwasat
thehighestexternalconcentrationofcalcium(Smmol l" 1 ). So,unlikeunivalentcations,CaatahighconcentrationstimulatesKinflux.Becauseofthehighcalciumcontentofthegypsumrootsandtheshorttimeofuptake,calciumdeficiencyisimprobable.
ThehypothesisofacalciumstimulatedKinfluxbywayofacalciumstimulatedorcalcium
inducedtransportcarrierorbyasubstratecalciumcomplexismoreprobable.
Theessentialandirreplaceableroleorfunction(s)ofCainmembranesandwalls
ofdifferentplantcellswasdemonstratedbyseveralinvestigators (Bangerth, 1970;
Goor,1968;Steveninck,1965).
Tochecktheeffectofthecalciumstatusoftherootonthebehaviour,e.g.influx
andeffluxofpotassium,maizeplantsweregrownfordifferenttimesonacalcium-free
mediumandthenusedforfluxexperiments (Experiment 13).
Experiment 13: (1) Potassium influx
in exoised roots of plants grown before
influx experiment for 0, 5, 8, 11, IS and 18 d on a calcium-free
ralized water). Influx measurements by both depletion
solution
and accumulation.
the
(demine(2) Potas-
sium desorption or exchange. After the influx period of 4 h, roots were then
-i
washed 5 times with fresh aliquots
5, 5, 5, 15 and 30 min.
of a 10 mmol I
unlabelled KCl solution
for
Withincreasingtimeofstarvationoftheintactplants,Cacontentofrootsand
shootsdecreased (Fig.18), andsteady-stateKinfluxwasdepressedconsiderably (Fig.19).
Presumablycalciumstarvationforatleast5dsignificantlyreducesinfluxofKto
theroot.ArelationbetweenthecalciumcontentoftherootandKinfluxintheroot,
asfoundinthisexperimentandpresentedinFigure20,maynotbequitecorrect,
% calcium effect
UOn
*
- '
o
+ ' '
x
*
105o-*
o
100
'X
-Ca
95-
90
Mr*
30
Fig.17.Experiment 12.EffectofdifferentadditionsofCatothe1mmol1
KClabsorption solutiononinfluxofK
.
,
,
,
,
inexcisedlow-saltmaizerootsover4h.
0.025 0.05 Q25 0,5 2,5 50
Absorptioncalculatedbydepletion(o)
mmolCaI
andaccumulation (x).Minuscalcium=100.
mmol Cakg-1DM
150
16
20
days- calcium
Fig. 18.Experiment 13.Contentofcalciumin
shoots (•) androots (x)ofmaizeplantsgrown
fordifferent times (0-18d)onamediumwithoutcalcium.
mmol Kkg"1 DM h"1
15
18
days-calcium
Fig. 19.Experiment 13.Steady-state influxof
potassiumfroma1mmolI - 'KC1absorptionsolutioninexcised low-saltrootsofmaizegrown
fordifferenttimes (0-18d)beforethe4-habsorptionexperimentonamediumwithoutcalcium.
Influxwasmeasuredbydepletion (A)andaccumulation(x).
becausethecalciumcontentusedinthisfigureisameanvalueofthewhole roottissue.
ProbablytheroottissueincludesaCa-richpart,developedduring enrichment,andpart
formedduring starvationthatwaslowinCa.ThelatterwillhavethefeaturesofCa
deficiencyandberesponsibleforthedeviationorinhibitionininfluxofK.
Time-curvesforinfluxanddesorptionofK,presentedinFigure21forroots
grownduringperiodsof0,8,and18dbefore influxexperimentonacalcium-free
solution,showthat:
-with increasing timeofCastarvationsteady-state influxofKwasinhibitedsignificantly;
-with increasing timeofstarvation,desorptionkeptalmostconstant,butthepattern
ofdesorptionduringexchangefor60minwasdifferent.
Just like influx,theflux (desorption)inrootslowinCaaswellasrichinCa
31
wasfastduringtheinitialphaseoftheexchangeperiod.Thedesorptionrateofthe
Ca-richrootmaterialkepthigherduringthesubsequentphaseofcytoplasmicefflux.A
higherrateofcytoplasmicexchangeofCa-richrootsmayindicatethatcalciumstarvation
alterstheoutercellularmembrane,theplasmalemma.Anincreaseinthesolutepermeabilityofthismembranemaybeexpressedbyfastreleaseorleakageofcytoplasmic
solutes,whiletheeffluxofsaltsbythewell-developedcellularmembranewithanormal
contentofCaismoregradualandhigheroveralongertime (Macklon&Higinbotham,
1970).Whethercalciumstarvationalsoaffectsstructureorpermeabilityoftheinner
membrane,thetonoplast,isunclear.Togetthisinformation,theexchangeperiodshould
havebeencontinuedmuchlonger.
5.2.3
Effect of the internal K status of the root
Onewouldexpect,asindeedfoundbyCram (1973b)andGlass (1975),thatinflux
ofionsbyrootsisnegativelycorrelatedwithinternalconcentrationofions.The
ionuptakeinplantsmayberegulatedbyaformoffeedbackcontrolinwhichthe
absorbedionactsastheregulatoroffurtheruptakethroughitseffectuponinflux.
Ifso,thedynamicsofsaltinfluxmustbedifferentforplantrootswhichhavebeen
starvedforinorganicionsforvaryingperiods (lowsaltroots)andforrootswhich
havebeengrowninsolutionsrichinmineralions (highsaltroots).Inthetwocompartmentmodelofplantcellswiththeplasmalemmaandtonoplastplaced 'inseries'
(Cram,1968;Pitman,1963),atleasttwodifferentcomponentsoftracerfluxinto
planttissuewillexist:$ Q C ,theinwardplasmalemmafluxand4> ,afluxbetweenthe
cytoplasmandvacuole,thetonoplastinflux.Accordingtothishypothesis,bothfluxes
*oca n d *cvw i l 1 dependo n t n e internalcytoplasmicandvacuolarionconcentrations.
Consequently,thedynamicsofthetotalflux,acomplexflux,willdependonthesalt
statusofthedifferentcellcompartmentsandthusalsoontheinternalsaltstatusof
mmolKkg"1DMh"1
10l
x
-'A
8aA '
"
'
s
a
X
6/x
/
'A
2-
20
32
40
60
80
100
mmolCakg"1DM
Fig.20.Experiment 13.Relationbetween
steady-stateinfluxofKover4handcontent
ofCainexcised low-saltmaizeroots.AbsorptionsolutionhadKC1atsubstanceconcentration1mmol1~'.Influxmeasurementbydepletion(&)andaccumulation (x).
mmol K kg" 1DM h _1
exchange-
Fig. 21. Experiment 13. A. Influx of K for k h in excised low-salt roots of maize grown
for 0 d (x), 8 (o) and 18 d (A) before the absorption experiment on a medium without
calcium. B. Rate of K exchange during a subsequent 1 h period. Exchange medium was a
solution 10 mmol l - 1 unlabelled KC1.
the entire root c e l l .
To investigate the effect of the internal potassium status of maize roots on the
subsequent influx of K, the following experiment was started.
Experiment 14: Potassium influx in excised maize roots in relation to the internal
K status of the root. Plants were grown on a complete nutrient solution (Table 1)
and transferred
identical
0, 2, 4, 6 and 8 d before the absorption experiment to a fresh
solution minus potassium. Potassium influx was measured for 4 h by both
depletion and accumulation with
Rb as tracer.
With increasing time of K s t a r v a t i o n , the Kcontent in the plant root dropped
rapidly, whereas the r a t e of steady-state influx of K increased almost linearly (Fig. 22).
As root c e l l s of plants grown on a potassium-free medium get more and more exhausted and
*ow a low i n t e r n a l concentration of K, [ K j / f K j and [Kj/[K v ] are leveled down. Especially the l a t t e r can be responsible for the enhanced influx of K (Fig. 22).
The relationship between the influx * o c and [K.] , the mean internal cellular
Potassium concentration, seems t o be an exponential one, similar to that observed by
Cram (1973b). P l o t t i n g * against 1/[K.J (Fig. 23) showed that this relationship is
iinear with a c o r r e l a t i o n ' c o e f f i c i e n t of 0.98. This supports the suggestion that the
influx of Kin maize roots i s regulated as a negative feedback system. Increasing
\ \ would automatically r e t a r d the uptake of K. Whether an additional feedback system
is
at work, and how the regulator signal i s then perceived and translated to control
33
mmolKkg"1DMh"1
days-K
Fig.22.Experiment 14.Steady-stateinflux
ofKover4h (x)andsubstancecontent(A)
ofKinexcised rootsofmaizeplantsgrown
for0-8dbeforetheabsorptionexperiment
onamediumwithoutpotassium.Absorption
solutionhadKC1atsubstanceconcentration
1mmoll-'.
influxisanopenquestion.Theplaceinthecell (membrane,compartment)wherethe
systemshouldbeoperativeisalsofarfromunderstood.
AnotherphenomenonobservedduringthisexperimentwastheeffectoftheKcontent
oftherootontheabsorptionofKduringtheinitialphase.InFigure24,influxcurves
havebeenpresentedforboththedepletionandaccumulationmethod.Thesecurvesshow
thatthefraction-ofpotassiumaccumulatedduringthe4habsorptionintheAFSand
desorbedduringthe1hexchangewassignificantlylowerat0dinthetreatmentwith-,
outKthanifKhadbeenwithheldfor2ormoredays.Thisdemonstratesthat2ormore
daysofKstarvationsignificantlyreducethefractionofK,accumulatedoradsorbedin
theAFS.
Thus,afterKstarvationfor2dorlonger,initialandsteady-stateKinfluxare
increasedduringasubsequentperiodofatleast4h;steady-stateKinfluxseemedtobe
inverselycorrelatedwithinternalKconcentration.
5.2.4 "Effect of a treatment with various inorganic and organic salt
solutions
Thissectiontreatseffectsoftreatmentoflowsaltrootswithdifferentsalts
onthesubsequentsaltuptake.Ingeneral,atransferoflowsaltplantstoahighsalt
mediumwillincrease,thesaltstatus_oftheplantandchangethesubsequentuptake
patternofionsingeneralorparticular.Forexample,somesortofadirectcoupling
orfeedbacksystemseemedtoexistbetweenKstatusoftherootandinfluxofK.In
thetestsheredescribed,salts (inorganicororganic)wereusedthatdonotincrease"
theinternalcellularsaltstatuspersebutallowedthefollowingthreephenomenato
bestudied:
"
.
1.Anioneffect.AsreportedbyEpsteirt'andHagen (1952)andHiatt (1967),theuptake
.ofcationsbythelow-concentrationmechanism (System1)wasindifferenttothenatureof
34
mmolKkg1D Mh"1
400
800
1200
mmolKkg"1DM
[Ki]
y=3230x-0,90
r=0,98
8i
AH
2.10"
410'r3
(mmolKkg"
6.10"
DM)"
Fig. 23.Experiment 14.Relationshipwith
steady-state influxofKover 4h.A. Substance contentofK inroots ([K.] ) .B.reciprocalvalueofKcontentofroots (1/[K.]) .
theanion,whileuptakebythehigh-concentrationmechanism (System2)wasmarkedly
influencedbytheanion.Soatexternalsaltconcentrationsexceeding 1mmoll" ,K
influxwilldependontheidentityoftheanion.According toHiatt (1968),anion
entering therootbythismechanismmustbeaccompaniedbyanionofopposite charge
andconsequentlyuptakeofKfromhighconcentrationsofK 2 S0 4willberetardedbecause
oftherelatively slowrateofS0 4 uptake.Hiatt (1968)suggesteddiffusiontobethe
high-concentrationmechanismofionuptake.Ifso,S0 4 inhibitstheabsorptionrateof
thecounterion (K)andthiscation/anion-interaction isonlyoperativeathighexternal
saltconcentrations.An inhibitedanioninfluxwouldthendecreasetheinfluxofthe
cationtoo,butonlyathighconcentrations.Tocheckthishypothesis,rootswere•
enrichedwithCaanddifferentanions.Subsequently,influxofKwasmeasured from
solutionscontainingKwithdifferentanions.
2.Carboxylateeffect.Evidencehasbeenpresented thatelectrostaticbinding ofcations
byorganicanions isinvolvedinionaccumulationbycertainunicellular organisms
35
mmolKkg"1DM
100
Fig. 24.Experiment 14.Potassiumabsorption
inexcisedmaizeroots,estimatedbydepletion(A)andaccumulation (o),andpotassium
present intheapparentfreespace (+). Beforethe4habsorptionexperiment,plants
weregrownfor0-8donamediumwithout
potassium.AbsorptionsolutionhadasubstanceconcentrationofKC1 1.0mmol1 _ 1 .
(Leggetetal.,1965).Breteler(1975)andDeKocketal. (1973)showedalsothatrateof
ionabsorption (NH^,K,NO.,)bythehigherplantcanbeaffectedbytheinternalconcentrationoforganicanionsorcarboxylatesintheroot.Intactplantsweregrownon
differentcalciumsaltsanddifferentinternalconcentrationsofcarboxylatewerecreated
bymodifyingionuptakeandassimilation (Torii&Laties,1966b).Whenrootsofhigher
plantsabsorbcationswithoutconcurrentanionabsorption,thecationsexchangeforH +
fromtherootsandequivalentquantitiesoforganicacidsaresythesized (Hiatt,1967;
Jacobson&Ordin,1954).Assimilationofnitrateandsulphateisalsoaprocessthat
generatesorganicacids (Dijkshoorn,1962;Higinbotham&Pierce,1974).
3.Aminoacid/proteineffect.Asreportedbefore,N0 3nutritionoftheplantwillenhance
productionofcarboxylatesbyreduction.Inthefirstplace,Nnutritioningeneralwill
resultinanincreasedcontentoforganicNasamides,aminoacidsandproteins.According
toHiatt (1968),theseaminoacidsandproteinsplayanimportantroleinionabsorption
byplantroots.IfDonnandistributionsareinvolvedinionabsorption,thetotalamount
ofimmobileanionsinthecytoplasmwillbeofinfluence.Proteinscouldbeinvolvedin
iontransportinamorespecificway.Anenhancedproteinsynthesisprobablyincreases
availabilityofsitesbindingKorcarriers.Apartfromthisfunctionofproteins
ascarriers,theseorganiccompoundsareinvolvedinthestructureofcellularmembranes.
Accordingtothefluid-mosaicmembranemodel,postulatedbySinger&Nicolson (1972),
globularproteinsarepartiallyembeddedinthemembrane,penetratingintothelipid
phasefromeitherside,whileothersarecompletelyburiedinit.Typesornumbersof
specificproteinsoneachsurfaceoftheasymmetricmembranewillcontributetothe
featuresofthemembrane.
Inadditiontothesethreespecificeffects,thechangesbroughtaboutbythe
varioussaltsolutionsmaybebothstructuralandfunctional.Anincreaseininternal
saltconcentrationwillinfluencestructureoftheprotoplasmiclayers,viscosityand
hydrationofthecytoplasmandhencemetabolism.
36
Experiment IS: Potassium influx
in excised maize roots after rearing plants on
gypsum and 96 h on a 6 mmol I
influx
from a 1 mmol I
solution
solution
Of*
by depletion
with
of Ca(NOJ„
CaClgor CaSO..
Potassium
of KCl, %KpSO. or KNO, was measured over 4 h
Rb as tracer
The resultsofExperiment 15 (Fig. 25) indicate thatinroots enriched witha
certain anion there wasnosignificant declineininfluxofKwithananion identical
to the one used during enrichment.Thisistrue for all three anions CI, SO, and NO,.
Althoughplants enriched with CaCl. and CaSO, did not significantly differinuptake
of K, irrespectiveofthe natureo fthe accompanying anion,enrichmentwith C a ( N O , ) 2
significantly increased influxo fKfor all three anions.Obviously,areduced anion
influx does not negatively affect influxofthe cation,atleastata concentration
of the absorption solutiono f1mmol1~ .
To checkwhether plant roots enrichedwithacertain anion really inhibited influx
of the same anion, the next experimentwas set up.
Experiment 16: Potassium and chloride influx
enrichment as in Experiment IS. Influx
in excised maize roots
Of*
was measured over 4 h
depletion
after
of K and CI from a 1 mmol I
with
Rb and
KCl solution
'IC
Cl as tracers for K and CI.
Figure26provestheinfluxofCltobedepressed significantlyafter treatmentof
the plantwithCaCl 2 .This indicates that,asexpected,enrichmentofthe rootwith
Cl reduced subsequentCl influx. This experiment indicatesalsothat treatmentwith
Ca(NO) increasedinfluxofClduring the influxexperimentaswellasinfluxofK.
Probablyatreatmentofthe intactgypsummaizeplantbyaC a ( N 0 3 ) 2 solutionfor96h
stimulates the growthofthe plantmore thantreatmentswith CaCl 2orCaSO^ solutions.
The developmentofnew and more activerootmaterialduring the C a ( N 0 3 ) 2 treatmentcan
probably resultinahigheruptakecapacityofthe root for bothcations and anions.
Because anioninfluxisnot expectedtoaffect the influxofthe cationatexternal
relativeKuptake
150i
LSD=29,2
LSD=11,9
LSD=14,1
100'
Cl S0A NO3
Cl 50/ NO3
Cl SO, NOj
50.
Ca(N0 3 ) 2
CaCl 2
CaS04
Fig. 25.Experiment 15.Uptakeofpotassium
over4hinto rootsofmaize plants after
treating intact plants for96honsolutions
with C a ( N 0 3 ) 2 , CaCl 2orCaSO^5mmol I" 1 .
Subsequent uptake fromasolutionofK C l ,
JK 2 S0^ and KNO3 1mmol 1~'. Potassium uptake from solutionofKClbyCaSU4 treated
roots takenasreference (100).
37
relative uptake
150i
LSD=22,2
LSD =22,9
100-
to
50
CI
K>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
^
</.
100-1
LSD::42,2
f-
504
1
K
46
%
Ca
.
Na
LSD=38.2
Cl
Fig.33. Experiment23.Influx(•) andefflux
(.W over4hofK,Ca,NaandClinexcised
low-saltmaizerootswithandwithout triacetin
at10 mol1~1inabsorptionsolutionswith
KCl,{CaCl2,NaClorKClat1mmol l - 1 .Fluxes
forcontroltreatments (minustriacetin)were
100.
5.2.6 Metabolic inhibitors
and potassium uptake
Cyanide (CN)andDinitrophenol (DNP),inhibitorofrespiratoryelectronflowand
uncouplerofoxidativephosphorylation,respectively,wereusedasmetabolic inhibitors
onpotassium fluxesbymaizeroots.AccordingtoLundegSrdh&Burstrom (1933),Luttge
&Laties (1966)andRobertson (1968)bothinhibitorsreducetheuptakeofionsfrom
themediumandtheirtransfertothevessels.2,4-Dinitrophenolwasfoundtouncouple
phosphorylation fromoxidative respirationandreducesaltuptake,whereaselectron
transferthroughthecytochromechainwasnotinhibitedandoxygenuptakewas increased
(Robertsonetal., 1951).PerhapssaltuptakeisdependentonATP formationorelse
bothactivetransport andATPformationdependonacommonprocesswhichisinhibited
byDNP (Robertson, 1968).CN, ontheotherside,inhibitsorblockselectrontransport,
atthesametimereducing ionuptakeandtransport.
Althoughtheworkingmechanismofthetwoinhibitorsisquite-wellunderstood,it
certainlyisnotalwayseasytodistinguishbetweentheeffectofinhibitorsonspecific
metabolicprocessesandundesirableorunknownside-effects,suchasthealteration
ofthemembranepotentialandmembranepermeability.Higinbothametal. (1970) and
Pierce&Higinbotham (1970)foundafastdecreaseinthemembranepotentialofoat
coleoptilesinthepresenceofcyanideataconcentrationof1mmol1 .Asimilarrapid
depolarizationofthemembranepotentialappearstooccurinvariousalgae,fungiand
bacteria (Slayman, 1974).Studying themembranepermeability,Marschneretal.(1966)
reported thatKwas lostfrommaizerootsectionsmuchmoreunderunaerobicconditions
thanwithnormal aeration.Electronmicroscopyindicatedthatthecytoplasmofthe
anaerobicrootswasderangedandmuchlessdensethaninrootsreceivingair.In
general,aerobicmetabolismappearstoberequiredtomaintaintheintegrityofcells.
Therolewas investigatedofmetabolicinhibitorsininfluxandsimultaneous
effluxofpotassiuminmaizeroots,inordertounderstandtheireffectonthe
permeabilityofcellularmembranes.Firsttheeffectofconcentrationoftheinhibitor
DNPwas tested.
Experiment 24: Potassium influx in exoised lew salt maize roots from an absorption
solution, containing 1 mmol I1 KCl in addition to 0.0, 10 ,5.10
,10 , or
10~3 mol l'1 DNP. The experiment lasted 4 h; 86Rb was used as a tracer for K.
(1) K influx was calculated for all DNP concentrations from accumulation. (2)
Influx, of K was plotted against time for DNPat 10~4 mol I by the depletion
method.
Potassium influx (Fig.34)demonstrated theDNPeffectclearly.With increasing
DNPconcentrationintheabsorptionsolution,Kinfluxdroppedfasttoalowlevel.
AtaDNPconcentrationof10 - 4moll " \ potassium influxwasreducedtoabout 101of
thecontrol.Theabsorptioncurve (Fig.35)fora10 - 4moll - 'DNPconcentration,
demonstratesthatatthisconcentrationtheinhibitorstartsdepressingKinfluxwithin
15-30minafterstartingthe experiment.
TheeffectofDNPtreatmentonthepotassiumisothermwasstudiedinExperiment25.
47
mmol Kkg"1 DMh"1
4H
2J
OH
0
10"
5
5
5.10"
10"
A
10i-3
2.4 DNP mot 1"'
Fig.34.Experiment24.Rateofpotassiumabsorption
inexcisedlow-saltmaizerootsfroma1mmoll - 1KC1
absorptionsolutionwithdifferentconcentrationsof
DNP.Absorptionfor4h.
mmolKkg"1DMh"1
20-
16
12H
LSD=2,11
4h
Fig.35. Experiment 24.Rateofpotassiumabsorptioninexcisedlowsaltmaizerootsfor4h.Absorption^
solutionisaKC1solution1mmol1^
with^<j>)andwithout (x)DNPat10
mol1
Experiment 25: Potassium influx in excised low salt maize roots from an absorption
solution with KCl 0.10, 0.25, 0.60, 1.00, 1.50, 2.50, 5.00, 10.0 and 40.0 mmol I ,
—4
—1
i
with and without DNPat a concentration of 5.10 mol I . Influx after 4 n was
86
calculated from accumulation with
Rb as tracer.
- Theisothermsforthecontrol(minusDNP)hadadualcharacter(Epstein,1966; Laties,
1969;Fig.36);
-PotassiuminfluxwasreducedbyDNPatallexternalconcentrationsofKCl.
EffectofDNPonpermeabilityofrootcellmembranewasstudiedinthenext.
48
mmol K kg 1 DM h1
40
/
30-
/
20'
10
0'J
O-tyf
0.1
,
5
1
10
Experiment
influx
F i g . 36. Experiment 25. I n f l u x over 4 h of
potassium i n t o excised l o w - s a l t m a i z e . r o o t s
with (o) and without (x) DNP a t 5.10
mol
1 in the a b s o r p t i o n s o l u t i o n .
,
40
mmol K I"1
26: Potassium
experiment
influx
in excised
for 3 h in solutions
10~5, 10~4 and 10~3 mol I 1 . Potassium
low salt roots treated
of CaCl2 at 0.5 mmol I
influx
was calculated
before
the
and DNP at 0,
from depletion
and
Of*
accumulation
for 2 h with
Kb as
tracer.
Figure37confirmsthesuggestion thatblockedrespirationaffects permeability
ofthecellmembranewithinarelativeshort time.AtconcentrationsofDNP exceeding
10~ 4 moll"1 themajorityofthe'absorbed'potassiumisreturnedduringthe5min
exchange aftertheabsorptionperiod.Thisenhancedreleaseofpotassiumwith increased
concentrationofDNPoftheincubation solutionprovesthatwithincreasing concentration,
DNPbecomesmore andmore active alsoinaffectingmembranepermeability.Effectsof
DNParenotalways limitedtodirectrespirationorenergyeffects.Thefinaleffecton
ionabsorption canbecomposedoftwoormorecomponents,forexampleametabolic effect
andapermeability effect.
InthenextexperimenttheinhibitorCNwas tested.
Experiment
27: Simultaneous
roots from an absorption
by the standard
potassium
solution
method with
86
influx
and efflux
in excised
with KCl or KCNat 1 mmol I
Rb as
. Flux
low salt
maize
measurements
tracer.
Cyanideatasubstance concentrationof1m o l l"'depresses influxwithin15nun
ofaddition (Fig.38).Duringthesteady-statephase,influxwasdepressedbyafactor
3-SoCNinhibitstheinfluxofpotassium significantly too.However,theeffluxcurves
ofCNlookdifferent.WhereasDNPathighconcentrations increasesthereleaseof
accumulatedpotassium fromtheroot,CNdidnotaffecteffluxofpotassiumatall.
49
mmolKkg-1DM
mmolKkg"1DMh"1
LSD=6,58
25-1
100-n
A
V-.
20-
B0-\
60-
15- +—
®
10-
AO-I
\
\
5-
20
10"b 10"* 10"J
2MDNPmolr 1
Fig. 37. Experiment 26.Before theabsorption experiment, intact plants aregrownfor
3honsolutions of0.5mmol l - 1 CaCl 2 withDNPat0 (x),10 _ 5 (o),10" 4 (A)and10" 3 (+)
mol l - '. Substance concentrationofKC1inabsorption solutionwas1mmol I - '.A.Rateof
potassium absorption inexcised low-salt maize roots for2h.B.Potassium absorbed(x)
and accumulated (o)inexcised low-salt roots after 2h.(+)represents thefractionof
K returned duringwash/exchange lasting 5min after absorption.
2h
'0
PossiblyCNconcentrationwastoolowtoaffectmembrane permeability. Itwasimpractical
toobtaindataoninfluxandeffluxatvarious concentrationsofCN,sinceCNhadto
be appliedasapotassium salttoavoid complicationsofcationantagonism.A changein
concentrationofCNwould alsochangetheexternal concentrations ofK.
BothDNPandCNreduceKinfluxsignificantlyatsufficienthighconcentrations.
DNP affects root cellmembrane permeabilityatconcentrations exceeding 10 mol1 ;
at thisconcentrationthemembranebecomesmorepermeable (leaky)forpotassium.
CNdoesnotaffect effluxofKataconcentrationof1mmol l" orCNdoesnotaffect
root cellmembrane permeabilityatall,orelseaCNconcentrationof1mmol 1 may
havebeentoo low.
5.2.7
Effect of glucose supply to the roots and of duration of lighting
plants
Sincethecarbohydrate contentofplants generally increases inthelightand
decreasesinthedark (Breteler, 1974), therewillbeadaily fluctuationinthecarbohydrate contentoftheplantbecauseoftheday/night sequence.Moreover,inmost plants
the sugar content tendstochangeduringthegrowing season.Michaeletal. (1970)
foundadecreaseofthecarbohydrate contentofrootsofsugar-beetswith age.Titze
(1970)andZeid&Kiihn (1973)found significantvariations (mainly increases)inthe
carbohydrate contentofcarrotsandspringwheatduringthegrowing season.Inthe
literature,anumberofpapersonthedependenceoftheionuptakeonthesugar status
havebeenreviewed..According toHoagland&Broyer (1936), accumulationofbromideby
50
mmolKkg1DMh"
LSD=2,14
4h
Fig.38.Experiment27.Simultaneous
fluxesofpotassiuminexcisedlowsaltmaizerootsfor4h.Substance
concentrationinabsorptionmedium
ofKwas1mmoll - 'andofCN1 mmol
1~1(x)orzero (o).A.Influx.
B.Efflux.
segmentsofbarleyrootsthatwerelowinsugarcouldbeacceleratedbysupplyofsugar
totherootsintheabsorptionsolution.Pitmanetal. (1971)evenfoundaclosecorrelationbetweentheendogenouscarbohydratecontentofrootsandtherateofaccumulation
ofchloride.Consequently,boththelong-termanddiurnalchangesinsugarcontentof
aplant(rootorshoot)willprobablyaffectionuptakebytheplant.
Inthenextexperimentendogenoussugarcontentoftheexcisedmaizerootswas
alteredbyincubationoftherootsforacertaintimeinglucosesolutionsofdifferent
strength.
Experiment 28: Potassium influx
of the excised root Serial
in excised low salt maize roots,
for 17 h in solutions
after
of glucose contain^
1.0, 2.0, 3.0 and 5.0 % (W/V) of glucose. Influx of K from a 1 rmol I
solution
was 4 h.
was measured by depletion,
using
86
Rb as tracer.
incubation
0, 0.5,
KCl
The experimental
time
Incubationina0.5-1.0 % (W/V) glucose solution tendstogiveamaximum increase
inpotassium influx,whilewithafurther increaseinstrengthoftheincubationsolution,influxcurvesdecline (Fig.39).Ata 51 (W/V) glucose concentration,nofurther
stimulationbytheglucose treatmentoccurred. Inspiteofthehigh sugar contentof
these low-saltroots,theresultsprove thatamoderate supplyofglucose totheroot
mediumenhanceduptakeofpotassium.Thedecreaseofthestimulus at 2% {W/V} and
highercanprobablybeexplainedbythehigh osmoticpressure oftheincubation solution,
upto660kPaat S% {W/V). Thesehighosmoticpressures oftheexternalmediummay
changetheinternalcellularstructurebydehydrationanddisturbedmetabolism ingeneral.
Thereforeinfurtherglucoseexperiments,H (W/V) glucose solutionswereused togeta
maximumeffect.
Inthenextexperiment,theeffectofincubationtimeonglucose accumulationand
influxofKinrootswasstudied.
Experiment 29: Potassium influx in excised low salt roots after an incubation of
excised roots in a 17° (W/V) glucose solution for 0, 2, 4, 6, 8 and 10 h. Potassium
influx was measured by depletion over 4 h with Kb as tracer. Absorption
solution
was a 1 mmol I KCl solution.
Boththeendogenous sugarcontentofthemaize rootsandtheinfluxofK increased
almost linearlywith increasing incubation timeupto6h (Fig.4 0 ) .Incubationformore
than6hdidnotincreasewater soluble carbohydrate (WSC)contentanyfurtheroreven
madeitdecline.Paralleltothis,theincreaseinK influxdeclinedandalmost reached
equilibrium afteranincubationperiodof8-10h.Theclose relationbetween increase
inendogenousWSCcontentandinfluxofK totheroot,afteranincubationperiodof
0-6h,suggestsadirect linkbetweensugarcontentoftheorgans (rootorleaves)and
theirsaltabsorption.
mmolKkg"1DM
200'
175-
*r^*'
150
125-
100.
—i
1
4
5
% glucose
52
Fig. 39.Experiment 28.Potassiumabsorption
over4hinexcised low-saltmaizeroots
aftera16htreatmentofexcisedrootsin
solutionsofdifferent concentrationsof
glucose.Absorptionfroma1mmoll -1KCl
solution.
Measurementsduringtheuptakeexperimentsofthecontentofwatersolublecarbohydratesintheabsorptionsolutionshowedasignificantlossoreffluxofsugarfrom
theexcisedrootsduringthe4hofuptake (Fig.41).Althoughtheglucose-enriched
rootshadasignificantlyhighereffluxofWSCthancontrolroots (withoutglucose
incubation),thelatterhavearelativelyhighexcretionofsugarseitherfromthecut
endoftheexcisedroots (xylemefflux)orfromtheepidermalcells (radialefflux).This
releaseofsugarsbythecontrolrootsconfirmsthehighcontentofcarbohydrate (about
1(Hindrymatter)ofthislow-saltrootmaterial.Inspiteofthiscontent,aglucose
treatmentstillenhancesinfluxofK.Thesupplyofsugarstothemembranesmaybea
majorfactorratherthangeneralcontentofsugar,assuggestedbyBowling (1976).This
couldexplainwhyadditionalsupplyoffreshglucosetotherootsisstilleffectivein
enhancinginfluxofK.
Inthefollowingexperiments,potassiumuptakeofmaizeplantswasstudiedwith
darkandlightperiods.
mmolKkg"1DM
WSC(7.DM)
20
150
Fig.40.Experiment29.Absorption
over4hofK (x)andcontentof
watersolublecarbohydrates (WSC)
(o)inexcised low-saltmaizeroots
afteratreatmentofexcisedroots
inasolutionofglucose1% (W/V)
fordifferenttimes.Absorption
fromasolution1mmoll-'KC1.
100J^
gglucosekg"1DM
30-
«
O
20-
o
f
/
O
10-
n-
—
^ - X
y
X
X
r
4h
Fig.41.Experiment29.Amountof
glucosereleasedbyexcisedlowsaltmaizerootsduringabsorption
over4hfromasolution1mmoll - '
KC1.Excisedrootswith (o)andwithout (x)treatmentfor 16hinasolution 1% (W/V) glucose.
53
Experiment.30: Potassium influx
in excised low salt roots with and without an
incubation in a 1% (W/V) glucose solution
for 17 h. The roots w*e excised
maize plants either grown in continuous darkness, or up till
96 h of light
uptake experiment. Influx
of a 1 mmol I
of K was calculated
from depletion
from
before
KCl
solution for 6 h.
Influxoftherootsgrowninlightwassignificanthigherthaninonesgrownin
continuousdarkness (Fig.42A,B). Glucoseincubationdidnotaffectinfluxover6hof
rootsgrowninlight,whereasinfluxtorootsgrownindarknesswassignificantly
enhancedbyglucoseincubation.Rootsgrowninlightshowasteady-stateinfluxafter
about2h;rootsgrownindarkwithoutglucosedonotreachasteady-stateofionuptake.
Incontinuousdarkness,thesugarcontentoftheplantsmustbelowandpotassium
uptakemustbelimited,unlessstimulatedbyaglucoseincubation.Rootsofplants
growninlighthaveahighKuptake,whichisnotaffectedbyaglucoseincubation.This
indicatestherelativelyhighcontentofsugarintheroots.
Shiftsinpotassiumuptakerelatedtotheday/nightsequence,havebeenstudiedin
thenextexperiment.
®
1 -1
mmolKkg DMh'
LSD=2,54
32,
24
LSD=2,67
16
0
54
1
6h
Fig.42.Experiment30.InfluxofK
intoexcisedlow-saltmaizeroots
over6hwith(+)andwithout(x)
previoustreatmentfor16hof
excisedrootsina1% (W/V) glucose
solution.A.Intactplantskeptfor
9dindarkandthen4dinlight.
B.Intactplantskeptfor13d continuouslyindark.Absorptionsolutionwasa1mmol1 _1KClsolution.
Experiment 31: Potassium influx
during a 24-h cycle of day and night in
maize plants grown on a complete nutrient
Potassium influx
from a 1 mmol I
solution
intact
(Table 1) for three weeks.
KCl absorption solution was measured by
depletion
Oft
with
Rb.
Day/nightsequencesinpotassiumabsorptionaredemonstratedveryclearlyin
figure43A,B.Within2-3hofswitchingoffthelight,Kinfluxdeclinedsignificantly
(Fig.43A).Attheendofthedarkperiod,influxhadalmoststopped.Afterswitching
onthelightagain,potassiuminfluxacceleratedwithin2handreachedsteady-state
within4to6h.
Thisinfluxpatterndemonstratesmostclearlytheday/nightsequenceinKinflux
andthetransitionphasesafterswitchingonandoffthelight.Atthebeginningofthe
darkperiod,theplantderivesatemporaryenergysupplyfromareservepoolof
endogenoussugars;ontheotherhand,afterthestartofthelightperiod,itpresumably
takessometimebeforesugarproductionorsupplybecomeseffectiveforpotassiumuptake.
mmolKkg1DMh1
10
continuous light
i-K-r-x-rx-rx-
10-
£££
04
0
Fig.43.Experiment31.Rateofpotassiumabsorptionin"intactmaizeplants.A.During
transitionfrom lighttodarkness.B.Transitionfromdarknesstolight.Absorption
mediumisa1mmol1 _ 1KClsolution.• ,light;• ,darkness.
55
5.2.8
Temperature of the absorption solution and ion uptake
Temperaturecontrolstherateofmetabolism.AsreportedbyJacobsonetal.(1957)
andMengel&Herwig (1969),ionfluxesbyplantrootsdependonambienttemperature.
Sincetemperatureregulatesmanymetabolicprocesses,suchasrespiration (Mengel&
Herwig,1969),achangeintemperaturemustaffectionabsorptionbytheroot.Also
permeabilityofrootcellmembraneswillbeaffectedbydifferenttemperature treatments
oftheroot,ashasbeenfoundbymanyauthors.
Tostudytheeffectoftemperatureonabsorptionofcationsandanions,inparticularpotassium,thenextexperimentsweresetup.
Experiment 32: Absorption of potassium and chloride in excised low salt maize roots
from 1 mmol I KCl absorption solution at temperatures of 5, 12, 19, 26, 33 and
40 C. Absorption was measured for 8 h by depletion and accumulation without radioactive tracers.
Netabsorptionwasmeasured.Foratechnicalreason,nousecouldbemadeofradioactiveisotopesinthethermostaticbaths.Consequentlythetemperatureeffectoninflux
andeffluxcouldnotbeestijnatedseparately.
Sincetemperaturesoftheabsorptionsolutionsbelow7°Cand10°Cproducednegative
netabsorption (influx-efflux)ofKandCI (Fig.44),effluxexceeded influxunderthese
conditions.Probablylowtemperatureinhibitedioninfluxmore thanionefflux.AssuggestedalsobyMengel&Herwig (1969),theeffluxofpotassiumisalessmetabolic-linked
processthantheinflux,oreffluxmightevenbeadiffusionprocess.Atabout33°C,
netabsorptionrateofbothKandCIshowsanoptimum,whilewithafurtherincreasein
temperature,itdropsdrastically (Fig.44).Temperatureaffectsabsorptionofcationsand
anionsinadifferentway.Whileattemperaturesbelow 15-19 °C,chloride absorption
(whichmaybemainlydiffusionafteraperiodofchloridestarvation)exceedspotassium
absorption,withintherange 19-33°C,potassiumisabsorbedpreferentiallytochloride.
ThischangeinK/Clabsorptionwithdifferentexternal temperaturecanbesignificant,
bothdirectlybyachangedionuptake,butalsoindirectlybydifferenceinffl7H+efflux
bytheroot,resulting inwidedifferencesinexternalpH.
TemperatureeffectsoninfluxandsubsequentreleaseofKduringarinseandexchange
periodwerestudiedinthenextexperiment.
Expert
33: Influx of K in excised low salt maize roots from a 1 mmol C1 KCl
KCl
absorption solution at S and 22 °C. After influx for B h, roots were bathed five
t^es
each time in fresh aliauots of cold (4 °C) water followed by 10 mmol C1 *.-„
KCl
solution, for 1 h per treatment. Influx was measured by depletion with 86Bb as tracer
the s l ^
a
JTT "
eXt6mal t e
P
I t oLTtT ^
urmg
56
^
e m U r e f
™
8 9t03 23M
^ ^ -° '
n
22
°C to 5 °C, potassium influx during
I*" ™h"'-If - « — **»
40.45 and 16.15 mmol Kkg ' DMfor the high and the low
mmolKkg"1DMh"1
Fig.44.Experiment32.Rateofnetabsorptionover8hofpotassium (x)and
chloride (o)inexcisedlow-saltmaize
rootsfrom 1mmol1 KC1absorption
solutionsatdifferenttemperatures.
A.Absorptioncalculated fromdepletion.B.Absorptioncalculatedfrom
accumulation.
mmol K kg"1 DM h"1
influx
^ * - rinse* «-exchange•
|X
Fig.45.Experiment33.
InfluxofKintoexcisedlowsaltmaizerootsfromasolution1mmolI"1KCLat22(x)
and5°C (o).Afterabsorptionfor5h,rateofK
releasewasmeasuredduring
arinseandexchangeperiod
of 1heachinwaterandin
asolutionofKC1at 10mmol
1_1.
57
temperature,respectively.TheamountsofKaccumulated,calculated toremainafterthe
1hrinseand 1hexchangeperiod,were49.71and 14.49mmolkg DM,respectively.These
dataandtheshapeofthereleasecurves (Fig.45),indicatethat:
-potassiumaccumulatedatlowtemperature isretainedcompletelywithintheplantroot
cellsduring therinseandexchangeperiod;
- afterinfluxatahightemperature (22 C ) , potassiumreleaseduringthe subsequent
1hexchangemaynothavebeencomplete.
Thefirstobservationprovesthattheinfluxofpotassiumwas inhibited significantlyatalowtemperature,butalsothatthefractionabsorbed during the steady-state
phaseaccumulatedactively,eitherintothecytoplasmorintothevacuoleorintoboth.
58
6 Potassium transport through excised roots
Iontransportinrootswillbesubdividedinthisreportasfollows:
-ionaccumulationintheroottissue (short-distancetransport);
-centripetaltransporttothexylemandsubsequentupwardorxylemtransporttothe
shoot (long-distancetransport).
Chapter5discussedonestageoftheiontransport:accumulationofionsinroot
cells.
Thischapterwillpresentresultsofexperimentsdealingwithbothlong-distance
centripetaltransportofionstothexylemvesselsandsubsequentupwardmovementto
theleavesinthexylem.Thelatterhasbeenstudiedinexperimentsonexudationof
excisedroots,amethodusedbeforebymanyotherinvestigators (Anderson&Collins,1969;
Cooil,1974;Klepper,1967;Lauchlietal.,1971;Lauchli&Epstein,1971;Mein, 1973;^
Wallaceetal.,1967).Xylemexudationfromrootsischieflywater.Thegeneralopinionis
thatthiswaterisdriventhroughtherootfromtheexternalsolutiontothexylemvessels
byrootpressure,i.e.bytheosmoticpressuredifferencebetweentherootmediumand
thexylemfluid.
Thetranspirationcomponentiseliminatedindecapitatedrootsystems.
^
e
f
o
^
theequationforwatermovementacrosstheroottothexylem,describedbySlatyer (1967)
as:
J = L (AP-onflTAe)+ $Q
canbe'modifiedorreduced.Theresultistheonenormallyusedtodescriberootpressure
exudationofexcisedrootsystems (House&Findlay,1966):
(7)
J V = Lp k(-anRTAo
s )+» o
where J isthevolumefluxofwaterintotheroot,
v
I isthehydraulicconductivityoftheroot,
APisthetranspirationtension,
_
,,..„«. -f thp effectiveosmoticmembraneintheroot,
o isthereflexioncoefficientoftheeriective
„.,«.„,,ca1t.
„,-iQr„-^c<vfthecompletelydissociatedsalt,
n isthesumofthecationandanionvalenciesoftnecomp
y
R istheuniversalgasconstant,
T istheabsolutetemperature,
* «.-™Hiffprenceofsolutebetweenxylemsapandthe
Ac isthesubstanceconcentrationdifferenceor
s
externalsolution,
* isanon-osmotic (active)waterflux.
Th°issimplifiedmodelwithoneosmoticbarrier (membrane)assumes^ ^ » »
A «
J ofdecap^atedplantstobethesumofanosmoticcomponent(-y***.) « * » " £ "
component lQ. Consequentlythelongitudinalorupwardsaltflux,.willbeequaltothe
productof J andc , or:
59
J = J c. ,
S
V X
where o. isthesubstanceconcentrationofioniinxylemsap.Understeady-stateconditions,ifnosynthesisofnewadsorptionsitestakesplaces intheroot:
- J willequalthefluxoftotalsoluteorsaltabsorptionacross themembrane;
- ionabsorptionwillequalxylemtransport asnonetionaccumulation intherootwill
occuranymore.
Whereasalluptakeexperiments,describedinChapter5,werewithsingle excised
rootsofyounglowsaltmaizeplants,theexudationexperiments described inthenext
sectionswereperformedwithexcisedbranched rootsystemsofmaizeplants 5weeksold
aswellasexcisedyoungrootmaterialasused inpreviousabsorptionexperiments.The
latterallowedaccumulationandtransportdata (Chapters 5and6)tobecompared and
interpretedbetter.-Excisedcompleterootsystemswithcutstumpswereusedtocollect
moreexudate.Inthisway,moremeasurements (volumeofexudate,chemical composition
andosmoticpressure)couldbedonetocheckthevalidityofEquation7.
Thesaltconcentrationofthexylemsap,collectedbytheexudationmethod,is
consistentlyhigherthanoftherealxylemsapofintacttranspiringplants.Therefore,
theosmoticpressurecomponent indecapitatedrootsystems isoverestimated significantly.
Yet,onemayassumethatthenatureofmosttransportprocesseshasnotbeendisturbed
toofartodosomeinvestigationsontheabsorption-accumulation-transportmechanism
withdecapitatedmaizerootsystems.Moreover,takingaccount that'for24h theexcised
plantrootsdonotsufferfromlackofcarbohydrates andconsequently theuptakeofsalts
wasnotdifferentforexcisedandattachedroots,therateofupward salttransportwill
alsobeequalforintactplantsandexcisedrootsystemsunderequilibrium conditions.
Howeverupwardwaterflux J andconcentrations ofxylem saltwillbedifferentfor
intacttranspiringplantsandexcisedrootsystems.
6.1 TRANSPORTANDACCUMULATIONWITHTIME
Thissectionwillreviewresultsofexperiments onthechainofuptake,accumulation
andxylemtransportofpotassium ionsbydecapitatedmaizeroots,with particular
emphasisontheinteractionandtimecourseofthethreeprocesses.A root immersed
inasaltsolutioncanabsorbandaccumulate ions.Mechanisms ofinitialand stationary
ionuptakefromtheexternalsaltsolutionhavebeendealtwith indetail inChapter 5.
Asthecortexoftheyoungmaizerootoccupiesabout9(Hoftherootvolume (Anderson,
1975b),themajorityofionstakenupwillbeabsorbed andtemporarily stored incortex
cellsbeforebeingtransportedradiallyinwardtosupplythecellsofthesteleandthe
xylemvessels,whicheventually supplytheaerialpartsoftheplants.Therearetwo
parallelpathways formovementacrossthecortextowards thestele,onethrough the
extracellular spaceorapoplasmandtheotherthroughthesymplasm,fromthe cytoplasm
ofonecelltothenextbywayoftheplasmodesmata (Anderson, 1975b).This implies that
theionsupplytothexylemstreamisnotastraightforwardpassagefromtheexternal
solutiontothexylemstreambyoneofthetwopathways,themoresosincea significant
desorption/absorptionorexchangeofionsbetweenthesymplasmicthrough-put tothe
60
(8)
xylemandthevacuolesofthecorticalcellswilloccur.Accordingtothisexchange
mechanism,freshlyabsorbedions,presentinthesymplasmicstream,caneitherbeaccumulatedtemporarilyinthevacuolewithorwithoutanexchangeagainstequalionsalready
presentinthevacuole,orelsebetransporteddirectly (withoutanyexchange)or
indirectly (afteroneormoreexchangesteps)tothexylemvessels,afterpassingthe
endodermisandthestele.
thusaftertheinitialphaseofionabsorptionacrosstheplasmalemma,theradial
andsubsequentupwardtransportofsaltscanbedelayed,dependentontheintensityof
symplasmic/vacuolarsaltexchange.
Inthefollowingexperiments,exudationtrials,lasting60hwiththeirfeatures
suchasabsorption,accumulationandupwardtransportofsaltwerestudiedbothunder
non-equilibriumandequilibriumconditions,tofindoutmoreaboutsalttransportover
shortandlongdistanceswithintheroot.
Experiment 34: Uptake, accumulation and xylem transport of potassium in decapitated
root systems of maize plants. The experiment lasted 60 h in 10 mmol I solutions of
KNO , KCl and %K SO . Rubidium-86 was used as a tracer for potassium.
About24hafterbeginningoftheexperiment,thereisasignificantdeclineinthe
rateofKabsorption (A),intheexudationrate (B)andintheKconcentrationofthexylem
sap(C)(Fig.46).So24hafterdecapitationofthemaizeplants,thecarbohydratepool
oftherootsbecomesexhaustedandrootactivitydeclines.Consequently,afterexperimental
timesexceedingabout24h,steady-statedoesnotexistmore.Therefore,theuptake,
accumulationandtransportprocesseshavetobestudiedwiththismaterialfornomore
than24h,asinthefollowingfigures.Figure47demonstratesthat
1.absorptionofKwasinitiallyhigh,butreachedstationaryconditionsafterabout
S h(I);
2.thebulkofthefreshlyabsorbedpotassiumaccumulatedintherootcortexcellsduring
thefirst6-10h,butastheexperimentproceeds,cortexcellsseemedtobecomesaturated
andconsequentlytherateofKaccumulationdeclinedrapidlywithtime (III);
3.simultaneouslywithandcomplementarilytothisdeclineinKaccumulation,therateof
upwardKtransportincreasedexponentiallyduringthefirst10to15h(II);
4.afterabout15h,netaccumulationceasedandthereaftertheratesofabsorptionand
xylemtransportwerealmostequalandequilibriumorsteady-statewasreached..
Figure48A,B,Clikewiseshowsthatafterabout16h,steady-statewasreached.As
soonasthepotassiumconcentrationofthexylemsapo^ isconstant,A C Rwillbeconstant
tooandaccordingtotheEquations7and8boththevolumefluxofexudationJ and
y
thesubstancefluxofpotassiuminxylemJ Rmustbeconstantwithtimetoo.Figure
48A.Cindeedshowsaconstantflowofwaterandsalt (K)afteranadjustmentperiodof
about16h.Figure48A.BalsoprovesthatinitiallybothC R andJ^ werelow,butincreasedsimultaneously.Finally,thisalsoconfirmsthatwithin24h,mechanismsof
absorption,accumulationandxylemtransportofsaltsmovetowardssteady-stateby:
1.aconstantrateofionuptake;
? „ * r ••
,.-• *i,.,«-icn-mtiallvhiehandresultsinalowrateofxylem
2.arateofsaltaccumulationthatisinitiallyiiigii»»«
61
®
-1 -1
^jmol Kplant h
x
mmol K kg1 exudate
40H
i
400-
A. '
30320
240160-
?V
f
10-
/
0
0
V
f
20-
\
°-^s*.~^»
15
30
45
60
75h
80
0-0—o—o.
0
kg plant"1 h 1
0.075-
0.0600.0450.I.030- /
1
\
I<•. \
0.015
0.0
0
N(—\
15
30
45
60
75h
Fig.46.Experiment34.Absorption,accumulation
andxylemtransportofKindecapitated root
systemsofmaizeplants for60h.Experimental
solutionsofKN0 3 (x), KC1 (+)orJK2SO4(o)
were 10mmol1 .A.RateofKabsorption.B.
Rateofexudation Jv. C.ConcentrationofKin
theexudate.
umolKplant h
480
400
320-
240
160
25h
62
Fig.47.Experiment34.Rateofabsorption
(I),xylemtransport (II)andaccumulation
(III)ofpotassiumindecapitatedroot
systemsofmaizeplantsfor25h.Absorptionmediumisa 10mmol I"1KNO3solution.
kg kg"1 DM h"1
2.0
1.0
mmol K kg ' exudate
40-i
20-
mmolKkg"1DMh1
X
1
60-
©
1
40-X—
20-
0-
16
24h
Fig.48.Experiment34.Absorption,accumulationandxylem
transportover24hofKindecapitatedrootsystemsof
maizeplants5weeksold.Absorptionmediumisa10mmol
l - 'KNO3solution.A.Rateofexudation<7V.B.concentraC.UpwardtransportofKin
tionofKintheexudate
xylemsap J„
transport,butinarisingconcentrationofpotassiuminthesymplasticandxylemsap;
3.thisloadingwithpotassiumwillbeaccompaniedbyanenhancedrateofexudationand
salttransportontheonehandandadeclineinKloadontheotherhand.Thisenhancement
inKtransportwillcontinueuntilC R andJ Rareconstantand,moreover, ^ isequalto
therateofKabsorptionbytheroot.Atthispoint,steady-stateisreached.
6.2EFFECTOFKSTATUSOFTHEROOT
Duringradialsalttransport,silasticionscanbeabsorbedorexchangedagainst
thesamekindofionsinthevacuole.Becausevacuolesmaybeconsideredaslarge
reservoirsthataccumulateionsoraredepletedaccordingtoconcentrationgradients
withthecytoplasm,theexchangemechanismofsymplasticorcytoplasmicpotassiumwith
vacuolarpotassiumcanresultin:
"netaccumulationordepletionofpotassiuminthevacuole.Inbothcases,theconcentrationsbothinthevacuoleandinthesymplasm/cytoplasmwillchangeandnoconstant
salttransportwillbeachieved.
.
-absenceofanynetsalttransportbetweensymplasmandvacuole;inspiteofexchange
63
ofvacuolarandsymplasticsalts,thesaltconcentrationinbothcompartmentskeeps
constant.Freshlyabsorbedpotassiumwillbeexchangedagainstpotassiumalreadypresent
inthevacuoleandconsequentlypotassiumfromthevacuolewillbetransported,while
freshlyabsorbedpotassiumwillbeaccumulatedtemporarilyinthevacuolebeforebeing
transportedradiallytothexylemvessels.
Becausevacuolarandsymplasticsaltconcentrationwillregulateandcontrolboth
fluxes<(> and$ (Diagram 4), thesaltstatusofbothcompartmentswillalsoregulate
thesymplasticorradialsalttransport.Theeffectofloadingtherootcellswithpotassiumonsubsequentradialandupwardtransportofthisionicspecieswasstudiedin
thenextexperiment.Toeliminatetheradialdesorptionofpotassiumduringupward
transportinthecutstemofoldexcisedrootsystemsandtomaketransportdatacomparablewithuptakedataofChapter5,excisedlowsaltrootsofyoungmaizeplantswere
usedintheexperiment.
Experiment
35: Influx,
in excised
lew-salt
solutions
containing
Absorption
and rate of accumulation
maize roots after
—7
O.S mmol I
from a 1 mmol I
used as a tracer
treatment
CaSO. (control),
KCl solution
and upward transport
of
of the intact
for 24 h in
i mmol I
plants
—7
potassium
—
KCl or 10 mmol I
was measured for 10 h; Rubidium-86
1
KCl.
was
for K.
Figure49A,Bindicatesthatlow-saltrootmaterialcanaccumulateallfreshly
absorbedpotassiumforthefirst6h.Sothebulkoftheionsaredivertedfromthe
radialsymplasticsalttransportstream Rafterpassingtheplasmalemmaandareaccumulatedinthevacuole O ) .Theoppositeflux $ willthenbeloworevennegligible.
Afterwards,upwardtransportofpotassiumthroughxylemstarts,showingroughlyan
exponentialincreaseuntil10h.Thequestionwhethergrossexchangefluxes<j> and
$ proceedduringtheperiodcannotbeansweredbythisexperimentbecausepotassium,
cytoplasm-symplasm
xylem
outer
solution
>"oc
vacuoles
if
64
*0r
*R 2
Diagram4.Schemeofthedifferenttransfersintheroot (afterPitman,1971).
Subscripto=outersolution;c=cytoplasmsymplasm:v=vacuole;x=xylem. M =direct
symplastictransfer, i?
andfl,arenettransfers.
mmolKkg1DMh"1
mmolKkg"1DMh"
30T
10h
Fig.49.Experiment35.Influx (x),
upwardxylemtransport (o,»)and
accumulation (4,A)ofpotassiumin
excisedmaizerootsfor 10h,after
24htreatmentofintactplantson:
A,B.a0.5mmoll - 1 CaSO^solution
(control);C,D.a 1mmol1 _1KC1
solution;E,F.a10mmoll"1KC1
solution.Openandsolidsymbols
representKandK(total),respectively.AbsorptionmediumisaI
mmoll -1KC1solution.
accumulatedinthevacuoleduringtheinitialloadingperiodwillbelabelledwith
Rb.
Thereforefreshlyabsorbed,labelledpotassiummovingallthewaywiththesymplastic
streamisnotdistinctfromlabelledpotassiumdesorbedbythevacuole(* )andsubsequentlytransportedupwardbythexylemstream.
EffectsonKinfluxofloadingrootsfor24hinanunlabelledsolutionofKC1,
equaltothatusedduringthesubsequentabsorption-transportexperiment,arepresentedin
Figure49C,D.Figure49CprovesthattheserootspreloadedwithKC1supportanupwardxylem
fluxofpotassium,whichcanbemeasuredimmediatelyafterexcisingtheroots.Thisupward
flowconsistedofbothlabelledandunlabelledpotassium.Potassiumthathasbeenabsorbed
andstoredduringloading,asitisexchangedgraduallyagainstfreshlyabsorbed (labelled)
potassium,thenmovesradiallyandlongitudinallytothecutendoftheexcisedroots.A
fractionofthefreshlyabsorbedpotassiumisnotexchangedagainstvacuolarpotassium,but
istransporteddirectlytothexylemvesselsbytheradialsymplasticflowof,salt.
Sounlabelledandfreshlyabsorbedlabelledpotassiumaretransportedupwards
(Fig.49C),andthefractionoflabelledpotassiumintotalaccumulationofKincreasesat
theexpenseofunlabelledpotassium,alreadypresent(Fig.49D).Figure49C.Dalsoindicatesthatsteady-stateseemstobedisturbedforabout2-3h,butisrestoredthereafter.
65
Loadingofrootsfor24h inasolutionofKC1 10mmol 1~ _yieldstransportaccumulationcurves,deviating fromthoseofFigure49C,Dasfollows:
1.Loading intherelativelyhighexternalconcentrationofKraises the internal cellular
concentrationofKtoahigher levelthanresultsaftertransfertoalowexternal solution
ofK.Thereforethesymplastic (cytoplasmic)Kconcentration decreases,because ofa
retardedplasmalemmainflux$ . Next,thislowered cytoplasmic concentrationmakes the
concentrationgradientwiththevacuolesteeperandconsequently the ionflux ij> 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 $ , <t> 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<r
0.1
72
0.5
1.0
5.0 10.0
mmolKI"1
Fig.53.Experiment39.Steady-state
transportinxylemofpotassium indecapitatedrootsystemsofmaizeplants
fromabsorptionsolutionswithdifferent
substanceconcentrationsofKCl (x)and
K2SO4(o).Absorptionwasmeasured 18-24
haftertransfer.
aperiod characterized ordominatedby ionaccumulation ($ ,• )»withoutasignificant
radialorvascular flux.Probably,onlyunderthesenon-equilibrium conditionswillthe
fluxes<1> and 4 showadualuptake isotherm,while later,afterreachingrealsteadyoc
cv
stateconditions,bothfluxesarereduceddrastically andconsequentlydual characteristics
donotexist anymore.Infact,thismeans thatonlyoneoftheSystems 1and2partakesin
upwardKtransport. InChapter7itwillbediscussedwhichsystemthisis.
6.4 TRANSPORTANDACCUMULATION OFKASAFFECTEDBYTHEEXTERNALKCONCENTRATION
Thispart givesmore detailsabouttheconsecutiveprocessesofabsorption,accumulationandtransport ofpotassium andtheirresponsetotheexternalconcentrationofK
andthekindofcounterion.
Rootsystemswere transferred toexudationmediaofdifferent strengthandfor24h
thexylemtransportbythecutendsoftherootsystemswasestimatedperiodically from
samplesaccumulatedbycontinuous collection.Attheendoftheexperiment,totalabsorptioncouldbecalculated asthesumofionaccumulationplusiontransport.Intheexperiments,specialattentionwaspaidtotherelationshipbetweentheexternalpotassium
concentrationandthetimecourseofpotassiumtransport.According toexternalKconcentration,adrasticchange inconcentrationgradientwilldevelop immediatelyafterthe
transferofthe low-salt roots tothedifferentexudationmedia.Ionabsorptionandion
accumulation U and• )willbedependentonorberegulatedbytheexternalconcentration (Chapters).Howe'ver,howexudationandxylemtransportchangewithtimeandwith
external saltconcentration islittledescribed intheliterature (House&Findlay,1966;
Meiri, 1973).
Inthenext experiment,potassium transport isothermsweremadewithCI,N0 3 and S0 4
ascounterion.Inthisway,special informationwasobtainedontheroleoftheanionin
allthreeprocesses during anexperimentalperiodthatwassufficiently longtoreach
steady-state.Under theseconditionsnonetaccumulationoccurs,andabsorptionandxylem
transportofpotassiumproceed steadilyandatequalrates (Section6.2).
Experiment 40: Absorption, accumulation and xylem transport of potassium in decapitated root systems of maize plants 5 weeks old with Cl, SO, and Ws as counterionAbsorption media were solutions of KCl,% V ° 4 and W°3 °' °A' °-5> U°' S'°
10.0 nvnol I1. The experiment lasted 24 h, without use of radioactive
isotopes.
Asapart oftheNO,wasprobably converted intoorganicnitrogenafter a b s ° ^ ° n
andtotalNO,uptakewas'assumedtobethesumofNO3accumulatedandNO3transported,the
valuesplotted inthenitrateabsorptionisotherm (I)willprobablybeunderestimated.
Allthreepotassiumabsorptionisothermsweresimlar (Fig.54); onlyatexternal
concentrations ofKexceeding5mmoll"1 allthreequantitiestendedtoincrease Thus
, ,,„ Ficire53 noneofthesethreeshoweda
justlikethetransport isotherms,presented inFigure ;>:>, 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 <7Vduring steady-state.
0,8 -A-
0.1
0.5
1.0
5.0 10.0
mmolKI"'
salt concentration.Although An isconstant forall the tested external salt concentrations,itismarkedly lower forK 2 S 0 4 plants than forplants suppliedwith KC1or KN0 3 How andwhy plants reach constant An for totally different external concentrations
forany kind of salt,but reach equilibriumwith K 2 S 0 4 at significantly lower Anvalues is
stillunclear.The exudation rates (Fig. 56CJ differ fromexpected values,at least for
theKC1 and KN0 3 treatments .If^ =L ? (-oRTnAes)
+* Q (Equation 7 ) , and Ac aswell as
L p and aare constantwith different external concentrations, J should be constant too.
However,bothKC1 and KN0 3 treatments donot showa constant Jv withvarying K concentrationof themedium.Perhaps oneorbothmembrane characteristics L or awill vary
withvarying internal concentration.However, themodelwith a single osmoticbarrier as
used for theFluxEquation 6could alsobe toosimple.
76
6.5 EFFECTOFGLUCOSE,CNANDDNPONABSORPTIONANDXYLEMTRANSPORTOFPOTASSIUM
Accumulationofionsinthexylemsapstronglysuggeststhatthispolarorradial
transportofionsisanactiveprocess.Theelectricalpotentialdifferencebetweenthe
exudationsapandtheexternalsolution,measuredbyBowlingetal. (1966), indicates
thatanionsmove intothexylemagainsttheelectrochemicalgradient,also indicating
activetransport.Cationsontheotherhandmovewiththeelectricalpotentialgradient,
exceptpotassiumwhichisclosetoequilibrium.Consequently,thecentripetal flowof
ionsappearstoconsistofanactivetransportofanionsaccompaniedbycationstomaintainelectricalneutrality.
Although theconcentrationandthepotentialgradientssuggestanactivepolartransportofionsbetweentheexternalmediumandthexylemvesselsthroughsymplasmor
apoplasm,themechanismofiontransportduringtheintermediatesteps (e.g.during
transport throughapoplasm,symplasmorthefinalsecretionintothexylem)isstill
poorlyknowandconfused.Muchcriticismhasbeendirected inrecent literatureagainst
thetheoryofCrafts&Broyer (1938)thattheradialtransportofionscanbesplitup
inthreesubsequentstages:
1.activeabsorptionofionsattheepidermis;
2.transportthroughthesymplasmdownaconcentrationgradienttothestele;
3.leakageofionsfromthestelarcells intodeadxylemvessels.
Becauseoftheexistenceofafreespace,waterandsaltscanfreelypenetrate into
therootasfarastheendodermisthroughthecellwalls.Consequently,itisnotthe
surfaceareaoftherootthatisthefirststageofsaltuptake,butthetotalvolumeof
thecorticalcellwalls.Thus,activeabsorptionwillnotberestricted totheepidermis
asmentionedunderPoint 1,butwilltakeplacebytheouterepidermiscellsaswellas
bythecortexcellsasthesaltspassthecortexbywayoftheapoplasm.
According torecentmeasurementsofinternalcellularionactivitiesbyuseof
selectiveelectrodes,Point2hasalsobeencriticizedstrongly.According toDunlop&
Bowling (1971),nosignificantradialtrendexistsinpotassiumactivityacrossthemaize
root.Thus,noconcentrationgradientexistsfromtheepidermis tothesteleandconsequentlydiffusionasthedrivingforceforradialsymplasmictransportisdoubtfultoo.
Whetherstelartransportandthesubsequent finalstepofsalttransportintothe
xylemvessels ispassiveishardtosay.Thatisolatedstelarcellshavetheabilityto
accumulate ionsjustlikethecorticalcells (Dunlop&Bowling,1971)doesnotprovethe
existence ofanactivestelariontransport.Alsoinhibitors likeCCCP,DNPandCNhave
beenusedtostudythetransferofionsfromtheroottissuetothexyleminmaizeroots.
Howeverthesechemicals inhibitabsorptionandaccumulationofionsbytheroottoo
(Chapter 5). Consequently,inhibitionofiontransferintothexylemvesselscanbean
indirecteffectofthesecompoundsbytheirinhibitionofionabsorption.Thereisno
evidencewhetherthesiteofactionoftheseinhibitors isatthesteleoratthecortex.
Inthenextexperimentsabsorptionandxylemtransportofionsarestudiedsimiltaneouslywith time.AfteradditionofglucoseoroneoftheinhibitorsDNPorCN,the
ratesofabsorptionandxylemtransportweremeasuredcarefully.A differenceintimeor
intensityoftheresponseofabsorptionandtransportwouldindicatewhetherstelar
77
transportofionsisactive.
Experiment 41: Absorption and xylem transport
of potassium in excised low-salt maize
—3
—1
roots with and without an addition of glucose (1%W/V)or DNP (10
1 mmol I
KCl absorption solution
24 h after starting
mental time was 50 h; absorption and transport
as tracer by depletion and cup-technique,
mol I ) to the
the experiment.
Total
experi-
of potassium were measured with
respectively
(Section
Rb
3.2.2).
Figure57showsthatDNPstartedtoinhibitbothabsorptionandupwardtransport
within2h.TherateofKabsorptiondeclinedveryrapidlyandeventurnednegativeafter
the2h,whereasthecontroltreatment (withoutDNP)showedalmoststeady-stateKabsorption.ParalleltotheinhibitionofKabsorption,thetransportcurveforK(total)shows
asimilarshape.About2hafterDNPtreatmentstarted,Ktransportwasblockedcompletelyanddidnotrecover.
Afteradditionofglucosetotherootmedium,absorptionandtransportcurveswere
muchdifferentfromthosewithDNP(Fig.58).Therateofabsorptionofthecontrol
declinedwithtime,probablybyexhaustionofendogenouscarbohydrates,butaddition
ofglucosekeptKabsorptionalmoststationaryfor50h.However,thetimecurveofthe
K(total)transportshowedaremarkablephenomenon.Immediatelyafteradditionofglucose
totheexternalmedium,K(total)transportdroppedtozeroforabout4h,whilesubsequent
tothis'deadperiod'K(total)transportstartedagainandacceleratedtoahighlevel.
Onelineofreasoningmightbe,thattheactivecomponentofthistransportmightbe
mmolKkg-1DMh"1
Fig.57.Experiment41.Absorption
(o,»)andupwardxylemtransport
(A,A)ofpotassiuminexcisedlowsaltrootsover48h.Openand
filled symbolsrepresentcontrol
(withoutDNP)andwithDNPatsubstanceconcentration 10"^molI"1.
After24h,DNPwasaddedtothe
absorptionsolutionwithKCl at
1mmol1~1.
78
1%glucose
mmolKkg1DMh"1
•—.
^
i
0
8
16
24
»
•
^ - ^
Fig. 58.Experiment 41.Absorption
(o,»)andupward transportinxylem
(A,A)ofpotassiuminexcised lowsalt rootsfor48h.Openandfilled
symbols represent control (without
glucose) andwith 1% {W/V) glucose.
After24h, glucosewas addedto
the absorption solutionofKC1at
substance concentration 1mmol l - '.
32
the transferofions into thexylemvessels rather thangenerationofawater flux,which
couldbeasecondary osmotic effect.Ifso,factors thattemporarily inhibit flowofwater
andmatter,but allow iontransfer,mightproduceastoreofpotassiuminthevessels,
leadingtoamore concentrated exudate oncemass flow resumes.Figure58provides only
slight indications that thismightbethecase.
Inthenext experiment,therateofexudationand the concentrationofKinexudate
were studied against time,with andwithout additionofaninhibitorand glucose.
Experiment 42: Potassium concentration
of exudate and rate of exudation of
root systems of maize plants 5 weeks old, transferred
The three treatments
I
KCN were started
used as a tracer for
(1) control
(no addition),
to a 10 mmol I
KCl
decapitated
solution.
(2) 1%(W/V) glucose and (3) 1 mmol
12 h later and the experiment continued 64 h. Ruhidium-86 was
potassium.
Exudation curves (Fig.59A)for all three treatments illustrate thatCN depresses
the rateofexudation significantly and almost immediately afteradditionofinhibitor.
Glucose immediately almosthalts exudation for aboutSh.After this 'deadperiod',
exudationrestarts and soonreachessteadyrate.
After additionofglucose,concentrationofKinxylem seemedtodecrease somewhat
79
kg kg"1 DM
^^-^•x
6^ X
o
X
4--
2-
y
x ^
y
/
n- K
0
,^A-
/!'
i
i
A
_-A
- " ^
i
mmol Kkg""'
18-
mmol K kg" DM
60-
. /
40
x^
S
20H
o-U*«*5
o
=&
20
A
A-
40
GO h
Fig.59.Experiment42.Transportofwaterand
potassiumindecapitated rootsystemsfor64h.
AbsorptionsolutionwasasolutionofKC1at10
mmol1~1.(x)control;(o)and (A)meanadditions
of 1% (W/V) glucoseand 1mmol I"1 KCNtotheabsorptionsolution,respectively.A.Exudation
(cumulative).B.ConcentrationofKinexudates.
C.Upward transportofKinxylem (cumulative).
for5h,andthenincreasedtoahighvalue (Fig.59B).Ontheotherhand,concentrations
inxylemoftheCNtreatmentwaslessforabout24hthaninthecontrol.Afterwards
theywerealmostequal,butdecreasedgraduallywithtime.Thelackofanincreasein
concentrationinxylemduringthe5hafteradditionofglucosecouldbeinterpretedas
indicationthatstelartransportandfinaltransferofKintothexylemvesselsispassive.
ActivetransferoractiveabsorptionofKintothexylemvesselswithsimultaneous
depressionintherateofexudation (Fig.59A)wouldatfirstsightresultinincreased
Kconcentrationoftheexudatebutthiswasnotobserved.However,theslightand
short-liveddepressionoftheKconcentrationintheexudatecouldwellbecausedby
continuanceorincreaseofabsorptionofsaltsintothecytoplasmafteradditionof
glucose,sothatpotassiumwouldbewithdrawnfromthexylemaswellasfromthemedium.
Thiswillresultinanenhancedaccumulationofsaltsintothevacuolesofcortexcells.
However,aftersometimetheosmoticequivalentoftheroottissueswouldincrease,by
80
uptakeofglucoseandaccumulationofsalt,toanosmoticpressure exceeding thatof
theexternal solution.Absorption,lateral transportandexudationofwaterwould then
restart.Asaresultoftheincreased internal symplasticconcentrationofsaltin
cortexcells,theexudatewillberatherrichinK.About18hafterglucose addition
tothe10mmol1 absorptionsolution,newsteadyratesinxylemtransportofKwere
reached (Fig.59C).
TheresultsofExperiments41and42donotjustifyanystatementwhether stelar
transportisactiveorpassive.
6.6 EFFECTOFpHOFTHEEXTERNALMEDIUMONpHOFEXUDATE
TheexternalpHseemstoaffecttheuptakeofcationsandanions significantly
(Section 5.2.1.),propablybycompetitionbetweenH andcationsontheonehandanda
competitionbetweenOH-HCO,andanionsontheother.
IfthecompetitionbetweenH andK inionuptakebeaccepted,onewouldexpect
thecellularconcentrationofH tobeaffectedbythismechanism too.TheoreticalcalculationsaboutDonnandistributions (Section5.2.1),indicatethatexternal [ H + ] affects
the internal [ H ].Therelationwouldbeinfluencedby [A~] ,(theinternalactivityof
non-diffusible organic anions),thenatureoftheseorganicanions (pX),andequivalent
substanceconcentrationandnatureoftheexternalinorganicsalts.Theactivityandnature
oftheorganic ions,suchasorganicacids,aminoacidsandproteinswouldregulateand
buffertheinternalcellularpHor[H.l.
AccordingtomeasurementsbyBowling (1974),alinearrelationshipexistsbetween
externalpHandpHofthevacuolarsapofrootcellsof HeHanthus annuus. Withintherange
ofexternalpH4-8,vacuolarpHrangesbetween4.5-6.5.Thismeansthatlowandhigh
valueswillbebuffered,sothattheinternalvacuolarpHrangesbetweenabout5and6.5.
MeasurementsofvacuolarpHacrosstherootofHeHanthus annuus, byBowling (1974),prove
thatvacuolarpHofallroottissuesrosefrom5.5attheepidermisto6.5attheprotoxylem.
Inthenextexperimentxylemsapwascollected frommaizerootsystemsgrownon
absorptionsolutionsvaryinginpHbetween3.8and6.8anditspHwasmeasured.
Experiment 43: Maize root systems were grown on absorption solutions with 10 mmol I
KCl, KNO, and kK SO.. After the plants were decapitated (18, 42, 66 and 90 h after
transferring the intact plants to the different solutions), exudates were collected
for 10 h. Low, medium and high external pHwere achieved by the different uptake
patterns in the absorption solutions of KJ50., KCl and KNO,.
InagreementwithHiatt's (1967)andHiatt's (1968)findings,pHoftheK 2 S0 4 ,KN0 3
andKClsolutions,respectively,tendedtodecrease,toincreaseortoremainalmost
constantwithtime (Fig.60).Consequently,aftersometime,pHoftheexternalmedium
varied fromnearly3.5to6.8forthedifferentabsorptionsolutions.
MeasurementsofthepHoftheexudates,collectedatdifferenttimesandfordifferent
solutions,illustrate thatpHofthexylemsaprangedonlybetween5.2and5.8,whilepH
81
pHexudate
6.0i
5.0H
4.0-
i-^
3-0:
*^43.0
i
4.0
i
5.0
Fig. 60. Experiment 43. pH of exudates at different pH of absorption solution. Different pH
of external medium were achieved 18 ( 1 ) ,42 ( 2 ) ,
i
i
66 (3) and 90 (4) h after transferring plants on
6.0
7.0
10mmol l - ' solutions of J K 2 S 0 4 ( o ) ,KC1 (x) and
pH externalmedium K N 0 3 ( A ) .
of the external medium ranged between 3.8 and 6.8. As Bowling (1976) found, either a plant
root can exclude H from the root, or the root or plant cells have a more general buffering
capacity.
With a K 2 S 0 ^ supply to the plant root and the resulting low external p H , cation absorption will exceed anion absorption and excess o f internal positive charge will be compensated by organic acids, as shown in Table 11. These organic acids, mostly weak acids,
will buffer p H of the cell sap and the xylem sap markedly. As a result o f KNO nutrition,
with excess uptake of anion over cation and high external p H , an internal buffering
capacity will develop b y reduction of a fraction of the absorbed N O in the root and production of amino and other acids. Consequently, both organic acids and amino acids will
mostly and generally be the main internal buffering compounds. They will be able to
maintain internal pH of cell sap (vacuole and cytoplasm sap) as well as xylem sap within
certain limits.
82
7 Discussion
Ourdataforuptakeofpotassiumbytheroothavebeenpresented largely separately
fromdataonsubsequent transportwithintheroottissue,andwithoutanyfurtherdiscussion.Attentionwillnowbepaidtobasicideasaboutuptake andtransportofions
throughmaizeroots,inanattemptalsotofindlinksbetweenabsorptionand subsequent
transport acrosstheroottissueofbothsolventandsolute.
7.1ACCUMULATIONOFPOTASSIUMINTHEROOT
Exceptinafewexperiments,allexperimentswerewith low-saltroots.Theadvantages
ofusingrootsofyoungplantsgrownonagypsummediumarethreefold,e.g.
- avoidanceofisotopicexchangeduringabsorptionexperimentswithtracers;
- absenceofreleaseofpotassiumalreadypresentintherootbeforethebeginningofthe
absorptionexperiment;
- arelativelyhighrateofionuptake.
Butthese low-saltrootshaveachemicalcompositionquitedifferent fromrootscultivatedonacompletenutrientmedium.ThehighcontentofcarbohydrateandthelowcontentofsaltintheseCaSO,rootsmakecomparisonwithresultsobtainedwithhighsalt
rootsdifficult (Pitmanetal., 1971).
Astotheuptaketechniqueemployed,onemustdistinguishbetweenthetworather
differentmethods,thathavebeencalled 'uptakebydepletion'and 'uptakebyaccumulation'.Inshort-termexperiments,datafromthetwomethodsaredivergent. Insuch
experiments,thecontributionoftheinitialuptakeinthetotaluptakecanbeveryhigh
(Section 5.1).Absorptiondataobtainedbytheaccumulationmethodincludeonlypartof
thesolutes.Itisthepartabsorbedduringthesteady-statephase,becausesaltspresent
intheAFSareremovedbyarinseandexchangetreatmentoftheroots (Epsteinetal.,
1963). Inmostexperiments,thedepletionmethodwaschosen,becausethetimecourseof
theabsorption,obtainedveryeasilybythismethod,givesinformationaboutboththe
initialandstationaryphaseofionuptake,whereasdataobtainedbytheaccumulation
methodarelimitedtotherateofstationaryionuptake.However,insomeexperiments,with
toosmalldepletions (concentratedabsorptionsolutions)orwithconditions interfering
withtheg-radiationcountingtechnique (suchascolouredsolutions),theaccumulation
methodwasused.
Ontheuseof86 Rb asatracerforpotassiuminplantphysiology,opinionsare
divided.West&Pitman (1967)reportadifferentbehaviourof Kand Rbinshort-term
absorptionexperimentswiththemarinealgae Viva and Chaetomorpha. Howeverresultsof
preliminaryexperiments (Chapter 4), andinvestigationsbyMaasandLegget (1968),
Marschner&Schimansky (1971),Mesbahuletal.(1971)andSchimansky (1970)prove the
83
suitabilityof86 Rbasaphysiologicalsubstitutefor K.Especiallyinshort-termexperimentswithrelativelylowconcentrationsofpotassiumintheabsorptionsolutions,
thistracersubstitutionisfullyjustified.
Thereasonwhyalmostalluptakeexperimentswerewithexcisedrootsinsteadofintact
plantsistopreventtheshootfrominterferingwiththeuptakecapacityoftheroot.
However,theworkwithexcisedrootsordecapitatedrootsystems,bathing completelyinthe
absorptionmediumasdescribedbyEpsteinetal. (1963),hasoftenbeencriticized,
because:
1.areleaseorleakageofpotassiumbythecutxylemvesselsofexcisedrootsandepicotyls,
whichareinopenconnectionwiththeabsorptionmedium,mightresultinincorrectdata
(Pitmanetal.,1971).Thisxylemorvascularfluxhasbeeninvestigatedinparticularby
exudationexperiments (Chapter6 ) ;
2.afterdecapitation,thedownwardphloemstreamofassimilatesfromshoottoroothas
beencutoff;thelattercanexhausttheexcisedrootmaterial (lackofcarbohydrates)and
subsequentlysaltabsorptionratetapersoff.
Resultsfromthedifferentexperimentsindicatethatvasculareffluxthroughexcised
rootswillbenegligibleiftheabsorptionexperimentisdoneeitherwith low-saltroots
duringanabsorptionperiodofupto8h,orwithhigh-saltrootsforupto6to8hwith
tracers.
TheseCaSO,rootsdonotseemtolackcarbohydratesduringshort-termexperiments.
Long-termabsorptionexperimentswithrootshavingahighersaltcontentdidnotshowa
reductioninuptakerateuntil20-24hafterdecapitation (Fig.46).Thesefindingsjustify
theuseofexcisedrootsinallshort-terminfluxexperimentswithtracers.Ontheother
hand,insimultaneousinflux-effluxexperimentstheuseofexcisedrootscanberisky.
Withthetechniqueemployed,theeffluxratewasmeasuredafterabsorptionforatleast10
h.Someasuredeffluxcouldbecomposedofbothatrue (Jackson&Stief,1965)andavasculareffluxcomponent.
7.1.1
Phases in the time course of the uptake
Aftertherootmaterialistransferredfromadilutedtoamoreconcentratedsolution,
thereisaperiodofrapiduptakeofpotassium,followedbyaperiodoflessrapidand
constantuptake (Chapter 5). Inliterature,thisrapiduptakeduringthefirstperiodis
calledinitialuptake,whereastheconstantuptakeduringthesubsequentphaseisindicated
assteady-stateorstationaryuptake.Notonlytimecoursesoftheinfluxshowthesetwo
phases,butalsoefflux.Rootstransferred fromalabelledabsorptionsolutiontoan
equivalentunlabelledsolutionshowarapidlossofactivity,followedbyanalmostconstantreleaseoflabel.
Inpotassiumabsorptionexperiments (Section5.1)withcontinuoustitration,an
intermediatetransitionphasewasdetected.AsreportedbyLuttge&Pallaghy (1972) and
Helleretal. (1973),afirstshortperiodofrapiduptakeisfollowedbyextremelyslow
potassiumuptakeduringasecondphaseofabout15min.Duringthesubsequentthirdphase,
uptakeratewasconstantwithtime.Thissecondphaseisnotarealtransitionalphase
betweenaprocessofinitialuptake (fillingofAFS)andasubsequentsecondprocessof
84
steady-stateuptake (Fig.7).ThefillingoftheAFSseemedtobecompletedafteronly
about 1h,whilerealaccumulationofpotassiumexistsfromthebeginningoftheabsorption
period.Alsothetimecourseofaccumulationitselfindicatestheexistenceofthissecond
phase,characterizedbyalowinfluxrateofpotassium.This impliesuptakekineticsmore
complicated thanpostulatedbythethree-compartmentalmodel (Cram,1968;Pitman,1963).
Thismodeloffreespace-cytoplasm-vacuoleinseriesdoesnotapplyunderallcircumstancesandforallrootmaterial.Morediscussionaboutthekineticsofthethree-phase
coursewillbepresentedinthenextsection.
Thesignificanceofthesethreephasesintheuptake-timecurveliesmainlyin
deriving informationaboutthesystemofionabsorption.Undernormalsteady-state conditionsofplantgrowth,thestationarysaltuptakewillbebyfarthemost importantcomponent.Onlyundernon-equilibrium,e.g.justaftersupplyoffertilizerorwater,afast
accumulationordepletionofsaltintheroottissue (AFS)willoccur,comparablewiththe
initialsaltflux.Theinitialuptakecomponentallowsthefollowingphasestoproceed
andregulatessupplyofsaltstobeabsorbedduringthesubsequentphase(s)ofionabsorption.Insaltabsorptionexperimentswithlow-saltroots,theinitialphasemustbeseen
moreorlessasan'artificial'period (artefact)beforerealionaccumulation.
7.1.2
Uptake
kinetios
Asmostattentioninthisstudyisconcentratedonthestationaryuptakeandsteadystateaccumulationofsaltswithintheroot,theinitialphaseofsaltabsorptionwill
beonlybrieflydiscussed.Onlythosefeaturesoftheinitialuptakewillbepresented
whichparticipateinorareassociatedwithsubsequentphasesofsaltabsorption.Some
speculations aboutthemechanismofthesecondphasewillbepresented togetherwith
discussionsonthetransportkineticsofsaltsduringthethirdphaseofsteady-state.
7.1.2.1 Initialuptakeandapparentfreespace
AsreportedbyBriggsetal. (1961),Grobler (1959),Nobel (1970)andVervelde (1952),
initialuptakeorthefillingofAFSisaphysico-chemicalprocess.Soluteandsolventmove
readily fromtheexternalsolutionintothewaterfreespace (WFS)oftheroottissues,
incontrasttotransporttothepartoftheroottissueorrootcellsintowhichthesolvent,butnotthesolute,readilypenetrates.Thisspace,theDonnanfreespace (DFS),in
thecellwallandprobablypartofthecytoplasm,showsthefeaturesofaDonnandistribution.
Experiment9indicatesthatabsorptionofcationsduringinitialuptakeishighly
positivelycorrelatedwiththepHofthemedium.Thiscontrastswiththeabsorptionof
anionsduringthisphase,whichislowandnearly independentofpH.Thismayindicate,
thatcompoundssuchasfixedcarboxylgroupsorcarboxylates,aminoacidsorproteinsare
responsibleforthefastabsorptionoradsorptionofcationsduringtheinitialphase,
becausethedegreeofdissociationofthesecompoundsandthusthenumberofnegative
chargesisstronglydependentofpH.TheoreticalcalculationsaboutDonnandistributions,
madefordifferentpHoftheexternalmediumandfordifferentpXofthehypothetical
85
indiffusibleorrestrainedacidsgaveresultsalsoinagreementwiththeexperimentaldata.
IncontrastwiththeresultsofIghe&Pettersson (1974),datagatheredinExperiments27,30and33showthatinitialuptakeofpotassiumisnotsignificantlyaffected
bythetemperatureoftheabsorptionsolutionnorbytreatmentoftherootswithglucose
orinhibitorsasCNandDNP.Soinitialuptake,contraryto.steady-state,isabindingof
ionsintheAFSoftheroot,whichisnotdirectlylinkedtometabolism.However,Ighe&
Pettersson (1974)andPettersson (1971)postulatedtheexistenceofacloserelationship
betweenexchangeablelabileboundsulphateandrubidium,andtherateofsubsequentuptake
oftheseions.
A changeinmembranepermeabilitylikewisedidnotaffectinitialuptake.Obviously,
a changedpermeabilityofcellularmembranesforelectrolytes,effectedbyacalcium
deficiencyoftheplantcellorbyatreatmentoftheexcisedmaizerootswithoneofthe
surfaceactivechemicalslikeglyceryltriacetate,didnotchangetheadsorptioncapacity
ofthecellsforpotassiumions.
Thismeansthatpermeabilitydoesnotplayasubstantialroleintheinitialadsorptionofpotassium.TheconclusioncouldbethattheAFSdoesnotextendtoanyappreciable
depthandmainlyoccupiesthecellwallandtheouterpartofthecytoplasm.Potassiumthen
doesnothavetotravelveryfarbeforebeingadsorbedandislittleretardedbyadiminishedpermeability.ThisidearunssomewhatparalleltotheideaofDainty &Hope (1959)
thattheAFSoccupiesthecellwallonly.
Asexpected,theamountofpotassiumadsorbedduringtheinitialphasewillbe
negativelycorrelatedtothepotassiumstatusoftheroottissue.
7.1.2.2 Steady-stateofionuptake
Potassiumaccumulatedduringthesteady-stateofionuptake,incontrasttothepotassiumintheAFS,isnotdirectlynorcompletelyexchangeableagainsta 10mmol1 KC1
solution (Figs.21and 45).Macklon&Higinbotham (1970)found,for4 2 K accumulatedinthe
freespace,cytoplasmandvacuoleofexcisedpeaepicotyls,halftimeexchangevalues (T,)
of2.53,69minand737h,respectively.Thismeansthat,onashort-term,thepassage
acrosstheplasmalemmabysolutesisalmostirreversible.Afterpassingthisbarrier,ions
canmoveto3differentdestinationsby
,
1.accumulationinthecytoplasm;
2.passingthetonoplastandaccumulationinthevacuole;
3.symplasmictranscellularradialtransport,andlong-distancelongitudinaltransport
throughxylem.
Inshort-termuptake (Chapter 5 ) , thenetsolutetransportduringsteady-statewill
becomposedofthefluxes$ o c ,<|> >* cv >*c >whilethelong-distancexylemtransport
canbeneglectedthere.
.
Potassiumuptakeisothermsshowclearlyadualcharacter (Fig.15).Ingeneral,the
lowerisotherm,hereafterreferredtoasSystem 1isoperationalatconcentrationsbelow1
mmol1 ,whereastheupperisotherm (System2)isoperativeatabove 1mmoll - 1 .Both
systemsarecharacterizedbydifferent K^andV m a ,butarealsodifferentlydependent
ontheanionspresent (Luttge,1973).Theclassicalinterpretationsofthedualisotherm
86
have,untilrecently,restedontheconceptofthecellasthreecompartments,cellwall,
cytoplasmandvacuole,betweenwhich thetransportofionsmustnecessarilybe sequential
(Torii&Laties,1966a).According tothisconcept,atlowerconcentrations oftheabsorptionsolution,therateofionuptakeisregulatedattheoutermembrane,theplasmalemma,
whileathighconcentrations,asaresultofahighrateofpassivediffusionofions
throughtheplasmalemma,therealbarrierforionuptakeisformedbythetonoplast
(System2 ) .
Criticismbothabouttheexistenceofthedualmechanismandthecorrectnessofthe
staticthree-compartmentmodeloftheplantcellhasrecentlychangedtheviewofmany
plantphysiologists abouttheuptakemechanismandthesiteofuptakeisotherms.Cram&
Laties (1971),Leighetal. (1973)andMacRobbie (1970)justifytheassumptionofadirect
pathway,possiblybywayofmembrane-boundvesicles,fromtheexternalsolutiontothe
vacuole.Inthisview,thecytoplasmdoesnotbehaveasasinglehomogeneouscompartment,
butasabiphasicone,composedofagroundcytoplasmandanumberofmicro-vesicles or
minivacuoles.A simplepicturepresentedbyCram (1973a)isthatofminivacuolesaccumulatingCIfromtheexternalsolutionorcytoplasm,restrictingexchangeofthisCIwith
thecytoplasmicCIwhile crossingthecytoplasm,andfinallydischarging theCIintothe
mainvacuole(s).
Theexistenceofsuchvesiclesorminivacuoles,provedbyfreeze-etchmicrographsby
Leighetal. (1973)canpossiblyexplainthediporsecondphase intheinflux-timecurves.
Despiteaslightaccumulationofpotassiumduringtheinitialphase,itprobably takes
sometimebefore themaincytoplasmicsalttransportbyvesiclesstarts,asdescribedby
Cram (1973a).Consequently,aftertheveryfastfillingoftheAFS,saltaccumulationwill
bediminished.Thiswillbeexpressedbyadipintheuptake-timecurve.
There isroomforabetterexplanationofthedifferences inmechanismand location
ofthetwosystems,leadingtothedualisotherms.Althoughnofinalproofisavailable
fromthepresentstudy,anattemptwillbemadetoformulatesuchanexplanation.
Theprobability thatathighsaltconcentrationsSystem 1andSystem2actsimultaneouslyandthatthetotalsaltabsorptionistheresultantofbothsystems,doesnot
favourtheideathatSystem 2isamodificationofSystem 1,whichmodificationthenwould
comeaboutwhentheexternalpotassiumchlorideconcentrationexceeds 1mmol1 .Suggestionsdepictingsuchamodificationasaqualitativechangeincellstructureorcell
contentscannotbesupportedtherefore.
Partoftheexplanationforthedualcharacteroftheisotherm,ifitisnotanartifactoflow-saltroots,isthatthetwosystemshavedifferent locations intheroot.The
ideathatthetwoSystems 1and2actatthetwomembranesofthecell,beingtheplasmalemmaandthetonoplast,respectively,inparallelorinseries,postulatedbyTorii&
Laties (1966a),Osmond&George (1969),infactissuchanexplanationonthebasisof
spatialdifferentiation.Oneadditionalpartoftheexplanationthatissuggestedhere,
isbasedonthevariationbetweenroottissues.Inlongitudinalaswellasinradial
direction,cellsaredifferentinstructure,sizeandfunction.Differenceswhichmight
easilycontributetothedualcharacterofuptakeisotherms,areexistentbetweenthe
youngnon-vacuolatedcellsofroottips (onlySystem 1)andmaturevacuolatedparenchymatouscells (bothsystems),ashasbeenprovedbeforebyTorii &Laties (1966a).The ideas
ofadifferentiationbetweencellcompartmentsandadifferentiationbetweentissueshave
incommonthedifferenceinsymplasmaticcompartmentandtheenclosedsapcompartment.
Thenextpartoftheexplanationsuggestedhereis,thatSystem 1mainlyconsistsof
exchangeofpotassiumfromthesurroundingDFSwithsymplasmaticcations (mainlyH ) ,
whereasSystem2istheseparationofvascularorvacuolarsapfromthesurrounding
symplasm.Theseparationjustmentionedmayproceedinthenormalgradualwayor,ifthe
externalconditionshavebeenchangeddrasticallyasinsomeexperiments,inamore
turbulentway.Butinbothcasestheseparationmeansawithdrawalofequivalentamounts
ofmineralcationsandanionsfromthesymplasmaticcompartmentwhich,duetotheprinciplesoftheDonnandistribution,containsfeweranionsthancations,themoresoasthe
externalsaltconcentrationisless.Theavailabilityofanionsthuslimitssapseparation.
At lowexternalconcentrationssapseparationmayalmostceaseinspiteofahighdifferenceinosmoticpressurebetweenexistingsapandtheexternalsolution.At lowexternal
concentrationSystem2worksslowly.Infact,System2may-havearatewhichismore
orlessproportionaltotheconcentrationofsymplasmaticanionsasdeterminedbythe
externalsaltconcentration.ThisalsoexplainswhypotassiumuptakeratesofSystem
2dependsonthekindandtheconcentrationsofanionsintheexternalsolution(Hiatt,
1968;Exp.34). ThusitisnotjustthetonoplastthatgovernstherateofSystem2,
butitistheconductanceoftheadjacentcytoplasmaticcompartment forsalts.Asthe
concentrationoftheexternalsolutionincreases,thepassageofanionsthroughthe
symplasmwillbe lessdiscriminated,atthesametimemodifying therateandnatureof
on-goingvacuolation.
ThedynamicsofKinfluxacrosstheplasmalemma ($ Jinrelationtointernalconcentration [K.] oftheexcisedrootswasexaminedinExperiment 14.AsGlass (1975)found
forbarleyroots,Figure23showsthattheplasmalemmainflux<f> isinverselycorrelated
with [Kj .Themarkeddecreaseintracerinfluxastheinternalionconcentration
increasessuggestsnegativefeedbackbythecytoplasmicorvacuolarionconcentrations.A
vasculareffluxfromtherootswithhighinternalpotassiumconcentrationpossiblyadded
tothemeasuredinfluxinhibition.
Somefactors,probablyinvolvedincellmembranebehaviouraffectinfluxandefflux
ofpotassiumquitedifferently.A reductionincationinflux<J> withdecreasingexternal
pHwasobserved.However,effluxunderthesameconditionsdidnotshowanypHeffect.So
duringtheseshort-terminflux-effluxexperiments,lowpHeffectsarenotcausedby
injuryandconsequentleakageoftheroottissue.TypesofinjurysuggestedbyRainset
al. (1964)(forinstancedenaturationofproteins,nucleicacids,phospholipidsandother
polymersinmembranes),willpossiblybeinvolvedonlyinthelongterm.Obviously,pH
affectsthepotassiumabsorptionbyH + -K +competition,atleastduringtheseshort-term
experiments.A secondbutlessprobableexplanationforthereducedKuptakeisreduction
ofinitialuptakeoradsorption.
Theideathatduringshort-termabsorptionexperimentsCaaffectstheuptakeofmonovalentionsmainlythroughitseffectuponpermeabilityoftheplasmalemma (Waisel,1962),
isnotveryattractiveinthelightofeffluxexperiments.ThehypothesisofKahn&Hanson
(1957)thatCapresentintheabsorptionsolutionincreasestheaffinitybetweenKanda
postulatedcarrierseemstobemoreprobableinshort-termexperiments (Vietseffect).
Rootsofplants,grownforlongerperiodsbeforetheexperimentonamediumwithoutcalcium,showadecrease inCacontentandastronglyreducedinfluxduringsubsequentabsorptionofK. Ifaneffluxorexchangeperiodof1hdirectlyfollowsthe4h influx,a
tendencyforslowandgradualK-RbexchangeintheCa-richrootscanbeobserved,whereas
theCa-starvedmaizerootshaveafast 'explosive'releaseofRb.Thismay indicate that
Cadeficiency intherootresultsinanenhancedpermeabilityoftheoutercellmembrane,
theplasmalemma.
Changesinmembranepermeabilityhavebeenachievedartificiallybythesurface-active
compoundglyceryl triacetate.Kuiper (1967)showedreleaseofsolutesbybeanrootstobe
promotedbysolutionsofacetylatedglycerolcompounds.Istudiedtheeffectofthis
surface-active chemicalonmembranepermeability indirectlybyinfluxandeffluxmeasurementsofsolutesbytheroot.Results (Fig.33)illustratethatroots,bathinginanabsorptionsolutioncontaining 10 mol1 triacetin,showasignificantincreaseininflux
andastrongreductioninefflux,bothresultinginasignificantincreaseinnetuptake
ofK.Theobligatorypresenceofthesurfactant intheabsorptionsolutionmakesit
reasonabletoassumethatthetriacetinisdirectlycoupledtoeitherthesoluteortothe
membrane (compounds).Theformationofasurfactant-solutecomplexandasubsequent
accelerated influxofthiscomplexthroughtheapolarinteriorofthelipidbilayerof
thecellmembrane,asdescribedfortheionophorevalinomycin-potassiumcomplexby
Kinsky (1970),isnotveryplausibleforthisglyceryltriacetate,becausethestructure
ofthischemicalistotallydifferentfromanannularorringlikecompoundlikevalinomycin.
Analternative andmoreprobablemodeofactionoftriacetinisincorporationofseveral
ofthesemolecules intothemembranetoformaphysicalchannelinthemembrane,atransit
porewhichadmitscationsandsmallunchargedmolecules.Amechanismfortheactivityof
acetylatedcompounds,assuggestedbyKuiper (1972),inwhichsurfaceactivechemicals
interferewiththehydrophobicregionsofthemembrane,resultinginamorehydrophyllic
characterofthemembraneandanincreasedpermeabilitytowaterandsaltisanother
possibility.Thenegative influenceoftreatmentoftherootsonsubsequentuptakefroman
absorption solutionwithoutsurfactantcanbetheresultofa 'wash-out'ofdissolvedmembranecompounds.Thiscouldresult inalossofstructureorinastructuralcollapseof
themembrane andasubsequent increasedeffluxoftherootcells.Sincetheeffectof
triacetinoninfluxandeffluxisquitedifferentfordifferent ionsmeansthatalsothe
selectivityofthebiologicalmembraneshasbeenchangedbythissurfactant.
Theeffectofmetabolicinhibitiononinfluxandeffluxofpotassiumwasstudiedwith
CNandDNP,whichinhibitrespiratoryelectron-flowanduncoupleoxidativephosphorylation,
respectively,andbyusingchilledabsorptionsolutions.Togetherwiththeresultsof
Luttge&Laties (1967),Drew&Biddulph (1971)andLiittge(1975),thedataconfirmthe
inhibitionofsoluteinflux.Specialattentionwaspaidtotheeffectoftheseagentson
efflux.AsopposedtoCNtreatment,treatmentoftherootsofintactplantswith increasing
concentrationsofDNPsignificantlyincreasedthefractionofK ( Rb)released immediately
duringa 'rinse-exchange'periodoftherootsaftertheuptakeexperiment.SohighconcentrationsofDNPinterferebyuncouplingenergytransfer,andalsohaveanindirecteffect
onroottissue,probablybyachangedmembranestructure.TreatmentofrootswithDNP
89
changes thenormalshapeoftheuptake isotherm (Fig.36).Themetabolicallydrivenpart
oftheionuptakeisswitchedoffandtheremainingpart oftheKuptake consists of
electrochemical transport.
Thetemperatureoftheabsorptionsolutionstrongly affects therateofnetKuptake.
Temperaturemainlymodified influxandnotefflux.
Astimulatoryeffectonpotassium influxwasachievedbyincubationofrootsina11
(W/V) glucosesolution.Theselow-saltrootswith theirhighcarbohydrate contentshowa
positiveeffectofglucose.With lightanddarktreatment ofintactplants,theglucose
effectisnotsignificantafteracontinuous lighting oftheplant,butaftera longdarkperiodglucosesignificantly increases therateofKuptakebyexcised roots.Thesedark
andlighteffects,justlikethedayandnightsequence inKuptake indicate thatglucose
isanenergysourceforsalttransport.Especially thehigh content ofcarbohydrate in
theCaS0 4rootsinrelationtorootsofnormally grownplants (Breteler,1975)makesthe
behaviourofthegypsumrootsquitedifferent fromhigh-salt roots.Forexample,a
carboxylate effectonpotassiumuptake,asfoundbyBreteler (1975),didnot showup
withthislowsaltmaterial.
7.2 TRANSPORTMECHANISMOFSALTINROOTS
Besidestheuptakeofsaltsintheroot,thetransport ofions fromtheroot (source)
totheupperpartoftheplant (sink)isanessentialprocess,becausethebulkofinorganicsaltscanreachthegrowingshootonlybyalongitudinal transport throughxylem.
Thefactthatbothuptakeandtransportofsaltsarecoupled,while the transportofsalts
isalsocoupledtotheuptakeandtransportofwater,makes itimpossible toconfinethis
transportmodelonlytolongitudinal transportofsalt.
^Mostofthetransportexperimentswerewithcompleterootsystemswith cutstump
derived fromplants 5weeksold,grownoncompletenutrient solution (Table 1),untilone
weekbefore thebeginningoftheexperiment.Withtheseoldercomplete rootsystems,large
volumesofexudatecouldbecollectedeasilyandaccurately. Ina fewexperiments,salt
transportwasstudiedwithexcised,low-saltroots,identical totherootmaterialusedin
theuptakeexperiments,togetanimpressionabout therateofvascularsalteffluxduring
uptakeexperiments.Instudyofsimultaneous transport ofwater and soluteswithdecapitatedrootsystemsaconsiderablepartofthewater transport,related to transpiration,
iseliminated.Consequently,theremainingxylem transport inexudationexperiments is
inducedbyosmosis.
While inuptakeexperiments,steady-statewasreached within 1h (Fig.7 ) ,forsalt
transportittook 10hormore.Exudationexperimentsmustcontinueforatleast 20-24h.
Becausealltransportexperimentswerewithsolutions ofonesaltandplantswere grown
forthepreviousweekonalow-saltmedium, [K] and [K] were indicators forn.
and itQ ,respectively (Munting, 1977).
°
90
X
7.3MODELOFSALTANDWATER TRANSPORT
Thecommonviewhowaroottransportswaterfromtheexternalsolutiontothexylem
streamhasbeendescribedbyAnderson (1975a),House&Findlay (1966)andSlatyer (1967).
Waterflow J acrosstheroottothexylemcanbedescribedforintactplantsanddecapitated
V
rootsystemsbyEquation6and7ofChapter6.
J = L (AP -onRTAc )+*
v p"•
s' o
J = L (-anflTAe)+4
v p
(6)
*• '
s o
Excisedrootsbehave likeanosmometer.Saltsareaccumulatedinthexylemsapsothat
theosmoticequivalentofthexylemsap(n.)exceedstheosmoticequivalentoftheexternal
medium (n) .Aswatermovesbyosmosisintothexylem,thexylemsapexudesoutofthe
stump.Consequently,thesubstance fluxofionsJ gisequaltotheproductofexudation
rate J andthesubstanceconcentrationofionsintheexudatec.,or
V
J = J .a.
s
V I
Equation7indicates thatthevolumefluxofwaterintothexylemdependsontheosmotic
equivalentdifferenceAnandonpermeabilityoftheroottowater.
Experiment34confirmedthevalidityofEquations 7and8.Immediatelyafterthe
startofuptake,afastinitialionuptaketogetherwithanegligiblesalttransportin
xylemresultintoafastaccumulationofsaltsandthebuild-upofanosmoticgradient.
However,thisprocessofsaltaccumulationwillenhancebothn.andJg.Thelatterwill
thenreducetherateofsaltaccumulation.AfteracertaintimeAt,netaccumulationstops,
becauseofthesteadilyrisingtransportinxylem.T(Fig. 47).Asteady-stateisthus
reached,characterizedby:
-equalratesofuptakeandtransport;
-asteady flowofwaterandofexudation J^;
- nonetaccumulationofsaltwithintheroot;
-aconstantconcentrationofKinxylem [K.].
Obviously,theplantachievessteady-statebythesubstanceconcentrationgradientof
salts,asreflectedintheosmoticequivalentdifferencebetweenxylemsapandrootmedium.
Undertheseconditions,thefluxofsaltinxylemisdependentonlyontherateofsalt
absorptionbytheroot,becauseamountsofsaltequaltothefreshlyabsorbedsalt have
tobetransportedupwardtokeepaconstantA V Anothercharacteristicoftransportis
thatinsteady-stateA. isalmostequalforallexternalconcentrations ofsaltwithintherange0.1-10mmolK 5l " . Sorootcellshaveamechanismthatregulates"Ptake,
accumulationandtransportofsalts,almostindependentlyoftheexternalconcentraion.
Despitethisconstant A C R thevolumefluxofwater J isnotreallyconstantforall
externalconcentrationsL therange0.1-10nrooll " . LookingbacktoEquation wrfh
a constantAHandtheassumptionofas»llandconstantV avariationin J canonly
betheresultofavariationinip,aorboth.Thus,assuggestedbyKlepper (1967),, p
91
seemsnottobeconstant fordifferentconcentrations ofsaltintherootmedium.
Theroleoftheaniononthetransportpatternofbothsoluteandsolventissignificant (Fig.56).Insteady-state,Ac K isconstant fordifferentexternal concentrations
ofsomekindsofsaltsuchasKC1orKN0 3 ,buttotallydifferent forothersalts like
K 2 SO A .PlantssuppliedwithK 2 S0 4reachequilibrium atmuch lowerAnthanplants supplied
withKC1orKNO .As in
J equations 7and 8,thelowAn withK„SO,reduced J andeven
l 4
v
more J .
s
IhedifferencebetweenplantswithKC1andK 2 S0 4 couldbecausedbythedifferences
inanionuptake.WithKC1,uptakeandtransportofcationsarealmostequaltothoseof
anions.WithK 2 S0 4 ,theabsorptionoftheassociatedanion isfarlessandorganicacids
aresynthesized inquantitiesequivalent tothecationexcess (Hiatt,1968;Luttge, 1973).
TheconcentrationbetweenKand S0 4 inxylemsapofK 2 S0 4plantswas indeeddifferent.
A considerablepartofthesaltsinthexylemsapispresentinorganicform,mainlyas
malates (Table 11). Thesecarboxylatesmayeitherbedirectly involved intheaccumulationandtransportofwaterandsolutesorindirectlybyachange in L ora.Another
anioneffectistherateoftranslocationoffreshlyabsorbedpotassium"fromtherootto
theupperpartoftheplant.As intobaccoroots (Wallace,1967),potassiumwithnitrate
ascounterionwastransportedmuchfasterinmaizerootsthanpotassiumaccompaniedby
CIorS0 4 (Table 10).Thisindicates thatK
,incontrasttoK fcl .A and
Y
+
,
nitrate
chloride
1S tra
sulphate'
nsp°rtedalmostdirectlyfromtheoutersolutionthroughapoplasm and
symplasmintothexylemstream,withoutfirstbeingmixedwithorexchanged against a
poolofpotassiumalreadypresentinthevacuolesofcorticalcells.Potassium froma
KC1saltwasmixedwithpotassiumfromthepoolbeforebeingtransferred totheexudate.
Theexistenceofthisexchangesupports totheviewsexpressed inSection 7.1,
indicating thaterrors inabsorptiondata,causedbyvascularefflux,arenegligible in
absorptionexperimentswithtracers.Avasculareffluxofpotassiumwasabsentduringthe
first 8hofanexudationexperimentwithexcisedlow-saltgypsumroots (Fig.49).Onthe
otherhand,maizerootsloadedwithpotassiumhaveaconsiderablevasculareffluxof
potassiumwithin 1to2h.However,sinceonlyasmallfractionofthexylemKis labelled
after6h,mostofthepotassiumpresentintheexudateisderivedfromapotassiumpool
alreadypresentintheroottissue,whilefreshlyabsorbed,labelledpotassium isaccumulated temporarilyintherootbeforebeingtransferred tothexylemstream.Inthisway,
allabsorptiondataforupto6handobtainedbytracertechniques arefreefromerrors
thatmightarisefromtheconsiderablevasculareffluxofKatthecutend.As foundby
Meiri (1973)andHodges&Vaadia (1964),loadingofrootswithapotassium saltreduces
thetimetoreachequilibrium intransportofK.Uptakeandtransport isotherms thenshow
thesamepattern (Fig.51).
Theisothermspresented inFigure51 lackadualcharacter,supporting theviewthat
dualitycouldbeanartefactwithexcised low-saltroots,orwithshort-term absorption
experiments,becauseSystem 1thenhasanabnormallypredominantposition.
Xylemsapwassimilarincompositiontoroot-cell sap.Concentration ofK inthecell
sapofrootsystems,after24hofabsorptionofKfromdifferent solutions (Experiment40),
was 17.3-33.3mmol1' andofxylemsap (Fig.55)itwas 18-34mmol T 1 .
Despitehighvariance,timecoursesofabsorption,accumulationandxylem transport
92
ofKfordecapitatedrootsystems (Fig.46,47,48),andforexcisedlow-saltroots
(Fig.49)showclearsimilarityofinterrelationships.Somaizerootsobeycertaingeneral
rulesasdescribedbefore,irrespectiveofsaltstatus,age,completenessorexcision,
branchingornot.
Absorptionratesofpotassiumbyexcisedlow-saltmaizeroots (Chapter5)wereabout
10mmolkg" DMh - 1 butfordecapitatedrootswere15-65mmolkg -1 DMh - 1 ,usually25-30
mmolkg DMh .Perhapsattachedanddetachedrootsmaydifferslightlyinionabsorption
andiontransportrates,atleastifexpressedonbasisofdrymassofroots.Substance
fluxofKandvolumefluxofwaterwerebothinhibitedimmediatelyaftertheadditionof
CNandDNPtotheabsorptionsolution (Exp.41).Afteradditionofglucose,anewsteadystatewasreached,becauseoftheincreasedinternalosmoticequivalentH..Inthisnew
situation,bothinflux<f> andxylemflux J werehigherthanbefore,becauseabsorption
wasstimulatedbyglucose.
Extrapolationofsaltandwatertransportinexcisedrootsandrootsystemswithcut
endstotransportinintactplantsisdifficult.Transpirationinintactplantwould
enhancethelongitudinalupwardwaterstreamanddilutetheinternalconcentrationof
salt (Munting,1977). Inintactplants,Equation6willapply,butthehydrauliccomponentwill,dependentontherateoftranspirationoftheshoot,accountforthemajority
ofthevolumefluxofwater J .Withthispassiveupwardflowofwater,themajorityof
theabsorbedsaltisalsotransportedupwards.Asaconsequence,theinternalconcentrationinentiretranspiringplantswillbelowandtheroleofosmosisinwaterandsalt
transportwillbesmallerinintactplantsthaninexcisedroots.Evenso,itwouldbe
nicetoknowmoreaboutthecontributionoftheosmosistosaltandwatertransport.
93
Summary
Uptakeofnutrients inrootsandsubsequenttransportofthesesubstances fromroot
toaerialpartsofplants formthefoundationsofmineralnutritionofplants.Especially
thefirststep,absorptionofionsintorootsisaprocess governedbyacomplexof
internalandexternal factors.Thesecondphase,transport ofions towardsxylemvessels
(radialtransport)andupwardtransportwithinxylemvessels (longitudinal transport),is
closely linkedtoionabsorption.Afterpassing theplasmalemmaofrootcellssaltsare,
eitherunchangedoraftertransformation,translocated toaerialpartsoftheplant.
Intheliterature,uptakeandtransportofminerals inplantshavebeendescribed
indetail.However,littleefforthasbeenpaid tosimultaneous studyofabsorptionand
transportofions.
Thisreportdealswitheffectsofvarious internalandexternal factorsonabsorptionofpotassium inmaizeroots (Chapter 5).Absorption -exudationexperiments aredescribedandsimultaneousabsorption,accumulationandxylem transportofpotassium in
maizerootsarediscussed (Chapter 6 ) .Mostexudationexperimentswerewithcompleteroot
systemscutfrommaizeplants 5weeksold.
Absorptionofpotassiuminexcised rootsofmaizeplants (about 10daysold)was
studiedafteraltering thepermeability,internal saltconcentrationandenergystatus
ofrootcells.Permeabilityofcellmembranes (plasmalemma,tonoplast)wasalteredby
incubatingmaizerootsinsolutionsdifferent inpHandtemperature,bywithholding calciumandbytreatingwiththeinhibitorscyanide (CN)anddinitrophenol (DNP)andthe
surface-active chemical triacetin.Energystatusoftherootswasalteredby treatment
oftherootsinsolutionsofDNP,CNandglucose.Cellularsaltconcentration ingeneral,
orconcentrations ofpotassium,organicnitrogencompoundsandinorganicandorganic
anionsparticularwerealteredbydifferenttreatmentsoftheroots.
Effectsonfluxesofpotassiuminexcisedmaizeroots lowinsalt (Chapter 5)seemed
tobemostlyrestricted toinfluxintheshort-term (4-10 h ) . Additionofthesurfaceactivechemicaltriacetintotheabsorptionsolutionsimultaneously increased influx,
decreasedeffluxandthusstimulatednetabsorptionofpotassium inthemaizeroot.Low
pHofabsorptionsolutionand lowtemperatureaswellascalcium starvationofrootssignificantlyinhibitedinfluxofpotassium.Ontheotherhand,increased endogenousconcentrationsofglucoseandaminoacidsincreased influxofpotassium.Besides theroleof
DNPasmetabolicinhibitor,thischemicalseemedathighconcentrations toaltermembrane
permeability.
Relativetouptakekinetics,titration-uptakeexperiments (Section 5.1)demonstrated a
three-phaseabsorption-timecurveratherthanthetraditional two-phaseone (initial
phaseandasteady-state).Oneofthefeaturesofthehigh-saltabsorption isotherm
(System 2), e.g.anion-dependent cationuptake,wasconfirmed inshort-term absorption
94
experiments,whereas inlongerabsorption-transportexperiments,nodualisotherms could
bedemonstrated.
Absorption -exudationexperimentswithcompleterootsystemsofmaizeplants 5
weeksold (Chapter6)indicated the following.
- Inpotassium-starved roots,absorbedpotassiumallaccumulated initially. Consequently,
cellularconcentration ofsaltandosmoticequivalent increasedwithtime.So exudation
rateandupward transport ofpotassium increased steadily. Insteady-state,upward transportofpotassium inxylemequalled absorption ofpotassium intheroot,whilenet
accumulationofpotassium stopped andconcentrations ofpotassium inxylem sap stayed
constant.
-Rootsrichinpotassiumdemonstrated aconsiderableupward transport ofpotassium in
xylemdirectly afterstarting theexperiment,becauseofthehigh internal concentration
ofsalt.However,experimentswith labelledpotassium (86Rb) indicated thatonlya fraction
ofthefreshlyabsorbed labelledpotassiumwas transported straighttoxylem andsubsequentlyupwards inthexylemvessels.Partofthefreshlyabsorbedpotassium exchanged
withpotassium alreadypresent intherootcells.Sopartofthefreshlyabsorbed potassium
wasstoredtemporarily inapool,probably invacuolesofcortexcells,andnetupward
transportofpotassiumkeptconstant.
-Thisexchangeoffreshlyabsorbedpotassiumwithpotassium alreadypresent inroot cells,
dependedonthenatureoftheanion.Sopotassiumwithnitrate ascounterion exchanged
moreslowlywiththepool thanpotassiumwithsulphate.
-Different external concentrations ofsaltresulted inequalosmoticequivalent differences
betweenouter solution andxylemsap.However,theosmoticequivalentinsteady-statewas
different forKN0 3 andKC1solutions fromthatforK.,S04solutions.Thedifferencewas
expressed inrateofexudationandoftransportofpotassiumupwardinxylem.
Althoughmnsport ofsalts inexcisedrootsystemsdiffers fromtransport inintact
transpiringplants insomerespects,thedatahavecontributed toabetterunderstanding
ofsalttransport inmaizeplants.Thissalttransport fromtherootsresemblesthepart
ofthe saltuptakeknown asSystem2.
95
Samenvatting
Deopnamevanvoedingsstoffendoordewortelenhettransportvandeze stoffenvanuit
dewortelnaardebovengrondsedelenzijndebelangrijksteprocessenindemineralenvoorzieningvaneenplant.Vooraldeeerstestap,deopnamevanionendoordeplantewortel,iseenprocesdatsterkafhankelijkisvantalvaninterneenexternefactoren.
Detweedefase,transportvanionennaardehoutvaten (radiaaltransport)enopwaarts
transportindehoutvaten (longitudinaal transport),sluitaanopdeopname.Nahetpasserenvandeplasmalemmavandewortelcellenwordenzouten,aldannietnaomzetting,
afgevoerdnaarbovengrondsedelenvandeplant.
Deopnameenhettransportvanmineralenenwaterindeplantwordenindeliteratuur
uitvoerigbeschreven.[Onderzoekingennaardedirectesamenhang tussenopnameentransport
van zoutenindeplant zijnechterschaars.
Ditverslagbehandeltenerzijdsdeinvloedvaneenaantal inwendigeenuitwendige
factorenopdeopnamevankaliumindemaiswortel (hoofdstuk 5 ) , anderzijdswordeneenaantalopname-exudatieproevenbeschrevenwaarindegelijktijdige opname,accumulatieen
transport (afvoer)vankaliumindemaiswortel gemetenzijn (hoofdstuk 6).Ditlaatstegebeurdeveelalmetbehulpvanvolledige,afgekniptewortelstelselsvanvijfwekenoudemaisplanten.
Deopnamevankaliuminafgekniptewortelsvanmaisplanten (ongeveer10dagenoud)
werdbestudeerdnadataldanniet ingegrepenwasindemembraanpermeabiliteit,hetinterne
zoutgehalteendeenergievoorziening vandewortelcellen.Wijzigingenindemembraanpermeabiliteit (plasmalemma,tonoplast)werdenbewerkstelligd doordompelingvandemaiswortelsinoplossingen (verderbuitenoplossingengenoemd)vanverschillende zuurgraad
entemperatuur,dooronthoudingvancalciumendooreenbehandelingmetderemstoffen
CNenDNPendeoppervlakte-actievestoftriacetin.Indeenergievoorzieningwerdingegrependoortoevoegingvanglucose,DNPenCNaandebuitenoplossing.Hetinterne,
cellulairezoutgehalteinhetalgemeen,ofhetgehalteaankalium,organischestikstofcomponentenenanorganischeenorganischeanioneninhetbijzonder,werdgevarieerddoor
middelvandiversevoorbehandelingenvanhetwortelmateriaal.
Influx-eneffluxmetingenvankaliumwezenuitdatgedurendedekortdurendeexperimenten (4-10h)effectenveelalbeperktbleventotdeinflux.Eenduidelijkewijziging
inzowelinfluxalseffluxvankaliumwerdverkregendoortoevoegingvantriacetin tijdens
deopname.LagepHenlagetemperatuurvandeopname-oplossing,evenalscalciumgebrekvan
dewortelremdendeinfluxvankaliumduidelijkaf.Daamaasthadeenverhogingvanhet
inwendigesuiker-enaminozuurgehaltedercellene e n positieveinvloedopdekaliuminflux.DuidelijkwerddatDNPnietalleeninvloedhadopdeenergievoorzieningvande
wortel,maareveneensopdemembraanpermeabiliteit (hoofdstuk5 ) .
96
Verderbleekdeopnamevankaliumbijhogeconcentraties indebuitenoplossingafhankelijktezijnvandeopnamesnelheidvanhetbegeleidend anion (systeem 2). Integenstelling tothettraditionele 2-£asen-beeld vandeopname-tijd-relatie,namelijkeen
initieleeneensteady-state-fase,werd inditonderzoek een3-fasen-verloopaangetoond
(hoofdstuk5).
Opname -exudatieproevenmetvolledigewortelstelselsvanvij£wekenoudemaisplanten
(hoofdstuk 6)wezenuitdat:
-bijkalium-armewortelsdeopgenomenkaliumaanvankelijk geheelaccumuleerdeinde
wortel.Alsgevolghiervannamhetinterne zoutgehalte indewortelendusdeosmotische
spanning toe.Ditresulteerde ineentoenamevandeexudatiesnelheid enhet opwaarts
transportvankalium.Eendynamischevenwichtwerdbereikt,waarbij opwaartse afvoervan
kaliumgelijkwasaandekaliumopnamedoordewortel.Vanafdatogenblikvond ergeen
nettoaccumulatievankalium indewortelmeerplaats.Bovendienwerddeze steady-statefasegekenmerktdooreenconstantekaliumconcentratieinhetxyleemsap.
-kalium-rijkewortels alsgevolgvaneenreedshooginterncellulair zoutgehalte vanaf
deaanvangvandeproef eenaanzienlijk opwaarts transportvankaliumvertoonden. Proeven
metgelabeldkalium (86Rb) wezenechteruitdatnietalleversopgenomenkaliumrechtstreeksviahetxyleemopwaarts afgevoerdwerd,maarvooreengedeelteomwisseldemet
kaliumdiereeds indewortelcel (vacuole)aanwezigwas.Opdezewijzewerdeengedeeltevandeversopgenomenkaliumtijdelijk indewortelcellenopgeslagen,terwijlde
nettoafvoervankaliumviahetxyleemconstantbleef.
- dezeomwisselingvanversopgenomenkaliummetreedsindewortelcel aanwezige kalium
afhankelijkwasvandeaardvanhetbegeleidend anion.Zobleekkaliummethet begeleidende
anionnitraat dezeomwisselingveelminder intensieftevertonendanbijde aanwezigheid
vansulfaat.
-verschillen inexterneconcentratiesvaneen zoutleiddentoteenzelfdeosmotischdrukverschil tussenbuitenoplossing enxyleemsap.Ditosmotischdrukverschilnamechtereen
anderewaardeaanbijdeaanwezigheidvaneenander zout.Ditleiddeondersteady-statecondities totgroteverschillen inopwaarts transportvankaliumenwatervoordeze
beide zouten.
Alhoewelhettransportvanzouteninafgekniptewortelstelsels insommigeopzichten
afwijktvandat innormale,intakte transpirerende planten,dragendeopdezewijze
verkregenresultatenbijtoteenverruimingvanhet inzicht inhetzouttransport inde
maisplant.Ditzouttransportvanuitdewortelsgelijkthetmeestophetalssysteem2
aangeduidedeelvande zoutopname.
97
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