starchlesskosskeb4

ControlofLeafExpansion:ADevelopmentalSwitchfromMetabolicstoHydraulics1[W][OA]FlorentPantin,ThierrySimonneau,Gae¨lleRolland,MyriamDauzat,andBertrandMuller*Laboratoired'EcophysiologiedesPlantessousStressEnvironnementaux,UMR759,InstitutdeBiologieIntegrativedesPlantes,INRA,34060Montpellier,FranceLeafexpansionisthecentralprocessbywhichplantscolonizespace,allowingenergycaptureandcarbonacquisition.
Waterandcarbonemergeasmainlimitingfactorsofleafexpansion,buttheliteratureremainscontroversialabouttheirrespectivecontributions.
Here,wetestedthehypothesisthattheimportanceofhydraulicsandmetabolicsisorganizedaccordingtobothdark/lightuctuationsandleafontogeny.
Forthispurpose,weestablishedthedevelopmentalpatternofindividualleafexpansionduringdaysandnightsinthemodelplantArabidopsis(Arabidopsisthaliana).
Undercontrolconditions,decreasesinleafexpansionwereobservedatnightimmediatelyafteremergence,whenstarchreserveswerelowest.
Thesenocturnaldecreaseswerestronglyexaggeratedinasetofstarchmutants,consistentwithanearlycarbonlimitation.
However,low-lighttreatmentofwild-typeplantshadnoinuenceontheseearlydecreases,implyingthatexpansioncanbeuncoupledfromchangesincarbonavailability.
From4dafterleafemergenceonward,decreasesofleafexpansionwereobservedinthedaytime.
Usingmutantsimpairedinstomatalcontroloftranspirationaswellasplantsgrownundersoilwaterdecitorhighairhumidity,wegatheredevidencethatthesediurnaldecreaseswerethesignatureofahydrauliclimitationthatgraduallysetupastheleafdeveloped.
Changesinleafturgorwereconsistentwiththispattern.
Itisconcludedthatduringthecourseofleafontogeny,thepredominantcontrolofleafexpansionswitchesfrommetabolicstohydraulics.
Wesuggestthattheleafisbetterarmedtobuffervariationsintheformerthaninthelatter.
Leafexpansionisamajorcomponentofplantper-formance.
Itenableslightcapture,whichpowerspho-tosynthesisandthusbiomassproduction.
Itisalsooneoftherstplantfunctionsaffectedbyenvironmentalstressessuchaswaterdecit(Hsiao,1973),makingitakeytargetforidentifyingtolerantgenotypesandspe-cies(TardieuandTuberosa,2010).
Tothisaim,under-standingtheprocessesdominatingthecontrolofleafexpansionisacrucialstep.
Amongthemultiplicityoffactorsinvolvedinleafexpansion,twomajorlimita-tionsemerge:abiophysicalcontrolmainlylinkedtowateruxestogrowingcells,andametaboliccontrollinkedtothesupplyofcarbohydrates(Dale,1988;Walteretal.
,2009).
Tosomeextent,plantscanberepresentedassystemsruledbybiophysicallaws,withwateruxesmodeledasOhm-likefunctionsofhydraulicconductancesandwaterpotentials(vandenHonert,1948).
InlinewiththeformalismofLockhart(1965),severalargumentssupporttheviewthatleafexpansionispredominantlydrivenbycellturgor,itselflargelyinducedbysoilwaterpotentialandtranspiration.
Notably,increasingsoilwaterdecit(Boyer,1968;Acevedoetal.
,1971)orevaporativedemand(Ben-Haj-SalahandTardieu,1997;Tardieuetal.
,2000)leadstogrowthinhibitionsthatcorrelateinspaceandtimewithturgordepres-sionsintheelongatingzoneofmaize(Zeamays)leaves(Bouchabkeetal.
,2006;Ehlertetal.
,2009).
Suchin-hibitionsunderlimitedwateravailabilityhavebeenattributedtoacollapseofwaterpotentialgradientsthatgovernwateruxestogrowingcells(Boyer,1988;TangandBoyer,2002,2008).
Hydrauliccontrolofgrowthisthusthoughttobemorerestrictiveduringthedaythanduringthenight,aperiodduringwhichstomatalclosureallowstherecoveryofleafwaterpotential(Ben-Haj-SalahandTardieu,1997).
Whilecellexpansionneedswatertoproceed,italsorequiresenergyandcarbonskeletonsandthereforereliesonassimilatessuppliedtothegrowingtissues(Dale,1985;SmithandStitt,2007).
Consistentwiththis,growthappearstobecloselycontrolledbycarbonmetabolismatdifferentscales.
Mostcurrentagronom-icalmodelsarebasedontheformalismofMonteith(1977),linkingbiomassaccumulationtoradiationinterceptedbyplantsandcarbonassimilationrate.
Likewise,stablecorrelationsareobservedbetweenorgangrowthratesandinterceptedradiationorcar-bohydrateavailabilityintheirgrowingparts,suchasinroots(Aguirrezabaletal.
,1994;Freixesetal.
,2002)orreproductiveorgans(Dosioetal.
,2011).
Finally,theleafgrowthrhythmofseveralspeciesatane1ThisworkwassupportedbytheFrenchMinistryofResearch(granttoF.
P.
)andbytheEuropeanCommission(FP6)throughtheAGRON-OMICSIntegratedProject(grantno.
LSHG–CT–2006–037704).
*Correspondingauthor;e-mailmuller@supagro.
inra.
fr.
TheauthorresponsiblefordistributionofmaterialsintegraltothendingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(www.
plantphysiol.
org)is:BertrandMuller(muller@supagro.
inra.
fr).
[W]TheonlineversionofthisarticlecontainsWeb-onlydata.
[OA]OpenAccessarticlescanbeviewedonlinewithoutasub-scription.
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org/cgi/doi/10.
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timescalecoincideswithuctuationsincarbohydrateavailability(WalterandSchurr,2005;Wieseetal.
,2007).
Toaccommodateuctuationsinphotosynthet-icallyactiveradiation,carbonavailabilityisbufferedbytransientstoragecompounds,especiallystarch,whichisaccumulatedduringthedayandusedasacarbonsupplyatnight.
Therateofnocturnalstarchbreakdownisundernecontroltoallowoptimumexhaustionofcarbonstoresbytheendofthenightwithoutenteringcarbonstarvation,whichhasdelete-riouseffectsonvariousmetabolicanddevelopmentalprocesses(Brouquisseetal.
,1991;SmithandStitt,2007).
Thisturnoverofstarchreservesisthoughttobeamajorintegratorintheregulationofgrowth(Sulpiceetal.
,2009).
Towhatextentleafontogenymayinterferewithmetabolicandhydraulicfactorsconstrainingleafgrowthisnotknown.
Still,expectationscanyetbedrawnfrombasicknowledgeofthesink-to-sourcetransitionindevelopingleaves.
Sinceyoungleavescriticallydependoncarbonimportfromolderleaves(Turgeon,1989),theirexpansioncouldbeexpectedtobemoredependentuponcarbonuctuationsascom-paredwitholderleaves,whichhaveapositivecarbonbalance.
Ontheotherhand,youngleavescouldbeprioritizedinsuchawaythatthecarbonsupplytothemismaintainedwhenthewholeplantcarbonavailabilityisdecreased(Minchinetal.
,1993;LacointeandMinchin,2008).
Inthesameway,possibleinter-actionsbetweenontogenyandhydraulicscanonlybespeculated.
Forinstance,watersupplythroughxylemvesselscoulddecreasewithleafmaturationasaresultofdecreasedhydraulicconductivity(Martreetal.
,2000,2001;MartreandDurand,2001;Nardinietal.
,2010).
Ontheotherhand,itisgenerallyadmittedthatcuticlethickenswithleafdevelopment(Richardsonetal.
,2007),suggestingthatthiscouldlimitpassivewaterloss(Kerstiens,2006)andfavorhydraulicstatusastheleafexpands.
Finally,althoughthemoleculareventsleadingtostomatalformationarenowwelldescribed(BergmannandSack,2007),theacquisitionofstomatalfunctionalityduringleafdevelopmentremainsunclear.
Thus,quantitativeargumentsthatdistinguishthepossiblerolesofwaterrelationsorcar-bonavailabilityonexpansionduringleafontogenyarelacking.
Inthisstudy,weaimedtodiscovertherelativecontributionsofwaterandcarbontothecontrolofgrowthwithrespecttoleafontogenyinthemodelplantArabidopsis(Arabidopsisthaliana).
Forthispurpose,weevaluatedataday/nighttimestepthedevelopmentalpatternofleafexpansioninmutantsimpairedinstarchsynthesisorbreakdownaswellasinmutantsderegu-latedinstomatalcontroloftranspiration.
Furthermore,plantsweregrownundervariouslevelsofsoilwatercontent,airhumidity,andirradiance.
Resultsconvergetoassociatehydraulicandmetaboliccontrolstodayandnightperiods,respectively.
Evidenceisthenpresentedthatduringitsdevelopment,theleafexperiencesrstmetabolicandthenhydrauliclimitation.
Bothgeneticandenvironmentalcuesacttomodulatemetabolicorhydraulicconstraintsandtoshiftinaconsistentwaythetimingwhenthemainlimitationswitchesfromcarbontowater.
RESULTSANDDISCUSSIONADualPatternintheWild-TypePlantsInArabidopsis,theexpansionofoneindividualleaffrominitiationattheshootapicalmeristemtogrowthcessationcanlastmorethan1month(Aguirrezabaletal.
,2006).
Becausewholeplantcharacteristics(e.
g.
sink-sourcebalance;Christopheetal.
,2008)canchangesubstantiallyoversuchalongperiod,wedesignedaprotocoltostudyday/nightchangesofrelativeexpansionrate(RER)atdifferentleafgrowthstagesoveramuchshortertimescale.
Briey,weusedtheexpansionof10to13serialleavesover24htoreconstructtheexpansionpatternofanindividualleafover8to10d.
Thisprocess,drivenbytheconceptofphyllochronage,isdescribedin"MaterialsandMethods"andfullydetailedinSupplementalMate-rialsandMethodsS1andinSupplementalFiguresS1toS3.
Wefocusedonadevelopmentalwindowencompassingthersthalfofleafexpansionafteremergence,aperiodduringwhichrelativeandthenabsoluteleafexpansionaresuccessivelymaximum(SupplementalFig.
S1)andthereforestronglycontrib-utetonalleafarea.
Underwell-wateredconditions(0.
35gwaterg21drysoil),acommonRERpatternemerged(Fig.
1)inbothecotypeColumbia(Col-0)andecotypeWassilewskija(Ws-3).
TwosuccessiveperiodscouldbedistinguishedalongtheoveralldecreasingpatternofRER.
Therstonetookplaceearlyafteremergenceforabout3dandshowedleafexpansionratebeingonaverage0.
15and0.
23d21higherduringlightthanduringdarkphasesforCol-0andWs-3,respectively.
Thesecondperiodoccurredatlaterstagesat5dfollowingemergenceandshowedRERbeing,bycon-trast,onaverage0.
16and0.
08d21higherduringthedarkphases.
Forthesakeofclarityandtoallowstatisticalanalysis,RERpatternswerethenparame-terizedbyttingasecond-degreepolynomialinde-pendentlytothedayandnightRER,asshownforCol-0intheinsetofFigure1.
Wehypothesizedthatlatealternations(RERnight.
RERday)reecthydrauliclimitationswhileearlyalternations(RERday.
RERnight)reectmetaboliclimitations.
Totestthesehy-potheses,wealteredthehydraulicandmetabolicstatusoftheleafusinggeneticandenvironmentalmanipulations.
DaytimeReductionsinLeafExpansionAreunderHydraulicControl:EvidencefromStomatalMutants,SoilWaterDecit,andHighAirHumidityDaytimedropsofleafexpansionasobservedhereinArabidopsisrecallresultsrepeatedlyobservedinmonocotssuchasmaize(Ben-Haj-SalahandTardieu,Pantinetal.
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1997;TangandBoyer,2002;Bouchabkeetal.
,2006),wheat(Triticumaestivum;Christ,1978),rice(Oryzasativa;Cutleretal.
,1980),fescue(Festucaarundinacea;SchnyderandNelson,1988;Durandetal.
,1995),orMiscanthus(Clifton-BrownandJones,1999)andalsoindicotssuchassunower(Helianthusannuus;Boyer,1968)orseveralhalophyticspecies(Rozemaetal.
,1987).
Eitherpermanentortransient,thesedropshavebeenattributedtoahydrauliclimitation,becausetheyoccurredespeciallyunderrestrictedwateravailability(i.
e.
undertranspiringconditionswithhigherampli-tudeundersoilwaterdecit).
Wethustestedifthedecreaseofexpansionduringdayscouldbeduetoanimpairmentofleafwaterpotentialinducedbytran-spiration.
Weanalyzedtheabscisicaciddecient4(aba4)mutant(Northetal.
,2007)andthe9-cis-epoxycarotenoiddioxygenase6overexpressor(NCED6-OE)transformant(Lefebvreetal.
,2006)reputedtohavehighandlowstomatalconductance,respectively,duetomodiedcapacitiestosynthesizeabscisicacid(ABA).
Stomatalconductanceoftheaba4mutantwassignicantlyhigherinourconditionsascomparedwiththewild-type,whilephotosynthesisratewasnotaffectedbythemutation(Fig.
2A,inset).
Hence,weexpectedtheeffectofthehydraulicconstraintonleafexpansiontobeampliedinthismutant.
Inaconsistentway,aba4showedearlierandmorepronouncedRERreductionsduringlightphases(Fig.
2A).
Fromday5afteremer-genceonward,thediurnalRERwasonaverage0.
31d21lowerthanthenocturnalRERinaba4ascomparedwith0.
16d21daytimereductioninthewildtype.
Contrastingwithaba4,stomatalconductancewasstronglyreducedinNCED6-OEand,stillinlinewithourhypothesis,itdisplayedastronglyalteredRERpattern(Fig.
2A).
LeafexpansionduringdayswasessentiallymaintainedsimilartonightRERat5dfollowingemergence(only0.
01d21loweronaverage).
Toascertainthehydraulicoriginofgrowthlimitationinthedaytime,weexposedbothCol-0andWs-3accessionstotwolevelsofsoilwaterdecit.
Applyingamoderatesoilwaterdecit(0.
23gwaterg21drysoil)toCol-0(Fig.
2C)orWs-3(Fig.
2D)ampliedthediurnaldepressions(0.
37d21lowerRERduringdaysthanduringnightsafter5d),withdaydropsoccurringearlierthanunderwell-wateredconditions.
Underseveredecit(0.
18gwaterg21drysoil),diurnalreductionsinRERwereevenmorepronounced,especiallyinCol-0,whereleafexpansionvirtuallyceasedduringdays(0.
30d21lowerRERinthedaytimeatanydevelopmentalstage).
Bycontrast,whenthevaporpressuredecit(VPD)waslowered(downto0.
3kPainsteadof0.
8kPa)inordertoimprovetheleafwaterstatus,day/nightalternationsofleafexpansionwereminimized,withaattenedpatternintheearlystagesandadiurnalRERbeingonly0.
08d21lowerthanduringthenightafter5donaverage(Fig.
2B).
Overall,theconsistencyoftheresultsobtainedusingstomatalmutants,lowevaporativedemand,orsoilwaterdecittreatmentsstronglysupportsthehypothesisthatdiurnalreductionsinleafexpansionobservedduringthelaterphasesofleafdevelopmentinthewell-wateredwild-typeplantsareunderhy-drauliccontrol.
Besidesactingonstomatalclosure,alteredABAcontentinstomatalmutantscouldhaveimposedlong-termadditionaleffects,forinstanceonhydraulicconductivity,possiblythroughchangesinaquaporinexpression,aswellasonnonhydraulicprocessessuchascellwallpropertiesorcelldivision(forreview,seeTardieuetal.
,2010).
Thesesideeffects,togetherwiththeslightlybutsignicantlylowernetphotosynthesis(Fig.
2A,inset),couldpartlyexplainthegloballylowerRERinNCED6-OEplantscom-paredwithothergenotypes.
NightDepressionsAreLinkedtoaMetabolicControlofLeafExpansion:EvidencefromStarchMutantsUsingasimilarcombinationofenvironmentalandgeneticapproaches,wetestedifearlynocturnalde-pressionsofgrowthwereduetoacarbonlimitation.
Inlinewiththishypothesis,experimentsatelevatedCO2havesuggestedthatleafgrowthissourcelimitedatnight(GrimmerandKomor,1999;RasseandTocquin,2006).
AsthestarchpoolisthemaincarbonsourceatnightinArabidopsis,weusedfourmutantswithimpairedabilitytostorestarchortouseitatnight.
Thestarchlessphosphoglucomutase(pgm)mutantcannotsynthesizestarch(Casparetal.
,1985),whereasthestarchaccumulatorstarchexcess1(sex1)isnotabletoFigure1.
Expansionpatternsofthewild-typeplantsunderwell-wateredconditions.
ThedayandnightRERsweremonitoredonCol-0(blackcircles)andWs-3(crosses)fromleafemergencetomiddevelopment(see"MaterialsandMethods";SupplementalFig.
S1).
TheinsetshowsthesmoothingprocessfortheRER.
AsshownhereforCol-0,asecond-degreepolynomialwasttedindependentlytothedayRER(dottedline)andtothenightRER(solidline).
Then,thegrowthpatternsweredrawnfromleafemergencetoleafmiddevelopmentbyjoiningthepredictedvaluesforthesuccessivelightanddarkperiods.
Thesameunitsareusedforthemaingraphandtheinset.
Blackrectanglesandgraybandsindicatethenightperiods.
Valuesshownaremeans6SE(n$10).
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degradeit(Casparetal.
,1991).
Maltose,themajorproductofstarchbreakdownatnight,cannotexitthechloroplastinmaltoseexcess1(mex1;Niittyla¨etal.
,2004).
Finally,maltosecytosolicmetabolismisim-pairedindisproportionatingenzyme2(dpe2;Chiaetal.
,2004;LuandSharkey,2004).
Asaresult,starchturn-overwasstronglyaffectedinthesemutants(Fig.
3,AandB,insets).
Strikingly,allmutationscausedastrongreductionofleafexpansionatnight(Fig.
3,AandB),especiallyintheearlystages,withRERduringtherst3dfollowingemergencebeing0.
80,0.
58,0.
49,and0.
48d21loweratnightthanindaytimeforpgm,sex1,mex1,anddpe2,respectively.
Bycomparison,earlyreduc-tionsinRERatnightforCol-0andWs-3wereonly0.
15and0.
23d21lowerthandaytimeRER.
Lateronduringleafdevelopment,thedifferencesbetweenwild-typeplantsandstarchmutantstendedtovanish.
Thisstronglysupportsthehypothesisthatearlynightreductionsinleafexpansioninthewild-typeplantsareundermetaboliccontrol.
Ampliedgrowthinhibitionsduringthenightinthestarchmutantscouldhaveresultedeitherfromthelackofcarbonorthetriggeringofthesignalingcascadeassociatedwithlowlevelsofsugars(SmithandStitt,2007).
Bycon-Figure2.
Effectsofhydraulicchangesonexpansionpatterns.
Pointsrepresentobservedandlinesrepresentsmoothedvalues.
Blackrectanglesandgraybandsindicatethenightperiods.
Valuesshownaremeans6SE(n$10).
A,Kineticsofstomatalmutants.
Expansionpatternsareshownforaba4,amildABA-decientmutant,andofNCED6-OE,anABAoveraccumulator.
Theinsetshowsstomatalconductance(Gs)andnetphotosynthesis(Pn).
LettersindicatesignicantdifferencesbetweengenotypesafteraKruskal-Wallistest.
Diurnalstomatalconductanceofmutantswasaffectedasexpected(n=10).
Netphotosynthesiswasonlyslightlyaffectedintheoveraccumulator(n=4).
B,EffectsoflowVPDontheexpansionpatternofCol-0.
Well-wateredCol-0wasgrowneitherunderstandardVPD(0.
8kPaduringtheday;black)orlowVPD(0.
3kPaduringtheday;turquoise).
CandD,Kineticsofwater-stressedplants.
ExpansionpatternsareshownforCol-0(C)andWs-3(D)plantssubjectedtoamildwaterdecit(0.
23gwaterg21drysoil)oraseverewaterdecit(0.
18gwaterg21drysoil)andthewell-wateredcontrol(0.
35gwaterg21drysoil).
Notetheamplicationofdiurnaldepressionsunderconditionslimitingwateravailability(aba4;waterstresses)andtheirdiminutionwhenwaterlossbytranspirationisreduced(NCED6-OE;lowVPD).
Pantinetal.
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Figure3.
Effectsofmetabolicchangesonexpansionpatterns.
Pointsrepresentobservedandlinesrepresentsmoothedvalues.
Blackrectanglesandgraybandsindicatethenightperiods.
Valuesshownaremeans6SE(n$10).
AandB,Kineticsofstarchmutants.
A,Expansionpatternsofpgm,astarchlessmutant,andofmex1,amutantimpairedinmaltoseexport(thepredominantrouteforcarbohydratesupplyatnight),bothintheCol-0background.
B,Expansionpatternsofsex1,astarchaccumulator,andofdpe2,whichisaffectedintheconversionfrommaltosetoSucatnight,bothintheWs-3background.
Insetsshowstarchturnover.
Thestarchcontent(inmmolofhexoseequivalentsperunitofdryweight[DW])wasmeasuredattheendofthedayandattheendofthenight.
LettersindicatesignicantdifferencesbetweenvaluesafteraLSDtestadjustedusingtheBonferronimethod(n=4).
CtoE,Kineticsofplantsunderlowlight.
ExpansionpatternsareshownforCol-0(C),pgm(D),andsex1(E)subjectedtolowlight(PPFDat70mmolm22s21versus220mmolm22s21forthecontrol).
F,Effectofirradianceonnetphotosynthesis(Pn).
LettersindicatesignicantdifferencesbetweentreatmentsafteraKruskal-Wallistest(n=6).
G,EffectofirradianceonSLA.
LettersindicatesignicantdifferencesbetweentreatmentsafteraKruskal-Wallistest(n=4).
Notetheamplicationofearlynocturnaldepressionsinstarchmutants,furtherincreasedunderlowlight.
Theweakeffectonthewildtypeisdiscussedinthetext.
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trast,wild-typeplantsdevelopbuffersystemsagainstcarbonstarvation,suchasdescribedinthewholerosetteofCol-0,whosecarbonbudgetisadjustedduringthedaytoavoidexhaustionduringthedarkperiod(Gibonetal.
,2004,2009;Bla¨singetal.
,2005).
BufferingCarbonStarvationUnderverylowlight(30mmolm22s21),leafexpan-sioninwild-typeArabidopsishasbeenshowntobereducedatnight(Wieseetal.
,2007),butthatstudydidnotconsidertheinuenceofleafontogeny.
Tofurtherchallengethehypothesisofacarbonlimitationofleafexpansionduringtheearlystagesofleafdevelopment,Col-0,pgm,andsex1wereexposedtolow-lightcondi-tions(70mmolm22s21)for4d.
Surprisingly,thislow-lighttreatmenthadnoeffectonthegrowthpatternofCol-0(Fig.
3C)duringtheearlystagescomparedwithstandardlight(220mmolm22s21).
Atlaterstages,lowlighttendedtoincreasetheexpansionrate,butnightRERwasstillhigherthandayRERby0.
16d21asunderstandardlight.
Bycontrastandasexpected,starchmutants(Fig.
3,DandE)underlowlightdisplayedthroughoutdevelopmentafurtherdecreaseofnightRERcomparedwithstandardlight,whiledaytimeRERwasincreasedatlaterstagesasinthewildtype.
Theseresultsraisethequestiononhowthewildtypemanagedtomaintainleafexpansiondespiteseverelightreduction.
Decreasingthelightbytwo-thirdslowerednetassimilationratebyasimilarpro-portion(Fig.
3F),butthespecicleafarea(SLA)nearlydoubledunderlowlight(Fig.
3G).
Thisimpliesthattheplantessentiallymaintainedsurfaceexpansiondespitelowercarbonavailabilitybyadjustingleafthicknessordensity.
AnincreaseinSLAunderlowlightisclassicallyobservedinavarietyofspeciesandawiderangeofconditions,includingspeciesinnat-uralhabitats(Boardman,1977),cropplants(Tardieuetal.
,1999),orArabidopsis(PigliucciandKolodynska,2002;CooksonandGranier,2006).
Similarlyintobacco(Nicotianatabacum),SLAandcarboncontributiontostructuralweightadjustedtophotosyntheticcapacity(Fichtneretal.
,1993).
Starchdynamicscouldalsohaveadjustedinresponsetothecarbonbalance,asdemon-stratedundervariousdaylengths(Gibonetal.
,2004,2009)andunderlowirradiance(ChattertonandSilvius,1981).
Together,ourresultstwiththegeneralviewthatplantsuseanarsenalofresponsesallowingthemtone-tunethebalancebetweensurfaceexpansionandstructuralgrowth.
Thisabilitytooptimizelightinterceptionunderlimitedirradiancebyreducingthecarboninvestmentperunitofleafarea(i.
e.
prioritizingsurfaceexpansion)representsastrongecologicalad-vantage(Poorteretal.
,2009).
Ourstudyalsosuggeststhatbufferingsystemsagainstcarbonuctuationsaremoreefcientinmaintainingleafexpansionundercarbon-limitingconditionsthanthemechanismsre-sponsibleforattractingwatertogrowingcellswhencompetitionforwaterishigh.
Thismakessensebe-causemaintainingsurfaceexpansionrepresentsanadvantageunderlowlightandadisadvantageunderwaterstress,withrespecttophotosynthesisandtran-spiration,respectively.
Howthisresponseisachievedisnotknown.
Nevertheless,veryefcientbufferingsystemsareactingtopreventcarbonstarvation,redi-rectgeneexpression,andslowdowngrowth(SmithandStitt,2007).
Thesebufferingsystemsforcarbonarelikelytobemediatedbysugarsensing(SmithandStitt,2007;Stittetal.
,2007)andtorelyonshort-termpooldynamics(e.
g.
starch;Gibonetal.
,2004,2009)oronlong-termchangesincarboninvestmentsintostruc-tures(Boardman,1977;Poorteretal.
,2009),asseeninourstudythroughamuchhigherSLAintheleavesofshadedplants.
Undermoredrasticcarbonconditions,asinstarchmutants(thisstudy)orunderverylowlight(Wieseetal.
,2007),limitedcarbonavailabilitymayultimatelyimpactleafexpansion.
SevereWaterStressRestorestheWild-TypePhenotypeinStarchMutantsToevaluatethepossibleinteractionsbetweencarbonandhydrauliclimitations,weexposedthestarchmu-tantstosoilwaterdecits.
Inallmutants,aseverewaterdecitresultedinastronglyreducedexpansionduringdayphasesandapracticallyunalteredexpan-sionduringnights.
Asaresult,allmutantsdisplayedapatternthatresembledthatofthewell-wateredwild-typeplants(SupplementalFig.
S4).
Thisisconsistentwiththesuperimpositionofthegradualinuenceofhydrauliclimitationsinthedaytimeoverthegrowthpatternofstarchmutants,characterizedbynightre-ductionofRER.
Additionally,besidespenalizingthehydraulicstatus,droughtcouldalsohaveimprovedthecarbonstatusofthestarchmutants.
Thisinterpre-tationisinlinewitharecentreview(Mulleretal.
,2011)showingthatdroughtimprovesthecarbonsta-tusofplantsduetoahighersensitivitytodroughtforleafgrowththanforcarbonassimilation,leadingtocarbonaccumulation(foranexemplicationinArabi-dopsis,seeHummeletal.
,2010).
Onthewhole,theabilitytorestorequalitativelythewild-typephenotypewithstarchmutantsunderwaterstressindicatesthatexpansionpatterns,includingthoseofthewild-typeplants,resultfromadynamicandplasticcombinationofmetabolicandhydraulicconstraints.
ADevelopmentalSwitchfromMetabolicLimitationstoHydraulicLimitationsofLeafExpansionInordertoanalyzethewholedatasetwithinacommonstatisticalprocedure,expansionpatternswerenormalizedaccordingtothetimeaxisandaclusteringwasperformedbasedonthedifferencebetweennightanddayRERs,ascomputedbyttingapolynomialindependentlytothedayandnightvalues(Fig.
4A).
ThestatisticalsignicanceofthisdifferenceforeachtreatmentateachtimestepisplottedinFigure4B.
Vertically,thisclusteringresultedinanorderedcontin-uumofgenotypicandenvironmentalconditionslead-Pantinetal.
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ingtosituationsrangingfrompredominantnightde-pressions(topofFigure4Aingreen)topredominantdaydepressions(bottomofFigure4Ainpurple)duringleafdevelopment.
Consistentwithouranalysislinkingnightdepressionstometabolicconstraint,allstarchmutantsunderwell-wateredormoderatewaterstressconditions(exceptdpe2)clusteredinthetoppartofFigure4A,withhighlysignicantnightreductionsofRERextendingseveraldaysafterleafemergence.
pgmandsex1underlowlightwererankedattheupperextremeoftheclustering.
Underseverewaterdecit,growthpatternsofallstarchmutantsshifteddownwithinthegroupoftheirwell-wateredwildtypes,inbothCol-0andWs-3backgrounds.
LeafexpansionofNCED6-OEwaspoorlyaffectedbywatershortageandclusteredatanysoilwatercontentwiththelowVPDtreatment,formingagroupcharacterizedbyweak,nonsignicantday/nightuctuationsofexpansionas-sociatedwithprobablelowuctuationsoftranspira-tion.
Thelastgroup,locatedatthebottomofthecluster,withstrong,highlysignicantdepressionsofdaytimeRER,wascomposedofallgenetic(aba4mutant)orenvironmental(soilwaterdecits)situationsthatin-creasedhydrauliclimitation.
Thehorizontalcolorgra-dientclearlyindicatedthatnightdepressionsoccurredpreferentiallyintheearlystageswhereasdaydepres-sionsweremorepronouncedinthelaterstages,withaswitchingpoint(whiteandreddiagonalsinFig.
4,AandB,respectively)followingthebalancebetweenmetabolicandhydraulicconstraint.
Asawhole,theconsistentrankingofthisclusteringgatheredfromenvironmentalandgeneticperturbationsshowsthatduringthecourseofontogeny,thecontrolofleafexpansionswitchesfrommetabolicstohydraulics.
Theseresultsimplythatevenunderwell-wateredconditions,leafexpansionbecomesmoreandmoreFigure4.
Heatmapoftheexpansionpatterns.
A,HierarchicalclusteringanalysisofthedifferencebetweennightanddayRER.
Thetimeaxisofallkineticswasnormalizedaccordingtoleafmiddevelopment(t50).
Then,asecond-degreepolynomialwasttedindependentlytothedayandnightRERs,andthedifferencebetweennightanddaywascomputedfor100successiveiterationsbetweent0(emergence)andt50.
These100variableswereusedtoclassifythekineticsusingtheEuclideandistances.
Thecomputedvariableswerethenassociatedwithacolor.
ClosenesstogreenindicatesadayRERsuperiortothenightRER,whileclosenesstopurpleindicatestheopposite.
Theleftpartoftherectanglesbesidebranchesofthedendrogramisacolorcodeforthegenotype,whiletherightpartisfortheenvironment.
Thereisnoaddedrightpartwhengenotypewasgrownundercontrolconditions(wellwatered,standardlight).
Blackisforthewildtype.
Tan(starchmutants)andbrown(lowlight)areforsupposedenhancedmetabolicconstraints.
Gold(aba4),orange(moderatewaterstress),andred(severewaterstress)areforsupposedenhancedhydraulicconstraints.
Steelblue(NCED6-OE)andturquoise(lowVPD)areforsupposedreducedhydraulicconstraints.
Notethatmutantsandenvironmentaltreatmentssupposedtomodifymetabolicorhydraulicconstraintsclusteredinaconsistentway.
Notealsothatmetabolicconstraintsarepredominantlyassociatedwithearly,nocturnalRERdepressions,whereashydraulicconstraintsareassociatedwithlater,diurnalRERdepressions.
B,SignicanceofthedifferencebetweennightanddayRER.
Condenceintervalsofthepolynomialregressionswerecalculatedatseveralcondencelevelsforeachiteration.
GreenindicatesthatthedifferencebetweennightanddayRERisveryhighlysignicant,whileredindicatesnosignicantdifference.
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limitedbywateruxesastheleafdevelops.
Watersupplytothegrowingtissues,therefore,appearsasakeypointtobalancetheincreasingcompetitionforwaterbetweengrowthandtranspirationastheleafexpands.
Theimpactongrowthofthiscompetitionhasbeenwelldocumentedinmonocotsbutalsoindicotssuchassunower(Boyer,1968),castorbean(Ricinuscommunis;Poireetal.
,2010a),andseveralhalophyticspecies(Rozemaetal.
,1987).
However,toourknowledge,ourstudyistherstonereportingaprogressiveestablishmentofhydraulicconstraintonleafgrowthduringitsontogeny.
Anincreasinghy-draulicconstraintduringdevelopmentcouldbeme-diatedbyvariousmeans.
First,xylemarchitecturecouldbecomelimitingduetoadecreasedveindensityastheleafexpands,asrecentlyreportedinArabidop-sis(Rolland-Laganetal.
,2009).
Moreover,hydraulicconductivitycoulddecreasewithtissuematuration,asshownalongfescueleaves(Martreetal.
,2000,2001;MartreandDurand,2001)andindevelopingleavesofhorsechestnut(Aesculushippocastum;Nardinietal.
,2010).
Alternatively,aquaporin-mediatedwatertrans-portmaybecomegraduallylimiting,assuggestedinthemaizeleaffromthespatialpatternoftranscriptionlevelsalongthematurationzone(Hachezetal.
,2008).
Thegloballengtheningoftheextravascularpathwayastheleafexpandsmayalsoexertanincreasingre-sistancetowaterowwithinthewholeleaf(Cochardetal.
,2004;Brodribbetal.
,2007).
Lastly,stomatadynamicsandsensitivitytoABAcouldbedependentonleafage,assuggestedincotton(Gossypiumhirsu-tum;Jordanetal.
,1975).
However,thedevelopmentalpatternofstomatalorxylemfunctioningremainsunknownforthegrowingleavesofArabidopsis.
Asarstinsightintothemechanismsunderlyingahydraulicdevelopmentalswitch,wedeterminedtur-gorinyoung(1dafteremergence)andolder(8d)leavesinwell-wateredCol-0attheendofbothdayandnight.
Leafturgorincreasedwithdevelopment(Fig.
5A),whichtswithearlierobservationsinmaize(TangandBoyer,2002;Bouchabkeetal.
,2006).
Inthosestudies,thiswasattributedeithertoadepressionofwaterpotentialinducedbythevolumetricgrowthortochangesinrheologicalparameterswithleafdevel-opment.
Moreinterestingly,ourresultsalsoshowedacleardevelopmentalswitchintheday/nightturgordifference:justafteremergence,turgorwassigni-cantlyloweratnight(0.
31MPaduringthedayversus0.
18MPaatnight;P,0.
05),whileasignicantdropinturgorwasobservedduringthedayatthelaterstage(0.
44MPaduringthedayversus0.
56MPaatnight;P,0.
01).
SimilardropsindaytimeturgorwererecentlyreportedinArabidopsisleavesusingapatch-pressuretechnique(Acheetal.
,2010).
Ourstudysuggeststhatveryyoungleavesarenotsubjectedtothisdaytimedropandthatthisdevelopmentalswitchintheday/nightturgorcouldbeinvolvedinthedevelopmentalswitchintheday/nightleafexpan-sion.
Whentheclusteringwasrestrictedtotherstthirdofthekinetics(e.
g.
3dfollowingemergenceforthewild-typeplantsundercontrolconditions),wild-typeplantsclusteredforemostwiththestarchmutants(SupplementalFig.
S5).
Thisimpliesthatleafgrowthsensitivitytometaboliccontroloccursintheearly,presumablyheterotrophicstages.
Accordingly,leafexpansionsensitivitytoshadinginmaize(Ben-Haj-SalahandTardieu,1996;Mulleretal.
,2001),sunower(GranierandTardieu,1999;Tardieuetal.
,1999),orArabidopsis(CooksonandGranier,2006)wasen-hancedintheyoungleaf.
Insinkorgans,thegrowthpatternwasalsoalteredatnight,asshownforArabi-dopsisroots(YazdanbakhshandFisahn,2010)orpotato(Solanumtuberosum)internodes(Kehretal.
,1998).
Furthermore,closerelationshipsbetweenlocalcarbohydrateavailabilityandgrowthrateobservedinseveralsinkorganssupporttheviewthatgrowthdependenceuponcarbonisanemergentfeatureofFigure5.
Ontogeneticshiftsinleafturgorandcarbohydratestores.
Measurementswereperformedattheendofdayandnightonleavesaged1and8dafteremergenceonthewell-wateredCol-0plants.
Valuesshownaremeans6SE.
A,Leafturgor.
Turgorpressurewasdeterminedasthedifferencebetweenleafwaterpotentialandosmoticpotential.
Mean,SE,andABCcondenceintervalwerecomputedusinganonparametricbootstrapmethod.
Singleanddoubleasterisksindicatesignicantdifferencesbetweendayandnightturgoratthe0.
95and0.
99condencelevels,respectively.
BandC,Carbohydratestores.
Thesolublesugar(B)andstarch(C)contentsweremeasured(inmmolofhexoseequivalentperunitoffreshweight[FW])attheendofdayandnight.
LettersindicatesignicantdifferencesbetweenvaluesafteraKruskal-Wallistest(n$6).
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sinkorgans(Mulleretal.
,2011).
Thus,thepredomi-nanceofcarbonlimitationatleafemergenceanditsdisappearanceduringleafdevelopmentcouldbelinkedtoaprogressivesink-to-sourcetransition.
Inseveraldicots,thesink-to-sourcetransition(themo-mentwhentheleafbecomesanetcarbonexporter)hasbeenshowntotakeplacewhentheleafis30%to60%fullyexpanded,butthisviewhasnotbeenrevisitedforalongwhile(forreview,seeTurgeon,1989).
Asfarasweknow,theimpactofsink-to-sourcebalanceonpatternsofleafexpansionhasneverbeendemon-strated.
Theyoungleafmustrelyoncarbohydrateimportfromolderleaves,becauseitsmetabolicre-quirementsaremaximalwhenitsproductioncapacityisminimal(Turgeon,1989).
Indeed,whilephotosyn-theticmachineryisdeveloping(Dale,1985),relativeexpansionrateandrelativecelldivisionrateareattheirhighestlevels(CooksonandGranier,2006),in-creasingcarbonneedsforrespiration(Dale,1985)andcarboncostssuchasthoseassociatedwiththesynthe-sisofnewstructuralcompoundsandcellequipment,includingwalls,nucleicacids,andproteins(Piquesetal.
,2009).
Recenttranscriptomicdataalongadevel-opmentalgradientinmaizeleafsupportthisinterpre-tation(Lietal.
,2010).
ThisisalsoexempliedinthestudyofSchurretal.
(2000),wheretheyoungleavesofcastorbeanshowedalowchlorophyllcontent,anegativenetassimilationrate,andanintenserespira-tionrate,whileleafgrowthratewasreducedatnight.
Toevaluateifleafcarbonbudgetscouldbeaffectedbyontogeny,wemeasuredcarbohydratestoresofyoung(1dafteremergence)andolder(8d)leavesinwell-wateredCol-0attheendofdayandnight.
Solublesugarcontents(Fig.
5B)werenotsignicantlydifferentbetweentheendofthedayandtheendofthenight.
Starchcontent(Fig.
5C)attheendofthedaywasthreetofourtimesthatofsolublesugarsandwasalmostexhaustedattheendofthenight,suggestingthatstarchisamajorcontributortotheleafdiurnalcarbonbalance.
Interestingly,theamountofstarchstoredattheendofthedayinthejustemergedleafwas40%lowerthanintheolderleaf.
Thecarbohydratestoresattheendofthedaywerethuslowerintheyoungleafdespiteitsmaximalgrowthrate,whichtswiththeideaofasteepermetabolicconstraintintheyoungleaf.
Besideshydraulicsandmetabolics,thecircadianclockmayplayanintertwinedroleinthecontrolofleafgrowth.
Recently,itwasfoundthatunderextendedlight,leafexpansionrateatashorttimescaleinArabidopsiskeepsonalternatingwithasimilarperiodasundernormalday/nightconditions(Poireetal.
,2010b),highlightingacircadiancontrolofleafexpan-sion.
Thedevelopmentalswitchoftheday/nightpat-ternpresentedinthisstudyanditsmodulationprovideevidencethatontogeny,genetics,andtheenvironmentcanallovercomeendogenousrhythms.
Theextenttowhichtheclockcouldinterferewithourresultsisnotthescopeofthisstudy,butwehypothesizethatcircadianrhythmcouldacttoamplifyandanticipateleafresponse,notablytometabolicorhydrauliccon-straints.
Indeed,theclockhasbeenshowntoorches-tratethetranscriptionofpathwaysrelatedtocentralprocesses,includingphotosynthesis,carbonmetabo-lism,orwaterinuxthroughaquaporins,togetherwithcellwalldynamics(Harmeretal.
,2000).
Further-more,sugarsthemselvescanmodifytheexpressionofclock-regulatedgenes(Bla¨singetal.
,2005),whileconversely,starchbreakdownisundercircadiancon-trol(Grafetal.
,2010).
StomatalconductanceandphotosynthesisarealsosubjecttoapartialcircadiancontrolinArabidopsis(Doddetal.
,2005).
Recently,itwasshownthatwaterdynamicsaswellasaquaporingeneexpressioninArabidopsisrootsoscillatedwiththecircadianrhythm(Takaseetal.
,2011).
Finally,rootxylempressure(Henzleretal.
,1999)andleafhydrau-licconductance(Nardinietal.
,2005)havebeenshowntobeclockdependent.
Thus,weraisethehypothesisthattheinuenceoftheclockonthedualpatternsreportedhere,althoughnotexcludable,couldbeatleastpartlyintegratedwiththemetabolicandhydrau-licprocessesdiscussedabove.
CONCLUSIONWhethercarbonorwateristhemainlimitationofleafgrowthisamatterofdebateintheliterature.
Here,weprovidegeneticandenvironmentalevidencethatthecontrolofleafexpansionswitchesfrommetabolicstohydraulicsduringthecourseofleafontogenyinArabidopsis.
Wedemonstratethatthisdevelopmen-talswitchisassociatedwithconsistentontogeneticchangesinday/nightleafturgorandstarchavailabil-ity.
Carboninuenceonleafexpansionoccursmainlyatnightduringtheearlyphasesofleafdevelopment,maybeduetoalimitedlocalstarchavailabilitywithrespecttoanintensecarbondemand,andisbufferedbyne-tuningstructuralgrowth.
Theseadjustmentsmaintainsurfaceexpansion(andthusenergycapture)underlimitinglightconditions,partlyuncouplingleafexpansionfromcarbonavailability.
Bycontrast,astheleafdevelops,hydraulicsexertincreasingconstraintduringthedaytimebyalteringleafturgor.
Toourknowledge,theestablishmentofanincreasingwaterlimitationofleafgrowthhasnotbeenreportedbefore.
Althoughofyetunknownnature,theselimitationscouldbeduetoadecreasingcapacityofthehydraulicnetworktosupplywatertothegrowingtissues.
Towhatextentthisdevelopmentalswitchcanbeextrap-olatedtootherspeciesremainsanopenquestion.
Thesink-to-sourcetransitionofleavesisageneralfeatureinplants.
Ifthistransitionisthecauseoftheweaken-ingofmetabolicconstraintonleafexpansion,itistemptingtospeculatethatthisresponseisconservedacrossspecies.
Bycontrast,iftheontogeneticestab-lishmentofhydraulicconstraintonleafexpansionisrelatedtothehydraulicnetworkarchitecture,whichishighlyvariableacrossspecies,thelevelofhydraulicconstraintonleafexpansionisexpectedtobespeciesControlofLeafExpansion:FromCarbontoWaterPlantPhysiol.
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dependent.
Asuperimpositionofmolecularandphys-iologicalinformationduringleafdevelopmenttothegrowthkineticsreportedhereisnowrequiredtofurtherdeciphertheconcertedactionsofcarbonandwateronleafexpansion.
MATERIALSANDMETHODSGrowthConditionsSeedsofArabidopsis(Arabidopsisthaliana)weresowninpotslledwithamixture(1:1,v/v)ofloamysoilandorganiccompost.
Oncegerminated,theyweregrowningrowthchambersata10-hphotoperiodunderaphotosyntheticphotonuxdensity(PPFD)of220mmolm22s21.
AirtemperatureandVPDwere21°Cand0.
8kPa,respectively,duringthedayand17°Cand0.
3kPaatnight.
Eachpotwasweighedtwiceadayandwateredwithone-tenth-strengthHoaglandsolution,sothatitssoilwatercontentwasmaintainedatawell-wateredlevel(0.
35gwaterg21drysoil)equivalenttoapredawnwaterpotentialof20.
3MPa(Hummeletal.
,2010).
Whenplantshad10visibleleaves,irrigationwassuspendedfortheplantsexposedtowaterstress,untilsoilwatercontentreachedatargetvaluecorrespondingtoamildwaterdecit(0.
23gwaterg21drysoil,20.
7MPa)oraseverewaterdecit(0.
18gwaterg21drysoil,21.
1MPa).
Firstmeasurementsoccurredwhenthesoilwatercontenthadstabilizedtothetargetvaluefor4d.
Forthelow-VPDexperiment,thetargetVPDwasmaintainedat0.
3kPaduringthedayand0.
1kPaatnightfrom4dbeforethebeginningofmeasurements.
Forthelow-lighttreatment,aneutralshadingveilreducedPPFDto70mmolm22s21,beginningalso4dbeforemeasurements.
PlantMaterialTheaba4mutantisimpairedinneoxanthinsynthesisanddisplaysamildphenotypecomparedwithotherABA-decientmutants,asABAcanbeproducedbyanalternativepathway(Northetal.
,2007).
Bycontrast,NCED6-OEoverexpressesa9-cis-epoxycarotenoiddioxygenaseleadingtoABAover-accumulation(Lefebvreetal.
,2006).
Thestarchlessmutantpgmlackstheplastidphosphoglucomutase(Casparetal.
,1985).
Bycontrast,thesex1mutant(Casparetal.
,1991)accumulatesalargeamountofstarch,asitisimpairedinana-glucanwaterdikinaseinvolvedintheearlystepsofstarchbreakdown(Ritteetal.
,2002).
Themex1mutantisimpairedinamaltosetransporteratthechloroplastenvelope,whichrepresentsthepredominantrouteforcarbohy-drateexportfromchloroplastsatnight(Niittyla¨etal.
,2004).
Thedpe2mutantisaffectedinthecytosolicdisproportionatingenzymeinvolvedinthecon-versionfrommaltosetoSucatnight(Chiaetal.
,2004;LuandSharkey,2004).
AllmutantswereintheCol-0background(N1092),exceptsex1anddpe2,whichwereintheWs-3background(N1638).
Accordingly,bothaccessionswereusedinourexperiments.
MonitoringLeafGrowthattheDay/NightTimeScaleInordertoevaluateleafgrowthlimitationsduringitsdevelopment(whichcanlastmorethan1month;Aguirrezabaletal.
,2006)regardlessofwholeplantchangeslikeoraltransition(Christopheetal.
,2008),wedevelopedanapproachwheretheexpansionofserialleavesoveronediurnalandonenocturnalphaseisusedtoinfertheexpansionofareconstructedleafoversuccessivedays.
Ourapproach,justiedinSupplementalMaterialsandMethodsS1andfullydescribedinSupplementalFiguresS1toS3,consistedofthreeconsecutivezenithalphotographs:therstoneatthebeginningofadayperiod,thesecondoneattheendofthesamedayperiod,andthethirdoneattheendofthesubsequentnight.
Thiswasrepeated3dlaterundermaintainedenvironmentalregimesasareplicate.
Duringeachacquisition,photographsweretakenwithinlessthan15minutilizingthephenotypingautomatondevelopedbyourgroup(Granieretal.
,2006).
WeextractedtheareaofsuccessiveleavesfromthedigitalphotographsusingasemiautomatedprogramdevelopedontheImageJsoftware(Rasband,2009).
Thelastdigita-lizedleafwaschosentorepresentmorethanhalfoftheareaofthelargestvisiblematureleaf(e.
g.
13leavesinthewell-wateredCol-0).
Whenappropri-ate,aseriesofindependentphotographswastakentoconsiderhyponastyandtheareaofeachleafwascorrectedforitsangle(SupplementalFig.
S3).
Foreachleafrank,theRERwascomputedasthelocalslopeofthenaturallogarithmofthearea(S)asafunctionofthermaltimetocorrectforthelineareffectsoftemperature(Granieretal.
,2002)andrecalculatedatareferencetemperatureof20°C,asmostlyencounteredintheliterature(SupplementalFig.
S1,step5).
Hence,thediurnal(respectivelynocturnal)RERbetweenti,thebeginningoftheday(respectivelynight),andtj,theendoftheday(respec-tivelynight),wascomputedasfollows:RERtitjlnSjSitjtiTTbase*dt3TrefTbasewhereTisthetemperature,Tbaseisthebasetemperatureof3°C,andTrefisthereferencetemperature(setat20°C).
RERwasexpressedinmm2mm22d21at20°C(notedd21elsewhereinthetext).
Whencalculatedover24-hintervals,thedailyRERcontinuouslydecreasedasafunctionoftime,meaningthatleafareaincreasewasalwayslessthanexponential,inlinewiththesigmoidpatternofdailyevolutionofleafarea(SupplementalFig.
S1,step3).
Thephyllochronofeachgenotype3environmentcombinationwasdeducedfromleafnumbercounts(seebelow).
Theageofeachleaffromitsemergencewasthencomputedas:agephyllochron3leafrankfromthetop1timedelaysincefirstpictureallowingustogathertheserialleafranksinasinglereconstructedtimeseries.
Thisswitchfromaspatialpatterntoatemporaltimecoursewaspossiblebecausesuccessiveleavesdisplayedsimilarfeatures(GrootandMeicenheimer,2000)duringthis10-hphotoperiod,whichpostponedtheinuenceofower-ing.
TheleftpanelofSupplementalFigureS2Ashowsthedynamicsofleafemergenceforwell-wateredCol-0,andthedoublearrowshowstheperiodoftheexperiment.
TherightpanelofSupplementalFigureS2Ashowstheassociatedphyllochron(inverseofleafemergencerate)anditsstabilityduringtheperiodcorrespondingtotheemergenceoftheoldestdigitalizedleafuntilthedayofmeasurement(doublearrow).
SupplementalFigureS2Bshowsforwell-wateredCol-0andpgmthemeasuredareaforareconstructedleafagainstthelogisticalttingusingthedataobtainedfromasingleleaffollowedduringseveraldays.
R2valueswerehighandequivalentbetweenthereconstructedandthesingleleaf(0.
905against0.
935and0.
917against0.
896forCol-0andpgm,respectively).
Then,afterdiscretizationtoprovideaday/nightrepre-sentationofthedata,second-degreepolynomialswereindependentlyttedtothedayRERandtothenightRER(Fig.
1,inset;SupplementalFig.
S1,step6),therebysmoothingkineticsandallowingfurtherstatisticalanalyses.
Thegrowthpatternsweredrawnfromemergencetohalfexpansionoftheleaf(t50)beforegrowthsloweddown,involvingotherprocessesthantheonesanalyzedhere.
t50wasdeterminedforeachgenotype3environmentcombinationafterttingalogisticalfunction(Aguirrezabaletal.
,2006)onthenightdataset,asexempliedinstep3ofSupplementalFigureS1.
Weprovedthatunderwell-wateredconditions,theexpansionpatternofthereconstructedleafwasverysimilartothatobtainedifonesingleleafwasfollowedover9d(SupplementalFig.
S2C).
TherewasatendencyforthesingleleaftodisplayasmallerRERintheearlystages,maybeduetoawhole-planteffect(seethedifferencebetweentherstandlastimagesinbothanalyses),buttheday/nightdifferenceswerealmostconservedandbothtreatmentsclusteredcloselytogetherwhenthesingleleafwasintroducedinthehierarchicalanalysis(SupplementalFig.
S2D,thicklines).
Usingthisframework,wewereabletoobservehighlyreproduc-ibleuctuationsofexpansionratesduringleafdevelopmentinseveralreplicatedexperiments(SupplementalFig.
S1E).
PhysiologicalMeasurementsWecharacterizedmutantsorenvironmentsbyprovidinghydraulicormetabolicmarkersmeasuredonfullyexpandedleavesoronthewholeplant.
Stomatalconductancewascalculatedasthesumoftheconductancesmea-suredonbothsidesofafullyexpandedleafon10replicatesusingadiffusionporometer(AP4;D-TDevices).
ThenetCO2assimilationratewasmeasuredwithawholeplantchamberdesignedforArabidopsisandconnectedtoagasanalyzer(CIRAS-2;PPSystems)onfourtosixindividualplants.
SLAwasmeasuredasdescribedbyHummeletal.
(2010)onfourwholerosettes.
Starchandsolublesugar(asthesumofGlc,Fru,andSuc)contentswereanalyzedbyenzymaticassayasdescribedbyHummeletal.
(2010)usingmaterialharvestedattheendsofdayandnight,eitheronfullyexpandedleavesoffourindividualplants(Fig.
3)oronpoolsofabout50leavesaged1or8dthatwerereplicatedthreetimesindependentlyandatleasttwicetechnically(Fig.
5).
Leafturgorwasdeterminedonleavesaged1or8dattheendsofdayandPantinetal.
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nightasthedifferencebetweenwaterandosmoticpotential.
Forwaterpotentialmeasurements,leaveswereharvestedandimmediatelyinsertedinasealedchambercarryingathermocouple(C-52;Wescor)connectedtoawetbulbdepressionpsychrometer(Psypro;Wescor).
Afoliardiscwaspunchedforthe8-d-oldleaves,whichwerelargerthanthe7-mmdiametersampleholder.
Atleast25replicateswereperformed.
Forosmoticpotential,apoolofleaveswasharvestedinliquidnitrogenandstoredat260°C.
Sampleswerethentransferredinto0.
8-mmlters(NucleoSpinlters;Macherey-Nagel)insertedinacollectiontubeandcentrifugedat4°C.
Samplesof10mLoftheresultingsap(n=8fortheearlystageandn=48forthelaterstage)wereanalyzedusingavaporpressureosmometer(Vapro5520;Wescor).
StatisticalAnalysesAllgraphicsandstatisticalanalyseswereperformedwiththeRsoftware(RDevelopmentCoreTeam,2008).
MeancomparisonswereperformedwiththeLSDtestadjustedusingtheBonferronimethodorwiththeKruskal-Wallistestwhenheteroscedasticitywasdetected.
Fortheheatmap,thetimeaxisofallkineticswasnormalizedaccordingtotheirt50.
Then,asecond-degreepolynomialwasttedindependentlytothedayandnightRERs,andtheirdifferencewascomputedfor100successiveiterationsbetweent0(emergence)andt50toprovideacontinuousvisualizationofthedifferencedynamics.
These100variableswereusedforthehierarchicalclusteringofthekinetics,whichwasperformedwiththeEuclideandistances.
Condenceintervalsofthepolynomialregressionswerecalculatedatthe0.
5,0.
9,0.
95,0.
99,0.
999,and0.
9999condencelevelsforeachiteration.
ThelevelfromwhichdaycondenceintervalandnightcondenceintervaloverlappedsetthesignicanceofthedifferencebetweendayandnightRER.
Asturgorwasdeterminedusingtwovariablesmeasuredonindependentsamples,anonparametricstratiedbootstrapwasperformedtoobtainthemeanandSEofthecomputedvariable.
Thenonparametricapproximatebootstrapcondenceintervalswerethencalculatedatthe0.
9,0.
95,0.
99,and0.
999condencelevelstoevaluatethesignicanceofthedifferencebetweendayandnightateachinvestigatedstage.
SupplementalDataThefollowingmaterialsareavailableintheonlineversionofthisarticle.
SupplementalFigureS1.
ProcessfromplantimagestoRERpatterns.
SupplementalFigureS2.
Validationoftheleafreconstructionmethod.
SupplementalFigureS3.
Takinghyponastyintoaccount.
SupplementalFigureS4.
Effectofaseveremetabolicandhydraulicconstraintincombination.
SupplementalFigureS5.
Heatmapoftheexpansionpatternsfortherstthirdofthekinetics.
SupplementalMaterialsandMethodsS1.
Fromleafranktoleafage:usesandprinciples.
ACKNOWLEDGMENTSWethankSamuelZeemanandAnnieMarion-PollforsupplyingseedsofstarchandABAmutants,respectively.
WearegratefultoNathalieWuytsfordevelopingtheImageJprogramtoassistleafareaextraction.
Finally,weacknowledgeChristopheMaurelandtwoanonymousreviewersfortheirsuggestionstoimprovethemanuscript.
ReceivedMarch12,2011;acceptedApril4,2011;publishedApril6,2011.
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