Root cortical aerenchyma improves the drought tolerance of maize

Plant,CellandEnvironment(2010)33,740–749doi:

10.1111/j.1365-3040.2009.02099.x

Rootcorticalaerenchymaimprovesthedroughttoleranceofmaize(ZeamaysL.)

JINMINGZHU,KATHLEENM.BROWN&JONATHANP.LYNCH

DepartmentofHorticulture,ThePennsylvaniaStateUniversity,UniversityPark,PA16802,USA

ABSTRACT

Rootcorticalaerenchyma(RCA)reducesrootrespirationinmaizebyconvertinglivingcorticaltissuetoairvolume.WehypothesizedthatRCAincreasesdroughttolerancebyreducingrootmetaboliccosts,permittinggreaterrootgrowthandwateracquisitionfromdryingsoil.Totestthishypothesis,recombinantinbredlineswithhighandlowRCAwereobservedunderwaterstressinthe eldandinsoilmesocosmsinagreenhouse.Inthe eld,lineswithhighRCAhad30%moreshootbiomassat oweringcomparedwithlineswithlowRCAunderwaterstress.Rootlengthdensityindeepsoilwassigni cantlygreaterinthehighRCAlinescomparedwiththelowRCAlines.Mid-dayleafrelativewatercontentinthehighRCAlineswas10%greaterthaninthelowRCAlinesunderwaterstress.ThehighRCAlinesaveragedeighttimestheyieldofthelowRCAlinesunderwaterstress.Inmesocosms,highRCAlineshadlessseminalrootrespiration,deeperrooting,andgreatershootbiomasscomparedwithlowRCAlinesunderwaterstress.TheseresultssupportthehypothesisthatRCAisbene cialfordroughttoleranceinmaizebyreducingthemetaboliccostofsoilexploration.

Key-words:aerenchyma;drought;rootgrowth;rootrespira-tion;soilwatercontent;yield;Zeamays.

Abbreviations:DAP,daysafterplanting;RCA,rootcorticalaerenchyma;RILs,recombinantinbredlines;RWC,relativewatercontent;TDR,timedomainre ectometry.

INTRODUCTION

Droughtstressisaprimarylimitationtocropproduction(Boyer1982;Tuberosaetal.2007),andimportantagroeco-sytemsmayfaceincreasingdroughtriskastheresultofglobalclimatechange(Easterlingetal.2000).Itisestimatedthatby2025,over60%ofthehumanpopulationwillinhabitcountrieswithwatershortage(Arnel1999).Theidenti ca-tionandunderstandingoftraitsimprovingcropdroughttoleranceareessentialforthedevelopmentofmoredrought-tolerantcropsandcroppingsystems.Plantshave

Correspondence:J.P.Lynch.Fax:8148636139;e-mail:JPL4@psu.edu740

evolvedavarietyofmechanismstoadapttodroughtcon-ditions(Blum1996;Cattivellietal.2008).Inrecentdecades,researchershaveidenti edanumberoftraitsthatmighthelpplantsuseacquiredwateref cientlyandtoleratedes-iccation,aswellasasmallernumberoftraitsthatmayassistsoilwateracquisition(Sinclair,Bennett&Muchow1990;Bruce,Edmeades&Barker2002;Richards2006;Nelsonetal.2007).Forinstance,rootgrowthoftencontinuesunderseveredroughtstressthatcompletelyinhibitsshootelonga-tioninmaize(Westgate&Boyer1985;Sharp&Davies1989;Zhuetal.2007),whichisconsideredtobeamecha-nismofplantadaptationtowater-limitedconditions(Sharp&Davies1989;Sharpetal.2004).

Aerenchymaisthegeneraltermfortissuewithlargeintercellularspaces(Esau1977).Theformationofcorticalaerenchymainmaizeroots(‘RCA’)variesamonggeno-typesandisassociatedwithtoleranceofhypoxiacausedby ooding(Jackson&Armstrong1999;Manoetal.2006).MaizerootaerenchymacanalsobeinducedbysuboptimalnutrientavailabilitysuchasN,PandSde ciencyinwell-aeratedsolution(Konings&Verschuren1980;Drew,He&Morgan1989;He,Morgan&Drew1992;Fanetal.2003;Bouranisetal.2006).Ethylenecantriggeraerenchymafor-mationinmaizerootsatlowphosphorusavailability(Heetal.1992;Fanetal.2003)andinhypoxia(Drew,Jackson&Giffard1979;Evans2004).Justin&Armstrong(1991)reportedthatalowconcentrationofNAApromotesrootaerenchymaformationinmaizeplants,possiblybecauseofauxin-inducedethylenerelease.TheinductionofRCAbynutrientstressinoxygenatedrootspromptedthehypoth-esisthatRCAenhancesadaptationtonutrientstressbyconvertinglivingcorticaltissuetoairspace,therebyreduc-ingtherespiratoryandnutrientcostofsoilexploration(Lynch&Brown1998).Evidenceinfavourofthishypoth-esiswasreportedformaizeandbeanunderlowPstress(Fanetal.2003).

Traitsthataffectthemetabolicef ciencyofrootgrowthandsoilexplorationshouldbeimportantcomponentsofdroughttoleranceinmaize(Palta&Gregory1997;Liedgens&Richner2001;Bruceetal.2002).Studieshaveshownthatthemetaboliccostsofsoilexplorationbyrootsystemsarequitesubstantial,andcanexceed50%ofdailyphotosynthesis(Lambers,Atkin&Millenaar2002).RCAcouldbeconsideredtocontributetodroughttoleranceif

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themetaboliccostassociatedwithitsproductionandmaintenancewasexceededbythemetabolicbene tsofenhancedwateracquisition(Lynch&Ho2005).RCAdis-proportionatelyreducesrootrespirationinmaizebycon-vertinglivingcorticaltissuetoairvolume;20%aerenchymacutsrootrespirationinhalfinphosphorus-stressedrootsinbothwell-aeratedsolutioncultureandsoilculturesystems(Fanetal.2003).Reductionofrootmetaboliccostspermitsmorecarbonresourcestobeallocatedtogreaterrootgrowthandwateracquisition.Reducedrootcostsmayalsobebene cialbyincreasingcarbohydrateavailabilityduringreproductivegrowth,sincereproductionandrootsarecom-petingsinksforcurrentphotosynthate.Toourknowledge,evaluationoftheimportanceofRCAhasneverbeencon-sideredinthecontextofdroughttolerance.

Theobjectivesofthisresearchweretotestthehypoth-esesthat:(1)RCAdecreasestherespirationofmaizeroots;and(2)maizegenotypeswithabundantRCAhavegreaterrootgrowthandrootdepthunderdroughtstress,resultingingreaterwateracquisition.Weusedmaizerecombinantinbredlines(RILs)withacommongeneticbackgroundbutcontrastingRCAunderwaterstress.

polyethylene lm,whichwereusedtofacilitaterootsam-pling.Thegrowthmediumconsistedofamixture(volumebased)of50%mediumsize(0.5–0.3mm)commercialgradesand(QuikreteCompaniesInc.,Harrisburg,PA,USA),25%horticulturalsize#3vermiculite,and25%peatmoss(Whit-temoreCompaniesInc.,Lawrence,MA,USA).Twenty-ninelitresofthemixturewereusedineachcylinder.Twodaysbeforeplanting,thecylinderswerewateredwith5LofanutrientsolutionadjustedtopH6.0andconsistingof(inmM):NO3(7000),NH4(1000),P(1000),K(3000),Ca(2000),SO4(500),Mg(500),Cl(25),B(12.5),Mn(1),Zn(1),Cu(0.25)Mo(0.25)andEDTA-Fe(25).Eachcylinderreceivedtwoplants,andafter2dtheywerethinnedtooneplant.Theplantsweregrowninatemperature-controlledgreenhouseinUniversityPark,PA,USA(40°49′N,77°49′W),withaphotoperiodof14/10hat28/24°C(light/darkness).Maximummiddayphotosynthetic uxdensitiesreached1200mmolphotonsm-2s-1.Therelativehumiditywas40%.Thewell-wateredtreatmentconsistedofdailyirrigationof50mLofdeionizedwaterfor5weeks.Inthewaterstresstreatment,therewasnoirrigationfor5weekstoprogres-sivelyreducesoilwatercontent.Whenplantswerehar-vested5weeksaftertransplanting,soilwaterpotentialwas0.0MPaat10,70,and110cmdepthsinwell-wateredmeso-cosms(WP4-Tmeter,Decagon,Pullman,WA,USA).Inwaterstresstreatments,soilwaterpotentialreached-0.91 0.04MPaat10cmdepth,-0.34 0.02MPaat70cmdepth,-0.10 0.02MPaat110cmdepth(n=24).

MATERIALSANDMETHODSGreenhousemesocosmstudy

Plantmaterials

SixRILsofmaize(ZeamaysL.),numbers33,34,248,284,331and364fromtheintermatedB73xMo17population(IBM)wereobtainedfromDrShawnKaepplerattheUni-versityofWisconsin,Madison,WI,USA,originallysuppliedbyCharlesStuberandLynnSenioratNorthCarolinaStateUniversity(Senioretal.1996;Kaeppleretal.2000).OurpreviousscreeningforRCAinthispopulationindicatedthatRILs33,248and331hadlowRCAandRILs34,284and364hadhighRCAunderwaterstress(Jaramillo2010).

Respirationofrootsegments

Measurementsofrootsegmentrespirationweremadeinseminalroots.Thesamplewastaken15cmfromthebaseandconsistedofa15cmlongsamplewiththerootlateralsremovedbyaTe onblade.Excisedsegmentswerepatteddryandplacedina40mLcustomchamberconnectedtotheLi-6250IRGA.Thecontainerwiththesamplewaskeptat22°Cinawaterbathfor2minwhiletherespirationwasmeasured.Followingrespirationmeasurement,sampleswerestoredin25%ethanolforanatomicalanalysisasdescribedasfollows.

Experimentaldesign

Thegreenhouseexperimentwasarandomizedcompleteblockdesignwitha2¥6factorialarrangementoftreat-ments.Thefactorsweretwowaterregimes(water-stressedandwell-wateredconditions)andsixgenotypes(RILs33,34,248,284,331and364),andfourreplicatesstaggered1dbetweenreplicateswithtimeofplantingastheblockeffect.

RCAmeasurement

Rootcrosssectionswereobtainedusingaprotocolsug-gestedbyRosemaryWhite(CSIRO,Australia,personalcommunication;http://roots.psu.edu/?q=en/node/151).Inshort,thisconsistedoffree-handsectioningwithTe on-coatedrazorbladesofrootsegmentsrestingondentalwaxinsideadropofwater,withtheaidofadissectingmicro-scope.ThesectionswereobservedwithaNikonDiaphotinvertedmicroscope,theimageswereacquiredusingImageMaster(PhotonTechnologyInternational,Birming-ham,NJ,USA)andanalysedinMATLAB7.62008a(TheMathWorksCompany,Natick,MA,USA),usingcodewedevelopedforthispurpose.Thecodeconsistsofthreephases.PhaseIoftheanalysisinvolvesseparationofthecross-sectionfromanydebrisandfromthebackgroundof

Growthconditions

Seedswereselectedforuniformsize,surface-sterilizedin0.05%NaOClfor15minandimbibedfor24hinaerated1mMCaSO4.Theseedswereplacedindarknessat28 1°Cinagerminationchamberfortwodays.Beforetransplanting,theendospermwasremovedfromtheseed-lings.Seedlingsofsimilarsizeweretransplantedtomeso-cosmsconsistingofPVCcylinders15.7cmindiameterand155cmhigh.Thecylinderswerelinedinsidewithplasticsleevesmadeof4mil(0.116mm)transparenthi-density

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theimage.InPhaseII,aseriesofstepsisolatesthestelefromthecortex,allowingforseparateanalysesoftheseareas.InPhaseIII,datacollectionincludesareameasure-mentofdesiredfeatures.Correctidenti cationofthesefeaturesisbasedonnumberofpixelsandvaluethreshold-ingforedgedetection.Areasofthecross-sectionandaerenchymalacunaewereobtainedbypixelcounting.Mea-surementswereinmm2,andwerecalibratedfrompixelsusinganimageofa1mmmicrometertakenatthesamemagni cationastheanalysedimages.Thepercentageaer-enchymaasareaofthecortexwasestimatedforfourdif-ferentsectionsperreplicate.

UT,USA)spreadrandomlyacrossplotsandplacedat25and50cmdepthsinthesoilloggedbyaCR23Xdatalogger(CampbellScienti cInc.)inwaterstressandwell-wateredplotswithfourreplicatesthroughoutthegrowingseason.Leafrelativewatercontent(RWC)ofthesecondfullyexpandedleafwasdeterminedmid-dayat42DAPaccord-ingtothemethodofBarr&Weatherley(1962).

Rootdepth

Rootdevelopmentwithdepthwasmonitoredwithminirhizotrons(BTC100X,BartzTechnologyCo.,SantaBarbara,CA,USA).Oneclearacrylicminirhizotronrootobservationtubewithadiameterof5.1cmwasinstalledineachplotata30°anglefromperpendiculartoadepthofabout65cm.Rootlengthdensityandrootnumberperframewereimagedfrom10to50cmdepthinthesoilwithanintervalof10cmat42DAPandanalysedbyRoot ysoftwareversion2.0(https:///projects/root y).Destructiveharvestsat56DAPwereusedtomeasurerootlengthandnumberdensitywithdepthbysoilcoring(GiddingsMachineCo.,Windsor,CO,USA)from10to50cmdepthinthesoilwithanintervalof10cm.Thediameterofsoilcoreswas5.1cm.

Shootandrootgrowth

Rootswereseparatedfromthesoilbysubmergingcylindersinacontainer lledwithwaterandrinsingtherootscare-fullyindeionizedwater.Rootswereseparatedfromevery20cmsoildepth.Totalrootlengthinvarioussoildepthswereobtainedbyscanningwithimageanalysissoftware(WinRhizoPro,RégentInstruments,Québec,QuébecCityCanada).Maximumrootlengthofeachplantwasrecordedat35DAP.Theshootsandrootsweredriedat60°Cfor72hpriortodryweightdetermination.

Fieldstudy

Plantmaterials,experimentaldesignand eldconditions

MaizeseedsofthesixIBMRILshavingcontrastingRCAunderwaterstressinmesocosms,rsonExperimentalFarmofPenn-sylvaniaStateUniversityatRockSprings,PA(40°43′N,77°56′W)in2008.Theexperimentwasarandomizedcom-pleteblockdesignwithasplit-plotarrangementoftreat-ments.Therewerefourbiologicalreplicatesforeachgenotype,andeachreplicatehad30plantsgrowninthree2.1mrows.Theshelters(10¥30m)werecoveredwithcleargreenhouseplastic lm(0.184mm)(Grif nGreenhouse&NurserySupply,Morgantown,PA,USA)andwereauto-maticallytriggeredbyrainfalltocovertheplotsandexcludenaturalprecipitationthroughouttheentiregrowingseason.Thesheltersautomaticallyopenedquicklyafterrainfalls,exposingexperimentalplotstoambientradia-tionandtemperatureconditions.Adjacentnon-shelteredcontrolplotsweredrip-irrigatedasneededthroughoutthegrowingseasontoprovideunstressedcomparisons.ThesoilwasaMurrillsiltloam( ne-loamy,mixed,semiactive,mesicTypicHapludult).SoilpHwas6.2,andsoilnutrientlevelsofN,P,K,Ca,Mg,Zn,Cu,Swereoptimumformaizeproduc-tionasdeterminedbysoiltests.

RCA,shootdryweightat oweringstage,andindividualplantyield

Rootsandshootswereharvestedat owering.RCAwassampledfromtheoldestnodalrootinthematurationzoneat56DAPwithfoursubsamplesperreplicate.TissuesectioningandRCAquanti cationwereaspreviouslydescribed.Shootdryweightwascollectedwiththreesub-samplesperreplicate(n=4).Atmaturity,individualseednumberperplantandindividualseeddryweightwererecordedtocalculateindividualyieldwiththreesubsamplesperreplicate.

Statisticalanalysis

DatawereanalysedusingtheMinitabstatisticalpackage(MinitabInc.,UniversityPark,PA,USA).Rootdepth,RCA,shootandrootdryweight,speci crootrespiration,relativewatercontent,yield,individualseeddryweight,seednumberperplant,rootlengthandnumberperframewere rstsubjectedtoanovaformaineffectsand rst-orderinterac-tionsusingagenerallinearmodelthatincludedwaterregimeandgenotypefactors.Genotypeandwaterregimewereconsidered xedeffects,andreplicateswererandom.Fisher’sLeastSigni canceDifferencewasusedformultiplecomparisonsatP 0.05levelunderposthocanova.

RESULTS

Greenhousemesocosmstudy

WaterstressincreasedRCAofthesixlinesbyanaverageof382%at35DAPinmesocosms(Fig.1).Inwaterstress,lines34,284,and364(highRCAlines)averaged5.5%RCA,

Soilvolumetricwatercontentandleafrelativewatercontent

Soilvolumetricwatercontentwasmonitoredwithamulti-plexedTDR-100system(CampbellScienti cInc.,Logan,

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Figure1.Productionofrootcorticalaerenchymaofsix

genotypesat35dafterplantinginbothwell-watered(WW)andwater-stressed(WS)conditionsinthemesocosms.Datashownaremeans SEofthemean(n=4).Meanswiththesamelettersarenotsigni cantlydifferent(P 0.05).

Specific seminal root respiration (µmol s–1 g–1)

whilelines33,248,and331(lowRCAlines)averaged0.29%RCA.Inwell-wateredconditions,therewasnosig-ni cantdifferenceforRCAamongallsixlines(P 0.05).Waterstressreducedshootbiomassofthesixlinesby39%at35DAPinmesocosms(Fig.2).Underwaterstress,thethreelineswithhighRCAhad86%moreshootbiomass

864Shoot dry weight (g)

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Figure3.Speci cseminalrootrespirationperlength(upper

panel)orweight(lowerpanel)ofsixgenotypesat35dafterplanting(DAP)inbothwell-watered(WW)andwater-stressed(WS)conditionsinthemesocosms.Datashownaremeans SEofthemean(n=4).Meanswiththesamelettersarenotsigni cantlydifferent(P 0.05).

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Figure2.Shootdryweightofsixgenotypesat35dafter

plantinginbothwell-watered(WW)andwater-stressed(WS)conditionsinthemesocosms.Datashownaremeans SEofthemean(n=4).Meanswiththesamelettersarenotsigni cantlydifferent(P 0.05).

at35DAPcomparedwiththethreelineswithlowRCA(P 0.05)(Fig.2).

Waterstressreducedspeci crootrespirationperunitofrootlengthandweightby61%and94%,respectively(Fig.3).Inwaterstress,thehighRCAlineshad53–57%lessspeci crootrespirationperunitofrootlengthandweightthanthelowRCAlines(Fig.3).Thediameterofrootsegmentsusedforrespirationmeasurementswas0.43–0.45mmunderwaterstress,withnosigni cantdifferencesamonggenotypes(datanotshown).

Waterstresssigni cantlyincreasedrootlengthdensityindeepersoillayersat35DAPinmesocosms(P 0.05)(Fig.4).Underwaterstress,thehighRCAlineshadsigni -cantlygreaterrootlengthin100–140cmsoillayersthanthelowRCAlines(Fig.4).

Rootdryweightwassigni cantlyassociatedwithshootdryweight(Fig.5),butrootdryweightwasnotsigni cantlycorrelatedwithpercentageofrootcorticalaerenchymaforthesixmaizelinesat35DAPinbothwell-wateredandwaterstressconditionsinthemesocosms.Covariateanaly-sisshowedrootdryweightwasnon-signi cantforpercent-ageofRCA(F=0.65,P=0.57)(Fig.5).

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Figure4.Rootlengthdistributionin

soillayersofsixgenotypes(lowRCAlinesontheleft,andhighRCAlinesontheright)at35dafterplanting(DAP)inbothwell-watered(WW)and

water-stressed(WS)conditionsinthemesocosms.Datashownaremeans SEofthemean(n=4).**showssigni cantlydifferentpairedmeansinWSandWW(P 0.05).

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Fieldstudy

Volumetricsoilmoisturewasmaintainedaround30%atboth25cmand50cmdepthsinwell-wateredconditionsthroughoutthegrowingseason(Fig.6).Forwater-stressedplants,volumetricsoilmoistureprogressivelydecreasedfrom30%to10%at25cmdepth,andto15%at50cmdepthinrainoutshelters(Fig.6).

WaterstressincreasedRCAinthehighRCAlinesat56DAPinthe eld(Fig.7).Lines34,77,284and364(highRCAlines)averaged12.6%RCA,whilelines33,248and331(lowRCAlines)averaged1.98%RCA.Inwell-wateredconditions,therewasnosigni cantdifferenceforRCAamongthelines(P<0.05).Water-stressedplantshaddoubledrootlengthdensityandrootnumberdensityat40–50cmdepthinthesoilcomparedwithwell-wateredplantsat56DAPinallsevenlines(Fig.8).Rootlengthdensityat40–50cmdepthwassigni cantlygreaterinthefourhighRCAlinescomparedwiththethreelowRCAlinesat56DAP(Fig.8).ThefourhighRCAlinesaveraged3.4timestherootnumberdensityat40–50cmdepthcom-paredwiththethreelowRCAlinesunderwaterstress(Fig.8).Resultsfromminirhizotronmeasurementsfurthercon rmedthathighRCAlineshaddeeperrootingintermsofrootlengthperframeandrootnumberperframecom-paredwiththelowRCAlinesat42DAP(Fig.9).Therewassigni cantcorrelationbetweenrootlengthdensitymea-suredbysoilcoringandbytheminirhizotrontechnique(R2=0.63,P<0.01)(Fig.10).Rootnumberdensitywasalsosigni cantlycorrelated(R2=0.31,P<0.01)betweensoilcoringandminirhizotrontechniques(Fig.10).

Mid-dayleafrelativewatercontent(RWC)at42DAPinthehighRCAlineswassigni cantlygreaterthanforthelowRCAlinesinwater-stressedconditions,whiletherewasnosigni cantdifferenceamonggenotypesforRWCinwell-wateredconditions(Fig.11).

Shootbiomassofthesevenlineswasreducedbyanaverageof46%underwaterstressat oweringcomparedwithwell-wateredplantsinthe eld(Fig.12).Underwaterstress,thefourlineswithhighRCAhad43%moreshootbiomassat oweringcomparedwiththethreelineswithlowRCA.ThehighRCAlinesaveragedeighttimestheyieldofthelowRCAlinesunderwaterstress(P<0.001)(Fig.12).Theincreasedyieldunderwaterstresswascausedbyincreasesofbothkernelnumbersperplantandindividualkernelweight(Fig.13).

DISCUSSION

OurresultssupportthehypothesisthatRCAincreasesdroughttolerancebyreducingrootmetaboliccosts,permit-tinggreaterrootgrowthandwateracquisition.Inmeso-cosmsundercontrolledwaterstressconditions,threehighRCAlineshadlowerspeci crootrespiration,greatermaximumrootdepthandgreatershootbiomassthanthreelowRCAlines.Inthe eldunderwaterstressconditionsinrainoutshelters,fourhighRCAlineshadgreaterrootdepth,greaterleafrelativewatercontentandsubstantiallygreatergrainyieldthanthreelowRCAlines.

FortheanalysisofthephysiologicalfunctionofRCA,weusedRILs,whichpermitthecomparisonofcloselyrelatedgenotypeswithidenticalgeneticbackgrounds,yetwithout

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sevengenotypesinbothwell-watered(WW)andwater-stressed(WS)conditionsinthe eld.Datashownaremeans SEofthemean(n=4).Meanswiththesamelettersarenotsigni cantlydifferent(P 0.05).

Figure5.Correlationofrootdryweightandshootdryweight

(upperpanel),androotdryweightandpercentageofroot

corticalaerenchyma(lowerpanel)forsixmaizelinesat35DAPinthemesocosms.Covariateanalysisshowedrootdryweightwasnon-signi cantforpercentageofRCA(F=0.65,P=0.57).Datashownaremeansfrom4replicates.

arti ciallyinducedmutationsortransformationevents(Zhu&Lynch2004;Zhu,Kaeppler,Lynch2005a,b;Zhuetal.2006).EachRILisadistinctgenotype,andcomparisonofsixtosevenRILsallowstheanalysisofaphenotypeindistinctgenomes,therebyreducingtheriskofconfounding

0.40

effectsfrompleiotropy,epistasis,orothergeneticinterac-tions(Zhu&Lynch2004).RILsareparticularlyvaluableintheanalysisofphenotypictraitsgovernedbymultiplegenes,asisthecaseforRCAinmaize(unpublisheddata).

Weusedmesocosmsinagreenhouseandmovablerainoutsheltersinthe eldtoobtainprogressivereductionofsoilwatercontent,astypicallyoccursinseasonaldrought.Plantbiomassandyieldweresigni cantlyreducedbytheimposedstresstreatment(Figs2,12&13).Wealsofoundsigni cantlydeeperrootingintermsofrootlengthdensityandrootnumberdensityinwater-stressedcom-paredwithwell-wateredplantsofallgenotypesinthetwodifferentculturesystems(Figs4,8&9),asevidencedbysoilcoresandminirhizotronmeasurements(Figs8and9).Ourresultsareconsistentwithotherstudiesofrootingdepthincropsincludingmaizeunderwaterstress(Blum&Ritchie1984;Hoadetal.2001;Sharpetal.2004;Hoetal.2005).Deeperrootinginresponsetowaterstressenhanceswater

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contentat25and50cmdepthsinbothwell-watered(WW)andwater-stressed(WS)conditionsthroughoutthegrowingseasonin2008.Datashownaremeans SEofthemean(n=4).

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consistentwiththeresponsetolowphosphorusavailability(Fanetal.2003;Zhu&Lynch2004).HigherRCAlineshavemorecorticalairspaceinroottissues(Figs1&7),whichmayreducerootcarboncostforrootmaintenanceinhigherRCAlines.Thus,higherRCAlinesmayhavemorecarbonandenergyavailableforrootconstruction,permittingexplorationofdeepersoilsunderwaterstress(Figs3,4,6,9&10).Covariateanalysisshowedthattherewasnosigni -cantrelationshipbetweentotalrootdryweightandper-centageofRCA(F=0.65,P=0.57).Inotherwords,RCAwasnotdrivenbythesizeofrootsystemorthesizeoftheplant(Fig.5).WefoundhighRCAlineswithdeeperrootingunderwaterstresshadhigherleafrelativewatercontent(Fig.11).Takentogether,these ndingsdemonstrateRCAplaysanimportantroleforplantadaptationtodrought.Wefoundthatseminalrootrespirationperunitlength(y1)wassigni cantlyassociatedwithpercentageofRCA

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Figure8.Rootlengthdensity(upperpanel)androotnumberdensity(lowerpanel)at56dafterplanting(DAP)from40to50cmdeepsoilcoresegmentsforsevengenotypesinbothwell-watered(WW)andwater-stressed(WS)conditionsinthe eld.Datashownaremeans SEofthemean(n=4).Meanswiththesamelettersarenotsigni cantlydifferent(P 0.05).

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acquisition,sincedeepersoildomainsgenerallyhavegreaterwatercontentcomparedwithshallowsoildomains,asevidencedinthisstudybytimedomainre ectometry(TDR)measurementsinthe eld(Fig.6)andsoilwaterpotentialmeasurementsat35DAPinmesocosms.

Thereisincreasingevidencethatrootmetaboliccostsareanimportantcomponentofplantgrowthandadaptationunderlowphosphorusavailability(Zhu&Lynch2004;Lynch&Ho2005;Zhu,Kaeppler&Lynch2005c).Rootcostcanbeestimatedastotalbelow-groundexpenditureofcarbon,whichisusedforrootconstruction,rootmainte-nance,andionuptake(Lambers,Atkin&Scheurwater1996;Lambersetal.2002;Lynch&Ho2005).Inthisstudy,speci crootrespirationperunitoflengthandweightweresigni cantlyreducedbywaterstress(Fig.3),whichis

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Recombinant inbred lines

Figure9.Rootlengthperframe(upperpanel)androotcounts

perframe(lowerpanel)byminirhizotrontechniqueat42dafterplanting(DAP)at50cmdeepsoilforsevengenotypesinbothwell-watered(WW)andwater-stressed(WS)conditionsinthe eld.Datashownaremeans SEofthemean(n=4).Meanswiththesamelettersarenotsigni cantlydifferent(P 0.05).

©2010BlackwellPublishingLtd,Plant,CellandEnvironment,33,740–749

Aerenchymaimprovesmaizedroughttolerance747

Root length (cm) per frame at 50cm deep soil by minirhizotron

200

12

WSWW

a

b

f

cd

e

g

gh

bc

Shoot dry weight (g)

96300

10

20

30

40

50

Root length (cm) 40–50 cm deep from soil coring

r² = 0.632 (P < 0.01)

150

b

cd

100

i

50

j

Root counts per frame at 50 cmdeep soil by minirhizotron

5040

ab

cd

a

90Yield (g)/plant

r² = 0.309 (P < 0.05)

30201000

20

40

WSWW

60

e

30

ef

g0

33

248

331

34

77

284

364

6080

Root number 40–50 cm deep from soil coring

g

g

Figure10.Correlationsbetweenmeasurementsmadeat50cm

deepsoilbyminirhizotrontechniqueand40–50cmdeepbysoilcoringtechniqueforrootlength(upperpanel)androotnumber(lowerpanel)inthe eld.

Low RCAHigh RCA

Recombinant inbred lines

Figure12.Shootdryweightat oweringstage(upperpanel)

underwaterstress(x)(Figs1&3).Thelinearregressionformulawasy1=-0.014x+0.148(r2=0.94,P<0.001).Asimilarcorrelationwasfoundforseminalrootrespirationperunitweight(y2)andpercentageofRCAunderwaterstress(x)(Figs1&3).Thelinearregressionformulawas

andgrainyield(lowerpanel)ofsevengenotypesinwell-watered(WW)andwater-stressed(WS)conditionsinthe eld.Data

shownaremeans SEofthemean(n=4).Meanswiththesamelettersarenotsigni cantlydifferent(P 0.05).

100%

ab

Relative water content

WWWS

ab

abbcd

ab

90%

cd

e

d

80%

e

e

70%

332483313477284364

Low RCAHigh RCA

Recombinant inbred lines

Figure11.Leafrelativewatercontentat56dafterplanting

(DAP)forsevengenotypesinwell-watered(WW)and

water-stressed(WS)conditionsinthe eld.Datashownare

means SEofthemean(n=4).Meanswiththesamelettersarenotsigni cantlydifferent(P 0.05).

y2=-30.4x+293(r2=0.77,P<0.05).Theseindicatethatabout6%RCAinseminalrootshalvesrootrespirationperunitlengthorweightunderwaterstress.Fanetal.(2003)reportedthat20%RCAcutsrootrespirationperunitvolumeinhalfatlowphosphorusavailabilityinasolutionculturestudy.

Inmaize,anadditionalbene ttoreducingrootcostsisthatyieldlossestodroughtarerelatedtocarbohydrateavailabilityduringreproductivegrowth(Boyer&Westgate2004).ReducedrootcarbondemandinhighRCAgeno-typesmaybebene cialbyincreasingcarbohydrateavail-abilityduringreproductivegrowth,sincereproductionandrootsarecompetingsinksforcurrentphotosynthate(Figs3,12&13).

WeobservedthatwaterstressinducedgreaterexpressionofRCAindrought-tolerantlinesinbothseminalrootsof5-week-oldplantsinmesocosmsandnodalrootsof8-week-oldplantsinthe eld,comparedwiththesensitivelines(Figs1,2,7,12&13),althoughtheabsolutevaluesofincreasedRCAdifferedinvariousroottypes.Seminalrootsof5-week-oldplantsandnodalrootsof8-week-oldplantsaremajorcomponentsofrootsystemarchitecture.Thisresponsetowaterstressallowsmorecarbonallocationfor

©2010BlackwellPublishingLtd,Plant,CellandEnvironment,33,740–749

748J.Zhuetal.

700600Seed number/plant

500400

3002001000Individual seed dry weight (g)

0.2

thatRCAreducesthemetaboliccostsofsoilexploration,leadingtogreaterwateracquisitionindryingsoil.WesuggestthatRCAdeservesconsiderationasatraittoimprovethedroughttoleranceofmaizeinbreedingprograms.

ACKNOWLEDGMENTS

ThisresearchwassupportedbyUSDA-CSREESgrant207-35100-18365toJPL,SMKaepplerandKMB.WeacknowledgeDrShawnKaepplerattheUniversityofWis-consinforprovidingseedsandRobertSnyderfortechnicalassistance.WealsothankAmyBurtonforassistanceinquanti cationofaerenchyma.

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