Aerenchymaimprovesmaizedroughttolerance741

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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