78MetabolicprofilingofrootexudatesofArabidopsistha

MetabolicProfilingofRootExudatesofArabidopsisthalianaTRAVISS.WALKER,†HARSHPALBAIS,†KATHLEENM.HALLIGAN,‡FRANKR.STERMITZ,‡ANDJORGEM.VIVANCO*,†DepartmentofHorticultureandLandscapeArchitectureandDepartmentofChemistry,ColoradoStateUniversity,FortCollins,Colorado80523-1173Inadditiontoaccumulatingbiologicallyactivechemicals,plantrootscontinuouslyproduceandsecretecompoundsintotheirimmediaterhizosphere.However,themechanismsthatdriveandregulaterootsecretionofsecondarymetabolitesarenotfullyunderstood.Toenlightentwoneglectedareasofrootbiology,rootsecretionandsecondarymetabolism,aninvitrosystemimplementingroot-specificelicitationovera48-daytimecoursewasdeveloped.AfterrootsofArabidopsisthalianahadbeenelicitedwithsalicylicacid,jasmonicacid,chitosan,andtwofungalcellwallelicitors,thesecondarymetabolitessubsequentlysecretedwereprofiled.High-performanceliquidchromatographywasusedtometabolicallyprofilecompoundsintherootexudates,and289possiblesecondarymetaboliteswerequantified.Thechemicalstructuresof10compoundswerefurthercharacterizedby1Hand13CNMR:butanoicacid,trans-cinnamicacid,o-coumaricacid,p-coumaricacid,ferulicacid,p-hydroxybenzamide,methylp-hydroxybenzoate,3-indolepropanoicacid,syringicacid,andvanillicacid.Severalofthesecompoundsexhibitedawiderangeofantimicrobialactivityagainstbothsoil-bornebacteriaandfungiattheconcentrationdetectedintherootexudates.KEYWORDS:Arabidopsisthaliana;Brassicaceae;butanoicacid;trans-cinnamicacid;o-coumaricacid;p-coumaricacid;ferulicacid;p-hydroxybenzamide;methylp-hydroxybenzoate;3-indolepropanoicacid;syringicacid;vanillicacid;elicitationINTRODUCTIONThehiddenhalfofaplantsystemthrivesinadiverse,ever-changingenvironmentwithbacteria,fungi,andothermicroor-ganismsfeedingonanarrayoforganicmaterial(1).Thus,theareaofsoilsurroundingaplantrootrepresentsauniquephysical,biochemical,andecologicalinterfacebetweentherootsandtheexternalenvironment.Thisso-calledrhizosphereisinpartregulatedbytherootsystemitselfthroughchemicalsexuded/secretedintothesurroundingsoil.Rootexudatesincludelowmolecularweightsecondarymetabolitesandhighmolecularweightcompoundssuchasmucilageandproteins(2).Itisestimatedthatnearly5-21%ofphotosyntheticallyfixedcarboniseventuallytransferredtotherhizosphereintheformofrootexudates(3,4),butthemechanismsthatdriveandregulatethisprocessarenotfullyunderstood.Secondaryplantmetabolitesmaynotplayafundamentalbiochemicalroleinthenormalbuildingandmaintainingofplantcells(5),buttheycanclearlyplayanecologicalrole(6).Secondarymetabolismisimportantinmanyplantprocesses,includingroot-specificmetaboliteproductionandrootsecretion(7,8).Elicitorsaremoleculesthatstimulatedefenseorstress-inducedresponsesinplants(9).Thedetectionofnovelsecond-arymetabolitesuponrootelicitationsuggeststhatelicitationandsecondarymetaboliteprofilingmaybeharnessedasafunctionaltooltorevealsilentgenesinplantgenomes(10,11).Functionalgenomicapproacheshavebeenusedtocharacter-izethebiologicaldiversityanddevelopmentofplantsatthegenelevel.ProteomicandmRNAprofilingscomplementfunctionalgenomicsandprovideinformationabouttherelation-shipbetweengeneactivation,transcription,andsuccessfultranslation.Metabolicprofilingaimstoimproveourunderstand-ingoftheconnectionbetweengenes,proteins,andplantmetabolism(12-15).Accordingly,metabolicprofilingwillbeextremelyusefulforstudyingchangesingeneexpressionandtheensuingmetabolicandphenotypicresponseduringplantdevelopmentorinresponsetoenvironmentaland/orchemicalstress(16).Extensiveprimarymetabolicprofilingusinggaschromatographyandmassspectrometryofpotatotubersfromvarioustransgenicpotatolines(13)andArabidopsisthalianaleaves(12)hasallowedforthedetectionandquantificationofmorethan100polarcompounds(sugarsandaminoacids)withinpotatotubersand300polarcompoundsfromA.thalianaleafextracts.Theuseofdataminingtoolssuchashierarchicalclusteranalysisandprinciplecomponentanalysishasrevealedthepresenceofuniquemetabolicprofilesfordistinctgenotypes,demonstratingthevalueofmetabolicprofilingasanadditionaltoolinthecharacterizationofplantgenotypes(12,13).*Correspondingauthor[telephone[phone];fax[phone];[e-mail]].†DepartmentofHorticulture.‡DepartmentofChemistry.2548J.Agric.FoodChem.2003,51,2548-255410.1021/jf021166hCCC:$25.00©2003AmericanChemicalSocietyPublishedonWeb03/20/2003Toexploretwoneglectedareasinplantbiology,rootsecretionandsecondarymetabolitestudies,wedevelopedasystemtometabolicallyprofilesecondarymetabolitessecretedfromtherootsofA.thalianaduringits60-daylifecycle.Thismodelplantsystemhasthepotentialtoaccuratelycharacterizebiologi-callyactivesecondarymetabolitessecretedbyrootsduetodevelopmentalcuesorinresponsetopathogenattackorotherstresses(17).Weherereportthemetabolicprofilingandpartialcharacterizationofwater-saturatedhexanesolublecompoundssecretedfromtherootsofA.thaliana.MATERIALSANDMETHODSPlantMaterial.Seedsofwild-typeA.thalianaecotypeColumbia(Col-O)wereobtainedfromLehleSeeds(RoundRock,TX).PlantInitiationandGrowthConditions.SeedsofA.thalianaweresurfacesterilizedusingsodiumhypochlorite(0.3%v/v)for10-12m