distinctregionsrichineitherserine/threonine,acidicorproline/glutamineresidues.Thisarrangementissimilartothatfoundintheactivationdomainsofothertranscriptionalregulators(Johnsonetal.,1993).ThelatterhalfoftheBnNAC5-8carboxy-terminaldomain,comprisedoftheacidicandproline/glutamine-richre-gions,wassuf?cientforfullactivation.Recently,theproline-richactivationdomainofA.thalianabZIPtranscriptionfactorswasfoundtointeractwithtwoadditionalproteins,GPRI1andGPRI2.InadditiontocontainingaGARPDNA-bindingdomain,thesean-cillaryproteinsalsopossessedacidicandproline-richtranscriptionalactivationdomains(Tamaietal.,2002)suggestingthatrecruitmentoftheRNApolymerasetranscriptioncomplexbyproline-richregionsmaybemediatedbyproteininteractionsatmultiplelevels.TheNACdomainsofAtNAC1wereshowntoin-teractwithoneanother(Xieetal.,2000);however,wesawnoevidenceofhomodimerizationwithBnNAC14orBnNAC5-1.Rather,BnNAC14recruitedotherBn-NACproteinsthatwerecapableoffunctioningastran-scriptionalactivatorsdespitebeingunabletoactivatetranscriptiondirectlyitself.TheBnNACNACdomainwasfoundtobeimportantforheterodimerformationbasedontheobservationsthattheNACdomainit-selfwassuf?cientforinteraction,albeitatreducedlevelsascomparedtotheintactprotein,anddeletionoftheNAsubdomainabolishedinteraction.Inaddi-tion,threeofthe?veaminoaciddifferencesbetweentheNACdomainsofBnNAC5-8(stronginteractionwithBnNAC14)andBnNAC5-7(limitedinteraction),residewithinorincloseproximityto22theNAsubdo-main.ThesechangesconsistofGln>Arg2226andCys>Ile26intheα-helicalregionoftheNAsub-domainandGlu33>Gln33intheregionbetweentheNAandNBsubdomains.Twolinesofevidencesup-portedthenotionthatthevariablecarboxy-terminaldomainmayalsobeinvolvedinproteininteractioninadditiontotheirroleintranscriptionalactivation.First,althoughthevariabledomainsofBnNAC5-8andBnNAC485didnotinteractalonewithintactBn-NAC14,reportergeneexpressionincreasedmanyfoldwhenassociatedwiththeirrespectiveNACdomains.Second,deletionoftheBnNAC14VBsubdomainvir-tuallyeliminatedinteractionwithbothBnNAC5-8andBnNAC485.
FunctionalcharacterizationofBnNACproteinsNAC1isknowntostimulatetheexpressionofauxin-responsivegenes,suchasAIR3andDBP,and
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NAC1geneexpressionisinduceduponexposuretoauxin(Xieetal.,2000).However,auxinalsodir-ectsthedegradationoftheNAC1proteinviatheubiquitin-dependentpathwayandtheE3ubiquitinlig-ase,SINAT5,wasfoundtoubiquitinateNAC1inanauxin-dependentmanner(Xieetal.,2002).ANACexpressionwasinducedexclusivelybyabscicicacidandwasfoundtointeractwithanotherputativeE3ubiquitinligase,RH2a(Greveetal.,2003).Suchopposingmechanismsarebelievedto?nelyregu-latethehormonalresponseandconsequentlyplantdevelopment.Althoughwedidnot?ndsimilarin-teractingpartners,ectopicexpressionofBnNAC14inA.thalianagaverisetodevelopmentalabnormalitiesstrikinglysimilartothoseobservedwithNAC1,in-cludinghyper-developmentofthelateralrootsystem,largeleavesandthickerstems(Xieetal.,2000).Itisprobablethatdespitelittlesimilarityintheirrespectivevariableregionsbothplaysimilarbiologicalrolesinaccentuatingtheauxinresponse.Aswell,transgenicBnNAC14linesalsoexhibitedasigni?cantdelayinboltingandlackedadominanttaproot,suggestingthatitmayalsoin?uenceapicalmeristemformationsimilartoAtNAM(Soueretal.,1998;SablowskiandMeyerowitz,1998).
EctopicexpressionofBnNAC1-1,5-1,5-7,5-8and5-11representingtheATAF1-likeandATAF2-likegroups,resultedinphenotypesmorecloselyassoci-atedwithA.thalianacuc1andcuc2mutants(Aidaetal.,1997,1999).Thesephenotypesincludedlim-itedshootapicalmeristemformation,cup-shapedorcurledcotyledonsthatinsomecasesfailedtofullyseparate,andunderdevelopedandfusedsepalsandpetals.CUC1andCUC2arefunctionallyredundantandareexpressedinregionsboundingthemeristemandorganprimordia(Ishidaetal.,2000;Takadaetal.,2001).Theyarebelievedtocontrolembryonicshootapicalmeristemformationbyregulatingtheexpres-sionoftheshootmeristemless(STM)genesincecucmutantsdonotaccumulateSTMtranscripts.EcopticexpressionofCUC1(Aidaetal.,1999)alsoleadstoectopicexpressionofSTM(Takadaetal.,2000);how-ever,wedidnotobserveanyphenotypicabnormalitiesinahomozygousA.thalianaATAF2T-DNAinsertionline.ItremainstobedeterminedwhethertheBnNACATAF-likegenescanfunctionallycomplementeitherofthecucmutants.
WhiletheroleofcertainNACdomaingenesinplantdevelopmenthasbeenestablished,theirsigni?c-anceindefenseagainstbioticandabioticstressislessclear.TriticumGRABproteinsbindtothewheatdwarf
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geminivirusRepAproteinandpreventviralreplica-tionintissueculture(Xieetal.,1999).InteractionofA.thalianaNACdomainprotein,TIP,withtheturnipcrinkleviruscapsidproteinwasrequiredforinduc-tionofthehypersensitiveresponseandviralresistance(Renetal.,2000).WhileATAF1andATAF2expres-sionwasinducedbywounding(CollingeandBoller,2001),Alternariabrassicolainfectionandethylene(Schenketal.,2000),theyrespondeddifferentlytojasmonicandsalicylicacidtreatment(Schenketal.,2000).WehaveshownthatBnNACgenesarealsosubjecttodifferentialregulationinresponsetobothbiotic(insectherbivory,S.sclerotioruminfection)andabiotic(cold,mechanicalwounding)stresses.Itseemsplausiblethatfactorsregulatinghormonalcontrolofplantgrowthanddevelopmentwouldbealsobeaf-fectedbysuchstressesastheseprocessesarecloselytiedtoplanthealth.Infact,someplantsareabletotolerateandovercomeattackbyinsectherbivoresbyincreasinggrowthratethuscompensatingforthelossofvitaltissue(Hegedusetal.,2002).ThisrelationshipbetweenBnNACgeneexpression,developmentanddefenseresponsesiscurrentlybeingexamined.
Acknowledgements
ThisresearchwasmadepossiblewithfundsfromtheCanadianCropGenomicsInitiative.WethanktheSalkInstitute’sGenomicAnalysisLaboratoryforprovid-ingthesequence-indexedArabidopsisTDNAinser-tionmutants.FundingfortheSIGnALindexedinser-tionmutantcollectionwasprovidedbytheNationalScienceFoundation.
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