An Arabidopsis gene regulatory network for seconda

ARTICLEdoi:10.1038/nature14099AnArabidopsisgeneregulatorynetworkforsecondarycellwallsynthesisM.Taylor-Teeples1,2*,L.Lin3{*,M.deLucas1,2*,G.Turco1,2,T.W.Toal1,2,A.Gaudinier1,2,N.F.Young3,G.M.Trabucco3,M.T.Veling3,R.Lamothe3,P.P.Handakumbura3,G.Xiong4,C.Wang1,J.Corwin5,A.Tsoukalas2,6,L.Zhang7,D.Ware7,8,M.Pauly4,D.J.Kliebenstein5,K.Dehesh1,I.Tagkopoulos2,6,G.Breton9{,J.L.Pruneda-Paz9,S.E.Ahnert10,S.A.Kay9{,S.P.Hazen3&S.M.Brady1,2Theplantcellwallisanimportantfactorfordeterminingcellshape,functionandresponsetotheenvironment.Sec-ondarycellwalls,suchasthosefoundinxylem,arecomposedofcellulose,hemicellulosesandligninandaccountforthebulkofplantbiomass.Thecoordinationbetweentranscriptionalregulationofsynthesisforeachpolymeriscomplexandvitaltocellfunction.Aregulatoryhierarchyofdevelopmentalswitcheshasbeenproposed,althoughthefullcom-plementofregulatorsremainsunknown.Herewepresentaprotein–DNAnetworkbetweenArabidopsisthalianatranscriptionfactorsandsecondarycellwallmetabolicgeneswithgeneexpressionregulatedbyaseriesoffeed-forwardloops.Thismodelallowedustodevelopandvalidatenewhypothesesaboutsecondarywallgeneregulationunderabioticstress.Distinctstressesareabletoperturbtargetedgenestopotentiallypromotefunctionaladaptation.Theseinteractionswillserveasafoundationforunderstandingtheregulationofacomplex,integralplantcomponent.Plantcellshapeandfunctionareinlargepartdeterminedbythecellwall.Almostallcellshaveaprimarywallsurroundingtheplasmamem-brane.Specializedcelltypesdifferentiatebydepositingasecondarycellwalluponcessationofcellelongation.Inadditiontoprovidingmech-anicalsupportforwatertransportandabarrieragainstinvadingpath-ogens,thepolymerscontainedwithinthewallareanimportantrenewableresourceforhumansasdietaryfibre,asrawmaterialforpaperandpulpmanufacturing,andasapotentialfeedstockforbiofuelproduction.Secondarycellwallsaccountforthebulkofrenewableplantbiomassavailableglobally.Thesecondarycellwallconsistsofthreetypesofpolymer—cellulose,hemicellulosesandlignin—andisfoundinxylem,fibresandanthercells.Cellulosemicrofibrilsformamainload-bearingnetwork.Hemi-cellulosesincludexylans,glucans,andmannans.Ligninisacomplexphenylpropanoidpolymerthatimparts‘waterproofing’capacityaswellasmechanicalstrength,rigidityandenvironmentalprotection.Despitetheimportanceoftheplantsecondarycellwall,ourknowledgeofthepreciseregulatorymechanismsthatgiverisetothesemetabolitesislimited.Theexpressionofcellwall-associatedgenesistightlyspatio-temporallyco-regulated1,2.However,thepervasivefunctionalredund-ancywithintranscriptionfactorfamilies,thecombinatorialcomplexityofregulation,andactivityinasmallnumberofcelltypesrenderfunc-tionalcharacterizationfromsinglegeneexperimentsdifficult.AmodelofmasterregulatorshasbeenproposedwithNACdomainandhomeo-boxHD-ZIPClassIII(HD-ZIPIII)transcriptionfactorsinitiatingcellspecificationandsecondarycellwallsynthesisinArabidopsisthaliana.Inthismodel,VASCULAR-RELATEDNACDOMAIN6(VND6)andVND7aresufficientbutnotnecessarytoregulatexylemvesselforma-tion;additionally,theHD-ZIPIIItranscriptionfactorPHABULOSA(PHB)alsoregulatesvesselformation,andactsinahighlyredundantmannerwithfourotherHD-ZIPIIIfactors3.Inanthers,twoNACdo-maintranscriptionfactors,NACSECONDARYWALLTHICKENING1(NST1)andNST2,aresufficienttodrivethesecondarycellwallbio-syntheticprogram,butactredundantly4.Thus,regulationofthisprocessishighlyredundantandcombinatorial.However,nocomprehensivemapofinteractionshasbeendevelopedatcell-type-resolutionovertime,norhaveupstreamregulatorsbeenidentified.Wethereforechosetopursueanetwork-basedapproachtocomprehensivelycharacterizethetranscriptionalregulationofsecondarycellwallbiosynthesis.MappingthesecondarycellwallsynthesisregulatorynetworkTosystematicallymapthisregulatorynetworkatcell-type-resolution,weusedacombinationofhigh-spatial-resolutiongeneexpressiondata5andtheliterature1,6toidentifyfiftygenesimplicatedinxylemcellspec-ification.Theseincludedtranscriptionfactorsandenzymesinvolvedincellulose,hemicelluloseandligninbiosynthesisthatareexpressedinrootxylemcells(SupplementaryTable1;Methods).Selectionofbothdevelopmentalregulatorsanddownstreamfunctionalgenesallowedustointerrogateupstreamregulatoryeventsthatdeterminexylemspeci-ficationanddifferentiationassociatedwithsecondarycellwallsynthe-sis.Promotersequenceswerescreenedusinganenhancedyeastonehybrid(Y1H)assayagainst467(89%)ofroot-xylem-expressedtran-scriptionfactors7.Proteininteractionswereidentifiedfor45ofthepro-moters(SupplementaryTable2).Thefinalnetworkcomprises242genesand617protein–DNAinteractions(Fig.1a;).Thirteenofthetranscriptionfactorshavebeenpreviouslyidentifiedashavingaroleinxylemdevelopmentor*Theseauthorscontributedequallytothiswork.1DepartmentofPlantBiology,UniversityofCaliforniaDavis,OneShieldsAvenue,Davis,California95616,USA.2GenomeCenter,UniversityofCaliforniaDavis,OneShieldsAvenue,Davis,California95616,USA.3BiologyDepartment,UniversityofMassachusetts,Amherst,Massachusetts01003,USA.4DepartmentofPlantandMicrobialBiology,UniversityofCaliforniaB