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1Shorttitle:1CrPIF1–CrGATA1regulatevindolinebiosynthesis23Correspondingauthors:LingYuanandSitakantaPattanaik,DepartmentofPlantandSoil4SciencesandKentuckyTobaccoResearchandDevelopmentCenter,UniversityofKentucky,5Lexington,KY,USA.
6Email:lyuan3@uky.
eduandspatt2@uky.
edu7Phone:859-257-4806;859-257-19768Fax:859-323-1077910GATAandPIFtranscriptionfactorsregulatelight-inducedvindolinebiosynthesisin11Catharanthusroseus1213YongliangLiu1,2#,BarunavaPatra2,#,SitakantaPattanaik2,YingWang1andLingYuan1,214151KeyLaboratoryofSouthChinaAgriculturalPlantMolecularAnalysisandGenetic16Improvement,SouthChinaBotanicalGarden,ChineseAcademyofSciences,Guangzhou,PR17China182DepartmentofPlantandSoilSciencesandKentuckyTobaccoResearchandDevelopment19Center,UniversityofKentucky,Lexington,KY,USA20#Theseauthorshavecontributedequallytothiswork.
21Onesentencesummary:22AregulatorymoduleconsistingofPhytochromeInteractingFactor(PIF)andGATAtranscription23factorsregulateslight-inducedvindolinebiosynthesisinCatharanthusroseusseedlings.
24Authorcontribution:25L.
Y.
,B.
P.
,S.
P.
andY.
W.
designedtheresearch;Y.
L.
,B.
P.
,andS.
P.
performedexperiments;Y.
L.
26andB.
P.
analyzeddata;andY.
L.
,B.
P.
S.
P.
,Y.
W.
andL.
Y.
wrotethemanuscript.
2728Fundinginformation:29ThisworkissupportedpartiallybytheHaroldR.
BurtonEndowedProfessorshiptoL.
Y.
andby30theNationalScienceFoundationunderCooperativeAgreementno.
1355438toL.
Y.
3132PlantPhysiologyPreview.
PublishedonMay13,2019,asDOI:10.
1104/pp.
19.
00489Copyright2019bytheAmericanSocietyofPlantBiologistsPlantPhysiologyPreview.
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1104/pp.
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2ABSTRACT33Catharanthusroseusistheexclusivesourceofanarrayofterpenoidindolealkaloidsincluding34theanticancerdrugsvincristineandvinblastine,derivedfromthecouplingofcatharanthineand35vindoline.
Leaf-synthesizedvindolineisregulatedbylight.
Aseven-stepenzymaticprocessis36involvedinthesequentialconversionoftabersoninetovindoline;however,theregulatory37mechanismcontrollingexpressionofgenesencodingtheseenzymeshasnotbeenelucidated.
38Here,weidentifiedCrGATA1,anLLM-domainGATAtranscriptionfactorthatregulateslight-39inducedvindolinebiosynthesisinC.
roseusseedlings.
ExpressionofCrGATA1andthevindoline40pathwaygenesT16H2,T3O,T3R,D4H,andDATwassignificantlyinducedbylight.
Inaddition,41CrGATA1activatedthepromotersoffivelight-responsivevindolinepathwaygenesinplant42cells.
TwoGATC-motifsintheD4HpromoterwerecriticalforCrGATA1-mediated43transactivation.
TransientoverexpressionofCrGATA1inC.
roseusseedlingsresultedin44upregulationofvindolinepathwaygenesandincreasedvindolineaccumulation.
Conversely,45virus-inducedgenesilencing(VIGS)ofCrGATA1inyoungC.
roseusleavessignificantly46repressedkeyvindolinepathwaygenesandreducedvindolineaccumulation.
Furthermore,we47showedthataC.
roseusPhytochromeInteractingFactor,CrPIF1,isarepressorofCrGATA1and48vindolinebiosynthesis.
TransientoverexpressionorVIGSofCrPIF1inC.
roseusseedlings49alteredCrGATA1andvindolinepathwaygeneexpressioninthedark.
CrPIF1repressed50CrGATA1andDATpromoteractivitybybindingtoG/E-box/PBEelements.
Ourfindingsreveal51aregulatorymoduleinvolvingPIF–GATAthatgovernslight-mediatedbiosynthesisof52specializedmetabolites.
5354Keywords:GATAtranscriptionfactor,phytochromeinteractingfactor(PIF),vindoline55biosynthesis,transcriptionalregulation,Catharanthusroseus.
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3INTRODUCTION58Catharanthusroseus(Madagascarperiwinkle)istheuniquesourceofmorethanhundredof59terpenoidindolealkaloids(TIAs)includingthetwoanticancerdrugs,vincristineandvinblastine.
60BiosynthesisofTIAs(SupplementalFig.
S1)startswiththeproductionofstrictosidine,whichis61formedbythecondensationoftheterpenoidprecursorsecologaninandtheindoleprecursor62tryptamine(Courdavaultetal.
,2014;Panetal.
,2016;Thammetal.
,2016).
Strictosidineserves63asauniversalprecursorforvariousTIAs,includingajmalicine,serpentine,catharanthine,and64tabersonine.
Tabersonineissequentiallyconvertedtovindolinebyaseven-stepenzymatic65process,andgenesencodingthesevenenzymeshavebeencharacterized(Vazquez-Flotaetal.
,661997;St-Pierreetal.
,1998;Levacetal.
,2008;Liscombeetal.
,2010;Besseauetal.
,2013;Quet67al.
,2015).
Vincristineandvinblastinearederivedfromcouplingofvindolineandcatharanthine.
68ExpressionofTIAbiosyntheticpathwaygenesinfourdifferentcelltypesiselicitedby69environmentalcuesandphytohormones(Courdavaultetal.
,2014).
7071InC.
roseus,anumberoftranscriptionfactors(TFs)regulateTIAbiosynthesis.
TheseTFs72includetheApetala2/EthyleneResponseFactors(AP2/ERFs)ORCA2/3/4/5andCR1(Menkeet73al.
,1999;vanderFitsandMemelink,2000;Panetal.
,2012;Lietal.
,2013;Liuetal.
,2017;74Pauletal.
,2017),basichelix-loop-helix(bHLH)TFsCrMYC2,BIS1/2,andRMT1(Zhangetal.
,752011;VanMoerkerckeetal.
,2015;VanMoerkerckeetal.
,2016;Patraetal.
,2018),Cys2/His2-76typezincfingerproteinsZCT1/2/3(Pauwetal.
,2004),MYB-likefactorCrBPF1(vanderFitset77al.
,2000;Lietal.
,2015),G-boxbindingfactorsCrGBF1/2(Sibériletal.
,2001;Suietal.
,2018),78WRKYTFCrWRKY1(Suttipantaetal.
,2011),andthejasmonateZIM-domain(JAZ)proteins79(Patraetal.
,2018).
ORCA3,ORCA4andORCA5,whichformaphysicalcluster,regulatea80numberofgenesintheTIApathwaythroughoverlappingyetdistinctmechanisms(vanderFits81andMemelink,2000;Pauletal.
,2017).
CrMYC2activatesORCA3bybindingtoaqualitative82sequenceinthepromoter(Zhangetal.
,2011),whereasitindirectlyactivatesORCA4and83ORCA5(Pauletal.
,2017).
Inaddition,CrMYC2interactswithCrGBF1andCrGBF2to84modulateTIAbiosynthesis(Suietal.
,2018).
TheCrMYC2–ORCAcascadehaslimitedeffects85onexpressionofgenesintheiridoidbranchofTIApathway.
However,arecentstudyhas86demonstratedthattransientoverexpressionofade-repressedCrMYC2(CrMYC2D126N)inC.
87roseusflowerpetalssignificantlyactivatesexpressionofiridoidpathwaygenes(Schweizeretal.
,88https://plantphysiol.
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42018).
BIS1andBIS2aremajorregulatorsoftheiridoidpathway(VanMoerkerckeetal.
,2015;89VanMoerkerckeetal.
,2016).
TransientoverexpressionofCrMYC2D126N,BIS1,andORCA390significantlyinducetheindoleandiridoidpathwaygenes,resultinginincreasedaccumulationof91strictosidine,16-hydroxytabersonine,andhorhammericine(Schweizeretal.
,2018).
ThebHLH92TFRMT1andJAZproteinsmediatecrosstalkbetweeniridoidandterpenoidpathwaysto93balanceTIAaccumulation(Patraetal.
,2018).
However,ourknowledgeislimitedonregulation94ofgenesinvolvedinthesequentialconversionoftabersoninetovindolineinC.
roseusleaves.
95Combinatorialoverexpressionofwild-typeorde-repressedCrMYC2(CrMYC2D126N),alongwith96BIS1and/orORCA3doesnotinducetheexpressionofvindolinepathwaygenes(Schweizeretal.
,972018),suggestingthatotherTFsarelikelyinvolvedinregulationofthevindolinepathway.
9899InC.
roseus,whiletabersonineisproducedinleavesandroots,vindolineproductionisleaf-100specificandlightdependent(DeLucaetal.
,1986).
Inaddition,previousstudiessuggestthatthe101conversionoftabersoninetovindolineisphytochromedependent(AertsandDeLuca,1992).
102Lightregulatesamyriadofphysiologicalanddevelopmentalprocessesinplants,including103photoperiodism,photomorphogenesis,seedgerminationandshade-avoidance(Jiaoetal.
,2007).
104Lightalsoplayscrucialrolesinbiosynthesisofspecializedmetabolites.
TheTFsinbHLH,basic105leucinezipper(bZIP),andGATAfamiliesareknowntocontrollight-responsivegeneexpression106inplants(Richteretal.
,2010;Toledo-Ortizetal.
,2014;Klermundetal.
,2016).
GATATFs,107widelydistributedineukaryotes,arecharacterizedbytheclassIVzincfingermotif(CX2CX17-10820CX2C)(LowryandAtchley,2000).
InArabidopsisandrice(Oryzasativa),GATATFsare109dividedintofourconservedanddistinctclasses,AthroughD(Reyesetal.
,2004).
ClassB110GATAs(B-GATAs)arefurthersub-dividedintotwofamiliesbasedonthepresenceofconserved111LLM(Leucine-Leucine-Methionine)domainorHAN(HANABATARANU)domain(Behringer112andSchwechheimer,2015).
InArabidopsis,expressionoftwohomologousLLMB-GATAs,113GNC(GATA-nitrate-inducible-carbonmetabolism-involved)andGNL(GNC-like/cytokinin-114responsiveGATAfactor1),isinducedbylightknowntoregulatechlorophyllbiosynthesis,115chloroplastdevelopment,nitratemetabolism,seedgermination,floweringtime,hypocotyl116elongation,andstomatadevelopment(Richteretal.
,2010;Hudsonetal.
,2013;Richteretal.
,1172013a;Richteretal.
,2013b;Klermundetal.
,2016;Ranftletal.
,2016;Xuetal.
,2017;Bastakis118etal.
,2018).
InadditiontoGATATFs,asmallgroupofbHLHTFs,thePhytochromeInteracting119https://plantphysiol.
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5Factors(PIFs)playcrucialroleinlight-responsivegeneexpressionanddownstreambiological120processesthroughinteractingwithphytochromesinplants(LeivarandMonte,2014).
121Phytochromesarethereceptorofredandfar-redlightsignalsandexistineithertheinactivePr122formortheactivePfrform(Phametal.
,2018).
PIFsphysicallyinteractwiththeactivePfr,123leadingtodegradationbythe26S/ubiquitinproteasomesystem(26S/UPS).
ThePIFdegradation124triggersmassivetranscriptionalreprogrammingthatregulatesvariousbiologicalprocesses125(LeivarandMonte,2014;Paiketal.
,2017).
Light-mediatedanthocyaninaccumulationin126ArabidopsisisregulatedbyPIFs(Shinetal.
2007;Liuetal.
2015).
InArabidopsis,GNCand127GNLaredirecttargetsofPIFsintheregulationofseedgermination,floweringtime,hypocotyl128elongation,andstomatadevelopment(Richteretal.
,2010;Klermundetal.
,2016;Ranftletal.
,1292016).
Infungi,GATA-typeTFCsm1hasbeenreportedtoregulatebiosynthesisofthered130pigmentsbikaverinandfusarubinsinFusariumfujikuroi(Niehausetal.
,2017);itisunclear,131however,whetherGATATFsareinvolvedintheregulationofspecializedmetabolite132biosynthesisinplants.
133134Thelightandphytochromedependentnatureofvindolinebiosynthesisledustohypothesizethat135alight-associatedtranscriptionalcascadeisinvolvedintheconversionoftabersonineto136vindolineinC.
roseusseedlings.
Inthisstudy,wecharacterizedalight-inducedC.
roseusLLM137domainB-GATA,termedCrGATA1,whichpredominantlyexpressesintheleaf.
CrGATA1138activatesthepromotersofkeyvindolinebiosyntheticgenesinplantcells.
Inaddition,transient139overexpressionorvirus-inducedgenesilencing(VIGS)ofCrGATA1inC.
roseusseedlings140significantlyalteredvindolinepathwaygeneexpressionandvindolineaccumulation.
Wealso141demonstratedthataC.
roseusPIF(CrPIF1)actsasanupstreamnegativeregulatorofCrGATA1,142resultinginrepressionofvindolinebiosyntheticgenesinthedark.
De-repressionofCrGATA1,143presumablythroughdegradationofCrPIF1underlight,leadstoincreasedaccumulationof144vindoline.
OurfindingsrevealapreviouslyuncharacterizedregulatorymoduleinC.
roseus145involvingPIF-GATAthatgovernslight-inducedvindolinebiosynthesisinseedlings.
146147Results148VindolinepathwayisinducedbylightinC.
roseusseedlings149Conversionoftabersoninetovindolineisresponsivetodevelopmentalaswellasenvironmental150https://plantphysiol.
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6cues,suchaslight,inC.
roseusseedlings(DeLucaetal.
,1986).
Ofthesevengenesinvolvedin151theconversion,expressionofdeacetylvindoline-4-O-acetyltransferase(DAT)and152desacetoxyvindoline-4-hydroxylase(D4H)isinducedbylight(Vazquez-Flotaetal.
,1997;St-153Pierreetal.
,1998;Vazquez-FlotaandDeLuca,1998).
Todeterminethelight-responsive154expressionoftabersonine–vindolineconversiongenesandalkaloidaccumulation,seven-day-old155etiolatedC.
roseusseedlingswereexposedtocontinuouswhitelightfor1h,4h,10h,24h,48h,156and96h,andtheaerialpartsoftheseedlingswerecollectedforgeneexpressionandalkaloids157analysis.
Expressionoftabersonine-16-hydroxylase2(T16H2),tabersonine-3-oxygenase(T3O),158D4H,andDATshowedgradualincreaseuponexposuretolightthatpeakedat24h.
Expression159oftabersonine-3-reductase(T3R)washighestat48hoflighttreatmentanddeclinedthereafter160(Fig.
1A).
However,expressionof16-hydroxytabersonine-O-methyltransferase(16OMT)and3-161hydroxy-16-methoxy-2,3-dihydrotabersonineN-methyltransferase(NMT)didnotsignificantly162changeinresponsetolight(Fig.
1A),whichcorroborateswithearlierstudiesshowingthe163activitiesof16OMTandNMTwerelesssensitivetolight(St-PierreandDeLuca,1995;Levac164etal.
,2008).
WealsomeasuredtheexpressionoftwoupstreamTIApathwaygenes165(SupplementalFig.
S1),tryptophandecarbxylase(TDC)andstrictosidinesynthase(STR),in166dark-andlight-treatedseedlings.
Incontrasttotherapidlightinductionforthevindoline167pathwaygenes(within4h),expressionofTDCandSTRdidnotsignificantlychangebylight-168treatmentupto24h(SupplementalFig.
S2A).
Inaddition,wecomparedcatharanthine,169tabersonine,andvindolineinetiolatedandlight-treatedC.
roseusseedlings.
Wedidnotobserve170significantdifferencesincatharanthineaccumulationbetweendark-andlight-treatedseedlings171(SupplementalFig.
S2B).
However,wedetectedasignificantincreaseofvindolineanda172decreaseoftabersonineinseedlingsexposedtolightfor24and96h(Fig.
1B).
Wealso173comparedlevelsofcatharanthine,tabersonine,andvindolineinnormal,11-day-oldC.
roseus174seedlingsgrownundercontinuouslighttothoseofdark-grownseedlingsexposedtolight.
We175foundthatvindolinelevelsweresignificantlyhigherinlightgrownseedlings(SupplementalFig.
176S2C)comparedtotheetiolatedonesexposedtolightfor24and96h(Fig.
1B).
Takentogether,177theresultssuggestthatexpressionoffiveofthesevenvindolinepathwaygenesandvindoline178accumulationinC.
roseusseedlingsarelight-inducible.
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7Putativelight-responsivecis-elementsarepresentinthepromotersoflight-inducible182vindolinepathwaygenes183Thelight-inducibleexpressionofthefivevindolinepathwaygenespromptedustoanalyzetheir184promotersforpresenceofputativelight-responsivecis-elements.
Wethereforeretrievedthe185promotersequences(2kbupstreamofthefirstATGofcodingframe)ofthefivelight-responsive186genes,T16H2,T3O,T3R,D4H,andDAT,fromtheMedicinalPlantGenomicsResource(MPGR;187http://medicinalplantgenomics.
msu.
edu/)andscannedthepromotersequencesusingPlantCARE188(Lescotetal.
,2002).
Weidentifiedmultipleknownlightresponsivecis-elements,ofwhichthe189GATA,Box4,andBoxImotifsarepresentinthepromotersofallfivelight-responsive190vindolinepathwaygenes(SupplementalTableS1).
GATAandGATCcis-elementswerealso191foundinthepromotersoftwoupstreamvindolinepathwaygenes,TDCandSTR,althoughtheir192expressionwerenotsignificantlyinducedbylight.
TheGATA-motif,consistingofW-G-A-T-A-193Rsequence(inwhichWdenotesAorT,andRdenotesAorG),andtheGATC-motifare194bindingsitesforlight-inducibleGATATFs(LowryandAtchley,2000;Newtonetal.
,2001;195Sugimotoetal.
,2003;Manfieldetal.
,2007).
AlthoughtheBox4andBoxIelementshavebeen196identifiedtobepresentinmanypromotersoflight-induciblegenes,theTFstargetingthese197elementsareunclear.
Therefore,wefocusedonGATAfamilyTFsofC.
roseusfortheirpotential198rolesinregulatinglight-induciblevindolinebiosynthesis.
199200CrGATA1islight-inducibleandco-expressedwithvindolinepathwaygenes201Weidentified24putativeGATATFsinC.
roseusgenome.
WenextusedC.
roseus202transcriptomicresources(Góngora-Castilloetal.
,2012)(Accessionno.
SRA030483)toperform203co-expressionanalysisofGATATFsandgenesinvolvedinvindolinebiosynthesis.
Hierarchical204clusteranalysisoftheputativeGATATFsandthefivelight-induciblevindolinepathwaygenes205reveledthattwoGATATFs(CRO_T134526andCRO_T117711)aretightlyco-expressedwith206thevindolinepathwaygenes(SupplementalFig.
S3).
QuantitativeRT-PCR(RT-qPCR)was207performedtomeasuretheexpressionoftwoGATATFsandvindolinepathwaygenesinarial208partsandrootsofC.
roseusseedlings.
AsshowninFigure2A,thevindolinepathwaygenes,as209wellasCRO_T134526andCRO_T117711,arepreferentiallyexpressedinaerialpartsofthe210seedlings(Fig.
2A).
ExpressionofCRO_T134526andCRO_T117711werealsomeasuredinC.
211roseusseedlingsexposedtolightfor1h,4h,10h,24h,48h,and96h.
Underlight-treatment,only212https://plantphysiol.
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8CRO_T134526expressionwasinduced(Fig.
2B)andpeakedat4h,whileCRO_T117711was213repressed(Fig.
2C).
Next,weclonedtheCRO_T134526promotertodrivetheexpressionofthe214GUSreportergene.
Twolight-dependent(D4HandDAT)andalight-independent(CaMV35S)215promotersdrivingtheexpressionofGUSreporterwereusedascontrols.
Thepromoter-GUS216reporterplasmidswereusedforAgro-infiltrationofNicotianabenthamianaleaves.
GUS217activitiesweremeasuredindiscsofinfiltratedleavescollectedfromdarkorlightincubated218plants.
AsshowninFig.
2D,theGUSactivityinlight-treatedleaves,infiltratedwithCrGATA1-219GUS,D4H-GUSorDAT-GUS,wassignificantly(3-4-fold)higherthanthatofthedarkincubated220plants;however,wedidnotobservesignificantchangeofGUSactivityinCaMV35S-GUS-221infiltratedleavesoflightordarkincubatedplants,suggestingthattheCRO_T134526promoteris222lightinducible.
WeselectedCRO_T134526,hereafterdesignatedasCrGATA1,forfurther223characterizationandtoelucidateitsregulatoryroleinlight-regulatedvindolinebiosynthesis.
224225PhylogeneticanalysisshowedthatCrGATA1isinthesamecladewiththelight-inducible226ArabidopsisGATATFsGNCandGNL(SupplementalFig.
S4)(Manfieldetal.
,2007;227BehringerandSchwechheimer,2015).
LikeGNCandGNL,CrGATA1belongstoLLM-domain228containingB-GATAs.
AminoacidsequencealignmentrevealedthatCrGATA1shares39-41%229sequenceidentitywithArabidopsisGATATFs,GNCandGNL(SupplementalFig.
S5).
To230determinethesub-cellularlocalization,CrGATA1wasfusedin-frametoeGFP(enhancedGFP),231andthefusiongenewasexpressedintobacco(N.
tabacum)protoplasts.
WhilethecontroleGFP232accumulatedthroughoutthecell,CrGATA1-eGFPfusionproteinwaslocalizedtothenucleus233(Fig.
2E).
234235TransientoverexpressionofCrGATA1enhancesvindolineproductioninC.
roseusseedlings236TodeterminetheroleofCrGATA1invindolinebiosynthesis,wetransientlyoverexpressed237CrGATA1inC.
roseusseedlingsusingtheFASTmethod(Weaveretal.
,2014).
Expressionof238CrGATA1andthefivelight-responsivevindolinepathwaygenesweremeasuredintheaerial239partsofyoungseedlingsinfiltratedeitherwiththeempty-vectorcontrolpCAMBIA1300(EV)or240theoverexpression-vectorpCAMBIA1300-CrGATA1(CrGATA1-OX).
TheRT-qPCRresults241reveledthat,comparedtoEV,CrGATA1overexpressionresultedin2.
5-to5-foldincreaseinthe242expressionofT16H2,D4H,andDAT;expressionofT3OandT3Rremainedunchanged(Fig.
3A).
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9Inaddition,vindolineaccumulationwassignificantlyincreased,whiletabersoninewasdecreased,244inCrGATA1-OXseedlingsrelativetoEVseedlings(Fig.
3B).
Thesefindingssuggestthat245CrGATA1isapositiveregulatorofvindolinebiosynthesisinC.
roseus.
246247VIGSofCrGATA1reducesvindolinebiosynthesisinC.
roseusleaves248VIGSwasusedtorepressCrGATA1expressioninC.
roseusleavesaspreviouslydescribed249(LiscombeandO'Connor,2011).
ExpressionofCrGATA1andthefivelight-responsive250vindolinepathwaygenesweremeasuredinyoungleavesofC.
roseusplantsinoculatedeither251withthepTRV2empty-vectorcontrol(EV)ortheVIGSvectorpTRV2-CrGATA1(CrGATA1-252VIGS).
WeobservedthatCrGATA1expressionwasrepressedapproximatelyby63%inVIGS253leavescomparedtotheEVcontrol(Fig.
3C).
Inaddition,transcriptlevelsofT3O,T3R,andDAT254werereducedby43%-58%inVIGSleavescomparedtoEV(Fig.
3C).
ExpressionofT16H2255andD4HwerenotsignificantlyaffectedinCrGATA1VIGSlines.
Moreover,theamountof256tabersoninewassignificantlyelevatedwhilevindolinewasdecreasedinleavesoftheCrGATA1-257VIGSplantscomparedtotheEVplants(Fig.
3D).
TheresultsfurthersupportthatCrGATA1isa258positiveregulatorofvindolinebiosynthesisinC.
roseus.
TodeterminewhetherCrGATA1259affectsexpressionoftheupstreamvindolinepathwaygenes,wemeasuredtheexpressionofTDC260andSTRinCrGATA1overexpressionandVIGSlines.
Wedidnotdetectsignificantchangesin261transcriptlevelscomparedtoEV(SupplementalFig.
S6).
262263CrGATA1transactivatesthepromotersofvindolinepathwaygenes264TodeterminewhetherCrGATA1candirectlyactivatethepromotersofvindolinepathwaygenes,265weperformedN.
benthamianaleaf-basedtransactivationassays.
Thepromotersoffivelight-266induciblevindolinepathwaygeneswereclonedinthebinaryvectorpKYLX71-GUStodrivethe267expressionofGUSgene.
TheresultingplasmidsweremobilizedtoA.
tumefaciensand268individuallyinfiltratedintoN.
benthamianaleavestogetherwithEV(pCAMBIA1300)or269pCAMBIA1300-CrGATA1.
GUSactivityassayshowedthattransactivationoftheT16H2,T3R,270T3O,D4H,andDATpromotersbyCrGATA1were1.
8-2.
9foldscomparedtoEVcontrol(Fig.
2714A),suggestingthatthevindolinepathwaygenesareregulatedbyCrGATA1inC.
roseus.
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10GATC-motifiscrucialforCrGATA1activationoftheD4Hpromoter275PreviousstudieshavedemonstratedthatGATATFsbindtobothGATAandGATCmotifs276(Newtonetal.
,2001;Sugimotoetal.
,2003;Xuetal.
,2017).
TheD4Hpromoter,highly277activatedbyCrGATA1(Fig.
4A),wasthuschosenforidentificationofpotentialbindingsitesof278CrGATA1.
InsilicoanalysisrevealedthattheD4HpromotercontainstwoGATAandtwo279GATCmotifs(Fig.
4BandC).
First,wemutatedthecoresequenceofthetwoGATA-motifs(-280431to-426and-139to-134,relativetofirstATGincodingframe)toGGCAbyPCR-based281site-directedmutagenesis(Pattanaiketal.
,2010).
MutationofsingleorbothGATA-motifshad282noeffectonCrGATA1-mediatedactivationoftheD4Hpromoter(Fig.
4B).
However,283transactivationoftheD4HpromoterbyCrGATA1wasabolishedwhenbothGATC-motifs(-617284to-614and-541to-538,relativetoATG)weremutatedtoGAAA,(Fig.
4C),suggestingthat285GATC-motifsarecrucialforactivationoftheD4HpromoterbyCrGATA1.
286287Phytochromeislikelyinvolvedinlight-responsiveexpressionofvindolinepathwaygenes288Plantssenseredandfar-redlightsignalsthroughthephotoreceptorphytochrome(Franklinand289Quail,2010).
Previousstudieshaveshownthatthered-lightinducedenzymaticactivitiesofD4H290andDATinC.
roseusseedlingsarereversedbyfar-redlight(AertsandDeLuca,1992;291Vazquez-FlotaandDeLuca,1998),suggestingaphytochrome-dependentregulationofvindoline292biosynthesis.
Totestthishypothesis,expressionofCrGATA1andfivelight-induciblevindoline293pathwaygenesweremonitoredinC.
roseusseedlingsexposedredandthenfar-redlightfor294differentdurations.
WeusedC.
roseusChlH,PORCandRCAasexperimentalcontrols,astheir295orthologsinArabidopsisandricerespondtoredandfar-redlightinaphytochrome-dependent296manner(Liuetal.
,1996;Moonetal.
,2008;Inagakietal.
,2015)(Fig.
5).
Geneexpression297analysisshowedthatredlightsignificantlyinducedtheexpressionofCrGATA1andvindoline298pathwaygenes,exceptforD4H(Fig.
5).
Theredlight-mediatedinductionofvindolinepathway299geneswasreversedfollowingatwo-hourexposuretofar-redlight(Fig.
5),suggestingthe300involvementofphytochrome-dependentregulatoryfactorsinthevindolinepathway.
301302CrPIF1repressesvindolinepathwaygeneexpressionandalkaloidaccumulationinC.
303roseusseedlings304PIFsactasnegativeregulatorsinlightsignalingpathway.
PIFaccumulatesindarkanddegrades305https://plantphysiol.
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11uponinteractionwithphytochromesinlight(Phametal.
,2018).
WethusaskedwhetherC.
306roseusPIFsareinvolvedinthelightregulationofvindolinebiosynthesis.
Weidentifiedthree307putativePIFsintheC.
roseusgenome,designatedhereasCrPIF1,CrPIF3andCrPIF4/5.
CrPIFs308share35-41%aminoacidsequenceidentitywithArabidopsisPIF1,PIF3andPIF4,whilethey309share52-60%identitywithtomatoPIFs(SupplementalFig.
S7).
Inaddition,C.
roseusPIFsare310phylogeneticallyclosertotomatoPIFsthanthoseofArabidopsis(SupplementalFig.
S8).
Amino311acidsequencealignmentofPIFs(SupplementalFig.
S7)showedthatallthreeCrPIFscontainthe312conservedbipartitenucleuslocalizationsignal(NLS)andphytochromeBbinding(APB)motif,313indicatingtheirconservedregulatoryrolesinphytochrome-PIFpathwayinplants.
Simiartothe314tomatoPIFs(Rosadoetal.
,2016),CrPIFsappearedtobealsoregulatedattranscriptionallevel315astheirexpressionwereaffectedbylight(SupplementalFig.
S9).
Todeterminetheregulatory316rolesofCrPIFsinvindolinebiosynthesis,CrPIFswereindividuallyoverexpressedinC.
roseus317seedlingsusingtheFASTmethod.
CrPIF1overexpressionresultedinsignificantdownregulation318ofCrGATA1andthelight-responsivevindolinepathwaygenesindark(Fig.
6A).
However,319CrPIF3orCrPIF4/5hadnoeffectsontheexpressionofCrGATA1andthevindolinepathway320genes(SupplementalFig.
S10).
Inaddition,overexpressionofCrPIF1resultedinincreased321tabersonineanddecreasedvindoline(Fig.
6B),indicatingthatCrPIF1actsanegativeregulatorof322vindolinebiosynthesisinC.
roseus.
Todeterminethesub-cellularlocalization,CrPIF1was323fusedin-frametoeGFP(enhancedGFP),andtheresultingCrPIF1-eGPFwasexpressedin324tobaccoprotoplasts.
WhilethecontroleGFPaccumulatedthroughoutthecell,theCrPIF1-eGFP325fusionproteinwaslocalizedtothenucleus(Fig.
6E).
326327VIGSofCrPIF1increasesvindolinebiosynthesisinC.
roseusleaves328VIGSwasusedtorepressCrPIF1expressioninyoungC.
roseusleaves.
ExpressionofCrPIF1,329CrGATA1,andthefivelight-responsivevindolinepathwaygenes(T16H2,T3O,T3R,D4H,and330DAT)weremeasuredinleavesofthedark-incubatedVIGSplants.
Plantsinoculatedwiththe331pTRV2empty-vector(EV)servedascontrol.
CrPIF1expressionwasrepressedbyapproximately33270%inVIGSleavescomparedtotheEVcontrol(Fig.
6C).
CrPIF1-silencingresultedin333upregulationofCrGATA1andfivevindolinepathwaygenesby2-8foldsinVIGSlines334comparedtoEV(Fig.
6C).
Moreover,theamountofvindolinewaselevatedinleavesofthe335CrPIF1-VIGSplantscomparedtotheEVplants(Fig.
6D).
Theseresultsfurthersuggestthat336https://plantphysiol.
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12CrPIF1isanegativeregulatorofvindolinebiosynthesisinC.
roseusseedlings.
337CrPIF1repressesCrGATA1andDATthroughbindingtotheG/E/PBE-box338TodeterminewhetherCrGATA1andthevindolinepathwaygenesareregulatedbyCrPIF1,we339performedtransactivationassaysinN.
benthamianaleaves.
TheCrGATA1promoterwascloned340intotheplasmidpKYLX71-GUStodrivetheexpressionofGUSgene.
TheGUSreporter341plasmidsdrivenbythepromotersofCrGATA1andfivevindolinepathwaygeneswere342individuallyinfiltratedintoN.
benthamianaleavestogetherwitheitherEV(pCAMBIA1300)or343pCAMBIA1300-CrPIF1.
WeobservedthatpromoteractivitiesofCrGATA1,T16H2,andDAT344weresignificantlyrepressed(40-50%)byCrPIF1indark(Fig.
6F),suggestingthatCrPIF1isa345repressorofthevindolinepathway.
ItiswelldocumentedthatPIFsbindtoG-box(CACGTG)or346PBE-box(CACATG/CATGTG)inthetargetpromoters(Zhangetal.
,2013).
WeidentifiedaG-347box(-142to-137relativetofirstATG)intheCrGATA1promoter(Fig.
6G)andaPBE-box(-3481106to-1101relativetofirstATG)inDATpromoter(Fig.
6H).
TheG-boxintheCrGATA1349promoterandPBE-box(CACATG)intheDATpromoterweremutatedtoCAAAAGand350CACAAA,respectively.
TransactivationassaysusingN.
benthamianaleaveswereperformedto351evaluatetheeffectsofCrPIF1onthemutatedCrGATA1andDATpromoters.
Wefoundthatthe352repressiveeffectofCrPIF1ontheCrGATA1andDATpromoterswasabolishedbymutationsin353G-boxandPBE-box,respectively(Fig.
6Gand6H).
ThesefindingssuggestthatCrPIF1354repressestheactivitiesofCrGATA1andDATlikelybybindingtotheG-boxorPBE-boxmotifs355inthepromoters.
356357DISCUSSION358TIAbiosynthesisinC.
roseusisahighlycomplexandelaboratedprocessthatinvolvesmorethan35930differentenzymes,multiplecelltypesandsub-cellularcompartments.
AnumberofTFs360regulatingbiosynthesisofTIAs,suchasstrictosidinethatserveasprecursoroftabersonineand361catharanthine,havebeencharacterized(Menkeetal.
,1999;vanderFitsandMemelink,2000;362Suttipantaetal.
2011;Zhangetal.
,2011;Panetal.
,2012;Lietal.
,2013;VanMoerkerckeetal.
,3632015;VanMoerkerckeetal.
,2016;Pauletal.
,2017;Patraetal.
,2018;Schweizeretal.
,2018;364Suietal.
2018).
Bycomparison,ourknowledgeontranscriptionalregulationofthevindoline365pathwayandlight-mediatedTIAbiosynthesisisextremelylimited.
Here,weidentifiedand366characterizedCrGATA1andCrPIF1fortheirrolesinregulatingvindolinebiosynthesisinC.
367https://plantphysiol.
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13roseusseedlings.
368369TIAbiosynthesisisdevelopmentallyregulatedandisinfluencedbyenvironmentalcues,suchas370light.
Tabersonineispresentinbothleavesandroots,whereasvindolineispredominantlyfound371inleaves(DeLucaetal.
,1986).
Moreover,dark-grown,etiolatedC.
roseusseedlingsaccumulate372traceamountofvindolinewhichincreasesuponexposuretolight.
Correspondingly,thedark-373grownseedlingsaccumulateasignificantamountoftabersoninewhichdeclinesuponexposure374tolight(DeLucaetal.
1986).
Inaddition,lightinducestheexpressionofD4HandDATandtheir375enzymaticactivitiesinC.
roseusseedlings(DeLucaetal.
,1986;DeCarolisetal.
,1990;376Vazquez-FlotaandDeLuca,1998;St-Pierreetal.
,1998).
Ourgeneexpressionanalysisrevealed377that,inadditiontoD4HandDAT,expressionofT16H2,T3O,andT3Raresignificantlyinduced378bylightinC.
roseusseedlings(Fig.
1A).
Inaddition,theincreaseofvindolineaccumulationis379accompaniedbythedecreaseoftabersonineuponexposuretolight(Fig.
1B),furtherconfirming380theroleoflightinvindolinebiosynthesisinC.
roseusseedlings.
Lightisalsoknowntoaffectthe381biosynthesisofotherspecializedmetabolites,includingSGAintomato(Wangetal.
,2018)and382anthocyaninsinanumberofplantspecies(Liuetal.
2018).
Consistentwithleaf-specific383vindolineaccumulation,CrGATA1andthefivelight-responsivevindolinepathwaygenesare384preferentiallyexpressedinarealpartsoftheseedling(Fig.
2A).
Theleaf-specificexpressionof385CrGATA1andthevindolinepathwaygenesrelativetothoseinroots(Fig.
2A)aresignificantly386higherthanlight-inducedexpressionintheseedling(Fig.
1AandFig.
2B),becausethebasal387expressionofthesegenesinrootsareextremelylow.
388Cis-regulatoryelementspresentinthegenepromotersofmetabolicpathwaysoftenprovideclues389aboutthepotentialTFsinvolvedintheregulatorynetworkandhavebeenusedasatoolfor390identificationofregulators.
Forinstance,CrMYC2wasinitiallyisolatedusingtheG-boxelement391presentinC.
roseusSTRpromoterandlaterdemonstratedasaregulatorofTIAbiosynthesis392(Zhangetal.
2011;Schweizeretal.
,2018;Suietal.
2018).
ThepresenceofAT-richmotifsinthe393jasmonate-responsiveelement(JRE)oftheORCA3promoterledtotheidentificationoftheAT-394hookregulatorsinC.
roseus(VomEndtetal.
,2007).
Inaddition,presenceofputativeMYB395bindingsitesinabetalainpathwaygenepromoter(PolturakandAharoni,2018)andG-box/G-396box-likesequencesintomatoSGAbiosyntheticpathwaygenes(Cárdenasetal.
,2016)indicate397thepossibleinvolvementofMYBsandMYC2,respectively,inthepathwayregulations.
Two398https://plantphysiol.
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14R2R3MYBshavesincebeenidentifiedasregulatorsofbetalainbiosynthesisinbeet(Polturak399andAharoni,2018).
Ouranalysisofthevindolinepathwaygenepromotersrevealedthepresence400ofanumberoflight-responsiveelements,includingtheGATA/GATCandG/E-boxmotifs401(SupplementaryTableS1).
ThepresenceofGATA/GATCmotifsinthefivelight-responsive402promotersinvindolinebiosyntheticpathwayledustospeculatethatGATAfamilyTFsare403involvedinthelightregulationofvindolinebiosynthesis.
Inaddition,wenoticedthepresenceof404G/E-boxthatarerecognizedbybHLHTFs,includingPIFs,thatareknowntobeinvolvedin405light-responsivegeneexpressioninplants(Yadavetal.
,2005;Phametal.
,2018).
406407Transcriptomicandgenomicresourcesareinvaluableforidentificationofmissinggenes408encodingkeyenzymes,transporters,andregulatoryproteinsinC.
roseus(Geu-Floresetal.
,2012;409VanMoerkerckeetal.
,2015;Larsenetal.
,2017;Pauletal.
2017;Payneetal.
,2017;Caputietal.
4102018;Patraetal.
2018;Quetal.
2019).
Co-expressionanalysisofvindolinebiosynthetic411pathwaygenesandGATATFsidentifiedtwoputativecandidatesforfurthercharacterization412(SupplementalFig.
S3).
Similartothevindolinepathwaygenes(Vazquez-Flotaetal.
,1997;St-413Pierreetal.
,1998;Besseauetal.
,2013),CrGATA1ispreferentiallyexpressedinleaves(Fig.
2A)414andsignificantlyinducedbylight(Fig.
2B).
TransientoverexpressionandVIGSofCrGATA1415significantlyalteredtheexpressionofmostofthevindolinepathwaygenesandvindoline416accumulationinC.
roseus(Fig.
3).
ExpressionofT3OandT3RwerenotincreasedinCrGATA1417overexpressionwhileexpressionofT16H2andD4HdidnotsignificantlychangeinVIGSlines.
418Theseobservationssuggestthatotherregulatorsarealsoinvolvedinthegeneregulation.
Similar419observationshavebeenmadeforotherregulatorsintheTIApathway.
Forinstance,aprevious420studyhasshownthatCatharanthusMYC2(CrMYC2)regulatestheAP2/ERF,ORCA3,by421bindingtotheT/G-boxmotifinthepromoter.
However,CrMYC2overexpressiondoesnot422significantlyaffectORCA3transcripts,whereassilencingCrMYC2inCatharanthuscellsreduces423ORCA3expression(Zhangetal.
,2011).
Similarly,ORCA3isknowntoregulatetheexpression424ofTDC.
However,overexpressionofORCA3inC.
roseushairyrootsdoesnotsignificantly425induceTDCexpression(Peeblesetal.
,2009).
ExpressionofTDCandSTRwerenotaffectedby426overexpressionorVIGSofCrGATA1,indicatingthatCrGATA1doesnotregulatetheupstream427TIApathwaygenesinC.
roseusseedlings.
428TransactivationassayinN.
benthamianaleavesshowedthatCrGATA1activatesthepromoters429https://plantphysiol.
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15ofkeyvindolinepathwaygenes(Fig.
4),suggestingthatCrGATA1isanactivatorinvindoline430biosynthesis.
GATATFsareinvolvedinanumberofdevelopmentalandphysiologicalprocesses.
431However,littleisknownabouttherolesofGATATFsinlightregulationofspecialized432metabolism.
Twowell-characterizedArabidopsisGATATFs,GNCandGNL,arelight-inducible433andinvolvedinchloroplastbiogenesisandnitrogenmetabolism(Richteretal.
,2010;Hudsonet434al.
,2011).
GNCandGNLbindtheGATAmotifintheGLU1promoter(Hudsonetal.
,2011),435whereasthetobaccoGATATF,AGP1activatesNtMYC2bybindingtotheGATCmotifinthe436NtMYC2promoter(Sugimotoetal.
,2003).
Moreover,genome-widebindinganalysisofGNC437andGNLrevealsthatbothGATAandGATCcis-elementsareenrichedinthetargets(Xuetal.
,4382017).
WeidentifiedmultipleGATAandGATCcis-elementsinthepromotersofDAT,D4H,T3R,439T3O,andT16H2.
MutationtotheGATCelement,butnottheGATAelement(Fig.
4B),inthe440D4Hpromoterhadasignificanteffectonthetransactivation(Fig.
4C),suggestingthatCrGATA1441likelyrecognizestheGATCelementinactivationofD4H.
442443ActivitiesofD4HandDATincreasesignificantlyuponexposureofC.
roseusseedlingstored444light,andtheeffectisreversedbyfar-redlight(AertsandDeLuca,1992;Vazquez-FlotaandDe445Luca,1998).
Inaddition,transcriptlevelsofD4Hincreasefollowingexposuretoredlight446(Vazquez-FlotaandDeLuca,1998).
Phytochromesserveasreceptorsofredandfar-redlightin447plantsandexistintwodifferentforms,theinactivePrandactivePfr.
Intheabsenceofredlight,448theinactivePraccumulatesincytosol;however,uponperceptionofredlight,Prconvertstothe449activePfrthatissubsequentlytranslocatedtothenucleus(FranklinandQuail,2010).
Herewe450demonstratedthatexpressionofCrGATA1,T16H2,T3O,T3R,andDATinC.
roseusseedling451increaseuponexposuretoredlightanddecreasefollowingexposuretofar-redlight(Fig.
5),452suggestingaphytochrome-dependentregulationofvindolinebiosynthesis.
Wedidnotobservean453apparentincreaseinD4Hexpressionuponexposuretoredlight.
Thisismostlikelyduetothe454durationoftreatmentthataffectsitsexpressioninseedlings.
PIFsareknowntointeractwith455phytochromesandregulatelight-responsivegeneexpressioninplants.
PIFsregulatebiosynthesis456ofspecializedmetabolites,includinganthocyanins(Shinetal.
,2007;Liuetal.
,2015)andSGAs457(Wangetal.
,2018).
ForanthocyaninsbiosynthesisinArabidopsis,PIF3andHY5actas458activators(Shinetal.
,2007),whereasPIF4andPIF5functionasrepressorsasoverexpressionof459PIF4orPIF5reducesanthocyaninaccumulation(Liuetal.
,2015).
Weidentifiedthreeputative460https://plantphysiol.
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16PIFsinC.
roseusgenome.
TransientoverexpressionorVIGSofCrPIF1alteredvindoline461pathwaygeneexpressionandvindolineaccumulationinC.
roseusseedlings(Fig.
6A-D).
462PreviousstudiesdemonstratethatPIFsbindtotheG/E-box/PBEelementsinthetargetpromoters.
463ScanningofthepromotersofCrGATA1andvindolinepathwaygenesrevealedthepresenceof464putativePIFbindingsites(Fig.
6GandH).
TransienttransactivationassaysshowedthatCrPIF1465repressedthepromoteractivitiesofCrGATA1andvindolinepathwaygenes(Fig.
6GandH).
466Site-directedmutagenesisofthePIFbindingsitesintheCrGATA1andDATpromotersabolished467PIF-mediatedrepression,suggestingthatCrPIF1directlyregulatesCrGATA1andDATby468bindingtotheG/E-box/PBEelementsinthepromoters.
TheT16H2promoterdoesnotcontain469canonicalPBE(CACATG)motifs,butcontainsthreeE-boxmotifs(CAAATG,CAATTGand470CAGCTG)thatarehighlysimilartothePBE.
CrPIF1likelysuppressesT16H2expressionby471bindingtotheE-box(Fig.
6A).
AsTIAaccumulationinC.
roseusisdevelopmentallyregulated,472thedifferenceinthealkaloidcontentsobservedinthisstudybetweenoverexpression(Fig.
3B,4736B)andVIGS(Fig.
3D,6D)linesofCrGATA1orCrPIF1islikelyduetotheageofthe474seedlingsusedintheexperiments.
475476Inconclusion,ourworkelucidatesamechanismbywhichlightinfluencesvindolinebiosynthesis477throughthenewlycharacterizedCrGATA1andCrPIF1.
CrPIF1repressestheexpressionof478CrGATA1andvindolinepathwaygenesinthedark,resultinginreducedvindolineaccumulation.
479CrPIF1ispossiblydegradedinlightbythe26S/UPS,leadingtode-repressionofCrGATA1.
The480activationofCrGATA1leadstoupregulationofthevindolinepathwaygenesandincreased481vindolineaccumulationinC.
roseusseedlings(Fig.
7).
Otherregulatorsarelikelyalsoinvolved482invindolinebiosynthesis.
ItremainsunclearwhattransactivatorregulatesCrGATA1,which,483despiterepressionbyCrPIF1,expressesatlow(basal)levelinthedark.
Consequently,trace484amountsofvindolinecanbedetectedindark-grownC.
roseusseedlings.
Nevertheless,our485findingsrevealedapreviouslyuncharacterizedmolecularmechanismthatcontrolslight-486mediatedTIAbiosynthesisinC.
roseus.
TherolesofPIF-mediatedGATATFregulationis487perhapsgenerallyconservedinplantlight-regulatedbiosynthesisofspecializedmetabolites.
488489MATERIALSandMETHODS490491https://plantphysiol.
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17Plantmaterials,growthconditionandtreatments492Catharanthusroseus(L.
)G.
Donvar.
'LittleBrightEye'seedsobtainedfromNESeed493(neseed.
com)weresurface-sterilizedusing30%(v/v)commercialbleachfor6minutes,rinsed494fivetimeswithsterilewaterandincubatedindarkat28°Cforgerminationonhalf-strengthMS495medium.
Seven-day-oldetiolatedseedlingsweretreatedwithwhitelight(40μmolm-2s-1),red496light(31μmolm-2s-1)orfar-redlight(52μmolm-2s-1).
Forwhitelighttreatment,seedlingswere497incubatedfor1,4,10,24,48or96hours.
Forredandred/far-redlighttreatments,theseedlings498wereexposedtoredlightfor2and4hr,andthenexposedtofar-redlightfortwohours.
Aerial499partsofseedlingswerecollectedforRNAisolationoralkaloidextraction.
500501RNAisolation,cDNAsynthesisandquantitativeRT-PCR502RNAisolation,cDNAsynthesisandquantitativereverse-transcriptionPCR(RT-qPCR)were503performedaspreviouslydescribed(Pauletal.
,2017).
PrimersusedinRT-qPCRarelistedin504TableS2.
505506InSilicoanalysisofputativeregulatorsofvindolineinC.
roseus507ToidentifyC.
roseusGATATFs,sequencesofallprotein-codinggenesweredownloadedfrom508thelatestversionoftheC.
roseusgenomefromDryadDigitalRepository(Kellneretal.
,2015).
509BLASTsearchwaspreformedtoidentifytheputativeGATAandPIFTFs.
Tofurthervalidate510theBLASTsearchresults,phylogenetictreeswereconstructedandvisualizedusingNeighbor-511Joining(N-J)methodthroughMEGA5.
1software(Tamuraetal.
,2011).
Thestatisticalreliability512ofindividualnodesofthenewlyconstructedtreeswasassessedbybootstrapanalyseswith1,000513replications(Altschuletal.
,1997).
AlignmentsofaminoacidswereconductedbyMAFFT514method(KatohandStandley,2013).
515ToanalyzetheexpressionpatternsofC.
roseusGATAsandvindolinepathwaygenesinfive516differenttissues(seedling,matureleaf,immatureleaf,stemandroot)(Góngora-Castilloetal.
,5172012),hierarchicalclusteringwasperformedaspreviouslydescribed(Pauletal.
,2017).
518519520Sub-cellularlocalization521Todeterminethesub-cellularlocalizationofCrGATA1orCrPIF1,full-lengthcDNAwas522https://plantphysiol.
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18translationallyfusedwithN-terminusofenhancedGFP(eGFP)inapBlueScript(pBS)plasmid523asdescribedearlier(Suttipantaetal.
,2011).
ExpressionofeGFPwasdrivenbytheCaMV35S524promoterandtherbcSterminator.
Plasmidswereelectroporatedintotobacco(N.
tabacum)525protoplastsandvisualizedwithafluorescencemicroscope(NikonEclipseTE200,NikonCorp.
)526after20hofincubationindarkatroomtemperature.
ApBSplasmidexpressingonlyeGFP527servedasanegativecontrol.
528529PlasmidconstructionandtransientgeneoverexpressioninC.
roseusseedling530FortransientoverexpressioninC.
roseusseedlings,CrGATA1,CrPIF1,CrPIF3orCrPIF4/5531wereclonedintoamodifiedpCAMBIA1300vectorcontainingCaMV35SpromoterandrbcS532terminator.
C.
roseusseedlingsweretransientlytransformedwiththeplasmidsusingtheFast533Agro-mediatedSeedlingTransformation(FAST)methodwithsomemodifications(Weaveretal.
5342014).
Briefly,AgrobacteriumtumefaciensGV3101harboringtheplasmidwasgrownonLuria-535Bertani(LB)platescontaining100gml1kanamycin,50gml1gentamicinand30gml1536rifampicin.
Asinglecolonywastransferredto2mlliquidLBmediumcontainingsame537antibioticsandincubatedat250rpmand28°C.
OvernightgrownAgrobacteriumcellsweresub-538culturedin20mlliquidLBmediumfor16hat250rpmand28°C.
Agrobacteriumcultureswere539thencentrifugedandthepelletwasresuspendedininfiltrationbuffer(10mMMgCl2,10mM540MES,100Macetosyringone)atanOD600of1.
0,followedbyincubationat28°Cforatleast3h.
541Seven-day-oldC.
roseusseedlingswereimmersedintheAgrobacteriumculturesfor1h.
After542infiltration,seedlingswerewashedwithsteriledistilledwaterforfivetimesandlaidonpetri543disheswithautoclavedwetfilterpapers.
Afterthreedays,aerialpartsofseedlingswerecollected544forRNAextractionandalkaloidanalyses.
545546Virus-inducedgenesilencinginC.
roseusplants547Forvirus-inducedgenesilencing(VIGS)(Liuetal.
,2002),theplasmidspTRV2-CrChlH,548pTRV2-CrGATA1andpTRV2-CrPIF1weregeneratedbycloningpartialcodingsequencesof549CrChlH(400bp)orCrGATA1(358bp)orCrPIF1(183bp)inthemultiplecloningsites(MCS)550ofpTRV2vector.
C.
roseusseedlingswithtwopairsoftrueleaveswereusedtoperformthe551VIGSassayusingpinchmethodaspreviouslydescribed(LiscombeandO'Connor,2011).
552Harvesttimewasguidedbytheappearanceofphotobleachingofthecorrespondingleavesin553https://plantphysiol.
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19whichCrChlHwassilenced.
Asacontrol,plantswereinfectedwithanemptypTRV2vector554(EV).
Thenewlyemergingpairofleavesfollowinginoculationwereharvested,frozeninliquid555nitrogen,andthenstoredin-80°CuntilRNAandalkaloidextraction.
556557PlasmidconstructionandNicotianabenthamianaleafinfiltrationassays558ThereporterplasmidsforN.
benthamianaleafinfiltrationassaysweregeneratedbyreplacingthe559CaMV35SpromoterinamodifiedpKYLX71vectorcontainingtheGUSreporterandrbcS560terminator(Schardletal.
,1987)withT16H2(1129bp),T3O(703bp),T3R(1129bp),D4H(704561bp),DAT(1317bp)orCrGATA1(1189bp)promoter.
Mutantsofthefollowingcis-elements,562GATA-motifs(-430to-427,and-138to-135)andGATC-motifsinD4Hpromoter(-617to-614,563and-541to-538),G-boxinCrGATA1promoter(-142to-137)andPBE-boxinDATpromoter(-5641106to-1101)weregeneratedbysite-directedmutagenesis(Pattanaiketal.
,2010).
565pCAMBIA1300vectorscontainingCrGATA1orCrPIF1wereusedastheeffectorplasmids.
A566fireflyluciferase(LUC)reporter,drivenbyCaMV35SpromoterandrbcSterminator,wasused567asaninternalcontrolintheleafinfiltrationassays.
Infiltrationsolutionswerepreparedas568describedinFASTmethod.
Beforeinfiltration,effector,reporterandinternalcontrolsolutions569werecombinedat1:1:1ratiosandmixedwell.
InfiltrationofN.
benthamianaleaveswas570performedaspreviouslydescribed(KumarandBhatia,2016).
Twodaysafterinfiltration,leaf571discswerecollected,frozeninliquidnitrogenandgroundtopowderforproteinextraction.
LUC572andGUSactivitiesweremeasuredaspreviouslydescribed(Pattanaiketal.
,2010).
573574Alkaloidextractionandanalysis575Toextractalkaloids,light-treatedseedlings,Agro-infiltratedseedlingsorleavescollectedfor576VIGSassaywerefrozeninliquidnitrogenandgroundtopowder.
Sampleswereextractedtwice577inmethanol(1:100w/v)for24honashaker.
Pooledextractsweredriedusingarotary578evaporatoranddilutedinmethanol.
Sampleswereanalyzedwithhigh-performanceliquid579chromatography(HPLC),followedbyelectrospray-injectionintandemmassspectrometry,as580describedpreviously(Suttipantaetal.
,2011).
Theconcentrationsofthealkaloidswere581calculatedusingstandardcurve.
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20CrGATA1(MK801106),CrPIF1(ALI87040.
1),CrPIF3(ALI87041.
1)andCrPIF4/5585(ALI87042.
1)586587SUPPLEMENTALDATA588SupplementalFigureS1.
SimplifiedTIAbiosyntheticpathwayinC.
roseus.
589SupplementalFigureS2.
GeneexpressionanalysisandmeasurementofTIAsinC.
roseus590seedlings.
591SupplementalFigureS3.
Co-expressionofCrGATAgeneswithfivelight-induciblevindoline592pathwaygenesindifferenttissuesofC.
roseus.
593SupplementalFigureS4.
PhylogeneticanalysisofAtGATAsandCrGATAs.
594SupplementalFigureS5.
MultiplesequencealignmentofCrGATA1,andArabidopsisGNCand595GNL.
596SupplementalFigureS6.
TDCandSTRexpressionarenotalteredinCrGATA1overexpression597andVIGSlines.
598SupplementalFigureS7.
AminoacidsequencealignmentofPIFsfromC.
roseus,Arabidopsis,599andtomato.
600SupplementalFigureS8.
PhylogeneticanalysisofPIFsfromC.
roseus,Arabidopsisandtomato.
601SupplementalFigureS9.
ExpressionofCrPIFsinresponsetolightanddark.
602SupplementalFigureS10.
CrGATA1andvindolinepathwaygenesarenotalteredbyCrPIF3and603CrPIF4/5overexpressioninC.
roseusseedlings.
604SupplementalTableS1.
Lightresponsivecis-elementswithinpromotersoffivelight-induced605vindoline.
606SupplementalTablesS2.
Primersusedinthisstudy.
607608609ACKNOWLEDGEMENTS610WethankDr.
BruceDownieofDepartmentofHorticulture,UniversityofKentuckyfor611assistanceandadviceonred/far-redlighttreatmentofC.
roseusseedlings,Mr.
J.
MayandMs.
M.
612Combs(DepartmentofCivilEngineeringandEnvironmentalResearchTrainingLaboratories,613UniversityofKentucky)forassistanceonLC-MS/MS.
Thisworkissupportedpartiallybythe614HaroldR.
BurtonEndowedProfessorshiptoL.
Y.
andbytheNationalScienceFoundationunder615CooperativeAgreementno.
1355438toL.
Y.
616617Figurelegends618Figure1.
Light-inducedexpressionofvindolinepathwaygenesandvindolineproductionin619C.
roseusseedlings.
(A)Relativeexpressionlevelsofsevenvindolinepathwaygenesinaerial620partsofC.
roseusseedlings.
Seven-day-old,dark-grown,etiolatedseedlingswereexposedto621whitelightfordifferentlengthsoftime(1h,4h,10h,24h,48h,96h).
Transcriptlevelsof622https://plantphysiol.
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21T16H2,16OMT,T3O,T3R,NMT,D4HandDATweremeasuredbyRT-qPCR.
TheRPS9gene623wasusedasaninternalreferencegene.
(B)Accumulationoftabersonineandvindolineinaerial624partsofC.
roseusseedlings.
Here,"0hour"referstoseven-day-oldetiolatedseedlings,which625weregrowingindark(control)andthenexposedtowhitelightfor24hand96h.
Alkaloidswere626extractedandanalyzedbyLC-MS/MS,andtheconcentrationsofthealkaloidswereestimated627basedonpeakareascomparedtostandards.
Valuesrepresentmeans±standarddeviationfrom628threebiologicalreplicates.
Foreachreplicate,8-10seedlingswerecombined.
Statistical629significancewasdeterminedbytheStudent'sttest(*P<0.
05;**P<0.
01).
630631Figure2.
IdentificationofCrGATA1.
(A)ExpressionofCRO_134526(CrGATA1),632CRO_117711andfivelightinduciblevindolinepathwaygenesinaerialpartsandrootsofseven-633day-oldlight-grownC.
roseusseedlings.
(B)ExpressionofCRO_134526(CrGATA1)isquickly634inducedwhile(C)CRO_117711isrepressedbylightinarealpartsofC.
roseusseedlings.
For635lighttreatment,seven-day-oldetiolatedseedlingswereexposedtowhitelightfordifferent636lengthsiftime.
Transcriptlevelsofvindolinepathwaygenes,CrGATA1andCRO_117711were637measuredbyRT-qPCRwithRPS9asinternalreferencegene.
(D)D4H,DATandCrGATA1638promotersarelightinducibleinN.
benthamianaleaf.
CaMV35Spromoterisusedasnegative639control.
D4H,DATandCrGATA1promoterswereclonedinpKYLX71vector,respectively,to640drivetheexpressionofGUSgene.
Thepromoter-GUSplasmidswereinfiltratedinN.
641benthamianaleaves.
Plantswerekeptindarkorlightforthreedays.
GUSactivitieswere642normalizedbyluciferaseactivities.
Valuesrepresentmeans±SDfromthreebiologicalreplicates.
643StatisticalsignificancewasdeterminedbytheStudent'sttest(**P<0.
01).
(E)eGFPis644accumulatedthroughoutthecell(left)whereaseGFP-CrGATA1islocalizedtothenucleus645(right).
Theexperimentwasrepeatedtwotimesandarepresentativeresultisshownhere.
646647Figure3.
CrGATA1positivelyregulatesvindolinebiosynthesisinC.
roseus.
Transient648overexpressionofCrGATA1inC.
roseusseedlingselevatesexpressionofvindolinepathway649genes(A)andvindolineproduction(B).
(A)RelativeexpressionofCrGATA1andfivelight-650induciblevindolinepathwaygenesinemptyvector(EV)controlsandCrGATA1overexpression651(OX)seedlingsmeasuredbyRT-qPCR.
(B)MeasurementoftabersonineandvindolineinEV652controlsandCrGATA1OXlines.
(C)ExpressionofvindolinepathwaygenesinCrGATA1VIGS653https://plantphysiol.
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22leaves.
RelativeexpressionofCrGATA1andfivelight-induciblevindolinepathwaygenesin654emptyvector(EV)controlsandCrGATA1VIGSleavesweremeasuredbyRT-qPCR.
(D)655MeasurementoftabersonineandvindolineinEVcontrolsandCrGATA1VIGSlines.
Alkaloids656wereextractedandanalyzedbyLC-MS/MS,andtheconcentrationsofthealkaloidswere657estimatedbasedonpeakareascomparedtostandards.
ForRT-qPCR,TheRPS9genewasused658asaninternalreferencegene.
Inallcases,valuesrepresentmeans±SDfromthreebiological659replicates.
StatisticalsignificancewascalculatedusingtheStudent'st-test(*P<0.
05;**P<6600.
01).
661662Figure4.
CrGATA1transactivatespromotersoffivelight-induciblevindolinepathway663genesthroughGATC-motifs.
(A)TransactivationofT16H2,T3O,T3R,D4HandDAT664promoters(-pro),fusedtotheGUSreporter,byCrGATA1.
Effector(CrGATA1)andreporter665(promoter-GUS)constructswereco-infiltratedintoN.
benthamianaleaves.
Aplasmidcontaining666luciferasereporter,drivenbyCaMV35SpromoterandrbcSterminator,wasusedasa667normalizationcontrol.
LuciferaseandGUSactivitiesweremeasuredtwodaysafterinfiltration.
668GUSactivitywasnormalizedagainstluciferaseactivity.
Controlrepresentsthereporterwith669emptyvector(EV).
(B)SchematicdiagramshowingtheGATA-motifsintheD4Hpromoter.
670PointmutationsintheGATA-motifsareindicatedbyredletters.
MutationintheGATA-motif671hasnoeffectsontheactivationoftheD4HpromoterbyCrGATA1.
(C)Schematicdiagram672showingtheGATC-motifsintheD4Hpromoter.
MutationsintheGATC-motifsareindicatedby673redletters.
MutationintheGATC-motifaffectstheactivationoftheD4Hpromoterby674CrGATA1.
Datapresentedherearethemeans±SDofthreebiologicalreplicates.
Statistical675significancewascalculatedusingtheStudent'st-test(*P<0.
05;**P<0.
01).
676Figure5.
ExpressionofCrGATA1andfivelight-induciblevindolinepathwaygenesinC.
677roseusseedlingsexposedtoredandfar-redlight.
Seven-day-oldetiolatedC.
roseusseedlings678weretreatedwithredlightfor4h(measuredat2hand4h),followedbyfar-redlightfor2h.
679GeneexpressioninaerialpartsoftheseedlingswasmeasuredbyRT-qPCR.
TheCrChlH,680CrPORCandCrRCAwereusedaspositivecontrols.
TheRPS9genewasusedasaninternal681referencegene.
Datarepresentmeans±standarddeviationofthreebiologicalsamples.
Different682lettersdenotestatisticaldifferencesasassessedbyone-wayANOVAandTukeyHSD(P<0.
05).
683684https://plantphysiol.
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23Figure6.
CrPIF1negativelyregulatesCrGATA1andvindolinepathwaygenesthroughG-685boxorPBEbox.
(A)RelativeexpressionofCrGATA1andfivelight-induciblevindoline686pathwaygenesinemptyvector(EV)controlsandCrPIF1overexpression(OX)seedlingswere687measuredbyRT-qPCR.
Seven-day-oldseedlingswereinfiltratedandkeptindarkforthreedays688beforesamplecollection.
(B)MeasurementoftabersonineandvindolineinEVcontrolsand689CrPIF1OXlines.
(C)ExpressionofCrGATA1andvindolinepathwaygenesinCrPIF1VIGS690leaves.
RelativeexpressionofCrPIF1,CrGATA1andfivevindolinepathwaygenesinEVand691CrGATA1VIGSleavesweremeasuredbyRT-qPCR.
(D)Measurementoftabersonineand692vindolineinEVcontrolsandCrPIF1VIGSlines.
ForRT-qPCR,theRPS9genewasusedasan693internalreferencegene.
Fortabersonineandvindolinecontents,alkaloidswereextractedand694analyzedbyLC-MS/MS,andtheconcentrationsofthealkaloidswereestimatedbasedonpeak695areascomparedtostandards.
(E)Sub-cellularlocalizationofCrPIF1intobacco(N.
tabacum)696protoplasts.
eGFPisaccumulatedthroughoutthecell(left)whereaseGFP-CrPIF1islocalizedto697thenucleus(right).
(F)TransactivationofCrGATA1,T16H2,T3O,T3R,D4HandDAT698promoters(-pro),fusedtotheGUSreporter,byCrPIF1.
Transactivationassayswerecarriedout699byco-infiltrationoftheCrGATA1-expressionvectorwithapro-GUSconstructintoN.
700benthamianaleaves.
Theplantswereincubatedindarkafterinfiltration.
Aplasmidcontaining701luciferasereporter,drivenbyCaMV35SpromoterandrbcSterminator,wasusedasa702normalizationcontrol.
LuciferaseandGUSactivitiesweremeasuredtwodaysafterinfiltration.
703GUSactivitywasnormalizedagainstluciferaseactivity.
ControlrepresentsthereporterwithEV.
704(G)MutationintheG-boxsequence(toppanel)affectsthetransactivationoftheCrGATA1705promoterbyCrPIF1.
(H)MutationinthePBE-boxsequence(toppanel)affectsthe706transactivationoftheDATpromoterbyCrPIF1.
Alldatapresentedherearethemeans±SDof707threebiologicalreplicates.
StatisticalsignificancewascalculatedusingtheStudent'st-test(*P<7080.
05).
709710Figure7.
ModelsdepictingtheregulatoryrolesofCrPIF1-CrGATA1moduleinlight-711inducedofvindolinebiosynthesis.
Leftpanel:CrPIF1islikelyaccumulatedinthedarkand712repressestheexpressionofCrGATA1,T16H2andDATbybindingtotheirpromoters.
713RepressionofCrGATA1resultsindownregulationofvindolinepathwaygenes,T16H2,T3O,714T3R,D4HandDAT,leadingtolimitedvindolineproductionandincreasedtabersonine715https://plantphysiol.
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24accumulation.
OneormoreunknownTF(TF)mediatesthelow(basal)expressionofCrGATA1716despiteCrPIF1repression.
Rightpanel:CrPIF1ispossiblydegradeduponexposureofC.
roseus717seedlingstolight,whichismediatedbytheredlightsensedbyphytochrome.
De-repressionof718CrGATA1resultsintheactivationofvindolinepathwaygenesandvindolineaccumulation.
The719sevengenesinvolvedinconversionoftabersoninetovindolinearelistedintheinsertedbox.
720Genesinredareresponsivetolightinduction.
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RootsAerialparts051010010000200003000040000Relativeexpression**************1410244896Time(h)012345RelativeexpressionCRO_T134526(CrGATA1)********1410244896Time(h)012CRO_T117711Relativeexpression****012345RelativeGUSactivityeGFPeGFP-CrGATA1ABCDEDarkLightDarkLight******DarkLighthttps://plantphysiol.
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******036RelativeexpressionRelativeexpression012**TabersonineVindolineTabersonineVindoline0500100015002000ngmg-1DW02040602000500010000ngmg-1DW*****ABCDControlCrGATA1-OXControlCrGATA1-OX****ControlCrGATA1-VIGSControlCrGATA1-VIGShttps://plantphysiol.
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01234RelativeGUSactivity********ACEV+promoter-GUSCrGATA1+promoter-GUSGATCGATCGAAAGATCGATCGAAAGAAAGAAA-617-614-541-538WTMUT1MUT2M1M2D4H-proWTMUT1MUT2M1M201234RelativeGUSactivityEV+D4H-pro-GUSCrGATA1+D4H-pro-GUS***-1ATGATGATGATGBGATAGATAGGCAGATAGATAGGCAGGCAGGCA-430-427-138-135WTMUT1MUT2M1M2D4H-pro-1ATGATGATGATGWTMUT1MUT2M1M201234RelativeGUSactivityEV+D4H-pro-GUSCrGATA1+D4H-pro-GUS****https://plantphysiol.
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CrChlHD4HDATCrGATA1CrPORCCrRCAT3OT3RT16H201230120123Relativeexpression0120123024601230120120246RFR0246RFR0246RFRcbacbbabcbcabbaacbaacdcabcbabaaabbbacTime(h)https://plantphysiol.
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***EV+promoter-GUSCrPIF1+promoter-GUS00.
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CrGATA1CrPIF1CrPIF1T3O,T3R,D4HCrGATA1CrGATA1T16H2,DATCrPIF1RedlightCrPIF1CrPIF1T16H2,DATT16H2,T3O,T3R,D4H,DATCrGATA1TabersonineVindolineVindolineTabersonineTFCrGATA1TFBasalactivationofCrGATA1StrongactivationofCrGATA1TabersonineVindolineT16H216OMTT3OT3RNMTD4HDAThttps://plantphysiol.
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