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RESEARCHARTICLE
Phalaenopsis
Functional Characterization of
aphrodite
Flowering Genes PaFT1 and PaFD
SeonghoeJang1,2*,Sang-ChulChoi3,Hsing-YiLi1,2,GynheungAn3,ElmonSchmelzer4
1 BiotechnologyCenterinSouthernTaiwan,AcademiaSinica,TainanCounty,741,Taiwan,2 Agricultural
BiotechnologyResearchCenter,AcademiaSinica,Taipei,115,Taiwan,3 CropBiotechnologyCenter,
KyungheeUniversity,Yongin,446–701,Korea,4 Max-Planck-InstituteforPlantbreedingresearch,Cologne,
50829,Germany
* [email protected]
Abstract
WeshowthatthekeyfloweringregulatorsencodedbyPhalaenopsisaphroditeFLOWER-
INGLOCUST1(PaFT1)andPaFDsharehighsequencehomologiestothesefromlong-day
floweringArabidopsisandshort-dayfloweringrice.Interestingly,PaFT1isspecificallyup-
OPENACCESS
regulatedduringfloweringinductivecoolingtreatmentbutisnotsubjectedtocontrolbypho-
Citation:JangS,ChoiS-C,LiH-Y,AnG,Schmelzer toperiodinP.aphrodite.Phloemorshootapex-specificexpressionofPaFT1restoresthe
E(2015)FunctionalCharacterizationofPhalaenopsis latefloweringofArabidopsisftmutants.Moreover,PaFT1cansuppressthedelayedflower-
aphroditeFloweringGenesPaFT1andPaFD.PLoS
ingcausedbySHORTVEGATATIVEPHASE(SVP)overexpressionaswellasanactive
ONE10(8):e0134987.doi:10.1371/journal.
pone.0134987 FRIGIDA(FRI)allele,indicatingthefunctionalconservationoffloweringregulatorycircuitin
differentplantspecies.PaFT1promoter:GUSinArabidopsisshowedsimilarstainingpattern
Editor:MohammedBendahmane,EcoleNormale
Superieure,FRANCE tothatofArabidopsisFTintheleavesandguardcellsbutdifferentintheshootapex.A
genomiccloneorheatshock-inducibleexpressionofPaFT1issufficienttothepartialcom-
Received:March6,2015
plementationoftheftmutants.Remarkably,ectopicPaFT1expressionalsotriggerspreco-
Accepted:July15,2015
ciousheadinginrice.Tofurtherdemonstratethefunctionalconservationoftheflowering
Published:August28,2015 regulators,weshowthatPaFD,abZIPtranscriptionfactorinvolvedinfloweringpromotion,
Copyright:©2015Jangetal.Thisisanopen interactswithPaFT1,andPaFDpartiallycomplementedArabidopsisfdmutants.Trans-
accessarticledistributedunderthetermsofthe genicriceexpressingPaFDalsofloweredearlywithincreasedexpressionofricehomo-
CreativeCommonsAttributionLicense,whichpermits
loguesofAPETALA1(AP1).Consistently,PaFT1knock-downPhalaenopsisplants
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare generatedbyvirus-inducedgenesilencingexhibitdelayedspiking.Thesestudiessuggest
credited. functionalconservationofFTandFDgenes,whichmayhaveevolvedandintegratedinto
DataAvailabilityStatement:Thenucleotide distinctregulatorycircuitsinmonopodialorchids,Arabidopsisandricethatpromoteflower-
sequencesreportedinthispaperhavebeen ingundertheirowninductiveconditions.
submittedtoGenBankdatabaseundertheaccession
numbersKJ609179(PaFT1)andKJ609180(PaFD).
Funding:FundedbyDevelopmentProgramof
IndustrializationforAgriculturalBiotechnology
(DPIAB,Taiwan099S0030086-AAtoSJ),http://
dpiab.sinica.edu.tw/index_en.php.Thefundershad
Introduction
noroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript. Plantshaveevolvedmechanismstointegrateenvironmentalanddevelopmentalsignalsand
preciselycontrolthetransitionterminatingvegetativegrowthandinitiatingtheformationof
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. flowers.
PLOSONE|DOI:10.1371/journal.pone.0134987 August28,2015 1/29
FunctionalstudiesofPhalaenopsisFTandFDgenes
InArabidopsis,floweringistriggeredbymultiplepathways[1]convergingonafewintegra-
torssuchasFLOWERINGLOCUST(FT)[2].FTmRNAexpressionisinducedintheleaves
anditsproteinmovessystemicallytotheshootapicalmeristemwhereflowersbloomin
responsetolongsummerdays(LDs)[3,4]indicatingtheFTproteinactsasamajorflowering
hormone‘florigen’[5,6].Inaddition,FT-likegenesarewellconservedamongfloweringplants
andwerereportedtoplayaroleoffloweringactivatorsinplantsotherthanArabidopsis,
includingtomato[7],squash[8]andrice[9,10].TheFTmovementfromtheleavestomeri-
stemsrequiresinteractionwithapartnercalledFTIP,orFTinteractingprotein[11].Onreach-
ingthemeristem,FTinteractswithabZIPdomaintranscriptionfactor,FDandtogetherthey
activateexpressionofdownstreamgenesthatchangetheidentityofthemeristemtothatofa
flower.OneexampletargetoftheFT-FDcomplexisAPETALA1(AP1),ameristemidentity
gene;anothertargetisSUPPRESSOROFOVEREXPRESSIONOFCONSTANS1(SOC1),which
promotestheexpressionofanothermeristemidentitygenecalledLEAFY(LFY).BothAP1and
LFYexpressionarerepressedduringvegetativegrowth,andthisrepressionisreleasedupon
arrivalofFTinthemeristem[12].
Inrice,theexpressionofHeadingdate3a(Hd3a),ariceFTorthologueisup-regulatedonly
underinductiveshort-day(SD)conditions[13]and14-3-3proteinsactasintracellularrecep-
torsforHd3aproteins.Ahexamericflorigenactivationcomplex(FAC)composedofHd3a,14-
3-3proteinsandOsFD1activatesOsMADS15,aricehomologueofArabidopsisAP1leadingto
floralinduction[10].AnothermodelspeciesusedtostudySDfloweringresponse,morning
glory(Pharbitisnil),possessestwoFTorthologues,PnFT1andPnFT2[14].Theexpressionof
thegenesexhibitscircadianrhythmsthataresetbytheonsetofdarknessandareupregulated
attheendofthenightunderSDonlyifthenightissufficientlylong.Despitetheconserved
functionsofFThomologues,theirexpressionappearstobecontrolledbydifferentsystemsin
differentplantspecies.
Phalaenopsisisanepiphyticandmonopodialorchidwiththickandsucculentleaves.Itisan
ornamentalcropspeciesofgreateconomicvalue.Ingeneral,orchidscanbedividedintotwo
groups,sympodialandmonopodial,basedontheirgrowthmorphology.Sympodialorchids
suchasthosefromthegeneraCattleya,Cymbidium,DendrobiumandOncidiumgrowfroma
stemthatishorizontal.Monopodialorchidsontheotherhand,includingPhalaenopsis,
PaphiopedilumsandVandagrowverticallyasasingleuprightstemwithoneleaffollowing
anotheronoppositesidesofthecenter.Phalaenopsisaphroditesubsp.formosana,aTaiwanese
nativePhalaenopsisorchid,isconsideredtobeamodelPhalanopsisspecies[15,16].Likemany
otherfloweringplants,thefloweringoforchidsisaffectedbyseveralenvironmentalfactors
suchasphotoperiodandtemperature[17–19].MostPhalaenopsisspeciesarenativetoareas
closetotheequatorandthustheydonotneedaspecificphotoperiodtoinduceflowering
althoughspecificsdependontheorchidgenusandcanevendifferaccordingtospecies.
Instead,lowambienttemperatureisoneofthetriggersforthefloweringinitiationofPhalae-
nopsisorchidsincludingP.aphroditesubsp.formosana.Also,orchidsincludingPhalaenopsisis
arecrassulaceanacidmetabolism(CAM)plantslikepineapplesandcactithatcantoleratehigh
temperaturesusingwell-adaptedmetabolicstrategiesforgrowth[20].Commercially,orchid
seedlingsareproducedthroughembryocultureinvitro[21]orclonalpropagation[22].
Spikeinduction(initiationofinflorescences)ofPhalaenopsiswassignificantlyinhibited
whenitwasgrownunderaconstanttemperaturehigherthan28°C[23].Conversely,diurnal
fluctuationofhighdayandlownighttemperatureorcooltemperatureinthenightpromoted
spikeinduction[24–26].Currently,manygrowersuseveryexpensiveair-conditioningtocool
temperaturesdownto18–25°CinsidegreenhousestospikePhalaenopsisduringthewarm
periodoftheyear[27].Therefore,itisimportanttofunctionallycharacterizefloweringgenes
suchasFTandFDintheorchidforbetterunderstandingoffloweringunderinductive
PLOSONE|DOI:10.1371/journal.pone.0134987 August28,2015 2/29
FunctionalstudiesofPhalaenopsisFTandFDgenes
conditions.Suchunderstandingwouldalsobebeneficialfortheproductionofnewvarieties
whichdonotrequirecoolingforfloweringinthefuture.InArabidopsis,itwasreported
recentlythattheabundanceoftheSHORTVEGETATIVEPHASE(SVP)-FLOWERING
LOCUSM(FLM)complex,asafloralrepressorofFTexpressionisregulatedbyambienttem-
perature[28,29].Interestingly,twoMADS-boxfloralrepressors,FLM-ßandSVPwere
reportedtobedown-regulatedtranscriptionallyandpost-transcriptionallybyhighambient
temperature[28].
TheArabidopsisFLOWERINGLOCUSC(FLC)geneencodesaMADSdomainproteinthat
actsasarepressoroffloweringbysuppressingFTandSOC1[30,31].FRIGIDA(FRI)isaposi-
tiveregulatorofFLC[32].FloweringofArabidopsisisacceleratedbyprolongedexposureto
cold(vernalization)andFLClevelsprogressivelydeclineduringthecoldperiods.Therefore,
loss-of-functionmutationsineitherFRIorFLCeliminatethevernalizationrequirement.Most
commonlyusedlabstrainsofArabidopsissuchasColumbialackactiveFRIand/orFLCalleles,
andexhibitrapid-floweringbehaviorunderinductivelongdays(LD)[32,33].Incereals,which
lackFLC,thedaylengthandvernalizationarealsolikelytobeinterconnectedthroughFT-like
genes.Inthecasesofbarleyandwheat,thenaturallyoccurring‘vernalizationgenes’,VRN-H3
andVRN-B4respectively,havebeenshowntoencodeFTorthologues[34].Inaddition,OnFT,
PhFTandCgFT,FThomologuesfromorchidssuchasOncidiumGowerRamsey,Phalaenopsis
hybridFortuneSalzmanandCymbidiumgoeringii,respectively,wererecentlyreported[35–
37].InArabidopsis,distinctmolecularmechanismscontrolFTexpressionforsubsequentflow-
eringunderdifferentrangesofenvironmentaltemperature.However,Phalaenopsisorchids
includingP.aphroditesubsp.formosanaareknowntooriginatefromtropicalandsubtropical
areasofthesouthpacificislandswherephotoperiodisalmostconstantthroughouttheyear
[23].Theseorchidsgenerallydonotrequirevernalizationdespitelowambienttemperature
beingnecessaryforflowering.Therefore,ratherthanphotoperiodand/orvernalization,recog-
nitionandsignalingsystemsforlowenvironmentaltemperaturearelikelytobethemajortrig-
gersfortheinductionofFTexpressionandconsequently,thetransitionfromvegetativeto
reproductivegrowthinPhalaenopsisorchids.
HerewedemonstratedthatPaFT1thatencodesanorthologueofArabidopsisFTwasaccu-
mulatedduringthelowambienttemperaturetreatmentrequiredforfloralinductionofthe
monopodialorchidP.aphroditesubsp.formosana.Wefurtherdemonstratedthatthefunc-
tionalroleofPaFT1asafloralinducerisconservedintheorchid.Moreover,weshowedthat
PaFT1-interactingprotein,PaFDhasaconservedfloralactivationfunction.Sinceplantshave
adaptedthemselvestovariousenvironmentsforsuccessfulreproduction,eachplanthasdevel-
opeditsownstrategytocontrolthetimingoffloraltransition.Weprovideevidenceatleast
partiallysupportingthenotionthatdistinctregulationoftheFTandFDgenesmayhave
evolvedforrapidfloweringbyinductiveenvironmentalcuesinthemonopodialorchid,P.
aphroditesubsp.formosana.
MaterialsandMethods
PlantMaterialsandGrowthConditions
Phalaenopsisorchids(P.aphroditesubsp.formosana)atdifferentdevelopmentalstageswere
purchasedfromChainPortOrchidNursery(PingTung,Taiwan).Theorchidswereadaptedin
thegrowthchamberenvironment(16hlight,28°C/8hdark,25°C)for2weeksbeforestarting
eachexperiment.Wild-typeArabidopsis(A.thalianaecotypeCol)wasusedtogeneratetrans-
genicplants.ft-10,fd-3[38],soc1-2[39]andFRI-Col[40]weredescribedpreviously.SOC1:
GUSand35S:SVPseedswerekindgiftsfromDr.IlhaLee(SeoulNationalUniversity,Korea)
andDr.PeterHuijer(MPIZ,Germany),respectively.Generally,Arabidopsisplantsweregrown
PLOSONE|DOI:10.1371/journal.pone.0134987 August28,2015 3/29
FunctionalstudiesofPhalaenopsisFTandFDgenes
inthegrowthchamberunderLDconditions(16/8-hphotoperiodat100μmolm-2s-1)at
23°C.TwoJaponica-typericecultivars,Dongjin,aKoreancultivarandTainung67(TNG67),a
Taiwanesecultivarwereusedtoproducetransgenicriceplantsandthetransgenicplantswere
growninthegrowthchamberorintheoutdoorGMOgreenhouseoftheAcademiaSinicaBio-
technologyCenterinSouthernTaiwan.ForArabidopsisfloweringtimemeasurement,8to
12plantsperlinewerecountedfortotalleafnumberswhentheirfirstflowerswereatanthesis.
Daysofheadingof8–12riceplantspereachlineweremeasuredwhenpanicleswereemerged.
CloningofPaFT1GenefromP.aphroditesubsp.formosana
TotalRNAextractedfromyoungspikes((cid:1)2cminlength)ofP.aphroditesubsp.formosana
wasusedforcDNAsynthesisasdescribedbySuetal[16]andBilginetal[41].Synthesized
cDNAsanddegeneratedprimerswereusedfortheamplificationofPaFT1.Degeneratedprim-
0
ersforPaFT1wereasfollows:forwardprimer(DegF:5CHTTCTACACBCTYGTSATGGTA
0 0 0
G3)andreverseprimer(DegR:5CDGGSGCGTAMACYGTCTG3).TheamplifiedPCR
productwasclonedintopCR-BluntIITOPOvector(Invitrogen)andthesequenceofthepar-
tialPaFT1wasverifiedtohavehighsimilaritywithPEBPgenes.Internalgene-specificprimers
forPaFT1weredesignedfortheisolationofafull-lengthcloneofPaFT1byRACE(Rapid
AmplificationofcDNAEnds)usingSMARTRACEcDNAamplificationkit(BDBiosciences
0 0 0
Clontech).Gene-specificprimersfor5 and3-RACEofPaFT1are5GAGGATCACTTGGAC
0 0 0 0 0
TTGGAGC3 (GS-5RACE)and5GTTGTTTCATCAACTAGGCCG3 (GS-3RACE),respec-
tively.ThecDNAofPaFT1fullopenreadingframe(ORF)wasobtainedbyPCRwithattB-
linkedgene-specificprimersandthePaFT1entryclonewasconstructedbyBPreactionwith
pDONR201(Invitrogen).ThePaFT1Y86Hformwasgeneratedbypointmutagenesisusing
Stratagene’sQuikChangesite-directedmutagenesiskit.Thefollowingtwoprimerscontaining
mismatchedbasepair(fromTtoC,boldtypeandunderlined)wereusedforthemutagenesis:
0
5CTCAACTTAGAGAACACTTACACTGGTTAGand5'CTAACCAGTGTAAGTGTTCTC
TAAGTTGAG.
PromoterIsolationandGUSExpression
GenomicDNAofP.aphroditesubsp.formosanawasisolatedbyDNeasyPlantMinikit(Qia-
gen).GenomiccloneofPaFT1wasisolatedbyPCRwithPhusiontaqpolymerase(NEB)and
theexonsandintronswereannotatedcomparedwiththePaFT1cDNAsequence.Thepro-
moterregionofPaFT1wasisolatedbyinversePCR(iPCR)togetherwiththeaidofGenome
WalkerUniversalKit(Clontech).Thepromoterentryclonewasconstructedandsequentially
introducedintopMDC163[42](CurtisandGrossniklaus,2003)togeneratePaFT1promoter:
GUSconstruction.FortheexpressionofPaFT1genomicclonecontainingitsownpromoter
andcodingregion,thepAlligator2vectorwithout35SpromoterandthetripleHAwasusedfor
thedestinationvector.GUSstainingwithArabidopsisseedlingswasperformedasdescribedby
Anetal[43].
ExpressionAnalyses
TotalRNAsfromvariousorgansofP.aphroditesubsp.formosanawereextractedbyRNeasy
PlantMiniKit(Qiagen)andtreatedwithRNase-freeDNase(Invitrogen)followingthemanu-
facturer’sprotocoltoremoveanyresidualgenomicDNA.DNase-treatedRNAwassubjected
toreversetranscriptasereactionsusingoligo-dTprimerandSuperscriptIIreversetranscriptase
(Invitrogen)accordingtothemanufacturer’sinstructions.Thesameprocedurewasappliedto
cDNAsynthesesofArabidopsisandriceafterRNAextraction.SubsequentPCRwasperformed
withthefirst-strandcDNAmixtureandEX-Taqpolymerase(TakaraBiochemical,Japan).
PLOSONE|DOI:10.1371/journal.pone.0134987 August28,2015 4/29
FunctionalstudiesofPhalaenopsisFTandFDgenes
Quantitativerealtime-PCR(qPCR)wasperformedontheCFX96TMreal-timesystem(Bio-
Rad)usingMaximaSYBRGreenqPCRMasterMix(Thermo).Theprimersusedforquantifi-
cationarelistedinS1Table.ForPCR,eachsamplewasanalyzedintriplicate.Therunningpro-
tocolwas:denaturationat95°Cfor10min,annealing/extensionrepeated40times(95°Cfor15
sand60°Cfor30s,dataacquisitionwasperformed).Geneexpressiondatawerenormalizedto
theexpressionofhousekeepinggenes.FortheexpressionalanalysesofP.aphroditesubsp.for-
mosana,PaACT[16]andPaUBQ[16]geneswereusedfornormalizationbutonlythefigures
usingPaACTareshownasbothsetsofresultsweresimilar.Theprimersusedinthisstudyare
listedinS2Table.AtleasttwoindependentexperimentswereperformedforRNAextraction
inexpressionalanalyses.
InSituHybridization
YoungspikesofPhalaenopsisorchidwerecollectedandfixed,dehydrated,embedded,sliced
(10ìmthickness),andperformedhybridizationaspreviouslydescribedbyLinetal[44]with
slightmodification.Forpreparationofdigoxigenin(DIG)-labeledRNAprobes,weamplified
gene-specific261bpfragmentofPaFDusingthefollowingprimers:5'GTTCGTCCAACAGT
CTTC3'(forward)and5'GTTTCCAGACTTCTTCCATAC3'(reverse).TheDNAfragment
wasclonedintopGEM-Tvector(Promega)andeachsenseandantisenseprobewassynthe-
sizedbyT7andSP6RNApolymerases,respectively.Hybridizationwasperformedat63°Cor
66°Cwith20ngofDIG-labeledRNAprobe.
YeastTwo-HybridScreeningforPaFD
PaFT1full-lengthORFwasclonedin-frameinthepBD-GAL4Camvector(Stratagene)to
generateapBD:PaFT1vectorasabait.ForthecDNAlibraryconstruction,totalRNAwas
extractedfromyoungspikes((cid:1)2cminlength)ofP.aphroditesubsp.formosanaandpoly(A)
+RNAwasisolatedusingaPolyATractmRNApurificationkit(Promega).TheGAL4ADvec-
torlibrarywasconstructedusingaGAL4Two-HybridPhagemidVectorKit(Stratagene)
accordingtomanufacturer’sinstructions.ScreeningandX-galfilterassaywereperformedas
describedpreviously[45].
ThePaFDΔ1–53clonewasgeneratedbyPCRusingthefollowingattB-linkedprimers:5'GG
GGACAAGTTTGTACAAAAAAGCAGGCTGCATGGAAGAAGTCTGGAAACACATTG
ACand5’GGGGACCACTTTGTACAAGAAAGCTGGGTGTTAAAATGGCGCGGATGAA
GTTCTCTGAAG.PaFDT225A,S226A,S227Aandtriple(PaFDT225A,S226A,S227A)
cloneswerealsogeneratedbyPCRusingthefollowingprimers:5'GGGGACAAGTTTGTACA
AAAAAGCAGGCTGCATGTGGCTCCTATCTCCTGC(forward),5'GGGGACCACTTTG
TACAAGAAAGCTGGGTGTTAAAATGGCGCGGATGAAGCTCTCTGAAG(reversefor
T225A),5'GGGGACCACTTTGTACAAGAAAGCTGGGTGTTAAAATGGCGCGGATGCA
GTTCTCTGAAG(reverseforS226A),5'GGGGACCACTTTGTACAAGAAAGCTGGGTGT
TAAAATGGCGCGGCTGAAGTTCTCTG(reverseforS227A),5'GGGGACCACTTTGTAC
AAGAAAGCTGGGTGTTAAAATGGCGCGGCTGCAGCTCTCTGAAG(reversefortriple).
AmplifiedPCRproductswereclonedintopDNONR201viaBPreaction(Invitrogen).
ParticleBombardmentandBiFCAssays
ForcellularlocalizationofAtFT,AtFD,AtFDP,PaFT1andPaFDinArabidopsis,eitherYFP:
GWorCFP:GWvectorwasusedfortheflorescencefusionasdescribedpreviously[46].
ForBiFCassaysinArabidopsis,ArabidopsisFTandPaFT1cDNAwereclonedintopVYCE
vectorforAtFT:cYFPandPaFT1:cYFPfusions.AtFDandAtFDPcDNAswereclonedinto
pVYNEvectorfornYFP:AtFDandnYFP:AtFDPfusions,respectively[47].Bombardmenton
PLOSONE|DOI:10.1371/journal.pone.0134987 August28,2015 5/29
FunctionalstudiesofPhalaenopsisFTandFDgenes
ArabidopsisleaveswascarriedoutasdescribedbyShirasuetal[48].ForBiFCinPhalaenopsis,
cDNAsencodingthePaFT1andPaFDgeneswereintroducedintopE3136andpE3130,
respectively[49](http://www.bio.purdue.edu/people/faculty/gelvin/nsf/protocols_vectors.
htm).Bombardment-mediatedtransienttransformationofPhalaenopsisandgenerationof
imageswereperformedasdescribedbySuetal[16].
VIGSAssays
EachspecificfragmentofPaFT1,PaFDandGUSgeneswasclonedintopCymMVvector[50]
byinvitrorecombinationwithBPClonaseII(Invitrogen)togeneratepCymMV-GUS,
pCymMV-PaFT1andpCymMV-PaFD,respectively.ForgrowthofAgrobacteriumandleaf
injection,wefollowedtheproceduredescribedbyHsiehetal[51,52].
PlantTransformationandAnalysesofTransgenicPlants
ForthepSUC2:PaFT1andpKNAT1:PaFT1constructs,thePaFT1entryclonewasinsertedinto
thepSUC2:Gateway(GW)andpKNAT1:GWdestinationvectors,respectively[43,46].For
thepFD:PaFT1construct,the35SpromoterandthetripleHAofthepAlligator2vectorwas
exchangedforthe3.1-kbFDpromoterandthenPaFT1wasintroducedbyLRreaction.With
thesamestrategy,PaFT1wasalsofusedtoArabidopsisheatshockprotein(HSP)18.2promoter
[53]togeneratepHSP18.2:PaFT1.Allplasmidsforplanttransformationwereintroducedinto
AgrobacteriumstrainGV3101(pMP90RK)[54]andtransformedintoColumbiawild-type,ft-
10orft-10soc1-2doublehomozygousplantsbythefloral-dipmethod[55].Foroverexpression
oftargetgenesinrice,binaryvector,pGA3426wasusedandeachtransgeneconstructwas
introducedintoricegenomebyAgrobacterium-mediatedtransformation[56].
Results
IsolationandMolecularCharacterizationofPaFT1
WeisolatedPaFT1byacombinedreversetranscriptionPCR(RT-PCR)andRACEstrategy
fromyoungspikesofP.aphroditesubsp.formosana.Degeneratedprimersweredesignedbased
ontheconservedregionsoftheFTsequencesfromOncidiumorchids,riceandbarley(seethe
materialsandmethods).
ThePaFT1cDNAencodesa178aminoacidproteinwithacalculatedmolecularmassof
20.02kDaandatheoreticalpIof6.83.ThePaFT1proteinshowed70%,76%and89%identity
toArabidopsisFT,riceHd3aandOncidiumorchidOnFT,respectively.Thekeyaminoacidres-
iduesTyrandGlnthatareconservedamongtheFThomologueswerelocatedatpositions86
and141ofthePaFT1protein(S1Fig)[57–59].
ThePaFT1andotherFTproteinsequencesfromvariousplantspecieswereusedtocon-
structaphylogenetictree.ThePaFT1isolatedfromP.aphroditesubsp.formosanabelongedto
theFTfamilyofmonocotyledonousplantsandinterestingly,itwasgroupedwithtwoother
orchidFTs,OncidiumFT(OnFT)andCymbidiumFT(CgFT)(S1Fig).
ExpressionPatternofPaFT1
ThespatialexpressionpatternofPaFT1wasinvestigatedbyquantitativeRT-PCRanalyses.
ThePaFT1transcriptaccumulatedtohighlevelsindevelopinginflorescences(spikes)and
developingfloralbuds,andwasalsodetectedinvegetativeorganssuchasleavesandrootsas
wellasreproductiveorganssuchaslips,columns,pedicels.However,PaFT1mRNAwashardly
detectableinsepalsandpetals(Fig1).
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FunctionalstudiesofPhalaenopsisFTandFDgenes
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FunctionalstudiesofPhalaenopsisFTandFDgenes
Fig1.GrowthandfloweringofP.aphroditesubsp.formosanawiththeexpressionofPaFT1.A,Floral
budsatdifferentdevelopmentalstagesandthestructureofflower.S:sepal,P:petal,L:lip,C:column,Pe:
pedicel.Baris1cm.B,SpikingandfloweringofP.aphroditesubsp.formosanaunderLDandSDconditions
atconstant23°C.C,UnderLDconditions,lowtemperaturetreatmentisessentialtoinduceinflorescenceof
P.aphroditesubsp.formosana.Dayandnighttemperaturesareshowninparenthesis.D,Spatialexpression
ofPaFT1inP.aphroditesubsp.formosana.MaterialsforRNAextractionwereharvestedfromsixtoeight
plants.Bud1,bud2andbud3indicatetheB1,B2andB3,respectivelyinA.Spike1,spike2andspike3
indicate(cid:1)3cm,3–10cmand(cid:3)10cminlength,respectively.The3rd,4thand5thleaveswereusedforthe
leafRNAextraction.E,DailyoscillationofPaFT1expressionunderLDandSDconditions.Ineachtimepoint,
leavesof4plants(18monthsold)wereharvestedforRNAextraction.F,Theeffectofambienttemperature
onPaFT1expression.LT;lowtemperature(23°C/20°C),HT;hightemperature(28°C/25°C).Thirtysixmature
plants(34-montholdasthestage4)weregrownatHTandthensixteenplantsweretransferredtotheLT
conditions.AllleavesoffourplantswereusedfortheanalysisofPaFT1expression,ateachtimepoint.Allthe
sampleswereharvestedattheendoflight(ZT16).Twoindependentexperimentalresultsshowedsimilar
expressionpatterns.
doi:10.1371/journal.pone.0134987.g001
ToexplorewhetherPaFT1expressionoscillatedover24hours,thePaFT1transcriptwas
analyzedevery4hoursovera24-hourperiodunderbothLDandSDconditionsusingleavesof
18-month-oldorchids.ThehighestpeakofPaFT1expressionwasdetectedatzeitgebertime
(ZT)16irrespectiveofthephotoperiod.ThelowestlevelofexpressionofPaFT1wasobserved
atdawn(Fig1).SincecoolingtreatmentisnecessaryforspikingofP.aphroditesubsp.formo-
sana,theexpressionofPaFT1wasinvestigatedundertwodifferentconditions(Fig1).There
wasnocleardifferenceinspikinginductiontimebetweentheplantsgrownunderSDandLD
(Fig1).
Whenthematureplants(34monthsold)grownathightemperature(28°Clight/25°Cdark,
LD)weretransferredtoacoolertemperature(23°Clight/20°Cdark,LD),spikesproutinginiti-
ated4weeksafterthetransfer(Fig1).However,theplantsmaintainedatthehightemperature
didnotdevelopspikes.Duringthisperiod,wemeasuredmRNAlevelsofPaFT1andthree
putativeSOC1homologuesfromtheorchidatweeklyintervals.Theexperimentrevealedthat
PaFT1expressionwasincreasedatthetwo-weekpointinthetransferredplantsbutwasnot
changedintheplantsthatremainedathightemperature(Fig1).ThethreeputativeSOC1
homologues,PaSOC1-1,PaSOC1-2andPaSOC1-3,wereallhighlyexpressedatthespiking
stageunderlowambienttemperaturealthoughtheyshoweddistinctexpressionpatternsdur-
ingthetemperatureshift(S2Fig).
PaFT1PromotesFloweringinArabidopsisandRice
TotesttheactivityofPaFT1asafloweringregulator,thepSUC2:PaFT1transgene,whichover-
expressesPaFT1inthephloemcompanioncells,wasintroducedintoArabidopsisColumbia
wildtype(WT)plants.Thetransgenicplantsexhibitedearlyfloweringwithanaverageof6.3
fewerleavesthanthecontrolWT(Fig2).
InordertoinvestigatewhetherPaFT1wasabletorescuethelatefloweringphenotype
causedbytheftmutation,thepSUC2:PaFT1constructwasintroducedintotheft-10,ftnull
mutant[60].ThetransgenicftmutantplantsexpressingPaFT1inthephloemcompanioncells
floweredmuchearlierthantheparentalftmutantplantswithasimilartotalnumberofleaves
totheWT(Fig2).ExpressingPaFT1undershootapicalmeristem(SAM)-specificpromoters
suchaspKNAT1orpFDalsorescuedthedelayedfloweringphenotypeintheftmutant(Fig2).
TheseresultsindicatedthatexpressionofPaFT1inphloemcompanioncellsorSAMissuffi-
cienttopromotefloweringinArabidopsisplantsthatcompletelylackendogenousFT.How-
ever,asingleaminoacidchangefromtheconservedTyr-86toHisofPaFT1resultedinaloss
ofthefloweringpromotioncapability(Figs2and3)[57–59].
PLOSONE|DOI:10.1371/journal.pone.0134987 August28,2015 8/29
FunctionalstudiesofPhalaenopsisFTandFDgenes
PLOSONE|DOI:10.1371/journal.pone.0134987 August28,2015 9/29
FunctionalstudiesofPhalaenopsisFTandFDgenes
Fig2.Phloem-specificexpressionofPaFT1inArabidopsisandoverexpressionofPaFT1inricedriveearlyflowering.A,Comparisonofflowering
timebetweentransgenicplantscontainingpSUC2:PaFT1andwildtypeplants.ThirtyindependentT1plantswereusedforeachgenotype.Arrowsrepresent
themeanvalueoftotalleafnumberineachgenotype.P(cid:1)0.0001(Student’st-test).BandC,PaFT1drivenbyphloem-specificorshootapex-specific
promotersrescuesthelatefloweringphenotypeofArabidopsisftnullmutant,ft-10.ThisactivityisatleastdependentonTyr-86residue,oneoftheconserved
aminoacidsamongFTproteinsfromvariousplantspecies.DandE,EctopicexpressionofPaFT1inricealsocausedearlyflowering(Dongjincultivar,grown
underSDcondition).MagnifiedpaniclesareshownintheboxofEandfloweringtimedataisshowninD.TLNmeanstotalleafnumber.
doi:10.1371/journal.pone.0134987.g002
WealsointroducedthePaFT1geneintoDongjinrice.PaFT1expressionwasunderthecon-
trolofmaizeubiqutinpromoter,astrongconstitutivepromoterinmonocotplants[56].Three
independenttransgeniclinescontainingpUbi:PaFT1showedearlyheadingcomparedwith
controlplantscontainingtheemptyvector(Fig2).UnderSDcondition,transgenicriceplants
producedpanicleswithinonemonthaftersowingalthoughtheydidnotproducemanyhealthy
grains.ThisearlyfloweringphenotypecorrelatedwithincreasedexpressionoftworiceAP1-
likegenes,OsMADS14andOsMADS15(S3Fig).
ExpressionofPaFT1OvercomestheLateFloweringPhenotypeofFRI-
ColPlants
TotestthecontributionofPaFT1tothefloweringofFRI-ColthatcontainsanactiveFRIlocus
ofSanfeliu-2accession[61],thepSUC2:PaFT1constructwasintroducedintotheFRI-Col.
TransgenicplantsexpressingPaFT1floweredearlierthancontrolFRI-Colplantswithout
coldtreatment(Fig3).Asexpected,SOC1expressionwasincreasedinthetransgenicplants
andtheincreaseinSOC1transcriptswasalsoobservedintheft-10mutantscontainingthe
pSUC2:PaFT1construct(Fig3).However,PaFT1Y86H,amutantformofPaFT1couldnot
induceSOC1expression(Fig3).
ToconfirmwhetherPaFT1positivelyregulatesSOC1,weemployedaβ-glucuronidase
(GUS)reporterassay.WeusedpSOC1:GUS[60]plantstovisualizetheexpressionofSOC1.
pSUC2:PaFT1pSOC1:GUSplantswereproducedtoexaminewhetherSOC1promoter-driven
expressionofGUSwasaffectedbypSUC2:PaFT1.AhistochemicalGUSassayshowedstronger
GUSstainingintheshootapex,cotyledonsandthefirsttrueleafinthepSUC2:PaFT1pSOC1:
GUSplantscomparedwithpSOC1:GUSplants(Fig3).ThissuggeststhatSOC1expressionwas
reinforcedbyPaFT1andtheincreasedSOC1expressioncontributedtotheearlyflowering.
ThepSUC2:PaFT1transgenewasintroducedintoft-10soc1-2doublemutantstoevaluate
theeffectofthetransgeneinfloweringofthedoublemutants.ThepSUC2:PaFT1ft-10soc1-
2plantsfloweredearlierwithapproximately32fewerleavesthantheft-10soc1-2double
mutants,whereastheyfloweredlaterwithapproximately5moreleavesthanthepSUC2:PaFT1
ft-10plantsunderLDs(Fig3).ThisimpliesthatSOC1isnotauniquetargetofPaFT1forpro-
motionoffloweringinArabidopsis.Recently,soc1fulwasreportedtobeepistatictopSUC2:FT
inflowering[62].AnalysesofFRUITFULL(FUL)transcriptlevelsrevealedthatthegene
expressionwashigherinpSUC2:PaFT1ft-10soc1-2plantscomparedtothatinft-10soc1-2but
lowerthanthatofpSUC2:PaFT1ft-10plants(Fig3).ThisindicatesthatPaFT1inducesFUL
expressionandsequentiallypromotesfloweringinArabidopsislackingendogenousFTand/or
SOC1.
ExpressionofPaFT1ReducestheEffectofp35S:SVPinFlowering
SinceSVPhasbeenidentifiedasafloweringrepressormediatedbyambienttemperature,we
generatedpSUC2:PaFT1p35S:SVPplantsbyintroducingthetransgenepSUC2:PaFT1intothe
p35S:SVPbackgroundtoinvestigatewhetherphloem-specificexpressionofPaFT1isableto
overcomethefloweringrepressioneffectofp35S:SVP.Weobservedthattheexpressionlevelof
PLOSONE|DOI:10.1371/journal.pone.0134987 August28,2015 10/29