Regulation of FLOWERING LOCUS T by a MicroRNA in Brachypodium distachyonC W

16 Pages·2013·1.87 MB·English

ThisarticleisaPlantCellAdvanceOnlinePublication.Thedateofitsfirstappearanceonlineistheofficialdateofpublication.Thearticlehasbeen
editedandtheauthorshavecorrectedproofs,butminorchangescouldbemadebeforethefinalversionispublished.Postingthisversiononline
reducesthetimetopublicationbyseveralweeks.
FLOWERING LOCUS T
Regulation of by a MicroRNA in
Brachypodium distachyon
CW
LiangWu,a,1DongfengLiu,a,b,c,1JiajieWu,dRongzhiZhang,aZhengruiQin,aDanmeiLiu,aAiliLi,aDaolinFu,d
WenxueZhai,b,candLongMaoa,2
aNationalKeyFacilityforCropGeneResourcesandGeneticImprovement,InstituteofCropScience,ChineseAcademyof
AgriculturalSciences,Beijing100081,China
bInstituteofGeneticsandDevelopmentalBiology,ChineseAcademyofSciences,Beijing100101,China
cUniversityofChineseAcademyofSciences,Beijing100049,China
dStateKeyLaboratoryofCropBiology,ShandongAgriculturalUniversity,Taian271000,China
ORCIDIDs:0000-0003-4953-7258(L.W.);0000-0002-3377-4040(L.M.).
The highly conserved florigen gene FLOWERING LOCUS T (FT) functions at the core of the flowering pathways. Extensive
studieshaveexaminedthetranscriptionalregulationofFT;however,otherlayersofFTregulationremainunclear.Here,we
identified miR5200 a Pooideae-specific microRNA that is expressed in leaves and targets Brachypodium distachyon FT
orthologsformRNAcleavage.miR5200wasabundantlyexpressedinplantsgrownundershort-day(SD)conditionsbutwas
dramaticallyrepressedinplantstransferredtolong-day(LD)conditions.Wealsofoundthattheepigeneticchromatinstatus,
specificallythelevelsofhistonemethylationmarks,atmiR5200precursorlocichangedinresponsetodaylength.Moreover,
artificial interruption of miR5200 activity by target mimicry in B. distachyon altered flowering time in SD but not in LD
conditions,suggestingthatmiR5200functionsinphotoperiod-mediatedfloweringtimeregulation.Together,thesefindings
illustrateaposttranscriptionalregulationmechanismofFTandprovideinsightsintounderstandingofthemultipleconcerted
pathwaysforfloweringtimecontrolinplants.
INTRODUCTION (Kardailsky et al., 1999; Kobayashi et al., 1999). In Arabidopsis
thaliana, FT protein travels from the vasculature to the shoot
The transition from vegetative growth to flowering stage is
apextoformaneffectorcomplexwithFD,abZIPtranscription
acriticaleventforseedpropagationinplants.Thecorrecttiming factor, and thereby stimulates flower organ formation through
of flowering initiation depends on five genetic pathways: pho- activation of a subset of downstream floral genes, including
toperiod, vernalization,autonomy,hormones,andage(Fornara
APETALA1(AP1),FRUITFULL(FUL),andSOC1(Abeetal.,2005;
et al., 2010). These endogenous and environmental flowering
Wiggeetal.,2005;Corbesieretal.,2007;Mathieuetal.,2007).
regulatory cues ultimately regulate a set of integrator genes,
PositiveandnegativeregulatorscontrolFTtranscription.Under
suchasSUPPRESSOROFOVEREXPRESSIONOFCONSTANS1
long-day (LD) conditions, FT expression is mainly activated in
(SOC1),LEAFY,andFLOWERINGLOCUST(FT)(Kobayashiand
vascular bundles via transcriptional regulators CONSTANS (CO)
Weigel,2007).
andGIGANTEA(Suárez-Lópezetal.,2001;SawaandKay,2011;
Photoperiodic control of flowering in plants begins with the
Tortietal.,2012).COisdirectlyrecruitedtotheFTpromoteror
perceptionofdaylengthinleaves,followedbythetransmission
indirectlyactswithothertranscriptionalcofactorstoregulateFT
of afloral signal termed florigeninto the shootapex wherethe
expression (Wenkel et al., 2006; Kobayashi and Weigel, 2007).
flowersareinitiated(BäurleandDean,2006).Themobileflorigen
Theage-dependentSQUAMOSAPROMOTERBINDINGPROTEIN-
signal is encoded by FT and FT orthologous genes in various
LIKE3 can also directly act at the FT promoter and modulate
plant species (Lifschitz et al., 2006; Tamaki et al., 2007; Pin ambienttemperature-responsiveflowering(Wangetal.,2009;Wu
et al., 2010; Meng et al., 2011; Navarro et al., 2011; Wigge,
etal.,2009a;Kimetal.,2012).AsamajornegativeregulatorofFT,
2011).MutationinFTcausesaseriousdelayinflowering,whereas
FLOWERINGLOCUSCformsaMADSboxcomplexwithSHORT
overexpression of FT induces precocious flowering, indicating
VEGETATIVE PHASE, which directly inhibits FT expression to
thatFTisnecessaryandsufficienttoacceleratethefloraltransition
mediate the effects of exposure to cold conditions or ambient
temperatureinArabidopsis(Bastowetal.,2004;BäurleandDean,
2006;Liet al., 2008). Furthermore,two TEMPRANILLOproteins
1Theseauthorscontributedequallytothiswork.
(TEM1andTEM2)directlyrepressFTandmayhaveantagonistic
[email protected].
roleswiththeactivatorCOtodetermineFTlevels(Castillejoand
The authors responsible for distribution of materials integral to the
findingspresentedinthisarticleinaccordancewiththepolicydescribed Pelaz,2008).SeveralmiR172-targetedAP2-likegenesalsoaffect
in the Instructions for Authors (www.plantcell.org) are: Liang Wu the repression of FT transcription, but their modes of action re-
([email protected])andLongMao([email protected]). mainunclear(Mathieuetal.,2009).
CSomefiguresinthisarticlearedisplayedincoloronlinebutinblackand
In addition to transcription factors, trimethylation of H3K27
whiteintheprintedition.
WOnlineversioncontainsWeb-onlydata. (H3K27me3)anddemethylationofH3K4(H3K4me2)modifications
www.plantcell.org/cgi/doi/10.1105/tpc.113.118620 incisalsoepigeneticallyregulateFTtranscription(Farronaetal.,
ThePlantCellPreview,www.aspb.orgã2013AmericanSocietyofPlantBiologists.Allrightsreserved. 1of15
2of15 ThePlantCell
2008;Adrianetal.,2010).FTmight alsobecontrolled atpost- Therefore, we have renamed this miRNA miR5200a, and the
transcriptional levels, since the werewolf root hair patterning sequenceidentifiedbyZhangetal.(2009)asmiR5200c.Owing
mutant indirectly affected FT mRNA stability in Arabidopsis, but to the importance of FT-like proteins in plant flowering path-
themolecularbasisofthisprocessisstillelusive(Seoetal.,2011). ways,wefurtheranalyzedthismiRNAindepth.Todetermine
Compared with eudicots, the behaviors of FT during floral the conservation of miR5200 in other plant species, we per-
inductioninmonocotsarelargelyunknowntodate.Inrice(Oryza formedaBLASTsearchofallavailablesmallRNAdatabases.
sativa), a typical short-day (SD) plant, two FT-like proteins re- Weonlydetectedthe21-nucleotidemiR5200sequenceinthe
ferredtoasHeading-date3aandRICEFLOWERINGLOCUST1, sRNA data sets of a number of Pooideae species, including
which are respectively activated under SD and LD conditions, barley and B. distachyon (Schreiber et al., 2011; Hackenberg
have been proposed to be the SD- and LD-specific florigens etal.,2012).
(Komiya et al., 2009). Ectopic expression of the FT-like gene Since B. distachyon has emerged as a research model for
VERNALIZATION3 (VRN3) in wheat (Triticum aestivum) and Pooideaeplants,byvirtueofitspublishedhigh-qualitygenome
barley(Hordeumvulgare)activatedtranscriptionoftheAP1or- sequence and tractable genetic transformation systems (Vogel
thologous gene VRN1 and promoted early flowering (Trevaskis et al., 2010; Brkljacic et al., 2011), we investigated the struc-
etal.,2003;Yanetal.,2006). tureandfunctionsofmiR5200indetailinB.distachyon.
Withits rapidgrowth,smallgenome,and simplegrowthcon- Bioinformatic prediction of miR5200a stem-loop structures
ditions,Brachypodiumdistachyonhasbeenthoughtasasuitable foundthattwolocionB.distachyonchromosome1potentially
modelsystemforstudiesontemperatecerealsandbiofuelplants, encodedthemiR5200atranscribedgenes,andwenamedthese
suchasswitchgrass(Panicumvirgatum;Opanowiczetal.,2008); two genes MIR5200a and MIR5200b (Figure 1A). Interestingly,
however, so far, information about its flowering is quite limited like in wheat, B. distachyon MIR5200c and MIR5200b were lo-
(Higginsetal.,2010;Hongetal.,2010).B.distachyonaccessions cated at thesame butreverse complementary loci, suggesting
showsubstantialnaturalvariationinfloweringtime.Forexample, that both mature sequences and precursor structures of
the genome-sequenced accession Bd21 is a spring annual and miR5200 are conserved between B. distachyon and wheat
can flower without any vernalization, whereas the winter acces- (Abrouk et al., 2012; Lucas and Budak, 2012). RNA gel blot
sionBd1-1requiresvernalization,aperiodofcoldtreatment,for analysisvalidatedthatmiR5200waspresentinthethreePooideae
flowering (Schwartz et al., 2010). In addition, daylength greatly plants,butnotinrice(Oryzasativa),maize(Zeamays)orsorghum
influences the flowering time of B. distachyon accessions. For (Sorghum bicolor),nor indicotyledonous plantssuch asArabi-
instance, Bd21canflowerwithin45d ofgrowthunder LD con- dopsisthaliana(Figure1B).TheseresultssuggestthatmiR5200
ditionsbutwilldelayfloweringforhalfayearifgrowninSDen- is a recently evolved and Pooideae-specific miRNA (see Sup-
vironments(Schwartzetal.,2010;seeSupplementalFigures1A plementalFigures2Aand2B).
and 1B online). Thus, there may be special components that To better understand the complete gene structures of primary
preciselyregulatefloweringtimeinB.distachyon. miR5200(pri-miR5200),weusedRNAligase-mediatedrapidam-
The 21- to 24-nucleotide microRNAs (miRNAs), a class of plificationofcDNAends(RACE)toretrieve59and39sequencesof
DICER-LIKE (DCL)–dependent small RNAs (sRNAs), regulate thetranscript.LikecanonicalmiRNAs(Xieetal.,2005),wefound
awiderangeofbiologicalprocessesinmonocotsandeudicots that both MIR5200a and MIR5200b transcripts were 59 capped
(Voinnet,2009;Wuetal.,2009b).Inthisstudy,wecharacterized with 39 polyadenylation (see Supplemental Figures 3A and 3B
a Pooideae-specific miR5200 that guides sequence-specific online).Intriguingly,wedetected 14splicevariantsofMIR5200a,
cleavageofFTorthologoustranscriptsinB.distachyonleaves. but each variant had the same 59 segment (see Supplemental
We further demonstrate that miR5200 plays an important role Figure3Aonline).DistinctfromvariousMIR5200atranscriptsthat
in regulation of the photoperiod-mediated floral transition. Our carriedonetoseveralintrons,theshortMIR5200btranscripthad
findingsshowthatamiRNAdirectlymediatesFTposttranscriptional onlyoneexon(seeSupplementalFigure3Bonline).
modulationinPooideaeplants,whichmayalsosupplyanalterna-
tiveapproachforheadingdatebreedinginbarleyandwheat. miR5200TargetsFTOrthologousGenesandAccumulates
inLeaves
RESULTS SimilartotheinitialcomputationalpredictionofmiR5200atargets
inwheatandbarley(seeSupplementalFigures4Aand4Bonline),
using psROBOT sRNA analysis toolbox (Wu et al., 2012), we
HairpinStructuresandPrimaryTranscriptsofmiR5200
identified two B. distachyon FT-like genes (Bradi2g07070 and
WepreviouslyidentifiedasubsetofmiRNAsbydeepsequencingof Bradi1g48830), which were previously annotated as FTL1 and
smallRNAsinbreadwheat.One21-nucleotidemiRNA,whichwe FTL2(Higginsetal.,2010),asmiR5200atargets(Figure1C;see
namedmiR2032,wasidentified,anditstargetswerepredictedin Supplemental Figures 4C and 4D online). Further validation
silico to be FT orthologous genes (Wei et al., 2009). We found through59RNAligase-mediatedRACEshowedthatbothFTL1
that another miRNA, previously annotated in miRBase as and FTL2 mRNAs could be cleaved at miR5200 target sites
miR5200 in B. distachyon (Zhang et al., 2009), has only one invivo,suggestingthatthesetwoFTsarebonafidemiR5200a
nucleotide difference with the miRNA we named miR2032 in target genes (Figure 1C). Although a majority of the cleavage
bread wheat (Wei et al., 2009). In addition, miR2032 has been events of FTL1 occurred outside of the canonical miRNA
assigned to another miRNA in sea anemone in miRBase. cleavage site, it is unlikely that this cleavage was mediated by
RegulationofFLOWERINGLOCUSTbyaMicroRNA 3of15
Figure1. HairpinStructures,ExpressionPatterns,andTargetValidationofmiR5200.
(A)The59to39secondarystructuresofpre-miR5200predictedforB.distachyon.ThematuremiR5200andmiR5200*sequencesareindicatedinred
andblue,respectively.ThedifferentnucleotidesbetweenmiR5200aandb/carehighlightedbyboldletters.
4of15 ThePlantCell
secondary siRNAs because miR5200a is not 22 nucleotides in etal.,2009).TodeterminewhethermiR5200wasintegratedinFT
length and there are no bulged bases between the miRNA: regulationinB.distachyonflowering,weexaminedtheexpression
miRNA* (for miRNA star) duplex that are known to be required patternsofmiR5200inresponsetovernalizationanddaylength.
forsecondarysiRNAformation(Chenetal.,2010;Cuperusetal., Treating B. distachyon accession Bd21 under 4°C low tem-
2010;Manavellaetal.,2012). peraturefor2to4weekscanaccelerateplantflowering,butwe
SinceFTisnotexpressedattheshootapex,itisreasonableto observed that mature miR5200 did not change in 2- or 4-week
ask whether miR5200a is also specifically expressed outside the cold-treated Bd21 plants (see Supplemental Figure 6A online),
shoot apex to degrade FT mRNAs. To address this question, we suggesting that vernalization may not affect miR5200 in Bd21.
collectedB.distachyonleavesandshootapicesandperformedRT- Bd1-1 is a Turkish winter-habit B. distachyon accession and
PCR to determine miR5200a expression patterns in these tissues cannotflowerwithoutvernalization(Filizetal.,2009).However,2to
(Figure1D).Strikingly,wefoundthatbothMIR5200aandMIR5200b 4weeksofcoldtreatmentdidnotaltertheabundanceofmiR5200
dramaticallyaccumulatedinleaves,butneitherofthemappearedin inBd1-1either(seeSupplementalFigure6Aonline).Theseresults
shoot apex (Figures 1Eand 1F). By contrast, AP1 ortholog VRN1 reveal that the regulation of FT by miR5200 is likely to be in-
andGAPDHgeneswerepresentinbothtissues(Figures1Eand1F). dependentofthevernalizationpathwayinB.distachyon.
Together with the observation that DCL1 was expressed in both Next, we examined the amounts of miR5200 in plants grown
leaves and shoot apices, these results suggest that miR5200a is underdifferentdaylengths.AsshowninFigure3A,RNAgelblots
specificallyexpressedinleavestorepressFTmRNAs. showedthatmiR5200accumulatedtohighlevelsin8-h-lightSD
To verify that miR5200a targets FTs in vivo, we generated conditions but was nearly undetectable in 16-h-light LD con-
miR5200aoverexpression(miR5200-OE)transgenicB.distachyon ditions.RT-PCRandquantitativereal-timePCR(qPCR)analyses
plants to trace whether FTLs were inhibited when miR5200a also revealed that the primary transcripts of MIR5200a and
increased. We obtained 11 independent transgenic lines from MIR5200b were much higher in SDs than in LDs (Figures 3A to
separately generated calluses and all of them showed delayed 3C).TheseresultssuggestthatmiR5200maybecontrolledbythe
floweringtimephenotypesinLDenvironments(seeSupplemental photoperiod. Tofurtherassessthe relianceofmiR5200 onday-
Figures5Aand5Bonline).Basedupontheseverityoflateflowering length,wedetectedtheabundanceofmiR5200under4-,8-,16-,
time,theselinesweredividedintoTypesIandII(Figure2A):TypeI and 20-h-light conditions. We found that miR5200 had similar
didnotflowerevenafter180dandthuscouldnotformseeds; levelsunder4-and8-h-lightSDconditions,muchhigherthanin
TypeIIboltedmuchlaterthanthewildtype(110versus45d)and 16-and20-h-lightLDs(seeSupplementalFigure6Bonline).
producedfewerseedsthanthewild-typeplants(Figures2Aand Considering thatFTfamilygenesusuallyshowed diurnalex-
2C). Moreover, both types of miR5200-OE plants eventually pression rhythms, we examined the oscillation of pri-miR5200
grew much higher than the wild type (Figures 2B and 2C). andtargetsevery4hduringtheday-nightcycleinB.distachyon.
RT-PCR showed that the expression of FTL1 and FTL2 was Under SDs, both MIR5200a and MIR5200b displayed an ex-
dramaticallyreducedinTypesIandIItransgeniclines,whereas pression peak at 4 h before dawn (Figure 3D), and FTLs also
thatofanotherFT-likegene(FTL10)withoutthemiR5200atarget peaked at this time but in LDs (Figure 3E). Furthermore, as
site was comparable to the wild type (Figures 2D and 2E). We shown in Figures 3D and 3E, MIR5200a and MIR5200b were
also found that B. distachyon AP1 and FUL orthologous expressed at much higher levels, whereas FTL1 and FTL2 ac-
genes,VRN1andFUL2,whichmayactdownstreamofFT(Li cumulated to much lower levels at any time point in SDs than
and Dubcovsky, 2008), also displayed decreased mRNA levels LDsduringthe48-htime-courseexamination.
in miR5200-OE plants (Figures 2D and 2E). These results in- In Arabidopsis, FT mRNA levels gradually increased with
dicatethatmiR5200mediatesFTL1andFTL2mRNAdegrada- growth time, especially under LD conditions (Kobayashi et al.,
tionforposttranscriptionalregulation. 1999).Therefore,weaskedwhethertheabundanceofmiR5200
changedprogressivelywithFTmRNAincreasesunderSDsand
LDs in B. distachyon. Under SDs, FTL1 and FTL2 expression
PhotoperiodAffectsmiR5200Expression
levels remained constant, whereas miR5200 showed a clear
Theinitiationoffloweringintemperategrassesdependsondel- increasealongwithgrowthtime(Figures3Fand3G).UnderLDs,
icate regulation of FT, which plays a central role in balancing both FTL1 and FTL2 showed a gradual increase in expression
competingsignalsfromvernalizationandphotoperiod(Distelfeld along with the photoperiod time; however, we did not detect
Figure1. (continued).
(B)RNAgelblotanalysisofmiR5200inwheat,barley,B.distachyon,rice,maize,sorghum,andArabidopsis.NotethatmiR444isamonocot-specific
miRNA,whereasmiR172andmiR159areconservedbetweenmonocotsanddicots.U6wasusedasRNAloadingcontrol.nt,nucleotides.
(C)SchematicrepresentationofgenestructureandvalidationofmiR5200targets.Geneexons,introns,andtargetregionsareshownbyrectangles,
lines,andgreenbars,respectively.Arrowsindicatethecleavagesitesdetectedby59-RACE.
(D)PartsofleafandshootapexinB.distachyonthatweredissectedformiR5200spatialexpressionanalysis.
(E)SpatialexpressionsofmiR5200andtargetgenesinleafandshootapex.Theexpressionofpri-miR5200andindicatedgeneswasanalyzedby
RT-PCRwithamplificationofGAPDHmRNAasacontrol.
(F)qPCRanalysisofpri-miR5200andindicatedgeneexpressioninleafandshootapex.GAPDHwasusedasaninternalcontrolfornormalizationof
qPCRresults.Eachpointrepresentstheaverageofthreebiologicalreplicates,anderrorbarsindicateSD.
RegulationofFLOWERINGLOCUSTbyaMicroRNA 5of15
Figure2. miR5200OverexpressionSeverelyDelayedFloweringTimeinTransgenicB.distachyonPlants.
(A)Floweringtimeofwild-type(WT)Bd21andtheindicatedtwotypesofmiR5200-OEtransgenicT0plants.ThreeplantsfortypeIandeightplantsfor
typeIIwerescored.ErrorbarsindicateSD.
(B)PlantheightsofthewildtypeandtwotypesofmiR5200-OEtransgenicT0plants.ThreetypeIplantsandeighttypeIIplantsweremeasured.Error
barsindicateSD.
(C)PhenotypesofectopicmiR5200expressionT0transgenicB.distachyonplantsinLDs.Theleftplantis2-month-oldwildtype.Themiddleandright
plantsarerepresentativesof4-month-oldmiR5200-OEtransgenicplantswithstrongandweakphenotypesrespectively.Whitearrowspointtospikes.
(D) miR5200 (by RNA gel-blots) and flowering-time genes (by RT-PCR) expression in wild-type and the indicated miR5200-OE transgenic plants.
NumbersbeloweachmiRNAgelblotdenotefoldchangesrelativetothemiRNAlevelinwild-typeplants.
(E)qPCRanalysisofpri-miR5200andindicatedfloweringgeneexpressionsinwild-typeandtheindicatedmiR5200-OEtransgenicplants.GAPDHwas
usedasaninternalcontrolfornormalizationofqPCRresults.Eachpointrepresentstheaverageofthreetechnicalreplicates,anderrorbarsindicateSD.
[Seeonlinearticleforcolorversionofthisfigure.]
6of15 ThePlantCell
Figure3. PhotoperiodAffectsmiR5200Expression.
(A)MaturemiR5200(byRNAgelblots),pri-miR5200,andflowering-timegenes(byRT-PCR)expressionunderSDsandLDs.Thenumbersbeloweach
miRNAgelblotdenotefoldchangesrelativetothemiRNAlevelunderSDs.
(B)qPCRanalysisofpri-miR5200inB.distachyonplantsunderSDsandLDs.
(C)qPCRanalysisoftheindicatedfloweringgeneexpressionsinSDsandLDs.
(D)DiurnalexpressionpatternsofMIR5200aandMIR5200binB.distachyonunderSDsandLDs.
(E)DiurnalexpressionpatternsofFTL1andFTL2underSDsandLDs.Eachpointrepresentstheaverageofthreetechnicalreplicates,andtheerrorbars
indicaterepeatSD.Thewhiteandblackbarsalongthehorizontalaxesrepresentlightanddarkperiods,respectively.Thenumbersbelowthehorizontal
axesindicatethetimeinhours.
(F)and(G)Growthstage–dependentexpressionsofmiR5200(F)anditstargetgenes(G)inSDs.NumbersbeloweachmiRNAgelblotindicatefold
changesrelativetothemiRNAlevelin1-weekSDplants.w,weeks.
(H)and(I)Growthstage–dependentabundanceofmiR5200(H)anditstargetgeneexpression(I)underLDs.Eachpointrepresentstheaverageof
threebiologicalreplicates,anderrorbarindicatesSD.GAPDHwasusedasaninternalcontrolfornormalizationofqPCRresults,andU6wasserved
asloadingcontrolforRNAgelblots.NumbersbeloweachmiRNAgelblotindicatethefoldchangesrelativetothemiRNAlevelin1-week-oldplants
underLDs.
RegulationofFLOWERINGLOCUSTbyaMicroRNA 7of15
miR5200 expression even after 7 weeks (Figures 3H and 3I). progressivelydecreasedtoalowlevelduringthisperiod(Figure
These results suggest that miR5200 appears to specifically in- 4C), suggesting a negative correlation between FTL1 and
hibitFTL1andFTL2accumulationinSDs. miR5200 in this process. When these plants were returned to
High expression in SDs and low abundance of miR5200 in SDs for another 7 d, FTL1 and FTL2 displayed a moderate in-
LDsasdescribedaboveimpliedthatphotoperiodmayaffectFT crease along with a reduction of miR5200 (see Supplemental
expressionthroughregulationofmiR5200.Tofurtherpursuethe Figures 6C and 6D online). By contrast, the abundance of
effects of daylength on miR5200 and its targets, we examined mature miR5200 in SD-grown plants remained constant after
the dynamic changes of miR5200 by RNA gel blots, shifting transfertoLDsfor7devenasthepri-miRNAexpressionbegan
plantsfromLDstoSDsandviceversa.WhenLD-grownplants to decline within 3 d (Figures 4D and 4E). When this shift time
were moved from LDs to SDs for 1 d, both pri-miR5200 and wasextendedtoover14d,agradualreductionofmiR5200was
mature miR5200 were elevated and gradually accumulated to detected (Figure 4D). Interestingly, when these shifted plants
a high level in 7 d (Figures 4A and 4B). In the meantime, FTL1 weretransferredbacktoSDconditionsforanother7d,miR5200
Figure4. DynamicEffectsofDaylengthChangesonmiR5200,FTL1,andFTL2Accumulation.
(A)TimecourseofmiR5200expressionwhenB.distachyonplantswereshiftedfromLDstoSDs.L3wandL4windicatethatB.distachyonweregrown
underLDsfor3and4weeks.L1S,L3S,andL7Srepresent3-week-oldplantsthatweremovedfromLDstoSDsfor1,3,and7d,respectively.Numbers
beloweachmiRNAgelblotindicatefoldchangesrelativetothemiRNAlevelinwild-typeBd21plantsgrownunderLDsfor3weeks.
(B)qPCRexaminationofdynamicchangesofMIR5200aandMIR5200bin(A).
(C)qPCRanalysisofdynamiceffectsofdaylengthonFTL1andFTL2in(A).
(D)TimecourseofmiR5200expressionwhen3-week-oldSDplantsweretransferredtoLDsfor1,3,7,14,and21d.PlantsgrownunderSDsfor5and
6weekswereharvestedandusedascontrols.NumbersbeloweachmiRNAgelblotdenotefoldchangesrelativetothemiRNAlevelinBd21grown
underSDsfor3weeks.
(E)qPCRexaminationofdynamicchangesofMIR5200aandMIR5200bin(D).
(F)qPCRanalysisofdynamiceffectsofdaylengthonFTL1andFTL2in(D).ErrorbarmeansSD(n=3).SamplesforRNAextractionintheexperiment
werecollectedatZeitgebertime2.U6wasusedasloadingcontrolforRNAgelblots.GAPDHwasusedasaninternalcontrolfornormalizationofqPCR
data.
8of15 ThePlantCell
Figure5. HistoneModificationStatusatPri-miR5200LociinB.distachyonGrownunderDifferentDaylengths.
(A)and(B)Diagramsshowingthegenomiclocations,theadjacentcodinggenes,andtheregionsexaminedbyChIPassayforMIR5200a(A)and
MIR5200b(B).Grayboxesrepresentexons,andwhiteboxesrepresentintrons.BlueboxeslocatedingrayboxesindicatepredictedMIR5200aand
MIR5200bhairpinstructures.PositionsofmaturemiR5200aresetasblackbars.ChIPregionsareshownbyblacklines.
(C)and(D)RelativeabundanceofH3K4me3,H3K27me3,andH3K9acatMIR5200a(C)andMIR5200b(D)genomicregionsinB.distachyongrown
underSDsandLDs.NotethatthehighlevelsofH3K4me3andthedecreasedH3K27me3aroundregionscorrespondingtoMIR5200a(P4/P5/P6)and
MIR5200b(P6/P7)hairpinstructuresareassociatedwiththeactivationofMIR5200aandMIR5200bexpressionunderSDconditions.
expressionwasrecovered(seeSupplementalFigure6Eonline). resultofepigeneticcontrol,wefirstexaminedthestatusofDNA
Meanwhile, FTL1 and FTL2 displayed roughly complementary methylationatpri-miR5200lociunderSDandLDconditionsby
alterations during these shifts (Figure 4F; see Supplemental Chop-PCR (see Supplemental Figures 7A and 7B online). We
Figure 6F online), suggesting that these two FTLs are photo- observed no obvious differences of DNA methylation at
periodicallyregulatedbybothmiR5200andadditionalflowering MIR5200aandMIR5200bwhenB.distachyonwasgrowninSDs
factorsinresponsetodaylengthvariation. andLDs(seeSupplementalFigures7Cand7Eonline).Bisulfite
sequencing of three regions confirmed these results (see
Supplemental Figures 7D and 7F online), suggesting that the
ChangesofChromatinStructuresatPri-miR5200Loci
distinct patterns of MIR5200 transcription under different light
ResultinPhotoperiodicExpressionofmiR5200
conditionsarenottriggeredbyDNAmethylation.
Epigenetic mechanisms regulate numerous genes in flowering We then tested histone epigenetic status at MIR5200 loci
pathways,includingPolycombrepressivecomplexinhibitionof underSDsandLDsusingchromatinimmunoprecipitation(ChIP)
FT transcription (Farrona et al., 2008). To explore whether the assay. We designed a series of oligos at both MIR5200 genes
transcriptional regulation of miR5200 by photoperiod was the and their neighboring regions (Figures 5A and 5B). Although the
RegulationofFLOWERINGLOCUSTbyaMicroRNA 9of15
Figure6. DisruptionofmiR5200ActivityinB.distachyonPromotesFloweringunderSDs.
(A)AdiagramofMIM5200showingthedesignedsequencesformiR5200targetmimics.UBI,maizeubiquitinpromoter;NOS,nosterminator.
(B)Representativephotographoffloweringphenotypesin6-month-oldwild-type(WT)andindicatedT1MIM5200transgenicplantsgrowninSDs.The
whitearrowspointtospikes.
(C)Floweringtimeofwild-typeandtheindicatedT1MIM5200transgeniclinesgrowninSDs.ErrorbarshowsSD(n=8).Asterisksindicateasignificant
differencebetweenwild-typeandtransgenicplants(Student’sttest,Pvalue<0.05).
10of15 ThePlantCell
enrichmentofpositivehistonemarkH3K9acaroundMIR5200aand in B. distachyon (Figure 6A). We termed these target mimic
MIR5200blociwassimilarunderSDsandLDs(Figures5Cand5D), transgenicplantsasMIM5200.
wefoundthattherepressivehistonemarkH3K27me3surrounding AsmiR2032wasexpressedmuchhigherunderSDsthanLDs,
the predicted hairpin structure regions of MIR5200 genes was wefirstexaminedthefloweringdatesofMIM5200plantsinSDs.
enrichedmuchhigherunderLDsthanthatunderSDs(Figures5C We observed that six out of nine independent MIM5200 T1
and 5D), suggesting that the suppression of pri-miR5200 tran- positive lines flowered 10 to 30 d earlier than wild-type plants
scripts under LDs is caused by the high level of H3K27me3 en- whentheyweregrowninSDs(Figures6Band6C),whereasthe
richment. H3K4me3 has been reported to act as bivalent mark T1 segregated negative plants did not show any early heading
togetherwithH3K27me3attheFTlociinArabidopsis(Adrianetal., phenotypes compared with the wild-type plants (see
2010).OurChIPdatashowedthatcomparedwiththosegrownin Supplemental Figures 9A and 9B online). To illustrate that the
LDs, H3K4me3 around the MIR5200a and MIR5200b stem-loop phenotypes of MIM5200 plants in SDs were indeed caused by
areawasmuchenhancedwhenB.distachyonplantsweregrown theattenuatedmiR5200activities,threeindependentlineswere
under SDs (Figures 5C and 5D). Furthermore, we found that subject to further molecular analysis. As shown in Figures 6D
H3K4me3wasalsohighlyenrichedintheregionofMIR5200b1-kb and 6E, the expression of miR5200 was reduced in MIM5200
terminatorunderSDs, despite that similarH3K27me3 status was plants,whileFTL1andFTL2aswellastheirdownstreamgene
foundatthissiteinbothSDsandLDs(Figure5D). VRN1increased.Moreover,wedetectedthediurnalexpression
Takentogether,ourresultsindicatethatMIR5200aandMIR5200b patternsofFTL1andFTL2at4-hintervalsunderSDsandfound
genomicregionsare subject to changes in histone modification they exhibited significantly higher expression in MIM5200 than
underdifferentdaylengths.Theseresultssuggestthatthedistinct wild-type plants (Figures 6F and 6G). These findings strongly
expressionofmiR5200 for photoperiodicregulation of flowering suggest that these two FT genes in B. distachyon are indeed
underSDsandLDsisprobablycausedbyepigeneticchromatin regulatedbymiR5200tomakeplantsfloweratthecorrecttime
alterationsatMIR5200loci. inSDs.
To determine whether disruption of miR5200 activities in
DisruptionofmiR5200ActivitySpecificallyAltersFlowering B.distachyoncanaffectFTsunderLDconditions,weextracted
TimeinSDs total RNA from LD-grown wild-type and MIM5200 plants and
measured the transcripts of FTLs. We did not detect obvious
miR5200 functions in photoperiodic modulation of FTs; there- differencesinexpressionsofFTL1andFTL2orVRN1,indicating
fore, one interesting question is what the consequences are if thatinterruptionofmiR5200activitiesinLDsmaynotaffectFTs
themiR5200regulatoryactivityiscompromised.Toaddressthis (see Supplemental Figures 9C and 9D online). Consistent with
issue, we first attempted to generate transgenic lines that ec- theseresults,thediurnalexpressionofFTL1andFTL2wassimilar
topically expressed FTL1 and FTL2 as well as their miRNA- in MIM5200 and wild-type plants under LDs (see Supplemental
resistantmutantformsFTL1mandFTL2m,whichwereresistant Figures9Eand9Fonline).Furthermore,weobservedthatheading
tointeractionwithmiR5200(seeSupplementalFigures8A,8B, dates were nearly the same for wild-type and MIM5200 plants
and8Donline).Unfortunately,wewereunabletogeneratesuch under LD environments (see Supplemental Figures 9G and 9H
transgenicplants,asallofthemfloweredextremelyearlybefore online). These results imply that miR5200 has little effect on
they could form roots in the regeneration medium (see regulationofB.distachyonfloweringtimeinLDconditions.
SupplementalFigures8Cand8Eonline).Theseresultsindicate Takentogether,specificalterationofboltingtimeofMIM5200
thatbothFTL1andFTL2haveflorigenactivityinB.distachyon. plantsinSDssuggeststhatmiR5200playsanimportantrolein
Artificial miRNA target mimics, RNA fragments that have photoperiod-mediatedfloweringtimeregulation.
similar sequence as the miRNA target but cannot be cleaved,
can deplete miRNAs and thus reduce their function, providing
PhotoperiodicRegulationofmiR5200AppearstoBe
usefulreversegenetictoolstoinvestigatebiologicalfunctionsof PrevalentinthePooideae
individualmiRNAfamiliesindiverseplants(Franco-Zorrillaetal.,
2007; Todesco et al., 2010; Bergonzi et al., 2013; Zhou et al., Considering the roles of miR5200 in B. distachyon flowering
2013). To gain more insights into photoperiodic regulation of control stated above, we asked whether the photoperiodic
flowering by miR5200, we designed and generated artificial modulation of miR5200 was conserved among species in the
targetmimicsforthemiR5200familyandthenexpressedthem Pooideae.Toaddressthisquestion,weselectedfivePooideae:
Figure6. (continued).
(D) Expression of miR5200 (by RNA gel blots), targets, and other flowering genes (by RT-PCR) as well as Bradi1g13680 (miR169 target gene as
acontrol)inwild-typeandtheindicatedT1MIM5200transgeniclinesunderSDs.U6andGAPDHwereservedastheRNAgelblotandRT-PCRRNA
loadingcontrols,respectively.ThenumbersbeloweachmiRNAgelblotdenotethefoldchangesrelativetothemiRNAlevelinthewildtype.
(E)qPCRanalysisofmiR5200targetgenes,VRN1,andBradi1g13680 (miR169targetasacontrol)expressioninwild-typeandtheindicatedMIM5200
transgenicplantsunderSDs.
(F)and(G)DiurnalexpressionpatternsofFTL1(F)andFTL2(G)inwild-typeandtheindicatedMIM5200transgenicplantsunderSDs.Eachpoint
representstheaverageofthreetechniquereplicates,andtheerrorbarindicatesSD.Whiteandblackbarsalongthehorizontalaxesrepresentlightand
darkperiods,respectively.Numbersbelowthehorizontalaxesindicatethetimeinhours.