The B3-Domain Transcription Factor VAL1 Regulates theFloral Transition by Repressing FLOWERINGLOCUS T1[OPEN]

Yanjun Jing,a,2 Qiang Guo,a,b,2 and Rongcheng Lina,b,c,2,3

aKey Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, ChinabUniversity of Chinese Academy of Sciences, Beijing 100049, ChinacCAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Beijing 100093, China

ORCID IDs: 0000-0002-4772-7923 (Y.J.); 0000-0001-8346-3390 (R.L.).

Many plants monitor changes in day length (or photoperiod) and adjust the timing of the floral transition accordingly to ensurereproductive success. In long-day plants, a long-day photoperiod triggers the production of florigen, which promotes the floraltransition. FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana) encodes a major component of florigen, and FTexpression is activated in leaf veins specifically at dusk through the photoperiod pathway. Repression of FT mediated byPolycomb group (PcG) proteins prevents precocious flowering and adds another layer to FT regulation. Here, we identifiedhigh-level trimethylation of histone H3 at Lys 27 (H3K27me3) in the high trimethylation region (HTR) of the FT locus from thesecond intron to the 39 untranslated region. The HTR contains a cis-regulatory DNA element required for H3K27me3 enrichmentthat is recognized by the transcriptional repressor VIVIPAROUS1/ABSCISIC ACID INSENSITIVE3-LIKE1 (VAL1). VAL1directly represses FT expression before dusk and at night, coinciding with the high abundance of both VAL1 mRNA andVAL1 homodimer. Furthermore, VAL1 recruits LIKE HETEROCHROMATIN PROTEIN1 and MULTICOPY SUPRESSOR OFIRA1 to FT chromatin, leading to an H3K27me3 peak at the HTR of FT. These findings reveal a mechanism for PcG repression ofFT mediated by an intronic cis-silencing element and suggest a possible role for VAL1 in modulating PcG repression of FTduring the flowering response.

Polycomb group (PcG) proteins play important rolesin the transcriptional repression of developmentalgenes in multicellular eukaryotes ranging from plantsto humans (Simon and Kingston, 2013; Mozgova andHennig, 2015). PcG proteins form two main types ofcomplexes: polycomb repressive complex 1 (PCR1) andPRC2. The evolutionarily conserved PRC2 catalyzes thetrimethylation of histone H3 at Lys 27 (H3K27me3),whereas PRC1 mediates transcriptional repression viaH2AK119 monoubiquitylation and chromatin com-paction (Turck et al., 2007; Kim et al., 2012; Wang et al.,

2014; Mozgova and Hennig, 2015; Li et al., 2018). Giventhat PcG complexes, per se, do not specifically bind toDNA, several mechanisms are involved in recruitingPcG proteins to their target chromatin. In fruit fly(Drosophila melanogaster), multiple DNA-binding pro-teins recognize cis-regulatory regions known as Poly-comb response elements (PREs) and recruit PcG factors(Kassis and Brown, 2013). No consensus PRE sequencesexist in mammals. Instead, CpG islands and long non-coding RNAs play important roles in recruiting PRC2(Simon and Kingston, 2013; Mozgova and Hennig,2015).

In Arabidopsis (Arabidopsis thaliana), several cis-elements were initially identified based on their PRE-likeproperties (Xiao and Wagner, 2015). A 50-bp cis-repression element in the promoter region of LEAFYCOTYLEDON2, which harbors an RY motif, confersH3K27me3 deposition at transgenes (Berger et al., 2011).A PRE-like sequence in KNOX homeobox genes isbound by the ASYMMETRIC LEAVES complex, whichrecruits PRC2 to the target genes (Lodha et al., 2013). ThePRE-like element in the upstream region of the floralmeristem terminator KNUCKLES is required for itstranscriptional repression in a cell division-dependentmanner (Sun et al., 2014). Recent genome-wide studieshave uncovered putative PRE sequences in Arabidopsisand specific cis-motifs as consensus PREs (e.g. GAGAmotifs and telobox motifs) related to PcG recruitment,

1This work was supported by the National Key Research and De-velopment Program of China (2017YFA0503800), the Ministry of Ag-riculture of China (2016ZX08009-003), the National Natural ScienceFoundation of China (NSFC) (31870288, 31570310), and the YouthInnovation Promotion Association of the Chinese Academy ofSciences (Youth Innovation Promotion Association CAS) (2015064).

2Author for contact: rclin@ibcas.ac.cn.3Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Rongcheng Lin (rclin@ibcas.ac.cn).

Y.J. and Q.G. performed the experiments; Y.J. and R.L. designedthe experiments, analyzed the data, and wrote the manuscript.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.00642

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revealing an evolutionarily conserved model similar tothat inDrosophila (Xiao et al., 2017; Zhou et al., 2018). Inplants, long noncoding RNAs also bind to CURLYLEAF (CLF) or LIKEHETEROCHROMATIN PROTEIN1(LHP1) to recruit PcG factors to their targets (Heo andSung, 2011; Ariel et al., 2014). Nevertheless, it is un-known how changes in chromatin occur in response toenvironmental signals (He and Li, 2018).The B3-domain–containing transcription factors VI-

VIPAROUS1/ABSCISIC ACID INSENSITIVE3-LIKE1(VAL1) and VAL2 are thought to be widely involved inPRC1-mediated repression of specific target genes(Yang et al., 2013; Merini et al., 2017). VAL1/2 possess aplant-specific B3 DNA-binding domain that specificallyrecognizes Sph/RY elements (CATGCA) in the regu-latory regions of target genes (Suzuki et al., 1997;Swaminathan et al., 2008; Qüesta et al., 2016; Yuanet al., 2016; Chen et al., 2018). More than half of thegenes that are derepressed .4-fold in val1 val2 doublemutants possess at least one RY motif in their promoterregions or first introns (Suzuki et al., 2007). A PRE-likesequence comprising two to three nucleosomes aroundthe first intron of FLOWERING LOCUS C (FLC) con-tains two RY motifs that are recognized by VAL1/2.VAL1 recruits LHP1 and the apoptosis- and splicing-associated protein complex to deposit H3K27me3 at thenucleation region, thereby silencing FLC after vernali-zation (Qüesta et al., 2016; Yuan et al., 2016). VAL1directly interacts with MULTICOPY SUPRESSOR OFIRA1 (MSI1), the core PRC2 subunit, and specifi-cally binds to two RY elements in the promoter ofAGAMOUS-LIKE15 (AGL15), leading to the establish-ment of H3K27me3 and repressing seed maturation(Chen et al., 2018).In rice (Oryza sativa), the binding of VAL1/2 homo-

logs GERMINATION- DEFECTIVE1 and VAL2 to theintronic RY motif of ELONGATED UPPERMOST IN-TERNODE1 (Eui1) results in the recruitment of repres-sor complexes to down-regulate Eui1 expression viahistonemodification (Xie et al., 2018). Hence, the RY cis-element–mediated recruitment of chromatin-associatedproteins by VAL1/2 is likely a conserved negativeregulatory mechanism in plants. Structural analysis ofVAL1-B3 indicated that it formsH-bonds with all six bpin the RY motif (Sasnauskas et al., 2018). In addition tothe B3 DNA-binding domain, VALs contain three otherdomains, including the PHD-like (PHD-L) and cysteineand tryptophan residue-containing (CW) domains(which are associated with histone binding) and theethylene-responsive element binding factor-associatedamphiphilic repression (EAR) domain (Suzuki andMcCarty, 2008; Jo et al., 2019). PHD-L reads the meth-ylation state of histoneH3K27, whereas the CWdomainis responsible for the interaction between VAL1 andHISTONE DEACETYLASE19 (Zhou et al., 2013; Yuanet al., 2016).The transition from vegetative to reproductive

growth is a crucial developmental switch that ensuresreproductive success in flowering plants. This transi-tion is often synchronized with changes in seasonal

cues, such as day length or photoperiod, through thephotoperiod pathway (Andrés and Coupland, 2012).Photoperiod is perceived in leaves, leading to thetransmission of a major component of florigen encodedby FLOWERING LOCUS T (FT) from the leaves to theshoot apical meristem (Corbesier et al., 2007; Turcket al., 2008). Under long-day (LD) conditions, FT is ac-tivated, primarily by the transcriptional regulatorCONSTANS (CO; Andrés and Coupland, 2012; Songet al., 2015). Multiple transcription factors or regula-tors, including CO, directly bind to the proximal pro-moter of FT and regulate its expression (Andrés andCoupland, 2012). A chromatin loop brings the distalenhancer and proximal cis-elements of FT in closeproximity, leading to FT activation (Cao et al., 2014; Liuet al., 2014). Moreover, the direct binding of transcrip-tion factors to the introns and 39-untranslated region(39-UTR) of FT has also been demonstrated (Searle et al.,2006; Mathieu et al., 2009).Arabidopsis mutants of PcG factors, such as clf, em-

bryonic flower1 (emf1), emf2, and lhp1, display extremelyearly flowering, largely due to increased expression ofFT (Bratzel and Turck, 2015). CLF functions as anH3K27 methyltransferase that deposits H3K27me3 atFT, leading to its repression (Jiang et al., 2008). LHP1,SHORT LIFE (SHL), and EARLY BOLTING IN SHORTDAYS (EBS) are readers of the H3K27me3 marks oftarget genes; their binding to H3K27me3 on FT chro-matin results in its silencing (Turck et al., 2007; Zhanget al., 2007a; López-González et al., 2014; Li et al., 2018).Both LHP1 and the bromo adjacent homology (BAH)domain-containing proteins SHL and EBS directly in-teract with EMBRYONIC FLOWER 1 (EMF1), formingtwo plant-specific complexes. Both of these complexesserve as PRC1 that directly represses FT expression andthus regulates flowering (Wang et al., 2014; Li et al.,2018). PcG proteins likely repress FT expression be-fore dusk and at night, but it is unclear how the PcGcomplex silences FT expression.Here, we show that LHP1 and the repressive histone

markH3K27me3 are more highly enriched at the Lys 27(H3K27me3) region (HTR) within FT than in other re-gions.We identified a cis-regulatory DNA element (twoRY motifs) in the HTR that is recognized by the B3domain transcription factor VAL1. The levels of VAL1homodimer accumulation, the binding of VAL1 to FTchromatin, and VAL1 repression of FT expression werehigher at night than at dusk in LDs. Moreover, VAL1recruits the PcG proteins LHP1 and MSI1 to FT chro-matin, leading to the H3K27me3 peak at the HTR.

RESULTS

Identification of a High-Level H3K27 HTR at the FT Locus

The deposition of H3K27me3 occurs throughoutthe various regions of FT locus, including the pro-moter region, gene body, and (particularly) down-stream regions (Turck et al., 2007; Adrian et al., 2010).

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We examined whether a region in FT chromatin con-tains particularly high levels of H3K27me3. We pro-duced amplicons across FT locus from26.8 to 3.4 kb tofurther explore the contribution of PRC2 to the repres-sion of FT (Fig. 1A). We compared the H3K27me3profiles along FT in the leaves of wild-type Columbia(Col) and the mutant of the PRC2 core component CLFover a LD cycle. In Col, H3K27me3 was indeedenriched at various regions across the FT locus, with ahigh-level H3K27 HTR located from the second intronto the 39UTR (Fig. 1, A and B). In clf-28, the levels ofH3K27me3 across FT locus were strongly reduced, al-though substantial enrichment was still detected at theHTR (Fig. 1, A and B). The residual level of H3K27me3at the FT locus in clfmutants is likely due to the H3K27methyltransferase redundancywith SWINGER (Farronaet al., 2011).

LHP1 [also known as TERMINAL FLOWER2] bindsto targets that colocalized with H3K27me3 (Turck et al.,2007; Zhang et al., 2007a). LHP1 is expressed in vasculartissue, and LHP1 is a component of PRC1 that bindsto FT locus and represses its expression (Kotake et al.,2003; Adrian et al., 2010; Wang et al., 2014). H3K27me3levels at FT locus in Col and tfl2-1/lhp1-3were exploredby ChIP–quantitative qPCR (qPCR); however, no sig-nificant changes were observed (Supplemental Fig.S1F), which is probably due to that LHP1 and EMF1 orthe BAH-domain–containing proteins SHL and EBS acttogether in binding and maintaining H3K27me3 at FTlocus (López-González et al., 2014; Wang et al., 2014; Liet al., 2018). We generated LHP1p:LHP1-GFP transgenicplants in the tfl2-1 background and found that the ex-tremely early flowering phenotype of tfl2-1was rescued

in the homozygous lines, which showed similar FTexpression levels to that of Col (Supplemental Fig. S1,A–C), implying that LHP1p:LHP1-GFP is biologicallyfunctional. LHP1-GFP fusion proteins produced fluo-rescent signals in the nucleus (Supplemental Fig. S1D).We then performed ChIP assays using LHP1p:LHP1-GFP tfl2-1, in which the LHP1-GFP fusion protein wasenriched after immunoprecipitation (Supplemental Fig.S1E). LHP1-GFP was strongly enriched on FT chro-matin, especially at the H3K27me3 HTR (Fig. 1C). Fi-nally, ectopic FT activation is known to occur (but notectopically expressed outside of the phloem) in plantsin the absence of PRC2 and PRC1 function (Moon et al.,2003; Steinbach and Hennig, 2014; Wang et al., 2014).These observations indicate that PRC2 and PRC1 arerequired for proper FT expression.

Identification of Two RY Motifs as the Putative FTRepression Element

PcG proteins are recruited to their target chromatinand mediate the spread of H3K27me3 to the surround-ing regions (Mozgova and Hennig, 2015; Xiao andWagner, 2015). The role of PREs in the recruitment ofPcG to specific genes is conserved in Arabidopsis andDrosophila (Kassis and Brown, 2013; Xiao et al., 2017;Zhou et al., 2018).We therefore attempted to identify thecis-regulatory element in the HTR required for FT ex-pression. We generated a reporter construct in whichGUSwas located downstream of a cassette including FTenhancer and proximal promoter (hereafter referredto as EP-GUS; Fig. 2A; Liu et al., 2014). The putative

Figure 1. Enrichment of H3K27me3 and LHP1 at the FT locus. A, Schematic diagram of the FT genomic region. Exons and the59UTR (or 39UTR) are represented by black and gray boxes, respectively, whereas other genomic regions are represented by ablack line. Numbers indicate the positions of amplicons for the chromatin immunoprecipitation (ChIP) experiments. B, ChIPexperiments showing relative fold enrichments of H3K27me3 across the FT locus in wild-type (WT) and clf-28 seedlings at ZT8 inLD. C, ChIPanalysis of LHP1-GFPenrichment on FT chromatin of wild-type (Col) and LHP1p:LHP1-GFP tfl2-1 seedlings grown inLDs at ZT2. The signals from Col samples were set to 1. In (B) and (C), error bars represent SDs of at least three independentbiological replicates, and ACT2 (ACTIN 2) and/or Ta3 were used as controls.

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PRE-containing fragments (PUTs) were inserted be-tween the enhancer and proximal promoter to generateEP-PUT constructs (Fig. 2A). We then separated theHTR into intron 2 (PUT1), intron 3 (PUT2), and adjacentdownstream PUT3 (similar length to PUT1) and ex-amined their transcriptional activity in transgenicplants. The presence of PUT1, but not PUT2 or PUT3,was sufficient to repress GUS expression (Fig. 2, Band C).Drosophila PREs are nucleosome depleted, perhaps to

facilitate the assembly of the multiprotein PRC1 andPRC2 complexes (Mohd-Sarip et al., 2005, 2006).We then added additional amplicons around theHTR and performed ChIP and FAIRE (formaldehyde‐assisted isolation of regulatory elements) assays tonarrow down the putative PRE-containing region(Supplemental Fig. S2A). The HRTs were located insimilar regions in H3K27me3 ChIP normalized to eitherhistoneH3 or input (Supplemental Fig. S2, B andC).Weidentified a nucleosome-depleted region located in the

HRT from the latter half of intron 2 (amplicon 14) tothe 39UTR (amplicon f), in which PUT4 overlapswith PUT1, as a possible PRE-containing fragment(Supplemental Fig. S2D). Indeed, the use of PUT4, butnot PUT5, reduced GUS activity in the assay (Fig. 2, Aand B). Using a sequence truncation approach, wefound that the presence of a 183-bp PUT6 region wassufficient to repress GUS expression (Fig. 2, A–C). TwoRY motifs shown to trigger the epigenetic repression oftarget genes were foundwithin PUT6. The simultaneousmutation (EP-PUT6m) and deletion (EP-PUT6Δ24) ofthese two RY motifs in PUT6 led to the release of GUSrepression (Fig. 2C), suggesting that RY motifs mightfunction as FT silencing elements.PREs act as cis-motifs for PcG recruitment, leading to

the deposition of H3K27me3 not only on genes con-taining these motifs, but also on adjacent genes (Simonand Kingston, 2013; Xiao et al., 2017). We conductedChIP assays to explore whether the putative PRE-containing regions are involved in the enrichment of

Figure 2. Identification of the FT cis-silencing elements. A, Schematic representation of the sequence truncation series of theputative FT PREs (PUTs, blue boxes) fused to the GUS reporter gene. The PUT fragments were placed between the 548-bp en-hancer and the 527-bp proximal promoter to generate various EP-PUT cassettes and driveGUS expression. The two RY (TGCATG;R, purine; Y, pyrimidine) motifs are marked by arrowheads. B and C, GUS expression analysis of T1 transgenic seedlingsexpressing the indicated EP-PUTs. The total number of plants of each transgenic line analyzed is indicated above the columns.S1M indicate lines with strong or medium GUS staining, whereas W1N indicate lines with weak or none GUS staining. Theseventh set of leaves is shown. Scale bar5 5 mm. D, ChIP experiments showing H3K27me3 accumulation relative to H3 on theindicated amplicons in 10-d-old seedlings of the indicated genotype. Error bars are means 6 SD of one biological replicatewith three technical repeats for samples from at least three independent transgenic lines. Strong GUS staining lines were chosenfor EP:GUS, EP-PUT7:GUS, EP-PUT6m:GUS, and EP-PUT6Δ24:GUS, whereas weak GUS staining lines were used forEP-PUT1:GUS, EP-PUT4:GUS, and EP-PUT6:GUS. The signal from FT amplicon 15 was set to 1. The locations of the FTamplicons are shown in Figure 1.

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H3K27me3 at the GUS locus. In transgenic plants car-rying RY motifs, the levels of H3K27me3 on GUS werehigher than those of the wild type, in agreement withthe notion that the GUS reporter genes were silenced.By contrast, in transgenic plants with mutated or no RYmotifs, the levels of H3K27me3 at the GUS locus werelower than those of the wild type (Fig. 2D). Hence, theRYmotifs are required forH3K27me3 binding andGUSsilencing.

The Intronic RY Motifs of FT Confer Flowering Repressionin LDs

The fusion of the FT enhancer and the proximalpromoter sequence constitutes a short promoter (here-after referred to as the EP promoter) that respondsnormally to day length (Liu et al., 2014). We investi-gated the biological effects of the RYmotifs on the floraltransition in LDs by generating various FT genomicfragments driven by the EP promoter and introducingthem into ft-10 plants (Fig. 3A). All transgenic plantsexpressing FT fragments under the control of EP pro-moters flowered earlier than the parent line ft-10 (Fig. 3,A and B), suggesting that these constructs were bio-logically functional.

Plants with a deletion of intron 1 or intron 2(EP:FTg2800Δ intron 1 ft-10 and EP:FTg2800Δintron 2 ft-10) showed earlier flowering than EP:FTg2800 ft-10,whereas plants harboring a deletion of intron 3(EP:FTg2800Δ intron 3 ft-10) exhibited a floweringphenotype similar to that of EP:FTg2800 ft-10, sug-gesting that both intron 1 and intron 2 might harbor FTcis-repression elements.

We further examined the role of RY motifs by intro-ducing an FTg2800 transgene with these two RY motifsdeleted (FTg2800Δ24) into the ft-10 background andfound that these plants exhibited earlier floweringthan those harboring the wild-type FTg2800 transgene(EP:FTg2800 ft-10; Fig. 3). Taken together, these resultsindicate that the RY-motif–containing region serves asan FT-repressing element.

The Transcriptional Repressor VAL1 Binds toFT-Repressing RY Motifs

Plant-specific B3-domain transcription factors bind toRY motifs (Reidt et al., 2000; Braybrook et al., 2006;Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018;Sasnauskas et al., 2018). The B3 domain of VAL1 isnecessary and sufficient for its binding to RY motifs(Suzuki et al., 1997; Qüesta et al., 2016; Sasnauskas et al.,2018). We therefore performed electrophoretic mobilityshift assays (EMSAs) using recombinant VAL1-B3fragments tagged with glutathione S-transferase (GST).GST-VAL1-B3 caused an up-shift of the biotinylatedwild-typeRY1/2probe but not the RY1/2mutant probe.We also detected binding of VAL1 B3 to single RY sitesRY1 and RY2 (Crick strand of and just downstream of

RY1; Fig. 4, A–D). Moreover, the amounts of shiftedbands were substantially attenuated by the addition ofexcess unlabeled wild-type DNA but not by com-petitors with mutated RY motifs (Fig. 4, B and C).

Figure 3. Phenotypic analysis of plants harboring FT transgenes drivenby the recombinant EP promoter. A, Schematic diagram of the FTtransgenes. The number 2800 indicates 12800 bp relative to the startcodon. The exons and 39UTR are represented by black and gray boxes,respectively; the deletion regions are represented by dashed lines; andthe remaining genomic regions are represented by black lines. The FTtransgenes were driven by the EP promoter to which the FT enhancerand proximal promoter were serially fused. B, Total number of leaves atflowering for the indicated plants (in the ft-10 background) grown underLD conditions. Each red triangle represents a single plant (n $ 23);means (black horizontal lines) 6 SD (error bars) are shown. Statisticalsignificance was determined by one-way ANOVA and multiple com-parison by the Duncan method (P 5 0.05). Letters above the bars in-dicate significant differences. C, Distribution of flowering time in T1transgenic plants carrying the indicated FT genomic fragments in theft-10 background grown in LDs. In (B) and (C), ft-10 served as a control.

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Interestingly, the presence of double RY motifs facili-tated the formation of nonspecific complexes (multipleprotein copies bound to a single DNA, indicated byarrowheads in Fig. 4, A and B).We performed yeast one-hybrid assays to explore the

two RY motifs recognized by VAL1. Fusion proteinscomprising the GAL4 transcriptional activation domainfused to VAL1 (GAD-VAL1), but not GAD-VAL2,GAD-RAV1, GAD-RAV2, or GAD alone, activated theLacZ reporter gene driven by the RY-motif–containingPUT4 and PUT6 fragments (Fig. 4, E and F).Mutation ofany of the two RY motifs (RY1-m or RY2-m) in PUT6attenuated the binding of VAL1, whereas both RYmutations (PUT6-RY1/2-m) and deletions (PUT6Δ24)abolished the activation of the reporter gene by GAD-VAL1 (Fig. 4F). These results indicate that VAL1 rec-ognizes and binds to the RY motifs of the FT repressionelement. We aligned the intronic sequences of FT from

Arabidopsis and FT homologs from other plants ofthe Brassicaceae family, including Arabidopsis lyrata,Brassica rapa, and Brassica oleracea. This alignmentrevealed that both RY motifs are highly conserved inspecies of the Brassicaceae family (Fig. 4G), suggestingthey play important roles in regulating FT expression.To investigate whether VAL1 directly regulates FT

expression, we generated VAL1p:VAL1-glucocorticoidreceptor (GR) val1-2 transgenic plants, in which VAL1was fused with the GR and driven by the 3.4-kb nativeVAL1 promoter. In the presence of dexamethasone(DEX), the flowering phenotype of VAL1p:VAL1-GRval1-2 was rescued (Supplemental Fig. S3, A and B).AGAMOUS LIKE15 (AGL15) is a direct target of VAL1(Chen et al., 2018). Both val1-2 and VAL1p:VAL1-GRval1-2 plants exhibited higher levels of AGL15 than thewild type, whereas only VAL1p:VAL1-GR val1-2 wasrescued by DEX induction (Supplemental Fig. S3C),

Figure 4. VAL1 binds to the intronic RY motifsand represses FT expression. A to D, EMSAshowing that GST-VAL1-B3 protein, but notGST alone, specifically binds to the double(RY1/2-wt; A andB) or single (RY1-wt and RY2-wt) RY wild-type probes (C and D) but not themutant probes (RY1/2-m, RY1-m, and RY/2-m;B to D). Arrows indicate the specific complex,arrowheads indicate the nonspecific complex,and square brackets indicate free probe. E,GAD-VAL1, but not GAD-VAL2, GAD-RAV1,GAD-RAV2, or GAD alone, activates the ex-pression of the LacZ reporter gene driven by thePUT4 fragment (shown in Fig. 2) in yeast. F,GAD-VAL1 activates the LacZ reporter genedriven by the wild-type PUT6 fragment (shownin Fig. 2) in yeast. Single mutations (RY1m andRY2m) and simultaneous mutation (RY1/2m) ofthe RYmotifs attenuate or abolish the activationof LacZ gene expression. G, Evolutionary con-servation of the RY motifs in FT intronic se-quences from different Brassicaceae species.RY motifs (CATGCA, Crick strand TGCATG)are shown in red, and the Crick strand isunderlined. H, Transcript levels of FT inVAL1p:VAL1-GR val1-2 transgenic seedlings inLDs. The 10-d-old seedlings were harvested atZT4 after treatment for 4 h with or without DEX(20 mM) in the presence or absence of CHX(50 mM). Expression levels were normalized toIPP2. Means 6 SD from three biological repli-cates are shown (*P , 0.05, **P , 0.01, Stu-dent’s t test). I, GUS staining of the indicatedtransgenic GUS lines (as depicted in Fig. 2A) inthe 35S:VAL1-GR background. Seedlings weregrown in MS medium with or without 20 mMDEX (Mock, 0.1% [v/v] ethanol) for 6 d in LDs. Atleast two independent transgenic GUS lineswere examined with similar results. Scalebar 5 1 mm.

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demonstrating that VAL1-GR is functional. DEX treat-ment supplemented with or without cycloheximide, aprotein synthesis inhibitor, led to reduced FT expres-sion (Fig. 4H), suggesting that the repression of FT re-quires nucleus-localized VAL1. However, upon VAL1mutation, FT expression was down-regulated and theplants flowered late (Supplemental Fig. S3, D and E),arguing against the notion that VAL1 represses FTexpression.

MADS domain transcription factors FLC andAGL15,whose encoding genes are directly repressed by VAL1,act as FT transcriptional repressors to prevent flowering(Searle et al., 2006; Adamczyk et al., 2007; Qüesta et al.,2016; Yuan et al., 2016; Chen et al., 2018). We reasonedthat the defect in FT repression in the val1 mutantsmight be due to the secondary effects of VAL1 on theregulation of flowering. This notion is supported by thefinding that FT expression and flowering time werelargely rescued in val flc (Supplemental Fig. S3, D andE). GUS transcript levels and GUS staining activitywere explored in the randomly chosen T1 EP:GUS andEP-PUTs:GUS reporter lines. The attitudes of GUSstaining reflect GUS mRNA levels (Supplemental Fig.S3F), supporting that these lineswere effective.We thencrossed EP:GUS and EP-PUTs:GUS reporter lines toval1-2 and to plants harboring functional 35S:VAL1-GR(Fig. 4I; Supplemental Fig. S3, G and H). Indeed, GUSexpression driven by RY-motif-containing fragments(EP-PUT6:GUS) was derepressed in the val1-2 back-ground, whereas GUS expression driven by fragmentslacking RY motifs (EP-PUT6Δ24:GUS val1-2) showedalmost no effect upon VAL1 mutation (SupplementalFig. S3H). When introduced into 35S:VAL1-GR plants,the weak GUS activity of EP-PUT6:GUS was furtherreduced in response to DEX treatment compared withmock treatment, whereas little difference in GUSstaining was observed in the other reporter lines(bearing a mutated or no RY motif, i.e. EP-PUT6m:GUS35S:VAL1-GR and EP-PUT6Δ24:GUS 35S:VAL1-GR)with or without DEX treatment (Fig. 4I). Furthermore,we conducted luciferase-based transient assay. TheNicotiana benthamiana cells coexpressing 35S:GFP withPUT4:LUC (luciferase) or PUT4m:LUC displayed com-parable signals, whereas the cells coexpressing35S:VAL1-GFP/PUT4:LUC displayed attenuated sig-nals comparedwith those coexpressing 35S:VAL1-GFP/PUT4m:LUC (Supplemental Fig. S3, I and J), indicat-ing that VAL1 repressed the expression of FT RY-motif–containing fragment of PUT4:LUC. Collectively,these results support the notion that the transcriptionfactor VAL1 represses FT expression via its RY motifs.

VAL1 Directly Represses FT Expression before Dusk andat Night in LDs

The binding of the PcG factors CLF, LHP1, and EMF1and the histone mark H3K27me3 to FT locus is reducedat dusk compared with night and midday (Luo et al.,2018). We explored whether VAL1 is involved in the

photoperiodic regulation of flowering time. We exam-ined the diurnal expression patterns of VAL1 in LDsand found that VAL1 mRNA levels peaked at ZT0,declined during the day, reached a trough at ZT16, andsubsequently increased (Fig. 5A). VAL2 showed asimilar expression pattern, but with much weakerfluctuations, whereas VAL3 was expressed at very lowlevels throughout the period (Fig. 5A). Consistent withthis notion, microarray data from the eFP browsershowed that the expression of VAL1, but not VAL2 orVAL3, oscillates and reaches a trough at the end of theday (Supplemental Fig. S4; Michael et al., 2008). VAL1was expressed in leaf veins of both cotyledons and trueleaves (Fig. 5B).

To explore whether VAL1 binds to FT chromatin, wegenerated VAL1p:VAL1-GFP val1-2 transgenic plants inwhich VAL1-GFP was driven by the VAL1 native pro-moter. The representative lines exhibited similar flow-ering times and FT expression levels as those of the wildtype (Supplemental Fig. S5, A–C) and produced fluo-rescent signals in the nucleus (Supplemental Fig. S5D),implying that the VAL1-GFP fusion protein is biologi-cally functional. We conducted ChIP using represen-tative VAL1p:VAL1-GFP val1-2 seedlings harvested atZT16 and ZT24. At ZT24, VAL1-GFP was stronglyenriched on amplicon 14 (containing RY motifs) and onamplicon 15 to a lesser extent, whereas at ZT16, theenrichment of VAL1-GFP at FT was significantly re-duced (Fig. 5C). We measured the protein level ofVAL1-GFP using VAL1p:VAL1-GFP val1-2 seedlingsharvested at ZT4, ZT16, and ZT24 in LDs and foundthat the protein levels did not change obviously(Supplemental Fig. S5E), indicating that the reducedbinding ability of VAL1-GFP at FT locus at ZT16 mightnot be due to the VAL1-GFP protein abundance.

The MADS domain transcription factors FLC andAGL15, whose encoding genes are directly repressedby VAL1, act as FT transcriptional repressors to preventflowering (Searle et al., 2006; Adamczyk et al., 2007;Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018).To identify the particular time in LDs when VAL1represses FT expression and to avoid the effects of FLCand AGL15 (Searle et al., 2006; Adamczyk et al., 2007;Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018),we measured FT transcript levels using RNA extractedfrom dissected leaves of VAL1p:VAL1-GR val1-2 trans-genic plants simultaneously treated with DEX and cy-cloheximide (CHX; to reduce secondary transcriptionaleffects after VAL1 moves into the nucleus). The re-pression of FT byVAL1wasmuch strongerwhenVAL1activity was induced at midday (ZT6) and at the end ofthe night (ZT24) versus dusk (ZT16; Fig. 5D). Theseresults indicate that the ability of VAL1 to bind to andrepress FT depends on day length, with maximum ac-tivity at night, which coincides well with the findingthat VAL1 is strongly expressed at this time.

VAL1 likely forms homodimers that bind to the RYmotifs in its target loci (Qüesta et al., 2016; Yuan et al.,2016). We therefore generated VAL1p:VAL1-GFP35S:Myc-VAL1 val1-2 transgenic lines and performed

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coimmunoprecipitation assays. VAL1 indeed formedhomodimers in seedlings harvested at ZT16 and ZT24(Fig. 5E), as previously reported (Chhun et al., 2016).Intriguingly, the homodimerization of VAL1 was sig-nificantly higher at ZT24 compared with ZT16 (Fig. 5, Eand F). This finding is consistent with the observationthat higher levels of VAL1 binding to FT chromatin andstronger repression of FT byVAL1 occur at night versusthe end of LDs.

VAL1 Recruits PcG Proteins LHP1 and MSI1 to theFT Locus

LHP1 and MSI1 interact with VAL1 to repress theexpression of target genes involved in seed maturationand the control of flowering time (Yuan et al., 2016;Chen et al., 2018). MSI1 regulates the photoperiodic

floral transition (Steinbach and Hennig, 2014). Wetherefore investigated whether the enrichment of LHP1and MSI1 on FT chromatin is dependent on VAL1. Wegenerated 35S:Myc-MSI1 transgenic plants in whichMyc-MSI1 fusion protein was enriched after immuno-precipitation (Supplemental Fig. S6, A and B). We in-troduced the LHP1p:LHP1-GFP and 35S:Myc-MSI1constructs into 35S:VAL1-GR transgenic lines andmeasured the association of LHP1-GFP and Myc-MSI1with FT chromatin by ChIP assays. Both LHP1-GFP andMyc-MSI1 were enriched at the HTR of FT after35S:VAL1-GR was induced by DEX treatment com-pared with the mock control (Fig. 6, A and B). Thus,VAL1 binding to the RY motif in the HTR increasedrecruitment of both LHP1 and MSI1 to FT chromatin.MSI1 is a component of PRC2 that catalyzes

H3K27me3 (Köhler et al., 2003). In addition, LHP1reads the H3K27me3 marks deposited by PRC2

Figure 5. Analysis of VAL1 expression patterns and its binding to FT chromatin. A, VAL1, VAL2, and VAL3mRNA levels in 10-d-old (wild type [WT]) seedlings over a 24-h LD cycle. The mRNA levels were normalized to IPP2. Error bars indicate SD of threeindependent biological replicates. White and black bars indicate light and dark periods, respectively. B, Spatial expressionpatterns of VAL1p:GUS plants grown in LDs determined by histochemical staining for GUS activity. Scale bars 5 1 mm (thecotyledon) and 2 mm (the fifth set of true leaf), respectively. C, ChIPanalysis of VAL1-GFP enrichment at the FT locus in 10-d-oldwild type (Col) and VAL1p:VAL1-GFP val1-2 seedlings grown in LDs. The levels of immunoprecipitated genomic fragments weremeasured by qPCR; the signals from Col samples were set to 1. Ta3 was used as a negative control. Error bars represent SD fromthree independent biological replicates (*P, 0.05, Student’s t test). The locations of the FT amplicons are shown in Figure 1. D,FTmRNA levels in VAL1p:VAL1-GR val1-2 seedlings grown for 10 d in LDs. Seedlings were harvested at the indicated ZTs aftertreatment for 2 hwithDEX (20mM) in the presence or absence of CHX (50mM). Expression levelswere normalized to IPP2. Valuesare means6 SD of two biological replicates for samples from three independent transgenic lines. The signals fromCHX treatmentswere set to 1.E, Coimmunoprecipitation assay showing the homodimerization of VAL1 in 10-d-old seedlings expressing theVAL1p:VAL1-GFP and 35S:Myc-VAL1 constructs. The panel at bottom shows higher intensity coimmunoprecipitation signalsdetected by Myc antibody. F, Quantification of the homodimerization level of VAL1 after normalization to immunoprecipitatedGFP using ImageJ software. The level of VAL1 homodimer at ZT16 was set to 1. Means6 SD from three biological replicates areshown. In (D) and (F) Significance of differences from ZT16 was calculated using Student’s t test (**P , 0.01).

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(Turck et al., 2007; Zhang et al., 2007a). MSI1 interactswith LHP1; this interaction links LHP1 to PRC2 andfacilitates the recruitment of PRC2 to chromatin bearingH3K27me3 (Derkacheva et al., 2013). We examinedwhether VAL1 recruits LHP1 and MSI1 to promoteH3K27me3 deposition at the FT locus by measuringH3K27me3 levels on FT chromatin in 35S:VAL1-GRval1 seedlings. Upon DEX treatment, H3K27me3 wasenriched at the FT locus at the HTR compared with theuninduced control (Fig. 6C). FT transcript levels in35S:VAL1-GR versus LHP1p:LHP1-GFP 35S:VAL1-GRor 35S:Myc-MSI1 35S:VAL1-GR were also examined byqPCR; however, no significant changes upon DEXtreatment were observed (Supplemental Fig. S7),probably due to that the encoding genes of FT repres-sors, such as FLC and AGL15, were also down-regulated upon DEX treatment in LHP1p:LHP1-GFP35S:VAL1-GR and 35S:Myc-MSI1 35S:VAL1-GR(Supplemental Fig. S7).

val1/2 strong mutants exhibit embryonic traits asthose in the mutants severely compromised of PRC2 orPRC1 function (Supplemental Fig. S8A; Yang et al.,2013; Jo et al., 2019). We further examined the role ofVAL1/2 proteins in H3K27 trimethylation at FT locus.Doublemutation ofVAL1 andVAL2 caused a reductionof H3K27me3 levels, but did reach the levels as those inclf-28, indicating VAL1/2 proteins mediate H3K27me3deposition on FT chromatin (Supplemental Fig. S8B).Together, these findings indicate that VAL1 repressesFT transcription via facilitating the recruitment of LHP1and MSI1 and mediating H3K27 trimethylation at theFT HTR.

DISCUSSION

In this study, we demonstrated that the plant-specifictranscription factor VAL1 contributes to periodic PcGrepression of the florigen gene FT in Arabidopsis underLDs. VAL1 binds specifically to the two intronic RYmotifs at the HTR of the FT locus, likely as a homo-dimer, which accumulates to higher levels at night thanat dusk in LDs. Thus, LHP1 andMSI1 recruitment to the

HTR is facilitated by interacting with VAL1, leading toincreased levels of H3K27me3 on FT and its repressionat night.

The FT locus is widely bound by LHP1 and the re-pressive mark H3K27me3, especially at the down-stream region (Turck et al., 2007; Zhang et al., 2007b;Adrian et al., 2010). The H3K27me3 and LHP1 profilesacross FT in our ChIP assays largely overlap with thoseobtained in previous studies, with an HTR covering thesecond intron and the 39UTR (Fig. 1). PcG-induced si-lencing of FLC is best characterized in Arabidopsis.During vernalization, H3K27me3 levels increase locallyat the nucleation region of FLC in response to cold andspread over the entire FLC gene body following transferto warm conditions (Qüesta et al., 2016; Yang et al.,2017). The nucleation of H3K27me3 occurs in a regionof two or three nucleosomes around the first exon,which confers metastable epigenetic silencing of FLC inthe cold. The spreading of H3K27me3 across the FLClocus results in the stable silencing of FLC after transferto warm conditions (Yang et al., 2017). Both the PcGfactors LHP1 and CLF and H3K27me3 are enriched onFT chromatin (Luo et al., 2018). Consistently, we foundthat both LHP1 and H3K27me3 highly enriched atthe HTR of the FT locus (Fig. 1, B and C). We inferredthat the HTR may confer metastable epigenetic re-pression of FT during the photoperiodic response torapid light/dark cycles.

We narrowed down a putative PRE-containing re-gion based on molecular dissection of the HTR via GUSstaining and colocalization analysis of the HTR andnucleosome-depleted region revealed by ChIP andFAIRE (Fig. 2, A–C; Supplemental Fig. S2). RY motifswere shown to be involved in the epigenetic silencing oftarget genes. Further experiments (i.e. RY motif dele-tion and mutagenesis approaches) confirmed that RYmotifs are necessary to confer the repressive activity ofH3K27me3 deposition (Fig. 2D). The FAIRE assayrevealed four nucleosome-depleted regions across theFT locus. The proximal promoter region (amplicons 9and 10) carries two CO-responsive elements (COREs;containing CCACA) and is bound by the output ofthe photoperiod pathway (CO; Adrian et al., 2010;

Figure 6. VAL1-mediated PcG repression of FT expression. A to C, Association of LHP1-GFP (A), Myc-MSI1 (B), and H3K27me3(C) with the FT locus revealed by ChIP-qPCR. The 10-d-old seedlings were treated with or without 20 mM DEX for 4 h andharvested at ZT4. qPCR signals from DEX-treated samples were normalized to input (A and (B) and H3 (C), respectively. Thesignals frommock-treated samples (Mock) were set to 1. TA3was used as a negative control. Values in (A) to (C) aremeans6 SD ofthree biological replicates. The locations of the FT amplicons are shown in Figure 1.

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Tiwari et al., 2010; Song et al., 2012), suggesting thisregion might facilitate CO-dependent FT expression,consistent with our previous report (Jing et al., 2019). AFAIRE signal peak covered the latter half of intron 2 tothe 39UTR and overlapped with the HTR, harboringtwo RY motifs bound by VAL1. These findings suggestthat this region is accessible to the transcriptional re-pressor VAL1, which recognizes the RY motif, therebyenabling the recruitment of PcG proteins LHP1 andMSI1. Further experiments are needed to investigatewhether this region favors the assembly of the multi-protein complexes PRC2, as shown in Drosophila(Mohd-Sarip et al., 2005, 2006). The first FAIRE peak(amplicon 6) appears to be bound by chromatin-remodeling factor PICKLE (Jing et al., 2019), whereasin the region of the third FAIRE peak (amplicon 12), nocandidate transcription factors have been reported thatrecognize the target DNA sequences.An experiment using the short fusion promoter (EP

promoter) driving FT expression revealed that the twoRY motifs are required for the proper flowering re-sponse in LDs (Fig. 3A). Interestingly, plants harboringconstructs with a deletion of intron 1 or intron 2 of theFT genomic sequence (EP:FTg2800Δintron1 ft-10 andEP:FTg2800Δintron2 ft-10) exhibited earlier floweringthan those harboring a construct lacking a deletion(EP:FTg2800 ft-10), indicating that the polymorphismsin the cis-regulatory regions within intron 1 or intron2 negatively modulate its function.Several proteins, including FLC, can bind to the first

intron in FT to repress its expression (Golembeski andImaizumi, 2015). We cannot exclude the possibility thatintron 2 carries other cis-silencing elements besides RYmotifs. Similar to our results, introducing an FLCtransgene with a deleted RY motif (FLCD10) into the flcmutant background led to the up-regulation of FLC andresulted in late flowering (Yuan et al., 2016). The re-sponse of flowering to the disruption of the intronicnative RY motifs of FT remains to be examined. A re-cently discovered dominant rice mutant, dwarf Eui1(dEui1), exhibits a severe enclosed-panicle phenotype.The phenotypic defects in dEui1 are caused by increasedexpression of Eui1 in which T-DNA replaces the 135-bpfragment containing a cis-silencing element harboring anRY motif in the Eui1 intron (Xie et al., 2018).Unlike the repressive effect of VAL1 on FT expres-

sion, the val1 mutant exhibited late flowering and de-creased FT expression (Supplemental Fig. S3, D and E).One possible explanation is that two proteins of VAL1silencing targets, FLC and AGL15, are both direct re-pressors of FT (Searle et al., 2006; Adamczyk et al., 2007;Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018).This study provides several lines of evidence support-ing this hypothesis. First, introducing the flc mutationinto val1-2 strongly attenuated the late-flowering phe-notype of val1-2 and derepressed FT transcription(Supplemental Fig. S3, D and E). Second, GUS activityin the transgenic line EP-PUT6:GUS (harboring the GUSreporter gene driven by the RY-motif-containing fragmentPUT6) increased upon VAL1 mutation (Supplemental

Fig. S3G). Third, FT expression was much more stronglyrepressed in VAL1p:VAL1-GR val1-2 plants after DEX andCHX treatment compared with DEX treatment alone(Fig. 4H), indicating that the secondary effect of VAL1 onthe transcriptional regulation of FT was attenuated byDEX/CHX treatment.H3K27me3 was largely reduced in the val1/2 double

mutant, but not to the level of that in clf-28 (SupplementalFig. S8B), indicating that there may exist other proteinsother than VAL1/2 to mediate PcG repression of FT. FLCwas previously shown todirectly bind to thefirst intron ofFT and directly interacts with EMF1, and this interactionis required for FT repression (Searle et al., 2006; Wanget al., 2014), suggesting that other than VAL1-PcG, FLC-PcG is also required for FT repression. Further workwould be needed to determine how FLC-PcG and VAL1-PcG function in concert to repress FT expression.VAL1 recruits repressive factors or complexes to its

target loci. Both VAL1 and LHP1 can read H3K27me3marks and interact with the PRC2 component MSI1(Turck et al., 2007; Zhang et al., 2007a; Derkachevaet al., 2013; Yuan et al., 2016). Our results suggest thatVAL1 (and possibly VAL2) binds to RY motifs andrecruits LHP1 to FT chromatin, which may favor therecruitment of a PRC2 complex (including the indis-pensable subunit MSI1) to FT chromatin to depositH3K27me3, resulting in the H3K27me3 peak at theHTR. The abundance of VAL1 mRNA and VAL1homodimers (and possibly heterodimers with VAL2),the binding of VAL1 and H3K27me3 to the HTR, andthe repression of FT expression by VAL1 exhibited aperiodic pattern, with higher levels at night than atdusk. We also examined the enrichment of LHP1-GFP,Myc-MSI1, and the repressive histone mark H3K27me3at FT locus over a LD cycle using ChIP-qPCR. Whencompared with those at ZT4 and ZT24, the levels ofLHP1-GFP and H3K27me3, but not Myc-MSI1, at theHTR fragment (amplicon 15) were reduced at dusk(ZT16; Supplemental Fig. S8, C–E). These results, to-gether with observations that PcG factors repress FTexpression at night and from dawn to late afternoonunder LDs and that PcG factors (CLF, EMF1, and LHP1)moderately associate with FT chromatin at dusk (Wanget al., 2014; Luo et al., 2018), suggest that VAL1 functionsin PcG repression of FT at night and from dawn to lateafternoon in LDs. When CO accumulates from late af-ternoon through dusk, VAL1-mediated PcG repressionof FT is relieved to some extent; thus, FT is derepressed.Together, these findings indicate that the transcriptionalrepressor VAL1 recognizes RY motifs and triggers epi-genetic repression of the florigen gene FT, conferring thephotoperiodic control of flowering in angiosperms.

METHODS

Plant Material and Growth Conditions

All Arabidopsis (Arabidopsis thaliana) plants used in this study are of theColumbia (Col) ecotype, and all stable transgenic lines were generated by floraldip usingAgrobacterium tumefaciens strain GV3101 (Clough and Bent, 1998). The

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mutants ft-10 (GK-290E08; Yoo et al., 2005), flc-6 (SALK_041126; Schönrock et al.,2006), val1-2 (SALK_088606; Suzuki et al., 2007), val2-3 (SALK_059568C; Yanget al., 2013), clf-28 (Doyle and Amasino, 2009), and tfl2-1 (Larsson et al., 1998)were obtained from the Arabidopsis Biological Resource Center (ABRC; TheOhio State University, Columbus). Double mutants/transgenic plants weregenerated by genetic crossing, and homozygous lines were used. Surface-sterilized seeds were plated on half-strength Murashige and Skoog mediumcontaining 0.6% (w/v) phytoagar, 3% (w/v) Suc, and 0.05% (w/v) MES (pH5.7) and kept for 3 d at 4°C in the dark before being transferred to the growthroom. Plants were grown pots of soil in controlled conditions at 22°C, under LD(16-h light/8-h dark).

Plasmid Construction and Plant Transformation

The distal enhancer and proximal promoter of FT reported previously(Adrian et al., 2010; Liu et al., 2014) were fused and inserted into theHindIII andSalI restriction sites of a pBI101.1-derived plasmid to make the construct EP-GUS4-pBI. A 2.8-kb FT genomic fragment (FTg2800), DNA fragments con-taining a deletion mutant (FTg2800Δ24), and FT complementary DNA (cDNA)were amplified and inserted into the SalI and SmaI restriction sites of EP-GUS4-pBI in which GUS fragment was deleted, to make the constructs EP-FTg2800,EP-FTg2800Δ24, and EP-FTcDNA, respectively. These plasmids were trans-formed into the ft-10 mutant.

The distal enhancer and proximal promoter of FTwere amplified and insertedinto the SalI-EcoRI and EcoRI-SalI sites of pBI101.1-derived plasmid EP-GUS3-pBI,respectively, tomake the construct EP-GUS3-pBI. Candidate fragments (PUT1; 7,PUT6m, and PUTΔ24) were amplified and inserted into the EcoRI-XhoI site of EP-GUS3-pBI. The resulting plasmids were transformed into Col.

The 3.4-kb VAL1 promoter fragment and the cDNA encoding VAL1 wereamplified and inserted in sequence into the HindIII-SalI and EcoR1-SalI sites ofpBI101.1-derived plasmids pBI-GFP and pGI-GR, to make the constructsVAL1p:VAL1-GFP and VAL1p:VAL1-GR, respectively. The same VAL1 pro-moter fragment was also inserted into the HindIII-SalI site of pBI101.1 to createthe construct VAL1p-GFP. TheVAL1 cDNA fragment was also inserted into theEcoRI-SalI sites of pRI-Myc and pRI-GR, to give rise to the constructsMyc-VAL1and VAL1-GR, respectively. The 1.4-kb LHP1 promoter fragment and the LHP1cDNA fragment were amplified and cloned into the HindIII-EcoRI and EcoRI-SalI sites of pBI-GFP to obtain LHP1p:LHP1-GFP. The MSI1 cDNA fragmentwas amplified and cloned into the EcoRI-XhoI sites of the pVIP-Myc vector toobtain Myc-MSI1. These plasmids were transformed into Col or val1-2mutantsindicated in the text.

To prepare constructs for the yeast one-hybrid assay, the RAV1, RAV2,VAL1,and VAL2 full-length coding sequence fragments were ligated into EcoRI- andSalI-digested pJG4-5 vector (Clontech), to generate pGAD-RAV1, pGAD-RAV2,pGAD-VAL1, and pGAD-VAL2, respectively. PUT4 and PUT7 fragments of FTwere amplified and then cloned into the EcoRI-XhoI sites of the pLacZi2m vector(Jing et al., 2013), to generate the FT-PUT4:LacZ and FT-PUT7:LacZ constructs.The PUT7 fragment containing the deletion (PUT7Δ24) and mutated RY-motif(PUT7-RY1-m, PUT7-RY2-m, PUT7-RY1/2-m) were also inserted into the EcoRI-XhoI sites of the pLacZi2m vector to make the constructs PUT7Δ24:LacZ, PUT7-RY1-m:LacZ, PUT7-RY2-m:LacZ, and PUT7-RY1/2-m:LacZ, respectively.

To produce GST-tagged proteins, the cDNA encoding the VAL1-B3 domainwas cloned into the EcoRI-XhoI sites of pGEX-5X-1 to make VAL1-B3-GST.

To prepare constructs for luciferase-based transient assays, the fragments ofPUT4 and PUT4m were inserted into the EcoRI-SalI sites of the pCAMBIA1302-LUC vector to generate the constructs PUT4:LUC and PUT4m:LUC, respectively.

The primers are listed in Supplemental Table S1.

Yeast One-Hybrid Analysis

Yeast one-hybrid assays were performed following the Yeast ProtocolsHandbook (Clontech). Briefly, the activation domain fusion constructs werecotransformedwith various LacZ reporter plasmids into yeast strain EGY48. Todetect protein–DNA interactions, transformants were grown on SD/-Trp-Uradropout plates containing 5-bromo-4-chloro-3-indolyl-b-Dgalactopyranoside(X-gal) for blue color development.

Gene Expression Analysis

Total RNAwas extracted from10-d-old seedlings at various timepoints using anRNA Extraction Kit (Tiangen) according to the manufacture’s instruction. Total

RNAswere used to preparefirst-strand cDNAs byMoloneymurine leukemia virusreverse transcriptase (Invitrogen). Reverse transcription-qPCR was carried out in aLightCycler 480 (Roche) using a SYBR Premix ExTaq Kit (Takara) following themanufacturers’ instructions. Expression analysiswasperformedwith three technicalrepeats. The expression level was normalized to that of IPP2 or UBQ10 (internalcontrols). Primers used for gene expression analysis are listed in SupplementalTable S1.

GUS Histochemical Analysis

The rosette leaves or seedlings were incubated in 0.1 M sodium phosphatebuffer containing 50 mM K3Fe(CN)6, 50 mM K4Fe(CN)6, and 1 mM 5-bromo-4-chloro-3-indolyl-b-D-glucuronide at 37°C in the dark for 20 h. The staining re-actionswere terminated by replacing the buffer with ethanol. The samples weresubjected to various conditions as indicated in the text, and GUS staining im-ages were captured by a digital camera (Olympus).

Luciferase-Based Transient Assays

Agrobacterium-mediated transient assay was carried out as described pre-viously (Chen et al., 2008). The Agrobacterium strains GV3101 carrying the in-dicated constructs were incubated in Luria–Bertani medium at 28°C overnight.The culture was pelleted, washed twice, and resuspended in 10 mM MgCl2containing 0.2 mM acetosyringone to a final concentration of OD600 5 1.5. Thebacteria were kept at 28°C for 3–5 h without shaking. The Agrobacterium sus-pensions were coinfiltrated into fully expanded young Nicotiana benthamianaleaves with a needleless syringe. The plants were then grown at 22°C for 2 dunder LD conditions before LUC activity was examined. LUC images werecaptured using a NightSHADE LB985 plant imaging apparatus equipped witha CCD camera (Berthold Technologies). The bioluminescence intensities werecalculated using Indigo software (v2.0.3.0, Berthold Technologies). Three in-dependent replicates were used for the data analysis.

EMSAs

VAL1-B3-GST and the empty pGEX-4T-1 were transformed into E. coli BL21strain (DE3), and protein expression was induced by isopropylthio-b-galacto-side. The soluble GST fusion proteins were purified using GlutathioneSepharose 4B beads (GE Healthcare; for GST fusions). EMSAs were performedusing the LightShift Chemiluminescent EMSA kit (Thermo). Double-strandedDNA was generated by annealing sense and antisense oligonucleotides.Binding reaction conditions were as following: 13 Binding buffer (10 mM Tris,0.1 mM EDTA, and 5 mM B-mercaptoethanol, [pH7.5]), 5% (v/v) glycerol, 5 mM

MgCl2, 50 ng/ml Poly (dIdC), and 0.05% (w/v) Nonidet P-40, in a final volumeof 20 mL. Binding reactions were incubated at 25°C for 20 min. Free DNA andprotein-DNA complexes were separated on 5% (w/v) native polyacrylamidegels in 30 mM MES/30 mM His (pH 6.3) as reported (Sasnauskas et al., 2018), for90 min. Detection of biotin-labeled DNA by chemiluminescence was performedaccording to the manufacturer’s instructions.

ChIP Assay

ChIP experiments were performed as described previously with minormodifications (Bowler et al., 2004). The following antibodies were used: anti-GFP (Abcam, ab1218), anti-Myc (Abcam, ab32), anti-H3 (Millipore, 07-690), andanti-H3K27me3 (Millipore, 07-449). Briefly, 1 to 2 g of 10-d-old seedlings withvarious genetic backgrounds were harvested and fixed 15 min in 1% (v/v)formaldehyde under vacuum. Fixed tissues were homogenized, and chromatinwas isolated and sonicated to produce DNA fragments around 300 bp. Relativeenrichment of each fragment was determined with precipitated DNA samplesby qPCR using SYBR Green PCR master mix. The ChIP DNA sample wasquantified in triplicate. Primer pairs used for ChIP assays are listed inSupplemental Table S1.

FAIRE Assay

The FAIRE assay was performed as previously described (Omidbakhshfardet al., 2014; Jing et al., 2019). In short, 0.5 g 10-d-old seedlings were fixed in 1%(v/v) formaldehyde at room temperature under a vacuum for 8 min, followedby sonication, resulting in the production of 0.3–1 kb DNA fragments. Afterfour rounds of phenol–chloroform extraction, the purified DNA was eluted in

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200 mL Tris-EDTA buffer. Relative enrichment in the FAIRE-treated DNA wascalculated with DNA from uncrosslinked DNA samples serving as the control.The enrichment of fragmented genomic DNAwas normalized to that of the Ta3retrotransposon (internal control; Wu et al., 2015). All primer sequences arelisted in Supplemental Table S1.

Statistical Analyses

Statistical significance was determined by Student’s t test and one-wayANOVA. The means and SD are derived from independent biological samples.

Accession Numbers

Gene information from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL data libraries under the following accessionnumbers: FT, AT1G65480; CO, AT5G15840; FLC, AT5G10140; AGL15,AT5G13790; VAL1, AT2G30470; VAL2, AT4G32010; VAL3, AT4G21550; CLF,AT2G23380; LHP1/TERMINAL FLOWER22, AT5G17690; MSI1, AT5G58230;ACTIN 2, AT3G18780; Ta3, AT1G37110; UBQ10, AT4G05320; and IPP2,AT3G02780.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Characterization of LHP1p:LHP1-GFP tfl2-1 trans-genic lines and analyses of H3K27me3 levels in wild typeand tfl2-1.

Supplemental Figure S2. Analysis of H3K27me3 enrichments and chro-matin state of FT locus.

Supplemental Figure S3. Analyses of FT repression by VAL1.

Supplemental Figure S4. Expression patterns of VALs viewed by the eFPbrowser.

Supplemental Figure S5. Characterization of VAL1p:VAL1-GFP val1-2transgenic lines.

Supplemental Figure S6. Characterization of 35S:Myc-MSI1 transgenic line.

Supplemental Figure S7. Analysis of FT, FLC, and AGL15 expression uponDEX treatment.

Supplemental Figure S8. Phenotype and enrichment pattern comparisonof different genotypes.

Supplemental Table S1. Summary of the primers used in this study.

ACKNOWLEDGMENTS

We thank the Arabidopsis Biological Resource Center (The Ohio StateUniversity, Columbus) for providing the T-DNA mutants.

Received May 28, 2019; accepted June 25, 2019; published July 9, 2019.

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