the chromatin-remodeling factor pickle antagonizes polycomb … · prc1 activity (merini and...

The Chromatin-Remodeling Factor PICKLE AntagonizesPolycomb Repression of FT to Promote Flowering1

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

Changing daylength (or photoperiod) is a seasonal cue used by many plants to adjust the timing of their floral transitionto ensure reproductive success. An inductive long-day photoperiod triggers the expression of FLOWERING LOCUS T(FT), which promotes flowering. FT, encoding a major component of florigen, is induced in leaf veins specifically at duskthrough the photoperiod pathway; however, the modulation of FT expression in response to photoperiod cues remainspoorly understood. Here, we report that the balance between Polycomb group (PcG) and Trithorax group (TrxG) proteinssets appropriate FT expression in long days in Arabidopsis (Arabidopsis thaliana). In PcG mutant lines, FT was highlyderepressed, but FT expression was decreased to an almost wild-type level and pattern upon the additional disruption ofchromatin-remodeling factors PICKLE (PKL) and ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1), but not bydisruption of photoperiod pathway components. PKL interacts with ATX1 to mediate trimethylation of histone H3 onlysine-4 at the FT locus, leading to antagonistic effects of PKL and ATX1 on PcG proteins in the regulation of FTexpression. Therefore, the TrxG-like protein PKL prevents PcG-mediated silencing to ensure specific and appropriateexpression of FT, thereby determining the proper flowering response.

The transition to flowering is critical for reproduc-tive success in higher plants. In many species, flow-ering is timed on the basis of seasonal changes inphotoperiod, or daylength, through actions of thephotoperiod pathway (Andrés and Coupland, 2012;Song et al., 2015). Daylength signals are perceived inthe leaves, leading to the production of a mobile flo-rigenic hormone, termed florigen, which is trans-ported from the leaves to the shoot apical meristem topromote flowering (Corbesier et al., 2007; Jaeger andWigge, 2007; Mathieu et al., 2007). The key florigenhormone, encoded by FLOWERING LOCUS T (FT),also integrates flowering signals from several geneticpathways (He, 2012). Under long-day conditions (LDs),FT is expressed specifically in the phloem and at dusk

(Takada and Goto, 2003; Turck et al., 2008). FT acti-vation in LDs is predominantly controlled by thephotoperiod pathway output CONSTANS (CO), whichis also expressed exclusively in phloem (Samach et al.,2000; Takada and Goto, 2003; An et al., 2004).

Polycomb group (PcG) and Trithorax group (TrxG)proteins, which are involved in gene repression andactivation, respectively, form distinct complexes thatmaintain the specific transcription of key develop-mental regulatory genes (Pu and Sung, 2015; Xiao andWagner, 2015). PcG proteins assemble into two maintypes of complexes: Polycomb Repressive Complex1(PRC1) and PRC2. In Arabidopsis (Arabidopsis thali-ana), the catalytic core of PRC2 comprises CURLYLEAF (CLF), SWINGER, and MEDEA (Xiao andWagner, 2015; Förderer et al., 2016). The PRC1 complexcontains LIKE HETEROCHROMATIN PROTEIN1/TERMINAL FLOWER2 (LHP1/TFL2), EMBRYONICFLOWER1 (EMF1), and the catalytic core RING-domain proteins. PRC2 proteins trimethylate Lys-27of histone 3, whereas PRC1 components recognize theresulting H3K27me3 sites to maintain the repressivestate and induce histone H2A monoubiquitination(Merini and Calonje, 2015; Mozgova and Hennig,2015; Xiao and Wagner, 2015). Both LHP1 and thebromo-adjacent homology domain-containing proteinsSHORT LIFE and EARLY BOLTING IN SHORT DAYSdirectly interact with EMF1 and form two types of plant-specific PRC1 complexes (Wang et al., 2014; Li et al.,2018). It was recently shown in both vertebrates andplants that PRC2 does not act upstream of H2A

1This work was supported by the National Key Research and De-velopment Program of China (2017YFA0503800), the National Natu-ral Science Foundation of China (31870288), the Ministry ofAgriculture of China (2016ZX08009-003), and the Youth InnovationPromotion Association of Chinese Academy of Sciences (2015064).

2Author for contact: [email protected] authorThe 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:Yanjun Jing ([email protected]).

Y.J. and R.L. conceived the research; Y.J. designed the experiments;Y.J. and Q.G. performed the experiments; Y.J. and R.L. analyzed thedata and wrote the article.

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monoubiquitination and PRC2 can also be recruited byPRC1 activity (Merini and Calonje, 2015; Zhou et al.,2017).Because PcG proteins function in global transcrip-

tional repression, their activity must be restricted at thechromatin level by chromatin regulators. The TrxGfactors, which were initially defined as proteins thatcounteract the activity of PcGs, include COMPLEXPROTEINS ASSOCIATED WITH SET1 (COMPASS)and COMPASS-like proteins, ATP-dependent chromatin-remodeling factors, and other related proteins (de la PazSanchez et al., 2015; Pu and Sung, 2015). ARABIDOPSISHOMOLOG OF TRITHORAX1 (ATX1) is closely relatedto the Drosophila melanogaster Trithorax proteins. ATX1trimethylates H3K4 and recruits TATA-binding pro-tein and RNA polymerase II to the promoters of itstargets (Alvarez-Venegas et al., 2003; Ding et al., 2011).Chromatin-modifier proteins function via their inter-action with ATX1. ATX1 functions in the activation ofFLC to regulate the floral transition by binding to theFLC locus and interacting with WDR5a to form aCOMPASS-like complex (Pien et al., 2008; Jiang et al.,2011). The SAND (Sp100, AIRE-1, NucP41/75, DEAF-1)domain-containing protein ULTRAPETALA1 (ULT1)antagonizes the repressive activity of PcG factors andphysically associates with ATX1 to negatively regulateshoot and floral stem cell activity (Carles and Fletcher,2009).PcG protein-mediated epigenetic silencing of FT is

important for the regulation of flowering. ArabidopsisPcG factor mutants, such as clf, emf1, emf2, and lhp1,exhibit extremely early flowering, largely due to ectopicexpression of FT (Bratzel and Turck, 2015). CLF acts asan H3K27 methyltransferase that deposits H3K27me3at FT, thereby repressing FT expression (Jiang et al.,2008). LHP1, SHORT LIFE, and EARLY BOLTING INSHORT DAYS function as readers of the H3K27me3mark, and their binding to H3K27me3 on FT chromatinalso results in FT repression (Turck et al., 2007; Zhanget al., 2007b; López-González et al., 2014; Wang et al.,2014; Li et al., 2018). However, it is unclear how PcG-mediated silencing of FT expression is restricted bychromatin regulators. H3K4me3 is an active chromatinmark in the TrxG pathway, which acts antagonisticallyto PcG regulation (Pu and Sung, 2015; Xiao andWagner, 2015). Elevated H3K4me3 is associated withthe up-regulation of FT expression (Jiang et al., 2008).Furthermore, the Jumonji C domain-containing proteinPKDM7B mediates H3K4 demethylation in FT chro-matin and represses its expression (Yang et al., 2010).The functions of several ATP-dependent chromatin-

remodeling factors are similar to that of TrxG factors inArabidopsis (de la Paz Sanchez et al., 2015; Han et al.,2015; Pu and Sung, 2015). Remodelers use the energyderived fromATP hydrolysis tomodulate histoneDNAcontacts and work with other chromatin factors tocontrol accessibility to the chromatin (Han et al., 2015).The closely related SWI2/SNP2 ATPases BRAHMA(BRM) and SPLAYED (SYD) counteract CLF activity byantagonistically regulating the target genes APETALA3

and AGAMOUS (Wu et al., 2012). PICKLE (PKL) is achromatin-remodeling factor of the CHD3 subfamilycharacterized by two tandemly arranged chromodo-mains and one or two PHD domains (Han et al., 2015).PKL is involved in regulating diverse developmentalprograms, such as embryonic development (Ogas et al.,1999), seed germination (Fukaki et al., 2006; Perrucet al., 2007), root meristem activity (Aichinger et al.,2011), and light- and temperature-mediated seedlingdevelopment (Jing et al., 2013; Zhang et al., 2014; Zhaet al., 2017). PRC2 regulation of root meristem activitythrough H3K27me3-mediated repression is counter-acted by PKL through alteration of H3K27me3 depo-sition and expression of the PcG gene (Aichinger et al.,2009, 2011). Moreover, the pkl mutant exhibits delayedbolting and reduced expression of LEAFY and LEAFY -regulated and GA-responsive genes, indicating thatPKL is involved in flowering regulation (Hanson et al.,1999; Fu et al., 2016; Park et al., 2017). We recentlyshowed that LDs trigger the FT chromatin switch be-tween the active state at dusk and the inactive state atnight. PKL is strongly expressed in leaf vascular tissue,and its protein is responsible for the diurnal switch ofthe FT chromatin state (Jing et al., 2019). PKL interactswith CO to enhance its binding to the common regionsof FT chromatin. Thus, PKL and CO act synergisticallyto activate FT expression (Jing et al., 2019). Together,these findings indicate that PKL promotes FT expres-sion likely through facilitating access of CO to FTchromatin in response to environmental cues.Here, we show that the PcG factors (CLF, LHP1,

and EMF1) repress FT expression largely indepen-dently of the photoperiod pathway. PKL antagonizesthe activity of PcGs in regulating FT expression, in-teracts with ATX1, and modulates H3K4me3 levelsof FT chromatin. Thus, our results demonstrate thatPKL serves as a TrxG-like protein that counteractsthe repressive activity of PcG factors, ultimately leadingto the daylength-mediated control of flowering time inArabidopsis.

RESULTS

PcG Proteins Repress FT Expression LargelyIndependently of the Photoperiod Pathway

Besides photoperiodic regulation of FT expression,PcG-mediated chromatin silencing is also involved inthe transcriptional regulation of FT (Bratzel and Turck,2015). Therefore, we sought to determine interactionsbetween PcGs and photoperiod pathway components.We crossed photoperiodic pathway mutants co-9, gi-201 (gigantea), cry1/2 (cryptochrome), and ft-10 with thePcG mutants clf-28 and tfl2-1 to examine the geneticinteraction between them. When grown under LDs(16 h of light/8 h of dark), clf-28 and tfl2-1 plantsflowered earlier than the wild type (Columbia [Col]).The late-flowering responses of gi-201, cry1/2, and co-9were largely inhibited in the double mutants gi clf,

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gi tfl2, co clf, and co tfl2 and the triple mutant cry1/2 clf inLDs (Fig. 1, A–C; Supplemental Fig. S1, A–G). Consis-tent with our findings, previous studies also showedthat the co clf and co tfl2 double mutants flowered onlyslightly later than their respective parental lines, clf-28and tfl2-1 (Supplemental Fig. S1, D–G; Takada andGoto, 2003; Pazhouhandeh et al., 2011). Similarly,when grown under short days (SDs; 8 h of light/16 h ofdark), clf-28 flowered much earlier than Col, as ob-served in LDs, and co clf flowered almost as early as clf-28 (Supplemental Fig. S1J), likely because CO does notregulate flowering under SDs.

PcG factors function upstream of FT (Kotake et al.,2003; Lopez-Vernaza et al., 2012). We similarly foundthat loss of FT abolished the early-flowering pheno-types of clf-28 and tfl2-1 under LDs and that of clf-28under SDs (Supplemental Fig. S1, H, I, and K). Notably,both ft clf and ft tfl2 flowered later than ft-10(Supplemental Fig. S1, H and I), which is similar to aprevious result showing that EMF1-RNAi ft floweredlater than the ft mutant (Wang et al., 2014), indicatingthat PRC1 (LHP1/TFL2 and EMF1) and PRC2 (CLF)components share a similar mechanism of genetic in-teraction with FT.

We further monitored FT transcript levels every 4 hover a 24-h LD cycle. In contrast to the canonicalrhythmic FT expression pattern, which peaks at Zeit-geber time 16 (ZT16) in Col, FTwas highly expressed in

clf-28 and tfl2-1 (Fig. 1, E and F) in the early day and atnight, consistent with the reduced photoperiod sensi-tivity and early-flowering phenotypes in clf-28 and tfl2-1 (Fig. 1, A–C; Supplemental Fig. S1, J and K; Larssonet al., 1998). Moreover, FT expression in clf-28 and tfl2-1 over the 24-h LD cyclewas higher than that in thewildtype at dusk (Fig. 1, D–H), indicating that FT is not fullyactivated at dusk. However, FT expression remainedlargely high in the double mutants gi clf, gi tfl2, co clf,and co tfl2 and the triple mutant cry1/2 clf in LD, as intheir parent mutants clf-28 and tfl2-1 (Fig. 1, D–H). Inaddition, FT expression was highly derepressed in co clfand gi clf, as in clf mutants, over a 24-h SD cycle(Supplemental Fig. S1, L and M). These results indicatethat PcG proteins repress FT expression and preventprecocious flowering in a manner that is largely inde-pendent of the photoperiod pathway and suggest thatphotoperiodic pathway components are not involvedin antagonizing PcG repression of FT expression to alarge extent.

PKL and PcG Proteins AntagonisticallyRegulate Flowering

Several chromatin-remodeling factors, includ-ing PKL, act similarly to TrxG factors to regulatemultiple developmental processes in Arabidopsis

Figure 1. PcG proteins CLF and LHP1 genetically interact with components of the photoperiodic pathway. A to C, Total leafnumber of plants of the indicated genotypes grown under LDs. Error bars indicate SD of at least 12 plants. D to H, FT expression inthe indicated seedlings over a 24-h LD cycle. Seedlingswere grownunder LDs for 9 d. Relative expressionwas normalized to IPP2expression. The white bars above the graphs represent light periods and the black bars represent dark periods. Data are means6SD of three biological replicates.

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(Aichinger et al., 2009, 2011; Wu et al., 2012). PKL isstrongly expressed in leaf vascular tissue, and its mutantalleles exhibit late flowering, a phenotype opposite tothat of PcG mutant lines (Jing et al., 2019). We sought todeterminewhether PKL is involved in the antagonism ofPcG-mediated repression of flowering. To this end, weused pkl clf (Aichinger et al., 2009) and generated thedouble mutant pkl tfl2 by genetic crossing. Under LDs,the early-flowering phenotype caused by the CLF mu-tation (clf-29) was overcome in the pkl clfmutant (Fig. 2A;Supplemental Fig. S2A). Under SDs, the pkl clf plantsfloweredmuch later than the clf-29 plants (SupplementalFig. S2, D and E). The early-flowering phenotype of tfl2-1 was also largely inhibited by PKL mutation in the pkltfl2 mutant (Fig. 2B; Supplemental Fig. S2B). We thencompared FT transcript levels in thewild typewith thosein the single- and double-mutant seedlings grown underLDs. FT expression in the pkl clf and pkl tfl2 double mu-tants was largely repressed throughout the day andexhibited a pattern similar to that in the wild type, incontrast to its misexpression in the clf and tfl2 singlemutants (Fig. 2, C and D).EMF1 is expressed in leaf vascular tissue, and EMF1

forms a nuclear complex with LHP1 that regulatesflowering (Wang et al., 2014). We therefore introducedthe PKL mutation into an EMF1-RNAi transgenic line.EMF1-RNAi flowered quite early in LDs (Fig. 2E;Supplemental Fig. S2C; Wang et al., 2014), and thisphenotype was largely suppressed by the PKL muta-tion in pkl EMF1-RNAi plants (Fig. 2E; SupplementalFig. S2C). FT mRNA levels were also drastically re-duced in pkl EMF1-RNAi plants and were largely res-cued to the level observed in the wild type (Fig. 2F).These results indicate that PKL antagonizes the effectsof PcG proteins in regulating FT expression and floraltransition and suggest that PKL functions as a TrxG-likeprotein that controls flowering. Next, we introducedthe co-9mutation into pkl clf by crossing co clf-28with pklclf-29. Two independent pkl clf co triple mutant lines (no.

8 and no. 16) flowered slightly later than pkl clf in LDsand with similar timing to pkl clf in SDs (Fig. 2A;Supplemental Fig. S2E). Under both LDs and SDs, FTwas expressed at similar levels in pkl clf co as in pkl clf(Supplemental Fig. S2F). At ZT16 in LDs, FT transcriptlevels were lower in the pkl clf comutants than in the pklclf mutants (Fig. 2C), suggesting a possible synergisticrelationship betweenCO and PKL at the end of LDs andindicating that the antagonism between CLF and PKL islargely independent of CO. These findings support thenotion that PKL and PcG proteins play antagonisticroles in regulating flowering time.

H3K4me3 Is Involved in the PKL-Mediated Antagonism ofPcG-Mediated Regulation of FT Expression

CLF functions as a major H3K27 methyltransferasethat deposits H3K27me3 on FT, leading to its repression(Jiang et al., 2008; Shu et al., 2019). PKL repressesH3K27me3 levels on the chromatin of target genes andregulates various developmental processes (Aichingeret al., 2009; Jing et al., 2013; Zhang et al., 2014). Weassessed whether H3K27me3 is involved in the antag-onistic effects of PKL and CLF on flowering. To thisend, we performed chromatin immunoprecipitation(ChIP) assays to compare H3K27me3 levels on FTchromatin. In Col, H3K27me3 was indeed enriched atvarious regions across the FT locus, particularly indownstream regions (Fig. 3A, FT-I; Supplemental Fig.S3), in accordance with results from previous studies(Turck et al., 2007; Zhang et al., 2007a; Adrian et al.,2010). In clf-28 and EMF1-RNAi, H3K27me3 levelsat the promoters and gene body regions were muchlower than those in Col (Fig. 3A; Supplemental Fig.S3), indicating that CLF mediates the trimethylationof H3K27 at FT and EMF1 is required for maintainingthe H3K27me3 level. These data support the recentconclusion that PRC1 activity can also recruit PRC2

Figure 2. PKL antagonizes the repres-sive effect of PcG factors on flowering.A, B, and E, Total leaf number of plantsof the indicated genotypes grown un-der LDs. Error bars indicate SD of at least16 plants. Asterisks indicate significantdifferences calculated using Student’st test (*, P , 0.05 and **, P , 0.01). C,D, and F, RT-qPCR analysis of FT tran-script levels in seedlings of the indi-cated genotypes grown under LDs for9 d. The white bars above the graphsrepresent light periods and the blackbars represent dark periods. Relativeexpression was normalized to IPP2expression. Data are means 6 SD ofthree biological replicates.



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(Merini and Calonje, 2015; Zhou et al., 2017). How-ever, H3K27me3 levels in pkl clf and pkl EMF-RNAiwere similar to those in clf-28 and EMF1-RNAi, re-spectively (Fig. 3A; Supplemental Fig. S3).

RELATIVE OF EARLY FLOWERING6 (REF6) is aJumonji domain-containing protein that demethylatesH3K27me3 (Lu et al., 2011). The loss-of-function mu-tant ref6-3 flowers later than the wild type, whereas theflowering time of ref6 clf is similar to that of clf-28 (Luet al., 2011). We found that the high expression level ofFT in clf-28 was not suppressed by the introduction ofthe ref6 mutation (Supplemental Fig. S4). Two otherH3K27me3 demethylases, JUMONJI13 and EARLYFLOWERING6, act as upstream repressors in the pho-toperiodic flowering pathway (Noh et al., 2004; Zhenget al., 2019) and are functionally redundant with REF6(Yan et al., 2018). Further investigation would be re-quired to reveal whether H3K27me3 demethylases re-dundantly antagonize CLF activity to regulate FTexpression.

H3K4me3 is an active chromatin mark in the TrxGpathway that functions antagonistically to PcG reg-ulation (Pu and Sung, 2015; Xiao and Wagner, 2015).We therefore analyzed the association of H3K4me3with FT and the control loci and found that H3K4me3was moderately enriched at the FT locus, as previ-ously demonstrated (Farrona et al., 2011), comparedwith the known positive lociUBIQUITIN 10 (UBQ10),WRKYDNABINDING PROTEIN70, LIPID TRANSFERPROTEIN7, and FLC (Pien et al., 2008; Ding et al., 2011)and the negative locus ACT2 (Supplemental Fig. S5, Aand B). The enrichment of H3K4me3 at the FT locus wasno different in clf-28 than in Col (Fig. 3B), consistentwith a previous study showing no significant changesin H3K4me3 levels upon mutation of CLF (Farronaet al., 2011). Importantly, H3K4me3 was reduced to asimilar level in pkl and pkl clf, suggesting that H3K4me3is required for PKL-mediated activation of FT. H3K4me3and H3K27me3 levels were both reduced in pkl clf(Fig. 3), resulting in restored FT expression and flower-ing. Taken together, these results suggest that changes inH3K4me3 (in particular at the transcription start site[TSS]) correlate with PKL function in counteracting PcGfactors for FT activation.

PcG-Mediated Repression of FT Is Antagonized by ATX1

We then sought to further elucidate the functionalimportance of H3K4me3 in inhibiting PcG-mediatedH3K27 trimethylation. ARABIDOPSIS HOMOLOGOF TRITHORAX 1 (ATX1) is a methyltransferase thattrimethylates H3K4 and regulates distinct develop-mental processes by marking target genes with the ac-tive mark H3K4me3 and activating their expression(Alvarez-Venegas et al., 2003; Pien et al., 2008; Dinget al., 2011). Our phenotypic analyses revealed thatthe mutant atx1-2 flowered slightly earlier than Col, aspreviously reported (Fig. 4, A and B; Alvarez-Venegaset al., 2003; Berr et al., 2015). The presence of the atx1-2mutation largely suppressed the abnormal phenotypesof clf-28 (Saleh et al., 2007) as well as EMF1-RNAi,including stunted growth and early flowering (Fig. 4,A and B; Supplemental Fig. S5, C and D). ATX1 isexpressed in vascular tissue (Pien et al., 2008), and itsmutation led to reduced FT mRNA levels at dusk(Fig. 4, C and D). FT was highly derepressed in thePcG mutants clf-28 and EMF1-RNAi (Fig. 4, C and D).Importantly, FT expression in atx1 clf and atx1 EMF1-RNAi was restored to almost wild-type levels andpatterns over the 24-h LD cycle, although a smallpeak was detected in atx1 EMF1-RNAi at ZT4 (Fig. 4,C and D). These results suggest that mutation ofATX1 antagonizes the derepression effects of PcGloss of function, leading to a normal FT expressionand flowering phenotype, consistent with the notionthat ATX1 acts as a PcG antagonist. These resultsimply that the counterbalance between ATX1 andPcGs fine-tunes FT expression in response to thephotoperiod.

Figure 3. H3K4me3 is involved in the antagonistic interaction betweenPKL and PcG proteins. Relative enrichment of H3K27me3 (A) andH3K4me3 (B) is shown at the FT genomic locus, as revealed by ChIP-qPCR analysis. Nine-day-old Col, pkl-1, clf-29, and pkl clf seedlingswere grown under LDs, and samples were harvested at ZT16. Data arepresented as the ratio to that of H3. Error bars indicate SD of three bio-logical replicates. Depicted above the graphs is a schematic drawing ofgenomic FT, with black boxes for exons, a gray box for the 59 un-translated region, and thick lines indicating ChIP-examined amplicons.TSS, Transcription start site.


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ATX1 Directly Binds to FT Chromatin and RegulatesIts Expression

We then examined whether ATX1 directly regulatesFT expression. Genetic analysis showed that the atx1 ftdouble mutant flowered at about the same time as ft-10(Fig. 5A), indicating that FT acts downstream of ATX1.We generated 35S:ATX1-GR transgenic plants, inwhichATX1 was fused with the glucocorticoid receptor (GR)gene. In the presence of the GR agonist dexamethasone(DEX), GR fusion proteins were translocated from thecytoplasm to the nucleus. DEX induction of ATX1-GRresulted in up-regulation of FT expression (Fig. 5B),indicating that ATX1 promotes FT expression. We alsogenerated transgenic lines expressing 35S:ATX1-GFP inthe atx1-2 background. A representative 35S:ATX1-GFPatx1 plant showed similar flowering time and FT ex-pression to Col and expressed properly folded GFP inthe nucleus, implying that the ATX1-GFP fusion pro-tein is biologically functional (Supplemental Fig. S6,A–C).We then performed ChIP assays, in which the ATX1-

GFP fusion protein was enriched after ChIP assays us-ing this ATX1-GFP atx1 line. ATX1-GFP was stronglyenriched at FT chromatin, including the proximal pro-moter and gene body, but not at the ACT2 control locus(Fig. 5C). These results collectively suggest that theH3K4 methyltransferase ATX1 binds to the FT locusand activates its expression. In agreement with thisnotion, upon ATX1 mutation, H3K4me3 levels in thepromoter region and gene body of FT were reduced(Fig. 5D), in agreement with a previous report (Bu et al.,2014). This reduction in H3K4me3 might contribute to

the decreased transcript levels in FT detected in atx1-2.However, H3K4me3 levels did not clearly differ be-tween atx1-2 and atx1 clf (Fig. 5D). These results suggestthat the ATX1 activation activity and the PcG repres-sion activity antagonistically regulate FT expression atthe chromatin level.

ATX1 Interacts with PKL to Regulate the Floral Transition

Both PKL and ATX1 are highly enriched at the FTlocus (Fig. 5C; Jing et al., 2019) and antagonize the re-pression activity of PcG factors to set proper FT ex-pression and a normal flowering response. The activeH3K4me3 mark is involved in this antagonism. Previ-ous studies showed that chromatin proteins that an-tagonize PcG activity physically interact with ATX1(Carles and Fletcher, 2009; Jiang et al., 2011). Wetherefore investigated a possible interaction betweenPKL and ATX1. Homologs of PKL are widely distrib-uted in plants and animals and contain four conserveddomains: chromodomains, PHD domains, helicase/ATPase domains, and putative DNA-binding domains(Fig. 6A; Han et al., 2015). We found that ATX1 inter-acted with the N-terminal D1 fragment, but not thecentral D5 fragment, of PKL (Fig. 6, A and B). We ex-plored which PKL region(s) could interact with ATX1and found that the fragments containing the PHD do-main (D3 and D4) were sufficient for ATX1 binding(Fig. 6B).Next, we conducted a protein pull-down assay using

a recombinant His-PKL fusion protein. We found that

Figure 4. ATX1 counteracts the repressive activityof PcG factors on flowering. A and B, Total leafnumber of plants of the indicated genotypesgrown under LDs. Error bars indicate SD of at least13 plants. Significant differences compared withCol were calculated using Student’s t test (*, P ,0.05 and **, P, 0.01). C andD, RT-qPCR analysisof FT transcript levels in the indicated genotypesgrown under LDs for 9 d. Thewhite bars above thegraphs represent light periods and the black barsrepresent dark periods. Relative expression wasnormalized to IPP2 expression. Data are means6SD of three biological replicates.



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His-PKL was able to pull down ATX1-GFP from totalprotein extracts of 35S:ATX1-GFP atx1 transgenicseedlings (Fig. 6C), confirming its direct interactionwith ATX1. No interaction signal was detected whenHis-MBP was used as the bait (Fig. 6C). To confirm thePKL-ATX1 interaction in vivo, we performed a coim-munoprecipitation (co-IP) assay using 35S:ATX1-GFPatx1 plants and found that anti-GFP precipitated PKLfrom the transgenic seedlings (Fig. 6D). Furthermore,we conducted firefly luciferase (LUC) complementationimaging (LCI) assays inNicotiana benthamiana leaves.N.benthamiana cells coexpressing ATX1-nLUC and cLUC-PKL displayed strong fluorescence signals, whereasthose coexpressing cLUC-PKL and nLUC or ATX1-nLUC and cLUC displayed no signals (Fig. 6E), con-firming that the ATX1-PKL interaction occurs in vivo.

To explore the genetic interaction between PKL andATX1 in flowering time regulation, we introduced thepkl-1 mutation into atx1-2. The late-flowering pheno-type of pkl-1 was partly suppressed by atx1-2 in LDs(Fig. 6F). Further FT expression analysis revealed thatFT transcript abundance was decreased at similar levelsin atx1 pkl and the single mutants (atx1-2 and pkl-1)compared with that in the wild type (Fig. 6G), indicat-ing that ATX1 and PKL act together to regulate FT ex-pression. ATX1-GFP levels were also explored in35S:ATX1-GFP and 35S:ATX1-GFP pkl-1 by ChIP-quantitative (q)PCR; however, no significant changeswere observed (Supplemental Fig. S6E), which isprobably due to the functional redundancy between

PKL and its homologs. Taken together, these datasuggest that PKL interacts with ATX1 to mediate H3K4methylation at the FT locus.

DISCUSSION

In this study, we showed that photoperiod pathwaycomponents were not involved in the antagonism ofPcG proteins’ repressive effects on flowering regula-tion. PKL acts as a TrxG-like protein that counteractsthe effects of PcG proteins (including CLF, LHP1, andEMF1), thereby conferring adequate FT expression andproper flowering response. The findings that PKL in-teracts with ATX1 and ATX1 mediates H3K4 trime-thylation are in agreement with the antagonistic role ofPKL versus PcG in regulating FT expression. These re-sults collectively reveal that the opposing activities of PcGand TrxG proteins ensure the specific and appropriateexpression of FT, thereby regulating flowering time inresponse to daylength. We previously showed thatPKL mediates the periodic switch in the chromatinstate of FT and activates its expression specifically atdusk. The interaction between CO and PKL is requiredfor their binding to the common and specific CORE(CO-responsive element) regions of FT in response tophotoperiod (Jing et al., 2019). Thus, PKL not only con-tributes to CO-mediated induction of FT but also coun-teracts the repressive activity of PcG proteins, ultimatelyleading to proper FT expression and flowering response.

Figure 5. ATX1 binds to FT chromatin and pro-motes its expression. A, Total leaf number ofplants of the indicated genotypes grown underLDs. Error bars indicate SD of at least 10 plants.Significant differences compared with Col areindicated (Student’s t test, *, P, 0.05 and **, P,0.01). B, Transcript levels of FT in 35S:ATX1-GRtransgenic seedlings in LDs. Ten-day-old seedlingswere harvested after treatment for the indicatedperiods with or without DEX (20 mM). Expressionlevels were normalized to IPP2. Means6 SD fromthree biological replicates are shown. C and D,ChIP analysis of the enrichment of ATX1-GFP (C)and H3K4me3 (D) on the FT chromatin at ZT16.Seedlings were grown under LDs for 11 d. Dataare represented as either the ratio to that of input(C) or H3 (D).ACT2was used as a negative controlin C. Error bars indicate SD of three biologicalreplicates. TSS, Transcription start site.


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We provide genetic evidence that PcG proteins, in-cluding CLF and LHP1, repress the floral transition in aphotoperiod-independent manner and act upstream ofthe floral integrator FT. Consistent with this, previousstudies reported that the late-flowering phenotypes ofco are abolished upon PcG gene mutation or knock-down (Takada and Goto, 2003; Pazhouhandeh et al.,2011; Wang et al., 2014). In a combinatorial set of dou-ble or triple mutant lines with mutations of PcG andphotoperiod components, FTwas expressed at levels as

high as those in the PcG single mutants over 24-h LDcycles, indicating that photoperiod components are notinvolved in the antagonism of PcG-mediated FT re-pression (Fig. 1, D–H). It was previously shown thatPcG factors function upstream of FT (Kotake et al., 2003;Lopez-Vernaza et al., 2012). In LDs, ft clf and ft tfl2flower later than the parental line, ft-10 (SupplementalFig. S1, H and I). Similarly, EFM1-RNAi ft flowersslightly later than ft-10 (Wang et al., 2014). It is likelythat introducing PcG mutations into an ft mutant

Figure 6. PKL interactswith ATX1. A, Schematic diagram of PKL showing its domain structure. B, PKL interactswith ATX1 throughits PHDdomain in yeast cells. PKL (or PKL fragment D1, D2, D3, D4, or D5) and full-length ATX1were fused to theGAL4-bindingdomain (BD) and activation domain (AD), respectively. Transformed yeast cells were grown on synthetic dextrose (SD)/-Trp-Leu(-WL) and SD/-Trp-Leu-His-Ade (-WLHA) dropout plates. C, Pull-down assay. His-MBP or His-PKL proteins were incubated withprotein extracts from 11-d-old 35S:ATX1-GFP transgenic seedlings and further immobilized with Ni-NTA. The pull-down pro-ducts were probedwith anti-GFP or anti-His. D, In vivo interaction between PKL and ATX1 shown by co-IPassay. Protein extractsfrom 11-d-old seedlingswere immunoprecipitated (IP) with anti-GFPagarose. E, LCI assays showing that PKL interacts with ATX1.LUC images of N. benthamiana leaves coinfiltrated with the different plasmid combinations are shown. The pseudocolor barexhibits the range of luminescence intensity. Bar5 1 cm. F, Measurement of total leaf number of plants of the indicated genotypesgrown under LDs. Error bars indicate SD of at least 16 plants. G, FTmRNA levels in the indicated lines grown under LDs for 10 d.The relative transcript levels were normalized to that of IPP2. The white bar above the graph represents the light period and theblack bar represents the dark period. Data are means 6 SD of three biological replicates.



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derepresses the expression of other negative regulatorsof flowering.

FT possesses a long promoter region. Nuclear factorY (NF-Y) and NF-CO bind to the distal enhancer (lo-cated 5.7 kb upstream of the transcription start site) andthe proximal regions, respectively, and a chromatinloop forms to bring these two regions into contact(Adrian et al., 2010; Cao et al., 2014; Liu et al., 2014;Gnesutta et al., 2017; Luo et al., 2018). A recent studyshowed that NF-CO and NF-Y act synergistically toreconfigure the conformation of FT chromatin, leadingto relief of Polycomb silencing around dusk (Luo et al.,2018). Therefore, besides PKL and ATX1, NF-CO andNF-Y contribute another regulatory layer to counteractPcG silencing of FT expression. It will be interesting toexamine whether PKL and/or ATX1 interact with NF-CO to antagonize PcG repression of FT.

TrxG proteins counteract the repressive effect of PcGproteins (de la Paz Sanchez et al., 2015). Both SYD andBRM act as TrxG proteins, as mutations of SYD andBRM rescue the curly leaf phenotype of clf, and CLFand SYD have opposite effects on H3K27 trimethyla-tion of their target genes (Wu et al., 2012). PKL acts as anantagonist of PcG activity. PKL and PICKLE RE-LATED2 (PKR2) are directly required for the activationof PcG target genes and are indirectly required for therepression of these target genes in roots (Aichingeret al., 2009). Moreover, PKL represses the associationof H3K27me3 with target genes and counteracts CLF-mediated repression of these genes to help determineroot meristem activity (Aichinger et al., 2011).

Our findings demonstrate that PKL acts in an op-posite manner to PcG proteins to derepress FT ex-pression and restore proper flowering response (Fig. 2;Supplemental Fig. S2), further confirming the role ofPKL as a TrxG protein. The loss of PKL did not alter CLFexpression in LDs (Supplemental Fig. S7) or the en-richment of H3K27me3 at FT chromatin (Fig. 3A).Hence, PKL serves as a TrxG protein in a differentmanner from that described in previous reports(Aichinger et al., 2009, 2011). We propose that PKL actsas a TrxG-like protein by (1) promoting PcG gene ex-pression and, subsequently, the activation of PcG targetgenes (Aichinger et al., 2009); (2) preventing PcG-mediated repression of H3K27me3 (Aichinger et al.,2011); and (3) promoting ATX1-mediated activation ofH3K4me3. PKL may use different mechanisms tocounteract the effect of PcG proteins in specific devel-opmental contexts. The early-flowering phenotypes ofPcG mutants were not fully rescued by the pkl-1 muta-tion (Fig. 2, A, B, and E; Supplemental Fig. S2, A–E). Inthe case of clf-29, the effect of pkl-1 seems quite minor(Fig. 2A). There are two possible explanations for this.First, pkl-1 is an ethyl methanesulfonate-derived allelecausing alternate splicing that results in a deletion ofthree amino acids in the in PKL ATPase domain (Ogaset al., 1997; Li et al., 2005), whichmay not fully interruptflowering time regulation by PKL. Second, PKLmay actredundantly with other CHD3 chromatin remodelerproteins in regulating flowering. PKR1, PKR2, and

CHROMATIN REMODELING5 (CHR5) are homologsof PKL (Shaked et al., 2006). Indeed, the mutants pkr1-1,pkr2-2, and chr5-1 flowered as early as Col, whereas thedouble mutant pkl pkr2 and the triple mutants pkl pkr1pkr2 and pkl pkr2 chr5 flowered later than the pkl-1 singlemutant and exhibited lower FT expression than that inCol under LDs (Supplemental Fig. S8). Future studiesshould involve higher order mutants of pkl and its ho-mologs with PcG mutants to further elucidate the ge-netic relationship between PKL and PcG factors.Although the pkl-1 mutation introduction did not fullyrescue the early-flowering phenotypes of PcG mutants(especially clf-29; Fig. 2, A, B, and E; Supplemental Fig.S2E), the effects of PcGmutation on FT expression werelargely rescued by the single pkl-1. The discrepancybetween flowering phenotype and FT expression maybe due to PKL (together with its homologs) being in-volved in different flowering pathways. In agreementwith this notion, PKL promotes flowering in both LDsand SDs and is involved in other flowering pathwaysbesides the photoperiodic pathway (Jing et al., 2019).Moreover, analysis of available transcriptomic data setsrevealed that the floral regulators AGL8 and AGL24 aredown-regulated in the pkl mutant and up-regulated inlhp1 (Zhang et al., 2012; Li et al., 2018).

Previous studies have revealed that the chromatin-modifier proteins that antagonize PcG activity physicallyinteract with ATX1. The COMPASS-like componentWDR5a,which interactswithATX1, is involved inH3K4methylation in FLC chromatin and represses flowering(Jiang et al., 2011). The SAND domain protein ULT1 is aTrxG protein that is thought to function in a complexwith ATX1, since mutation of ULT1 affects H3K4me3enrichment at the AG locus and ULT1 interacts withATX1 in vitro (Carles and Fletcher, 2009). PKLphysicallyinteracts with ATX1 and regulates FT expression. PKLand ATX1 were enriched on the FT chromatin (Fig. 5C;Jing et al., 2019), and mutation of either PKL or ATX1results in reduced H3K4me3 on FT chromatin (Figs. 3Band 5D). Thus, PKL may function as a coactivator of theTrxG protein ATX1, which is involved in local H3K4methylation, to influence transcription at specific de-velopmental targets. Moreover, it would be interestingto assess whether ATX1-mediated trimethylation ofH3K4 is involved in CO-dependent activation of FTexpression.

H3K4me3 levels were similar in the pkl clf doublemutant and the pkl single mutant (Fig. 3B), counter-balancing the reduced enrichment of H3K27me3, andthis suggests that H3K4me3 mediates the antagonismbetween PKL and PcG in controlling flowering time.ATX1 is an H3K4 methyltransferase that activates ho-meotic genes to regulate flower development (Alvarez-Venegas et al., 2003; Pien et al., 2008). Although loss ofATX1 results in a slight reduction in global H3K4me3levels (Alvarez-Venegas and Avramova, 2005), intro-duction of an ATX1mutation largely rescued the early-flowering phenotypes and almost completely restoredFT expression in the PcG mutants (Fig. 4), supportingthe notion that ATX1-mediated H3K4me3 methylation


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selectively but not extensively regulates developmentalgenes.PcGs are recruited by transcription factors to regulate

different targets in plants (Mozgova and Hennig, 2015).Recent studies have defined putative genome-widePolycomb-response elements (PREs) in Arabidopsisand identified specific cis-acting motifs as the consen-sus PREs (e.g. GAGA motifs and telobox motifs) in-volved in recruiting PcG factors (Xiao et al., 2017; Zhouet al., 2018). Arabidopsis PcG factormutant lines exhibitextremely early flowering largely due to ectopic ex-pression of FT (Bratzel and Turck, 2015). Our data, to-gether with those of the earlier study showing that thebinding of the PcG factors (CLF, LHP1, and EMF1) andthe histone mark H3K27me3 to FT loci is reduced atdusk compared with that at night and midday (Luoet al., 2018), suggest that PcG proteins are needed forproper FT expression and are required for thephotoperiod-independent flowering response. It will beof great interest to determine how Polycomb-mediatedsilencing of FT is initiated.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) Col accession was used as the wildtype for all experiments. The mutant alleles and transgenic plants used in thisstudy include pkl-1 (Ogas et al., 1999), pkr2-2, pkl pkr2, and pkl clf (Aichingeret al., 2009), pkr1-1 and chr5-1 (Jing et al., 2013), gi-201 (Martin-Tryon et al.,2007), ft-10 (Yoo et al., 2005), co-9 (Baduel et al., 2018), cry1/2 (Guo et al., 1998),clf-28 (Doyle and Amasino, 2009), clf-29 (Bouveret et al., 2006), ref6-3 and ref6 clf(Lu et al., 2011), EMF1-RNAi (Wang et al., 2014), tfl2-1 (Larsson et al., 1998), andatx1-2 (Pien et al., 2008). The T-DNA mutants were confirmed by PCR geno-typing and sequencing. Double and triple mutants were generated by geneticcrossing and were verified by genotyping and sequencing. The seeds weresterilized and sown on one-half-strength Murashige and Skoog (MS) mediumsupplemented with 3% (w/v) Suc. After stratification at 4°C for 3 d, the seedswere grown in LDs (16 h of light/8 h of dark) or SDs (8 h of light/16 h of dark)under light-emitting diode (LED) lights (90 mmol m22 s21) at 22°C. The plantswere transferred to climate chambers on day 0. ZT represents the time after thelights were switched on. Flowering time was scored as leaf number of plants atbolting. The numbers of total leaves and rosette leaves were counted until theshoot reached ;1 cm in length after bolting.

Plasmid Construction and Generation of Transgenic Lines

To construct vectors for recombinant proteins, the MfeI-XhoI-digested full-length PKL complementary DNA (cDNA) was released and inserted into pET-28a (Novagen) digested with EcoRI and XhoI, to give rise to His-PKL. TheD1,D2, D3, D4, and D5 fragments of PKL were cloned previously in Jing et al.(2013). The MfeI-XhoI-digested PKL, D1, D2, D3, D4, D5, and D6 fragmentswere released and inserted into EcoRI-SalI sites of the pGBKT7 vector (Clontech)to give rise to pBD-PKL, D1, D2, D3, D4, D5, and D6, respectively. The full-length ATX1 coding sequence was amplified from Col cDNA and cloned intothe pEASY vector (TransGen), resulting in pEASY-ATX1. ATX1 was releasedfrom SmaI-SalI-digested pEASY-ATX1 and ligated into the SmaI-XhoI sites ofthe pGADT7 vector (Clontech) to generate pAD-ATX1. ATX1 was releasedfrom KpnI-SalI-digested pEASY-ATX1 and ligated into the KpnI-SalI sites of thepRI-GFP and pRI-GR vectors to generate 35S:ATX1-GFP and 35S:ATX1-GR,respectively. To construct vectors for LCI assays, the KpnI-SalI-digested ATXwas fused to pUC19-nLUC (Chen et al., 2008) to produce ATX1-nLUC, whereasthe KpnI-SalI-digested full-length PKL cDNA was fused to pUC19-cLUC (Chenet al., 2008) to produce cLUC-PKL. Agrobacterium tumefaciens strain GV3101cells carrying the constructs were transformed into Arabidopsis plants via the

floral dip method. All primers used in cloning are listed in SupplementalTable S1.

RT-qPCR Analysis

For reverse transcription (RT)-qPCR analysis, total RNAwas extracted fromseedlings at the indicated time points using an RNA Extraction Kit (Tiangen).Total RNAs were used to prepare first-strand cDNAs by M-MLV reversetranscriptase (Invitrogen). RT-qPCR was carried out in a LightCycler 480(Roche) using a SYBR Premix ExTaq Kit (Takara) following the manufacturer’sinstructions. RT-qPCR was performed with three technical repeats; the ex-pression level was normalized to that of IPP2 (internal control). Primers usedfor gene expression analysis are listed in Supplemental Table S1.

Yeast Two-Hybrid Analysis

Yeast two-hybrid assays were performed to examine protein-protein inter-actions according to the Yeast Protocols Handbook (Clontech). The respectivecombinations of fusion vectors were cotransformed into the yeast strain Y2HGold (Clontech). To assess the protein interaction, the transformed yeasts weresuspended in liquid SD/-Leu/-Trp to OD 5 1. Four microliters of suspendedyeast were spotted on SD/-Trp-Leu or SD/-Trp-Leu-His-Ade dropout plates todetect direct interactions between two proteins after 3 d of incubation at 30°C.

ChIP Assays

The ChIP assaywas carried out as described previously (Bowler et al., 2004).Seedlings (1–2 g) were vacuum infiltrated with 1% (v/v) formaldehyde for15 min, and cross-linking was quenched with the addition of 0.125 M Gly for5 min. Tissue was ground, and the chromatin was isolated and sonicated toproduce DNA fragments of approximately 300 to 500 bp. Precipitated DNAwas phenol/chloroform extracted, ethanol precipitated, and dissolved inwater.The following antibodies were used: anti-H3 (Millipore, 07-690), anti-H3K27me3 (Millipore, 07-449), anti-H3K4me3 (Millipore, 07-473), and anti-GFPmAb-agarose (MBL, D153-8). The DNA samples were quantified by qPCRto examine the enrichment of target sequences, represented as either the ratio tothat of histone H3 (for H3K27me3 and H3K4me3) or the ratio to input (forATX1-GFP binding). Primer pairs used for ChIP assays are listed inSupplemental Table S1.

Pull-Down Assays

His-MBP and His-PKL fusion recombinant proteins were induced andexpressed in Escherichia coli BL21 (DE3). The proteins were then purified usingNi-NTA Agarose (Qiagen) following the manufacturer’s instructions. Ap-proximately 2mg of purified bait proteins and 500mg of total proteins (extractedfrom 10-d-old 35S:ATX1-GFP seedlings grown in LDs at ZT16) were incubatedin binding buffer (50mMTris-HCl [pH 7.5], 100mMNaCl, and 0.6% [v/v] TritonX-100) at 4°C for 2.5 h. Following the addition of Ni-NTA Agarose, the sampleswere further incubated for 1 h. After washing with binding buffer, the pre-cipitated proteins were eluted by heating the beads at 90°C for 5 min in 23 SDSloading buffer and then size fractionated on an 8% SDS-PAGE gel and immu-noblotted with anti-His (Abcam, ab14923) and anti-GFP (Abcam, ab1218)antibody.

Co-IP Assays

Seedlings were grown in the LDs for 11 d, and samples were harvested atZT16. Twenty-five microliters of anti-GFPmAb-agarose (MBL, D153-8) wereadded into the total protein extracts and incubated for 2.5 h. The beads werewashed four times with protein extraction buffer (500 mM Tris-HCl [pH 7.5],150 mMNaCl, 150 mM KCl, 5 mM MgCl2, 5 mM EDTA [pH 8], 1% (v/v) Triton X-100, 10% (v/v) glycerol, and protease inhibitor cocktail [Roche]). The precipi-tated proteinswere eluted in 23 SDS loading buffer by boiling at 90°C for 5min.The proteins were separated on an 8% SDS-PAGE gel and detected by immu-noblotting with anti-PKL (Jing et al., 2013) and anti-GFP (Abcam, ab1218)antibodies.



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LCI Assays

The LCI experiments were carried out as described previously (Chen et al.,2008). The A. tumefaciens GV3101 strains carrying the indicated nLUC/cLUCconstructs were incubated in Luria-Bertani medium at 28°C overnight. Theculture was pelleted, washed twice, and resuspended in 10 mM MgCl2 con-taining 0.2 mM acetosyringone to a final concentration of OD600 5 1.5. Thebacteria were kept at 28°C for 3 to 5 h without shaking. The A. tumefacienssuspensions were coinfiltrated into fully expanded young Nicotiana benthamianaleaveswith a needleless syringe. Theplantswere then grown at 22°C for 2 dunderLD conditions before LUC activity was measured. LUC images were capturedusing a NightSHADE LB985 plant imaging apparatus equipped with a CCDcamera (Berthold Technologies). The experiments were repeated four times.

Statistical Analyses

Statistical significance was determined by Student’s t test. The means and SD

are derived from independent biological samples.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL data libraries under the following accessionnumbers: PKL (AT2G25170), PKR2 (AT4G31900), FT (AT1G65480), WRKY70(AT3G56400), LTP7 (AT2G15050), CO (AT5G15840), GI (AT1G22770), CLF(AT2G23380), LHP1/TFL2 (AT5G17690),EMF1 (AT5G11530),REF6 (AT3G48430),ATX1 (AT2G31650),ACT2 (AT3G18780),UBQ10 (AT4G05320),Ta3 (AT1G37110),and IPP2 (AT3G02780).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Genetic interaction of the Polycomb mutantswith photoperiodic pathway mutants.

Supplemental Figure S2. PKL antagonizes PcG-induced repression offlowering under both LDs and SDs.

Supplemental Figure S3. ChIP analysis of H3K27me3 enrichment at the FTlocus in the EMF1-RNAi and pkl EMF1-RNAi seedlings.

Supplemental Figure S4. REF6 demethylase does not function antagonis-tically with CLF to regulate FT.

Supplemental Figure S5. Analysis of H3K4me3 enrichment by ChIP assayand the genetic interaction between atx1 and PcG mutants.

Supplemental Figure S6. Characterization of 35S:ATX1-GFP atx1-2 trans-genic lines and analyses of ATX1-GFP levels in pkl-1.

Supplemental Figure S7. PKL mutation does not affect CLF expressionunder LDs.

Supplemental Figure S8. PKL and PKR2 act redundantly to regulate flowering.

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

ACKNOWLEDGMENTS

We thank Dr. Xiaofeng Cao (Institute of Genetics and DevelopmentalBiology, Chinese Academy of Sciences) for ref6-3, clf-28, and ref6 clf mutantseeds, Dr. Yuehui He (Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences) for EMF1-RNAi seeds, Dr. Claudia Köhler (Swiss FederalInstitute of Technology, Eidgenössisch Technische Hochschule Centre) for pkl-1 and pkl pkr2mutant seeds, and the Arabidopsis Biological Resource Center forthe T-DNA insertion lines.

Received May 15, 2019; accepted July 16, 2019; published August 3, 2019.

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