Research Report

Temporal-Specific Interaction of NF-YC and CURLY LEAFduring the Floral Transition Regulates Flowering1[OPEN]

Xu Liu,a,2 Yuhua Yang,a,b,2 Yilong Hu,a,b Limeng Zhou,a,b Yuge Li,a and Xingliang Houa,3

aKey Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement andGuangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academyof Sciences, Guangzhou 510650, ChinabUniversity of the Chinese Academy of Sciences, Beijing 100049, China

ORCID IDs: 0000-0003-1970-0608 (X.L.); 0000-0002-3964-2372 (X.H.).

The flowering time of higher plants is controlled by environmental cues and intrinsic signals. In Arabidopsis (Arabidopsisthaliana), flowering is accelerated by exposure to long-day conditions via the key photoperiod-induced factor FLOWERINGLOCUS T (FT). Nuclear Factor-Y subunit C (NF-YC) proteins function as important mediators of epigenetic marks in differentplant developmental stages and play an important role in the regulation of FT transcription, but the mechanistic details of thisremain unknown. In this study, we show that Arabidopsis NF-YC homologs temporally interact with the histonemethyltransferase CURLY LEAF (CLF) during the flowering transition. The binding of NF-YC antagonizes the association ofCLF with chromatin and the CLF-dependent deposition of H3 lysine-27 trimethylation, thus relieving the repression of FTtranscription and facilitating flowering under long-day conditions. Our findings reveal a novel mechanism of NF-YC/CLF-mediated epigenetic regulation of FT activation in photoperiod-induced flowering and, consequently, contribute to ourunderstanding of how plants control developmental events in a temporal-specific regulatory manner.

Flowering is precisely regulated by environmentaland endogenous signals (Andrés and Coupland, 2012).In Arabidopsis (Arabidopsis thaliana), a facultative long-day (LD) plant, flowering signals are perceived andtransmitted by differentmolecular pathways, includingphotoperiod, vernalization, thermosensory, aging, au-tonomous, and GA pathways, which mostly convergeto regulate the key flowering integrator gene FLOW-ERING LOCUS T (FT). FT, the long-sought florigen,functions as a long-distance signal to the shoot apicalmeristem, where it diurnally binds to FD for tran-scriptional activation of floral meristem identity genes(Kardailsky et al., 1999; Kobayashi et al., 1999; Abeet al., 2005; Wigge et al., 2005; Corbesier et al., 2007). InLD conditions, the main photoperiod-responsive reg-ulator CONSTANS (CO) promotes flowering by

directly activating the FT and another floral integratorgene, SUPPRESSOR OF OVEREXPRESSION OF CO1(SOC1; Putterill et al., 1995; Lee et al., 2000; Samachet al., 2000). In addition, FT also could be regulated byimportant regulators involved in other floweringpathways, such as FLOWERING LOCUS C (FLC),FLOWERING LOCUS M variants, and PHYTO-CHROME INTERACTING FACTOR4, etc., to modu-late flowering time (Michaels and Amasino, 1999;Kumar et al., 2012; Posé et al., 2013). So far, recentfindings have suggested that FT exerts a central func-tion in the flowering transition.

Flowering is genetically controlled by various epi-genetic factors that activate or repress the transcrip-tion of flowering genes (He, 2012). In Arabidopsis,the Polycomb Repressive Complex2 (PRC2) specifi-cally mediates the histone H3 Lys-27 trimethylation(H3K27me3) that plays extensive regulatory roles invarious developmental stages, including flowering(Holec and Berger, 2012). CURLY LEAF (CLF) is aconserved component in PRC2, and loss of function ofCLF causes an early-flowering phenotype with curledleaves (Goodrich et al., 1997). Previous extensive evi-dence supported that FT up-regulation is fundamen-tally required for the early flowering of clf, andCLF retains FT repression through promoting theH3K27me3 deposition in FT chromatin (Jiang et al.,2008; Adrian et al., 2010).

Nuclear Factor-Y subunit C (NF-YC), also termedHistone-Associated Protein5 (HAP5) and CCAATBinding Factor C (CBF-C), interacts with NF-YB(HAP3/CBF-A) to form a heterodimer analogous to the

1 This work was supported by grants from the National NaturalScience Foundation of China (no. 31301055), the Guangdong Scienceand Technology Department of China (no. 2015B020231009), and theNatural Science Foundation of Guangdong Province (no.2017A030313211).

2 These authors contributed equally to the article.3 Address correspondence to houxl@scib.ac.cn.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:Xingliang Hou (houxl@scib.ac.cn).

X.L. and X.H. designed the research; X.L., Y.Y., Y.H., L.Z., and Y.L.performed the research; X.L., Y.Y., and X.H. analyzed the data; X.L.and X.H. wrote the article.

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core histone 2A/2B dimer with the highly conservedhistone fold domain. The NF-YC/YB dimer, in turn,unites with NF-YA (HAP2/CBF-B) to constitute theNF-Y heterotrimer that canonically binds to theCCAAT box in eukaryotic promoters (Huber et al.,2012; Nardini et al., 2013). In plants, NF-YC subunitsare encoded by a multigene family and function asimportant participants in flowering time control(Petroni et al., 2012; Swain et al., 2017; Zhao et al., 2017).Arabidopsis NF-YC homologs have redundant roles inthe FT activation of photoperiod-dependent floweringby contributing to forming the canonical NF-YC-YB-YAand specific NF-YC-YB-CO complexes, which recog-nize the distal CCAAT box enhancers and the proximalCO-responsive elements, respectively, within the FTpromoter region (Kumimoto et al., 2010; Cao et al.,2014; Gnesutta et al., 2017). Recent studies have dem-onstrated that NF-Y factors recruit different histonemodifiers to regulate various developments in plants(Hou et al., 2014; Tang et al., 2017). However, howNF-Ymediates the epigenetic regulation of FT transcriptionremains unknown.

In this study, we demonstrate a CLF-dependent ep-igenetic mechanism in the NF-YC-mediated FT tran-scription regulation in Arabidopsis. NF-YC homologscounteract the H3K27me3 deposition in FT chromatinby temporally interacting with CLF to attenuate theassociation of CLF with the FT locus, thus derepressingFT transcription under LD conditions. Collectively, ourfindings support that NF-YC proteins function as im-portant modulators of epigenetic marks controllingflowering, promoting the understanding of how plantsprecisely control developmental events in a temporal-specific regulatory manner.

RESULTS

NF-YC Interacts with CLF

To further investigate the molecular roles of NF-YCproteins, we performed a yeast two-hybrid screening toidentify the potential NF-YC-interacting epigeneticpartners. Interestingly, CLF, the conserved histonemethyltransferase of PRC2, was found to interactstrongly with NF-YC3 and NF-YC9, and weakly withNF-YC4, in yeast (Fig. 1A). Then, the glutathione S-transferase (GST) pull-down assay showed that therecombinant proteins of His-NF-YC3, His-NF-YC4, andHis-NF-YC9 were coprecipitated by GST-CLF fusionproteins but not by the GST control (Fig. 1B), indicatingthe physical interaction between CLF and NF-YC pro-teins in vitro. Since NF-YC9 could genetically replacethe function of the other two homologs (Liu et al.,2016b; Tang et al., 2017), we chose NF-YC9 as the rep-resentative NF-YC gene for further investigation.

According to the conserved regulatory domains inNF-YC and CLF (Holec and Berger, 2012; Liu et al.,2016b), we designed various truncated versions ofNF-YC9 and CLF to identify the necessary domainsrequired for the protein interaction. This result indicates

that the highly conserved CXC-SET domain of CLFand the C-terminal end of NF-YC9 contribute to theprotein interaction (Fig. 1C). Bimolecular fluores-cence complementation (BiFC) analysis was con-ducted in tobacco (Nicotiana tabacum) leaf epidermalcells to examine the interaction between NF-YC9 andCLF in vivo. The fluorescence by NF-YC9-nEYFPtogether with cEYFP-CLF was localized in the cellnuclei (Fig. 1D). Coimmunoprecipitation (Co-IP) anal-ysis using the nuclear extracts of 9-d-old seedlings(clf-28 nf-yc9-1 35S:GFP-CLF pNF-YC9:NF-YC9-FLAG)further confirmed the binding of NF-YC9 to CLF inArabidopsis (Fig. 1E). Taken together, these data sup-port the direct interaction between NF-YC and CLFproteins in plants.

CLF Is Epistatic to NF-YC

As both CLF and NF-YC are involved in floweringcontrol (Goodrich et al., 1997; Kumimoto et al., 2010;Hou et al., 2014), but with little mutual regulation re-garding transcriptional levels (Supplemental Fig. S1),the interaction between CLF and NF-YC proteins led usto investigate whether these two proteins associatefunctionally to regulate flowering. We examined thegenetic connection between NF-Y and CLF by intro-ducing the nf-yc3-2 nf-yc4-1 nf-yc9-1 (nf-ycT) mutationsinto the clf-28 (clf) mutant background. Under LDconditions, the clf nf-ycTmutants exhibited similar earlyflowering and upward-curled leaves to clf mutants,whereas nf-ycT showed extreme late flowering, asreported previously (Fig. 2, A and B; Kumimoto et al.,2010). Meanwhile, mutation of CLF also rescued thelate-flowering phenotype in loss-of-functionmutants ofNF-YBs (nf-yb2-1 nf-yb3-1), another subunit of NF-Yinvolved in flowering control (Supplemental Fig. S2).These observations indicate that the genetic function ofCLF in flowering control is epistatic to NF-YC/B genes.To test this further, we examined the temporal expres-sion of several flowering genes in various geneticbackgrounds. Both FT and SOC1 were significantlyup-regulated in the Col wild type during the floraltransition under LD conditions. Remarkably, the dailyexpression of FT was down-regulated in nf-ycT buthighly up-regulated in clf and clf nf-ycT compared withthat in the wild type (Fig. 2C; Supplemental Fig. S3),supporting the epistatic role of CLF to NF-YC in FTexpression. By contrast, SOC1 transcripts were de-creased by nf-ycT in either the clf or CLF background,indicating that CLF does not mediate NF-YC-regulatedSOC1 expression (Fig. 2C). In addition, CO, encodingthe main regulator of FT transcription, was hardlyregulated by NF-YC and CLF (Fig. 2C). FLC, SEP3, andAG genes are known as the direct downstream targetsof CLF and are repressed by CLF in flowering (Schubertet al., 2006; Lopez-Vernaza et al., 2012). Our analysisshowed that, compared with that in the wild type, theexpression of these genes was increased remarkably inclf and clf nf-ycT but changed slightly (FLC) or not

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affected (SEP3 and AG) in the nf-ycT background(Supplemental Fig. S4), indicating that it is unlikely thatNF-YC acts as the main regulator of the CLF targets

FLC, SEP3, and AG. Therefore, these results suggestthat CLF and NF-YC mediate flowering time primarilyby regulating FT.

Figure 1. NF-YCs interact physically with CLF in vitro and in vivo. A, Yeast two-hybrid assays show the interactions between CLFand NF-YC proteins. Transformed yeast cells were grown on SD-Trp/-Leu/-His/-Ade or SD-Trp/-Leu medium. AD, Activationdomain; BD, binding domain. B, Pull-down assays show the physical interaction between GST-CLF and His-NF-YC fusionproteins in vitro. His-NF-YC proteins were incubated with immobilized GSTor GST-CLF, and immunoprecipitated fractions weredetected by anti-His and anti-GSTantibodies, respectively. Arrows indicate the specific bands of GST-CLF or GST; the arrowheadindicates the nonspecific bands. C, Domains required for the interaction of CLF and NF-YC proteins. Sketches show the domainsof NF-YC9 and CLF and their various deletions. Yeast two-hybrid assays show the interactions between CLF, NF-YC9, and theirderivatives. Transformed yeast cells were grown on SD-Trp/-Leu/-His/-Ade or SD-Trp/-Leu medium. D, BiFC analysis of the in-teraction between NF-YC9-nEYFP and cEYFP-CLF in tobacco leaf epidermal cells. The 49,6-diamino-2-phenylindol (DAPI)staining indicates the nucleus. Bars = 20 mm. E, In vivo interaction of NF-YC9 and CLF in Arabidopsis. Plant nuclear extracts from9-d-old clf nf-yc9 35S:GFP-CLF pNF-YC9:NF-YC9-FLAG seedlings under LD conditionswere immunoprecipitated by either anti-GFP trap or preimmune serum (IgG). The coimmunoprecipitated proteins were detected by anti-FLAG and anti-GFP antibodies.

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NF-YC Mediates CLF-Dependent H3K27me3 Deposition

CLF functions in the trimethylation of H3K27 atchromatin of flowering genes (Goodrich et al., 1997;Schubert et al., 2006; Jiang et al., 2008). To learnwhetherNF-YC affects the epigenetic function of CLF, we ex-amined the H3K27me3 levels at several selected FT lociin Col, nf-ycT, clf, and clf nf-ycT plants by chromatinimmunoprecipitation (ChIP) assays (Fig. 3A). Notably,H3K27me3 marks in the FT locus were increased in nf-ycT, whereas the loss of CLF significantly reduced thelevels of H3K27me3 in both the nf-ycT and wild-typebackgrounds (Fig. 3B). Furthermore, we examined thedeposition of H3K27me3 at the selected FLC, AG, andSOC1 loci. Consistent with the expression analysis,H3K27me3 levels in FLC and AGwere attenuated in clfand clf nf-ycT (Figs. 2C and 3B). By contrast, loss of NF-YC had only a slight effect or no effect on H3K27me3deposition in FLC and AG, respectively, but enhancedthat in SOC1 (Fig. 3B). These results indicate that

NF-YC may repress CLF-dependent H3K27me3 depo-sition to specifically regulate FT transcription.

Loss of NF-YC Function Elevates CLF Enrichment andH3K27me3 Deposition on FT Chromatin

We created nf-ycT clf GFP-CLF by introducing the nf-ycT mutations into clf 35S:GFP-CLF plants. Under LDconditions, GFP-CLF could rescue the early-floweringphenotype of clf and nf-ycT clf GFP-CLF exhibited a late-flowering phenotype similar to that in nf-ycT (Fig. 4A),suggesting that CLF activity is required for late flow-ering caused by NF-YC loss of function. We next con-ducted ChIP assays to examine the association ofGFP-CLF with target chromatin in clf GFP-CLF andnf-ycT clf GFP-CLF plants. NF-YC loss of function sig-nificantly promoted the binding of GFP-CLF to FT,which was concomitant with higher H3K27me3 levelsof FT chromatin and lower FT expression in nf-ycT clf

Figure 2. Genetic analysis of the interaction of CLF and NF-YC. A, Flowering phenotypes of Columbia (Col), clf, nf-ycT, and clfnf-ycT plants. The seedlingswere grown under LD conditions at 22°C for 28 d and selected for photography. B, Flowering times ofCol, clf, nf-ycT, and clf nf-ycT plants under LD conditions at 22°C. The number of rosette leaves in at least 10 plants at bolting wasused as an indicator of flowering time. Statistically significant differences are indicated by different lowercase letters (one-wayANOVA, P, 0.05). C, Quantitative real-time PCR (qPCR) analysis of FT, SOC1, and CO temporal expression in developing Col,clf, nf-ycT, and clf nf-ycT seedlings. The seedlings were grown under LD conditions at 22°C and collected for RNA extraction atthe 12th h in a 24-h period (16 h of light/8 h of dark). The TUBULIN2 gene (TUB2) was used as an internal control. Data representmeans 6 SD of biological triplicates.

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GFP-CLF compared with that in clf GFP-CLF plants(Fig. 4, B–D). By contrast, no difference was observedregarding GFP-CLF binding to SOC1 between clf GFP-CLF and nf-ycT clf GFP-CLF plants (Fig. 4, C and D).These results confirm the repressive role of NF-YC onCLF binding to FT chromatin.

Temporal-Specific Interaction of NF-YC and CLF RegulatesCLF-Dependent H3K27me3 Deposition in FT Chromatin

Generally, plants do not immediately enter theflowering transition without undergoing a period ofvegetative development. Hence, we wondered howNF-YC represses CLF function via protein-protein in-teraction to facilitate flowering at the right time. Toaddress this, the temporal pattern of the NF-YC-CLFinteraction was investigated by a time-course ChIPanalysis using developing clf GFP-CLF and nf-ycT clfGFP-CLF seedlings grown under LD conditions. Asplant growth progressed, both CLF binding andH3K27me3 deposition in FT decreased gradually dur-ing the flowering transition stage (9–11 d after germi-nation; Fig. 5A; Supplemental Fig. S5). It is worthnoting that NF-YC loss of function significantly atten-uated this decline (Fig. 5A). Furthermore, ChIP assaywith a transient expression system showed that the

binding of CLF to the FT region was decreased dra-matically by the full-length NF-YC9 but not by theC-terminal-deleted NF-YC9, which fails to interact withCLF (Fig. 1C; Supplemental Fig. S6, A and B). Theseobservations support that CLF function may be im-paired by the enhanced interaction of CLF and NF-YCduring the flowering transition stage. One possibility isthat the varied interaction pattern could be triggered bythe increasing abundance of NF-YC proteins duringplant growth. However, we did not observe obviousincreased NF-YC accumulation during the floweringtransition stage by immunoblot analysis of clf nf-yc9GFP-CLF NF-YC9-FLAG seedlings (Fig. 5B). In contrastto the decreased CLF binding and H3K27me3 levels(Fig. 5A), time-course Co-IP analysis showed an in-creasing interaction between CLF and NF-YC9 duringthe flowering transition (Fig. 5B), suggesting that otherunknown factors determined by flowering signalsmight be involved in the temporal regulation of theNF-YC-CLF interaction.

DISCUSSION

In yeast and mammals, NF-Y factors are deemed tomake a functional link between chromatin and tran-scription (Dolfini et al., 2012). Our recent studies have

Figure 3. ChIPanalysis of H3K27me3 levels in the relative flowering genes in various mutants. A, Schematic representation of thespecific primer positions in the FT, FLC, SOC1, and AG loci used in B as well as in Figures 4B and 5A. Exons are represented byblack rectangles and upstream regions and introns by black lines; the primer fragments used for amplification are indicated bygray lines. B, ChIP analysis of H3K27me3 levels of the relative flowering genes in Col, clf, nf-ycT, and clf nf-ycT plants. Plantnuclear proteins were extracted from 9-d-old seedlings under LD conditions. Relative fold enrichment values were calculated bynormalizing the amount of a target DNA fragment against that of a genomic fragment of a reference gene, Cinful-like, and thenagainst the respective input DNA samples. The enrichment of an ACTIN2 (ACT2) genomic fragment was used as the negativecontrol. Data represent means 6 SD of biological triplicates. Statistically significant differences between Col and mutants areindicated by different lowercase letters (one-way ANOVA, P , 0.05).

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revealed that NF-YC regulates plant developmentalstages via mediating epigenetic modifications on targetgenes (Hou et al., 2014; Tang et al., 2017), implying animportant function of NF-YC proteins in the epigeneticregulation in plants. In the photoperiod floweringpathway, NF-Y factors are crucial for the chromatinactivation of FT, which is simultaneously controlled byvarious epigenetic regulations (Gu et al., 2013; Bu et al.,2014; Cao et al., 2014). However, whether and howNF-YC mediates the epigenetic regulation of FT tran-scription remain unknown. Here, we reveal a temporalinteraction of NF-YC with the histone methyltransfer-ase CLF that specifically counteracts CLF-dependentH3K27me3 deposition to derepress FT transcription,thus promoting flowering under LD conditions. Theseresults illustrate a novel epigenetic regulatory model inthe photoperiod flowering pathway and expand ourunderstanding of how plants precisely control devel-opmental events in a temporal-specific manner.

In eukaryotes, the PRC2 members are structurallyand evolutionarily conserved, in which CLF orthologs

function as the core component to catalyze H3K27methylation. The conserved CXC and SET domains ofCLF are necessary for H3K27methyltransferase activityand sequence-specific binding (Cao et al., 2002; Mülleret al., 2002; Ketel et al., 2005). For example, the gain-of-function allele clf-59, harboring a Pro-to-Ser amino acidmutation in the CXC domain, results in vernalization-independent early flowering in Arabidopsis (Doyle andAmasino, 2009). Like CLF, NF-YC, with a histone folddomain close to the core histone 2A, is widely con-served in eukaryotic species and functions as a subunitof NF-Y heterotrimers (Dolfini et al., 2012). In thisstudy, the highly conserved CXC and SET domains ofCLF and the C-terminal end of NF-YC9 contribute totheir protein interaction (Fig. 1C), implying that thecombination of CLF andNF-YCmay frequently presentand function in different eukaryotic species ormay playdistinct roles in various biological processes. Upon ourobservations, the binding of NF-YC to the CXC-SETdomain of CLF impairs the DNA affinity of CLF, thusrestricting CLF function to target chromatins. However,

Figure 4. Analysis of NF-YC’s effect on the CLF enrichment and H3K27me3 deposition at FT loci. A, Late flowering caused by theloss of NF-YC genes requires CLF activity. At least 10 seedlings of Col, clf, nf-ycT, clf GFP-CLF, clf nf-ycT, and nf-ycT clf GFP-CLFplants grown under LD conditions at 22°C were investigated for flowering time determination. B, qPCR analysis of FT gene ex-pression. Nine-day-old seedlings were grown under LD conditions at 22°C and collected for RNA extraction at the 12th h in a 24-hperiod (16 h of light/8 h of dark). C, ChIP analysis of CLF binding to the FT locus (amplicons defined in Fig. 3A). D, Analysis ofH3K27me3 levels in FT chromatin. Nine-day-old clf 35S:GFP-CLF and nf-ycT clf 35S:GFP-CLF seedlings were grown under LDconditions at 22°C and collected for ChIPassay with anti-GFPand anti-H3K27me3 antibodies, respectively. Data represent means6SD of biological triplicates. Statistically significant differences are indicated by different lowercase letters (one-way ANOVA, P ,0.05). Asterisks indicate significant changes of enrichment fold between two genetic backgrounds (Student’s t test, P , 0.05).

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we cannot exclude the possibility that NF-YC might actas a potential catalyzed substrate of CLF to mediateunknown regulations.FT functions as a major output of the photoperiod

pathway regulator CO when plants respond to LDconditions (Andrés and Coupland, 2012). Recent stud-ies have demonstrated that NF-Y factors are involved inthe CO-FT regulatory cascade via direct interactionwith CO (Kumimoto et al., 2010; Gnesutta et al., 2017).The identification of NF-Y-mediated chromatin loopingat the FT locus and the novel sequence-specific NF-YB/YC/CO complex further support that CO regulating FTis dependent primarily on NF-Y(C) functions (Caoet al., 2014; Gnesutta et al., 2017). It was shown that CLFindirectly controls FT expression by epigeneticallyregulating FLC in different aspects, such as the auton-omous pathway (Doyle and Amasino, 2009; Lopez-Vernaza et al., 2012; Müller-Xing et al., 2014).However, CLF could bind directly to FT chromatin andmediate H3K27me3 deposition in FT under LD condi-tions (Fig. 5A; Jiang et al., 2008; Adrian et al., 2010).Similar to clf nf-ycT, clf co doublemutants also presenteda clf-like early-flowering phenotype (Pazhouhandehet al., 2011). Considering the biological interaction be-tween NF-YC and CLF presented in this study, it isquite possible that the CO-NF-Y module regulates FTtranscription by preventing CLF-dependent H3K27me3deposition in FT chromatin. Consistently, clf could rescue

the late-flowering phenotype caused by the loss ofNF-YB2/3 function (Supplemental Fig. S2); however,the question of whether NF-YC represses CLF togetherwith NF-YA/B and CO to facilitate flowering requiresfurther investigation.

Besides FT, SOC1 acts as another important integra-tor in flowering control (Lee et al., 2000; Samach et al.,2000). Our previous study showed that SOC1 is regu-lated directly by CO-NF-Y via the recruitment of anH3K27 demethylase, RELATIVE OF EARLY FLOW-ERING6 (REF6), under LD conditions (Hou et al., 2014).Although H3K27me3 levels in SOC1 decrease duringthe flowering transition, we did not observe that CLFaffects SOC1 gene expression in this study (Fig. 2C),suggesting that NF-YCmediates H3K27me3 depositionin SOC1 chromatin primarily by recruiting REF6 ratherthan by impairing CLF activity. In addition, we detec-ted gene expression and H3K27me3 levels of FLC,SEP3, and AG genes, which are known as CLF targets(Goodrich et al., 1997; Schubert et al., 2006; Lopez-Vernaza et al., 2012). These results showed that thesegenes were hardly regulated by NF-YC, implying thatother factors, but not NF-YC, might be involved in theirregulation by CLF. Furthermore, since the dynamics ofthe H3K27me3 mark regulated by CLF at many ge-nomic loci may be detected only in specific tissues andat specific developmental stages (Liu et al., 2016a), it ispossible that the NF-YC-CLF module also could

Figure 5. Temporal pattern analysis of NF-YC and CLF regulating CLF-dependent H3K27me3 at the FT locus. A, Time-courseChIPanalysis of CLF binding (top) and H3K27me3 levels (bottom) in FT chromatin (amplicons defined in Fig. 3A). Seedlings of clf35S:GFP-CLF and nf-ycT clf 35S:GFP-CLFwere grown under LD conditions at 22°C and harvested for nuclear protein extractionat the indicated days. The percentages indicate the relative enrichment of the FT-I fragment in clf 35S:GFP-CLF against that in nf-ycT clf 35S:GFP-CLF (designated as 100%). B, Co-IPanalysis of the temporal interaction between NF-YC9 and CLF in developingseedlings. clf nf-yc9 GFP-CLF NF-YC9-FLAG seedlings grown under LD conditions were harvested for nuclear protein extractionat the indicated days and then immunoprecipitated by anti-GFP trap. The coimmunoprecipitated proteins were detected by anti-FLAG and anti-GFP antibodies.

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contribute to the precise control of other developmentalevents.

In addition to H3K27me3, other histone modifica-tions, such as H3K4me3 and histone acetylation, alsoare involved in the regulation of flowering (He et al.,2003; Engelhorn et al., 2017). Generally, repressiveH3K27me3 and activation-relatedH3K4me3 oppositelyregulate the spatial and temporal expression of genesduring specific developmental processes. A recentgenome-wide study revealed that the H3K4me3 depo-sition prevails over H3K27me3, whereas H3K27me3reduction occurs later during early flower morpho-genesis (Engelhorn et al., 2017). Since NF-YC functionswith CO to promote FT transcription, whereas COactivates FT by recruiting MRG1/2 proteins, theH3K4me3/H3K36me3 readers (Kumimoto et al., 2010;Bu et al., 2014), it could be speculated that H3K4me3modification might be involved in NF-YC-mediatedflowering control. It was reported that several histonedeacetylases (HDAs) regulate flowering by the tran-scriptional repression of flowering repressor genes suchas FLC, MAF1, MAF4, and MAF5 (Wang et al., 2014).Although our previous work revealed that NF-YCregulates photomorphogenesis by HDA15-mediatedhistone acetylation (Tang et al., 2017), no evidencesupports the idea that NF-YC-HDA15 is involved inflowering regulation. However, it is worthwhile in-vestigating whether NF-YC regulates flowering viarecruiting other HDAs related to the flowering re-sponse, such as HDA6/9/19 (Wang et al., 2014).

It is intriguing that a temporal-specific interaction ofNF-YC and CLF was observed by time-course Co-IPassays (Fig. 5B). The increasing interaction during theflowering transition under LD conditions results inattenuated CLF binding and H3K27me3 depositionin FT chromatin, which convincingly explains thephotoperiod-induced FT up-regulation. In the photo-period pathway, the periodic change of CO protein a-bundance by light is well known to underlie therhythmic expression of FT within a day. However, thisdoes not fully explain FT transcripts increasing day byday under LD conditions, which was attributed previ-ously to FT accumulation or other positive feedbackregulations (Suárez-López et al., 2001). Here, thetemporal-specific NF-YC-CLF module provides aplausible hypothesis for increasing FT transcript a-bundance. In this scenario, FT may be induced by anNF-YC-triggered decrease of H3K27me3 when seed-lings are ready for flowering. Because we did not ob-serve a significant increase of NF-YC protein levelduring the flowering transition, it raises the question ofhow the appropriate timing of the NF-YC interactionwith CLF is regulated.We propose that othermediatingfactors or posttranslational modifications, which areinduced by the flowering signals, may be included inthis process. Previous work reported a functional in-teraction between the cullin-RING ubiquitin ligase(CUL4-DDB1) and the PRC2 complex (Pazhouhandehet al., 2011). CO is known to interact with NF-YC, andturnover of the CO proteins is triggered by COP1-SPA

complexes, a central repressor of light signaling, in thedark (Jang et al., 2008; Chen et al., 2010). In addition, itwas reported that the CUL4-DDB1 complex is requiredfor FT expression and associates with the COP1-SPAcomplex (Pazhouhandeh et al., 2011). The interactionof NF-YC and CLF may be mediated by CUL4-DDB1,COP1-SPA, or CO in a light-dependent manner, whichis worthwhile for future investigation.

Taken together, we reveal the temporal-specific reg-ulatory module NF-YC-CLF, through which NF-YCdirectly counteracts CLF activity to promote FT ex-pression and flowering at an appropriate time. Thesefindings provide novel insight into the molecularmechanisms of how plants precisely control specificdevelopment events by the temporal regulation of ep-igenetic factors.

MATERIALS AND METHODS

Plant Material and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) plant materials nf-yc3-2 nf-yc4-1nf-yc9-1 (nf-ycT) and nf-yc9 NF-YC9-FLAG were described previously (Houet al., 2014; Liu et al., 2016b). clf-28 (SALK_139371) seedswere obtained from theArabidopsis Biological Resource Center. clf GFP-CLF plants were generated bycrossing 35S:GFP-CLF (Schubert et al., 2006) with clf-28 in the Col background.The nf-ycT clf GFP-CLF and clf nf-yc9 GFP-CLF NF-YC9-FLAG plants weregenerated by crossing clf GFP-CLFwith nf-ycT and nf-yc9 NF-YC9-FLAG plants.Plant growth conditions were described previously (Hou et al., 2014), underwhich the flowering transition time of the Col wild type has been defined tooccur during 7 to 11 d after germination (Liu et al., 2008). The average numberof rosette leaves calculated from a minimum of 10 plants at bolting was used asan indicator of flowering time.

Yeast Two-Hybrid Assay

The coding regions of NF-YC3, NF-YC4, NF-YC9, CLF, and truncated ver-sions of NF-YC9 and CLF were amplified and cloned into pGBKT7 (bindingdomain) and pGADT7 (activation domain) vectors (Clontech), respectively. Theprimers used are listed in Supplemental Table S1. Yeast two-hybrid assayswereperformed using the Yeastmaker Yeast Transformation System 2 (Clontech).Yeast AH109 cells were cotransformed with the selected binding domain andactivation domain constructs. The yeast transformants were grown and se-lected on SD/-Trp/-Leu or SD/-Trp/-Leu/-His/-Ade medium.

BiFC Analysis

The coding regions of NF-YC9 and CLF were cloned into the pGreen binaryvectors containing C- or N-terminal fusions of EYFP to generate 35S:NF-YC9-nEYFP and 35S:cEYFP-CLF constructs. Plasmids were cotransformed into to-bacco (Nicotiana tabacum) leaf epidermal cells with Agrobacterium tumefaciensstrain GV3101-psoup as described previously (Tang et al., 2017). The YFP sig-nals were detected by an inverted fluorescence microscope (Leica) in tobaccoleaf grown for 48 h after infiltration, while 49,6-diamidino-2-phenylindolestaining was used as the nuclear localization indicator.

Pull-Down Assay

Using the designed primers (Supplemental Table S1), the coding regions ofNF-YC3, NF-YC4, NF-YC9, and CLFwere amplified and cloned into the pQE30(Qiagen) and pGEX-4T-1 (Pharmacia) vectors to produce His-NF-YC3, His-NF-YC4, His-NF-YC9, and GST-CLF constructs, respectively. These GST and Hisrecombinant constructs and the empty pGEX-4T-1 plasmid were transformedinto Escherichia coli Rosetta cells, and then recombinant protein expression wasinduced by isopropyl-b-D-thiogalactoside (Sigma). The purified His-NF-YC3,His-NF-YC4, and His-NF-YC9 proteins were incubated with immobilized GST

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or GST-CLF using glutathione-Sepharose beads (Amersham Biosciences) andanalyzed using immunoblots as described previously (Liu et al., 2016b).

Co-IP Assay

The clf nf-yc9 GFP-CLF NF-YC9-FLAG seedlings grown in LD conditionswere harvested at the indicated growth stages and ground in liquid nitrogen.Nuclear proteins were extracted as described previously (Hou et al., 2014) andthen incubated with Protein G PLUS/Protein A-Agarose Suspension (IP10;Calbiochem) plus anti-GFP antibody (ab290; Abcam) or preimmune serum(IgG) in Co-IP buffer (50 mM HEPES, pH 7.5, 150 mM KCl, 10 mM ZnSO4, 5 mM

MgCl2, and 1% [v/v] Triton X-100) at 4°C overnight. After immunoprecipita-tion, the beads were washed and eluted with SDS loading buffer. The precip-itated proteins were resolved by SDS-PAGE and immunodetected by anti-GFPand anti-FLAG (F3165; Sigma) antibodies.

Gene Expression Analysis

VariousseedlingsgrowninLDconditionswereharvestedat the indicatedgrowthstages for total RNA extraction using the E.Z.N.A. Total RNA Kit I (Omega) andreverse transcribed to cDNAusingMMLV-RTase (Promega). qPCRwas performedusing a LightCycler 480 thermal cycler system (Roche)with KAPASYBR Fast qPCRKitMasterMix (KapaBio). Thedifferencebetween the cycle thresholdof target genesand the cycle threshold of the control gene was calculated by the relative quantifi-cation method (22△△Ct) and used to evaluate quantitative variation. The primersused for gene expression analysis are listed in Supplemental Table S1. All expressionanalyses were performed with at least three biological replicates (three replicates ofsamples were taken from the same batch of seedlings, and total RNAwas extractedfrom the pooled three to five seedlings per independent replicate). The above ex-periments were independently performed at least three times, and representativeresults are shown. The P value in data statistics was calculated by two-tailed Stu-dent’s t test and one-way ANOVA.

ChIP Assay

ChIP assays were performed primarily as described previously (Hou et al.,2014). The clf GFP-CLF and nf-ycT clf GFP-CLF seedlings grown in LD condi-tions were collected at the indicated growth stages and fixed for 40 min in 1%formaldehyde. Then, the nuclear proteins were extracted, and chromatin wasisolated and sonicated to create DNA fragments approximately 500 bp in lengthon average. After that, the sonicated chromatin was immunoprecipitated byGFP-Trap (ChromoTek) or Protein G PLUS Agarose (16-201; Millipore) plusH3K27me3 antibody (07-449; Millipore) at 4°C overnight, and the remainingchromatin was used as an input control. The precipitated DNA was recoveredand quantified by qPCR with KAPA SYBR Fast qPCR Kit Master Mix (KapaBio). Relative ChIP enrichment was calculated by normalizing the amount of atarget DNA fragment against that of a Cinful-like genomic fragment and thenagainst the respective input DNA amount. The primers used for the ChIP assayare listed in Supplemental Table S1. All ChIP analyses were performed in threebiological replicates (three replicates of sampleswere taken from the same batchof seedlings, and chromatins were extracted from the pooled 20 to 30 seedlingsper replicate). The above experiments were independently performed at leastthree times, and representative results are shown. The P value in data statisticswas calculated by two-tailed Student’s t test and one-way ANOVA.

Accession Numbers

Accession numbers are as follows: NF-YC3 (AT1G54830), NF-YC4(AT5G63470), NF-YC9 (AT1G08970), CLF (AT2G23380), CO (AT5G15840), FT(AT1G65480), FLC (AT5G10140), AG (AT4G18960), SEP3 (AT1G24260), TUB2(AT5G62690), Cinful-like (AT4G03770), and ACT2 (AT5G09810).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Expression analysis of NF-YC3, NF-YC4, NF-YC9, and CLF genes in nf-ycT, clf, and Col plants.

Supplemental Figure S2. Loss of NF-YB has a minimal effect on the earlyflowering of clf mutants.

Supplemental Figure S3. Quantitative analysis of FT gene circadian ex-pression in nf-ycT, clf, clf nf-ycT, and Col plants.

Supplemental Figure S4. Expression analysis of FLC, AG, and SEP3 genesin nf-ycT, clf, clf nf-ycT, and Col plants.

Supplemental Figure S5. Time-course ChIP analysis of GFP-CLF bindingand H3K27me3 levels at the ACT2 locus.

Supplemental Figure S6. ChIP analysis of CLF binding to the FT locus inthe presence of the C-terminal-deleted NF-YC9.

Supplemental Table S1. List of primers used in this study.

ACKNOWLEDGMENTS

We thank the Arabidopsis Biological Resource Center for providing themutant seeds used in this study.

Received March 8, 2018; accepted March 19, 2018; published March 29, 2018.

LITERATURE CITED

Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y,Ichinoki H, Notaguchi M, Goto K, Araki T (2005) FD, a bZIP proteinmediating signals from the floral pathway integrator FT at the shootapex. Science 309: 1052–1056

Adrian J, Farrona S, Reimer JJ, Albani MC, Coupland G, Turck F (2010)cis-Regulatory elements and chromatin state coordinately control tem-poral and spatial expression of FLOWERING LOCUS T in Arabidopsis.Plant Cell 22: 1425–1440

Andrés F, Coupland G (2012) The genetic basis of flowering responses toseasonal cues. Nat Rev Genet 13: 627–639

Bu Z, Yu Y, Li Z, Liu Y, Jiang W, Huang Y, Dong AW (2014) Regulation ofArabidopsis flowering by the histone mark readers MRG1/2 via inter-action with CONSTANS to modulate FT expression. PLoS Genet 10:e1004617

Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, Jones RS,Zhang Y (2002) Role of histone H3 lysine 27 methylation in Polycomb-groupsilencing. Science 298: 1039–1043

Cao S, Kumimoto RW, Gnesutta N, Calogero AM, Mantovani R, Holt BF III(2014) A distal CCAAT/NUCLEAR FACTOR Y complex promotes chro-matin looping at the FLOWERING LOCUS T promoter and regulates thetiming of flowering in Arabidopsis. Plant Cell 26: 1009–1017

Chen H, Huang X, Gusmaroli G, Terzaghi W, Lau OS, Yanagawa Y,Zhang Y, Li J, Lee JH, Zhu D, et al (2010) Arabidopsis CULLIN4-damaged DNA binding protein 1 interacts with CONSTITUTIVELYPHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA complexes to reg-ulate photomorphogenesis and flowering time. Plant Cell 22: 108–123

Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A,Farrona S, Gissot L, Turnbull C, et al (2007) FT protein movementcontributes to long-distance signaling in floral induction of Arabidopsis.Science 316: 1030–1033

Dolfini D, Gatta R, Mantovani R (2012) NF-Y and the transcriptional ac-tivation of CCAAT promoters. Crit Rev Biochem Mol Biol 47: 29–49

Doyle MR, Amasino RM (2009) A single amino acid change in the en-hancer of zeste ortholog CURLY LEAF results in vernalization-independent, rapid flowering in Arabidopsis. Plant Physiol 151: 1688–1697

Engelhorn J, Blanvillain R, Kröner C, Parrinello H, Rohmer M, Posé D,Ott F, Schmid M, Carles CC (2017) Dynamics of H3K4me3 chromatinmarks prevails over H3K27me3 for gene regulation during flowermorphogenesis in Arabidopsis thaliana. Epigenomes 1: 8

Gnesutta N, Kumimoto RW, Swain S, Chiara M, Siriwardana C, Horner DS,Holt BF III, Mantovani R (2017) CONSTANS imparts DNA sequencespecificity to the histone fold NF-YB/NF-YC dimer. Plant Cell 29:1516–1532

Goodrich J, Puangsomlee P, Martin M, Long D, Meyerowitz EM,Coupland G (1997) A Polycomb-group gene regulates homeotic gene ex-pression in Arabidopsis. Nature 386: 44–51

Gu X, Wang Y, He Y (2013) Photoperiodic regulation of flowering timethrough periodic histone deacetylation of the florigen gene FT. PLoS Biol11: e1001649

Plant Physiol. Vol. 177, 2018 113

NF-YC and CLF Interact to Regulate Flowering

https://plantphysiol.orgDownloaded on February 17, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

He Y (2012) Chromatin regulation of flowering. Trends Plant Sci 17:556–562

He Y, Michaels SD, Amasino RM (2003) Regulation of flowering time byhistone acetylation in Arabidopsis. Science 302: 1751–1754

Holec S, Berger F (2012) Polycomb group complexes mediate develop-mental transitions in plants. Plant Physiol 158: 35–43

Hou X, Zhou J, Liu C, Liu L, Shen L, Yu H (2014) Nuclear factorY-mediated H3K27me3 demethylation of the SOC1 locus orchestratesflowering responses of Arabidopsis. Nat Commun 5: 4601

Huber EM, Scharf DH, Hortschansky P, Groll M, Brakhage AA (2012)DNA minor groove sensing and widening by the CCAAT-bindingcomplex. Structure 20: 1757–1768

Jang S, Marchal V, Panigrahi KC, Wenkel S, Soppe W, Deng XW,Valverde F, Coupland G (2008) Arabidopsis COP1 shapes the temporalpattern of CO accumulation conferring a photoperiodic flowering re-sponse. EMBO J 27: 1277–1288

Jiang D, Wang Y, Wang Y, He Y (2008) Repression of FLOWERING LOCUSC and FLOWERING LOCUS T by the Arabidopsis Polycomb repressivecomplex 2 components. PLoS ONE 3: e3404

Kardailsky I, Shukla VK, Ahn JH, Dagenais N, Christensen SK, NguyenJT, Chory J, Harrison MJ, Weigel D (1999) Activation tagging of thefloral inducer FT. Science 286: 1962–1965

Ketel CS, Andersen EF, Vargas ML, Suh J, Strome S, Simon JA (2005)Subunit contributions to histone methyltransferase activities of fly andworm polycomb group complexes. Mol Cell Biol 25: 6857–6868

Kobayashi Y, Kaya H, Goto K, Iwabuchi M, Araki T (1999) A pair of re-lated genes with antagonistic roles in mediating flowering signals. Sci-ence 286: 1960–1962

Kumar SV, Lucyshyn D, Jaeger KE, Alós E, Alvey E, Harberd NP, WiggePA (2012) Transcription factor PIF4 controls the thermosensory activa-tion of flowering. Nature 484: 242–245

Kumimoto RW, Zhang Y, Siefers N, Holt BF III (2010) NF-YC3, NF-YC4and NF-YC9 are required for CONSTANS-mediated, photoperiod-dependent flowering in Arabidopsis thaliana. Plant J 63: 379–391

Lee H, Suh SS, Park E, Cho E, Ahn JH, Kim SG, Lee JS, Kwon YM, Lee I(2000) The AGAMOUS-LIKE 20 MADS domain protein integrates floralinductive pathways in Arabidopsis. Genes Dev 14: 2366–2376

Liu C, Chen H, Er HL, Soo HM, Kumar PP, Han JH, Liou YC, Yu H (2008)Direct interaction of AGL24 and SOC1 integrates flowering signals inArabidopsis. Development 135: 1481–1491

Liu J, Deng S, Wang H, Ye J, Wu HW, Sun HX, Chua NH (2016a) CURLYLEAF regulates gene sets coordinating seed size and lipid biosynthesis.Plant Physiol 171: 424–436

Liu X, Hu P, Huang M, Tang Y, Li Y, Li L, Hou X (2016b) The NF-YC-RGL2module integrates GA and ABA signalling to regulate seed germinationin Arabidopsis. Nat Commun 7: 12768

Lopez-Vernaza M, Yang S, Müller R, Thorpe F, de Leau E, Goodrich J(2012) Antagonistic roles of SEPALLATA3, FT and FLC genes as targetsof the polycomb group gene CURLY LEAF. PLoS ONE 7: e30715

Michaels SD, Amasino RM (1999) FLOWERING LOCUS C encodes anovel MADS domain protein that acts as a repressor of flowering. PlantCell 11: 949–956

Müller J, Hart CM, Francis NJ, Vargas ML, Sengupta A, Wild B, Miller EL,O’Connor MB, Kingston RE, Simon JA (2002) Histone methyltransferaseactivity of a Drosophila Polycomb group repressor complex. Cell 111:197–208

Müller-Xing R, Clarenz O, Pokorny L, Goodrich J, Schubert D (2014)Polycomb-group proteins and FLOWERING LOCUS T maintaincommitment to flowering in Arabidopsis thaliana. Plant Cell 26: 2457–2471

Nardini M, Gnesutta N, Donati G, Gatta R, Forni C, Fossati A, Vonrhein C,Moras D, Romier C, Bolognesi M, et al (2013) Sequence-specific transcrip-tion factor NF-Y displays histone-like DNA binding and H2B-like ubiquiti-nation. Cell 152: 132–143

Pazhouhandeh M, Molinier J, Berr A, Genschik P (2011) MSI4/FVE in-teracts with CUL4-DDB1 and a PRC2-like complex to control epigeneticregulation of flowering time in Arabidopsis. Proc Natl Acad Sci USA108: 3430–3435

Petroni K, Kumimoto RW, Gnesutta N, Calvenzani V, Fornari M,Tonelli C, Holt BF III, Mantovani R (2012) The promiscuous life ofplant NUCLEAR FACTOR Y transcription factors. Plant Cell 24:4777–4792

Posé D, Verhage L, Ott F, Yant L, Mathieu J, Angenent GC, Immink RG,Schmid M (2013) Temperature-dependent regulation of flowering byantagonistic FLM variants. Nature 503: 414–417

Putterill J, Robson F, Lee K, Simon R, Coupland G (1995) The CONSTANSgene of Arabidopsis promotes flowering and encodes a protein showingsimilarities to zinc finger transcription factors. Cell 80: 847–857

Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z,Yanofsky MF, Coupland G (2000) Distinct roles of CONSTANS tar-get genes in reproductive development of Arabidopsis. Science 288:1613–1616

Schubert D, Primavesi L, Bishopp A, Roberts G, Doonan J, Jenuwein T,Goodrich J (2006) Silencing by plant Polycomb-group genes requiresdispersed trimethylation of histone H3 at lysine 27. EMBO J 25: 4638–4649

Suárez-López P, Wheatley K, Robson F, Onouchi H, Valverde F, Coupland G(2001) CONSTANS mediates between the circadian clock and the control offlowering in Arabidopsis. Nature 410: 1116–1120

Swain S, Myers ZA, Siriwardana CL, Holt BF III (2017) The multi-faceted roles of NUCLEAR FACTOR-Y in Arabidopsis thalianadevelopment and stress responses. Biochim Biophys Acta 1860:636–644

Tang Y, Liu X, Liu X, Li Y, Wu K, Hou X (2017) Arabidopsis NF-YCsmediate the light-controlled hypocotyl elongation via modulating his-tone acetylation. Mol Plant 10: 260–273

Wang Y, Gu X, Yuan W, Schmitz RJ, He Y (2014) Photoperiodic control ofthe floral transition through a distinct polycomb repressive complex.Dev Cell 28: 727-736

Wigge PA, Kim MC, Jaeger KE, Busch W, Schmid M, Lohmann JU,Weigel D (2005) Integration of spatial and temporal information duringfloral induction in Arabidopsis. Science 309: 1056–1059

Zhao H, Wu D, Kong F, Lin K, Zhang H, Li G (2017) The Arabidopsisthaliana nuclear factor Y transcription factors. Front Plant Sci 7: 2045

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