Nuclear factor Y-mediated H3K27me3 demethylation of the SOC1 locus orchestrates flowering responses of Arabidopsis

Hou, Xingliang; Zhou, Jiannan; Liu, Chang; Liu, Lu; Shen, Lisha; Yu, Hao

doi:10.1038/ncomms5601

Article
Published: 08 August 2014

Nuclear factor Y-mediated H3K27me3 demethylation of the SOC1 locus orchestrates flowering responses of Arabidopsis

Xingliang Hou¹,
Jiannan Zhou²,
Chang Liu²,
Lu Liu²,
Lisha Shen² &
…
Hao Yu²

Nature Communications volume 5, Article number: 4601 (2014) Cite this article

12k Accesses
196 Citations
2 Altmetric
Metrics details

Subjects

Abstract

Nuclear factor Y (NF-Y) is a conserved heterotrimeric transcription factor complex that binds to the CCAAT motifs within the promoter region of many genes. In plants, a large number of genes code for variants of each NF-YA, B or C subunit that can assemble in a combinatorial fashion. Here, we report the discovery of an Arabidopsis NF-Y complex that exerts epigenetic control over flowering time by integrating environmental and developmental signals. We show that NF-Y interacts with CONSTANS in the photoperiod pathway and DELLAs in the gibberellin pathway, to directly regulate the transcription of SOC1, a major floral pathway integrator. This NF-Y complex binds to a unique cis-element within the SOC1 promoter to modulate trimethylated H3K27 levels, partly through a H3K27 demethylase REF6. Our findings establish NF-Y complexes as critical mediators of epigenetic marks that regulate the response to environmental or intrinsic signals in plants.

You have full access to this article via your institution.

Download PDF

Spatial co-transcriptomics reveals discrete stages of the arbuscular mycorrhizal symbiosis

Article Open access 08 April 2024

Karen Serrano, Margaret Bezrutczyk, … Benjamin Cole

Transcription bodies regulate gene expression by sequestering CDK9

Article Open access 08 April 2024

Martino Ugolini, Maciej A. Kerlin, … Nadine L. Vastenhouw

N6-methyladenine in DNA antagonizes SATB1 in early development

Article 15 July 2020

Zheng Li, Shuai Zhao, … Andrew Z. Xiao

Introduction

The transition from vegetative to reproductive development is crucial for reproductive success of flowering plants. In Arabidopsis, the timing of this transition is controlled by a complex network of flowering genetic pathways in response to developmental cues and environmental signals¹. The interactions among these flowering pathways regulate the expression of two floral pathway integrators, FLOWERING LOCUS T (FT) and SUPPRESOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), which in turn activate the genes involved in the formation of floral meristems^2,3,4,5. The major flowering pathways include photoperiod and gibberellin (GA) pathways that promote flowering in response to seasonal changes in day length and the endogenous phytohormone GA, respectively.

CONSTANS (CO) plays a central role in mediating photoperiod-dependent flowering⁶. When CO messenger RNA (mRNA) expression regulated by the circadian clock coincides with the exposure of plants to light under long days (LDs), the CO protein is stabilized in the nucleus and upregulates downstream genes, including FT and SOC1, to promote flowering^3,7,8. Both CO in the photoperiod pathway and PHYTOCHROME INTERACTING FACTOR4 in the thermosensory pathway directly activate the mRNA expression of FT that encodes the protein as part of the florigen in leaves^3,9,10,11. Long-distance movement of FT protein from leaves to the shoot apex through the phloem system allows FT to interact with a bZIP transcription factor FD in the shoot apical meristem for activating floral meristem identity genes^10,12,13. SOC1 is expressed in leaves and shoot apical meristems in response to multiple flowering pathways, and encodes a MADS-box transcription factor that acts as a key flowering promoter through regulating another floral meristem identity gene, LEAFY^{2,3,4,5,14,15,16}. Although several studies indicate that CO activates SOC1 either directly or indirectly via FT in the photoperiod pathway^3,10,17,18, so far there is no evidence to support direct regulation of SOC1 transcription by CO or FT.

The GA pathway also plays an important role in the promotion of flowering in Arabidopsis under both LDs and short days, although its effect under LDs is usually masked by the photoperiod pathway^19,20. Molecular genetic analyses of GA and photoperiod pathways suggest that the photoperiod and GA pathways are coordinated to promote flowering under LDs^15,20,21. The GA effect on flowering is genetically mediated by five DELLA proteins, GIBBERELLIC ACID INSENSITIVE (GAI), REPRESSOR OF ga1-3 (RGA), RGA-LIKE1 (RGL1), RGL2 and RGL3, which are key GA signalling repressors whose degradation is triggered by GA^22,23. As transcriptional regulators, DELLA proteins have been shown to exert their function by recruiting other transcription factors rather than directly binding to their target genes^24,25,26,27.

It has been suggested that photoperiod-dependent flowering involves nuclear factor Y (NF-Y) complexes, which are conserved combinatorial transcription factors in eukaryotes consisting of NF-YA, NF-YB and NF-YC subunits^{28,29,30,31,32}. NF-Y and its binding element, the CCAAT box, are among the first set of trans-acting factors and cis-elements identified in eukaryotes³³. The Arabidopsis genome contains 10 NF-YA, 13 NF-YB and 13 NF-YC subunits, which could theoretically form 1,690 unique heterotrimeric NF-Y transcription factors, potentially enabling the specific control of a variety of target genes^32,34. In this study, we report the discovery of a complete NF-Y complex consisting of three different subunits that regulates flowering time in response to photoperiod and GA pathways in Arabidopsis. NF-Y subunits interact with CO and DELLAs to directly regulate SOC1 expression. The NF-Y complex binds to a unique cis-element in the SOC1 promoter through its NF-YA2 subunit, and modulates H3K27me3 dynamics at SOC1 partly via RELATIVE OF EARLY FLOWERING 6 (REF6), a H3K27 demethylase. Our findings uncover that the combinatorial NF-Y transcription factor plays a critical role in mediating epigenetic control of the floral transition through its interaction with key regulators in the photoperiod and GA pathways, thus orchestrating plant responses to environmental and intrinsic flowering signals.

Results

DELLAs and CO interact with NF-Y subunits

DELLA proteins function as key transcriptional regulators that mediate GA effect on plant development. To further investigate the function of DELLA proteins, we performed a yeast two-hybrid screening to identify RGA-interacting partners using an inflorescence cDNA library (CD4-30 from ABRC)²⁶, and found that RGA interacted with NF-YC3 and its closest homologue NF-YC9, two Arabidopsis NF-YC family members^32,34, in yeast. This result, together with a recent finding describing the involvement of NF-YC3 and NF-YC9 in CO-mediated control of flowering²⁸, prompted us to investigate whether DELLAs and CO coordinately interact with NF-Y subunits to regulate flowering time.

To systematically study the protein interactions of representative NF-Y subunits with RGA and CO, we selected two NF-YA (A1 and A2), three NF-YB (B1, B2 and B3), and three NF-YC (C1, C3 and C9) homologues because they are either relevant with flowering time control or expressed in the vasculature similarly to CO^{28,29,30,31,34}. Yeast two-hybrid assays revealed that in addition to NF-YC3 and NF-YC9 found in initial yeast two-hybrid screening, RGA also interacted strongly with NF-YA1 and NF-YA2, and weakly with NF-YB2 (Fig. 1a). CO not only interacted with all NF-YC subunits examined, but also interacted weakly with NF-YB2 (Fig. 1a). Glutathione S-transferase (GST) pull-down assays verified the protein interactions of the selected NF-YB and NF-YC subunits (NF-YB2 and NF-YC9) with RGA and CO, but did not show the interaction between RGA and the selected NF-YA subunit (NF-YA2) (Fig. 1b) as revealed by yeast two-hybrid assays. We further performed bimolecular fluorescence complementation (BiFC) analyses, and found the interactions of all selected NF-Y subunits (NF-YA2, NF-YB2 and NF-YC9) with RGA and CO in the nuclei of tobacco cells (Fig. 1c, Supplementary Fig. 1). Therefore, these results consistently demonstrate that RGA and CO interact with NF-YB and NF-YC subunits, while the interaction of NF-YA subunits with RGA and CO may depend on other endogenous factors in plants, such as NF-YC subunits, as discussed later.

**Figure 1: CO and RGA interact with NF-Y subunits *in vitro* and *in vivo*.**

To test whether the selected NF-Y subunits that interact with RGA and CO could form heterotrimeric NF-Y transcription factors, we performed yeast two-hybrid assays to examine the interactions among these NF-Y subunits. NF-YC3 and NF-YC9 interacted with both NF-YA and NF-YB subunits (Supplementary Fig. 2), whereas no interactions between NF-YA and NF-YB subunits were observed (Fig. 1d, upper panel). Yeast three-hybrid assays further showed that NF-YC9 was required for the interactions between NF-YA and NF-YB subunits (Fig. 1d). BiFC analysis confirmed the interactions of NF-YC9 with NF-YA2 and NF-YB2 in the nuclei of tobacco cells (Fig. 1c). These results, together with a recent study on the bilateral interactions between NF-Y subunits³⁵, suggest that NF-YC subunits directly interact with NF-YA and NF-YB subunits, and might be necessary for the interaction between NF-YA and NF-YB subunits for the assembly of the trimeric NF-Y complex.

As NF-YC9 interacted with other NF-Y subunits, RGA and CO in yeast and tobacco cells (Fig. 1a,c, Supplementary Fig. 2), we created pNF-YC9:NF-YC9-FLAG plants to examine the interaction of NF–YC9 with other proteins in Arabidopsis. pNF-YC9:NF-YC9-FLAG rescued the late-flowering phenotype of nf-yc3-2 nf-yc4-1 nf-yc9-1 (ref. 28) (Table 1), indicating that NF-YC9-FLAG retains the biological function of NF-YC9. The nf-yc9-1 pNF-YC9:NF-YC9-FLAG line was crossed with various plants containing other tagged transgenes and the resulting homozygous progenies were used for co-immunoprecipitation analyses. Our results revealed the in vivo interaction of NF-YC9 with RGA, CO, NF-YA2 and NF-YB2 (Fig. 1e, Supplementary Fig. 3). Taken together, these data suggest that RGA and CO interact with NF-Y subunits, and that NF-YC9 is a common partner interacting with other proteins involved. Similar to RGA, other DELLA proteins, such as GAI, RGL1 and RGL2, also interacted with NF-Y subunits (Supplementary Fig. 4), implying widespread interactions between DELLAs and NF-Y.

Table 1 Flowering time of various plants in this study.

Full size table

Interactions among CO, GA signalling and NF-Y

Because previous studies have shown that CO, GA signalling, and individual NF-YB/C subunits are all involved in the control of flowering time under LDs^{20,21,28,29,30,31}, we proceeded to study how their interactions affect flowering. We first crossed nf-ya2-1 and nf-yc9-1 (ref. 28) with nf-yb2-1 nf-yb3-1 that showed late flowering³⁰. The resulting quadruple mutant nf-ya2-1 nf-yb2-1 nf-yb3-1 nf-yc9-1 exhibited even later flowering (Table 1), indicating that the heterotrimeric complex of the NF-Y transcription factor comprising YA, YB and YC subunits play an important role in determining flowering time.

As NF-Y subunits interacted in vivo with RGA and CO (Fig. 1), we hypothesized that they could coordinately regulate certain common targets in both photoperiod and GA pathways. Thus, we tested the temporal expression of SOC1 and FT, both of which act downstream of photoperiod and GA pathways. Both SOC1 and FT were consistently downregulated in co-1, the GA-deficient mutant ga1, nf-yb2-1 nf-yb3-1, and nf-ya2-1 nf-yb2-1 nf-yb3-1 nf-yc9-1 during the floral transition, whereas there was no consistent change in CO expression in these mutants (Fig. 2a). Furthermore, induced CO activity upregulated SOC1 and FT in a steroid-inducible co-2 35S:CO-GR line as previously reported³ (Fig. 2b, Supplementary Fig. 5). Similarly, induced RGA activity downregulated SOC1 and FT in a steroid-inducible 35S:RGA-GR line³⁶ in the background of ga1-3 gai-t6 rga-t2 (Fig. 2b, Supplementary Fig. 5), in which null mutants of RGA and GAI, two major DELLA proteins mediating GA effect on flowering, suppressed the non-flowering defect of ga1-3 (ref. 22). These results demonstrate that CO, GA signalling and NF-Y all affect SOC1 and FT expression under LDs.

**Figure 2: CO, GA signalling and NF-Y regulate flowering.**

We then investigated genetic interactions among CO, GA signalling and NF-Y. Consistent with the role of CO in the promotion of flowering, co-1 showed late flowering, while SUC2:CO-6HA showed early flowering (Fig. 2c, Table 1, Supplementary Fig. 6). nf-yb2-1 nf-yb3-1 exhibited the comparable late-flowering phenotype to nf-yb2-1 nf-yb3-1 co-1, and almost completely suppressed early flowering of SUC2:CO-6HA (Fig. 2c, Supplementary Fig. 6, Table 1). Consistently, upregulation of SOC1 and FT expression in SUC2:CO-6HA was greatly attenuated in a nf-yb2-1 nf-yb3-1 background (Supplementary Fig. 6). These results suggest that NF-Y predominantly mediates CO effect on downstream flowering promoters. In contrast, ga1 ( Columbia (Col) background) and ga1-3 (Landsberg erecta (Ler background)), both of which exhibited late flowering to different extents, did not affect early flowering of overexpression of CO (Fig. 2d, Table 1). Furthermore, both co-1 ga1 (Col background) and co-2 ga1-3 (Ler background) hardly flowered under our experimental period (Fig. 2e, Table 1). These results support that CO and GA signalling partially function in parallel pathways to control flowering under LDs. Since CO controlled flowering in a NF-Y-dependent manner, nf-yb2-1 nf-yb3-1 ga1 expectedly exhibited the non-flowering defect similar to co-1 ga1 under LDs (Fig. 2f, Table 1).

NF-Y subunits are associated with the SOC1 promoter

The interaction of NF-Y subunits with RGA and CO and their effects on SOC1 and FT expression provoked us to speculate whether NF-Y serves as a convergent point to mediate flowering through regulating SOC1 and FT expression in response to both photoperiod and GA pathways. To this end, we first examined whether NF-Y subunits are directly associated with SOC1 and FT regulatory regions. Chromatin immunoprecipitation (ChIP) assays of nf-yc9-1 pNF-YC9:NF-YC9-FLAG and nf-yb2-1 pNF-YB2:NF-YB2-6Myc in which pNF-YB2:NF-YB2-6Myc rescued the late-flowering phenotype of nf-yb2-1 (Table 1) revealed that both NF-YC9-FLAG and NF-YB2-6Myc were associated with the genomic region near the SOC1-5 fragment with the highest enrichment fold (Fig. 3a). We further created 35S:NF-YA2-6HA, 35S:NF-YC3-6HA and 35S:NF-YC9-6HA transgenic plants, all of which showed slightly early flowering and dwarf or semi-dwarf stature (Supplementary Fig. 7, Table 1), indicating that these NF-Y subunits mediate similar developmental processes. ChIP assays of these materials demonstrated that these NF-Y subunits tagged with 6HA bound to the same SOC1 genomic region as NF-YC9-FLAG and NF-YB2-6Myc (Fig. 3a, Supplementary Fig. 8). These results and the observation on the interactions among these NF-Y subunits suggest that three different types of subunits form the heterotrimeric NF-Y complex that recognizes the same binding region in the SOC1 promoter.

**Figure 3: CO and GA signalling regulate *SOC1* expression through NF-Y binding to the *SOC1* promoter.**

We further tested whether CO and RGA also bind to SOC1 by ChIP analysis of SUC2:CO-6HA and ga1-3 gai-t6 rgl1-1 rgl2-1 rga-t2 35S:RGA-6HA³⁷. Association of CO-6HA with SOC1 spanned a ~1.7 kb promoter region from SOC1-1 to SOC1-9 fragments with an enrichment peak at around SOC1-7 (Fig. 3a). In contrast, association of RGA-6HA with the SOC1 genomic region was not detectable (Supplementary Fig. 8). These observations imply that CO and NF-Y co-localize at the SOC1 promoter, whereas the interaction between RGA and NF-Y could be separate from NF-Y association with the SOC1 promoter.

Notably, although ChIP analysis of SUC2:CO-6HA revealed the association of CO-6HA with a 9-kb FT genomic region with an enrichment peak at the FT-14 fragment, there was no significant enrichment detectable for NF-YA2, NF-YB2, and NF-YC9 (Supplementary Fig. 9). Thus, these NF-Y subunits might affect FT expression in an indirect manner.

The association of CO-6HA with both SOC1 and FT genomic regions implies that CO could directly regulate both genes as previously suggested^3,18. This is supported by additive effects of ft soc1 mutants on delaying flowering under LDs^17,38. We also found that SOC1 was significantly upregulated in ft-10 SUC2:CO-6HA compared with ft-10 (Supplementary Fig. 10). This result, together with the ChIP result showing direct binding of CO to SOC1, suggests that CO partially activates SOC1 expression independently of FT.

NF-YA2 directly binds to the NFYBE of the SOC1 promoter

To identify the subunit(s) of the NF-Y complex that interacts with the SOC1-5 fragment, we performed the electrophoretic mobility shift assay (EMSA) using the biotinylated SOC1-5 fragment as a probe, and found that only NF-YA2 specifically bound to SOC1-5 (Supplementary Figs 11 and 12). This is consistent with a previous study showing that only NF-YA subunits contain the essential histidine residues required for NF-Y binding to DNA³⁹.

We further revealed that NF-YA2 only bound to the middle subfragment of SOC1-5 that contains a 15-bp motif flanked by a pair of inverted repeat sequences (Supplementary Figs 11 and 13), which could facilitate the protein-DNA interaction⁴⁰. We then performed EMSA using this 15-bp motif and its mutated versions as probes, and found that NF-YA2 bound to the native motif rather than the mutated version (Mut1) (Fig. 3b,c). This motif (5′-TTCACAAACACCATT-3′) was, thus, designated as the NFYBE.

NF-Y binding mediates the regulation of SOC1 by CO and GA

To test in vivo whether CO and GA signalling regulate SOC1 through NF-Y binding to the NFYBE in the SOC1 promoter, we used an established pSOC1:β-glucuronidase (GUS) construct in which the GUS reporter gene was driven by a 2-kb SOC1 promoter upstream of the translational start site⁵. Based on this construct, we further created a mutated reporter gene cassette containing the Mut1 version of the NFYBE, 5′-TTCAgtAAgACgATT-3′ (Fig. 3b). Among 30 lines of transformants harbouring the mutated version, 28 lines displayed significantly reduced GUS staining at 4 and 9 days after germination compared with pSOC1:GUS plants (Fig. 3d,e), suggesting that the NFYBE is critical for promoting SOC1 expression during the floral transition.

We then crossed the representative native and mutated pSOC1:GUS plants with nf-yb2-1 nf-yb3-1 and 35S:CO. GUS staining of pSOC1:GUS was significantly weaker in both 4- and 9-day-old nf-yb2-1 nf-yb3-1 seedlings than that in wild-type seedlings, whereas there was no significant difference in the intensity of GUS staining for the mutated pSOC1:GUS in two different genetic backgrounds (Fig. 3d,e). Thus, mutagenesis of the NFYBE abolishes NF-Y capacity in upregulating SOC1, confirming that NF-Y promotes SOC1 expression through binding to the NFYBE. As expected, pSOC1:GUS displayed increased GUS staining in 35S:CO compared with wild-type seedlings, but the mutated pSOC1:GUS displayed the same weak staining in both 35S:CO and wild-type backgrounds (Fig. 3d,e). Furthermore, GA treatment resulted in increased GUS staining in 9-day-old pSOC1:GUS seedlings compared with non-treated seedlings, but had no effect on the mutated pSOC1:GUS seedlings.

To directly test whether the NFYBE affects flowering, we transformed a 6.6-kb native SOC1 genomic fragment and its derived version with the mutated NFYBE into soc1-2. Most of the independent soc1-2 lines transformed with the native fragment exhibited comparable flowering time as wild-type plants, whereas almost all the soc1-2 lines transformed with the mutated version flowered later than wild-type plants (Fig. 3f), demonstrating that mutagenesis of the NFYBE compromises the SOC1 effect on promoting flowering. Taken together, these observations strongly support that NF-Y binding to the NFYBE partly contributes to the upregulation of SOC1 in response to CO and GA signalling during the floral transition.

GA signalling enhances NF-Y binding to the SOC1 promoter

As our genetic analyses suggest that CO and GA signalling function in parallel pathways to control flowering under LDs (Fig. 2e, Table 1), we then investigated the relative contribution of GA and CO to modulating flowering. GA treatment accelerated flowering of wild-type plants in both Col and Ler backgrounds under LDs (Supplementary Table 1). Consistently, GA-deficient mutants, ga1-3 (Ler background) and ga1 (Col background), exhibited late flowering, while loss of function of DELLA proteins in gai-t6 rga-t2 rgl1-1 rgl2-1 exhibited early flowering (Fig. 4a, Table 1). These data demonstrate that GA signalling contributes to the promotion of flowering under LDs even when the photoperiod pathway mediated by CO plays a dominant role. However, in the absence of CO, the fundamental effect of GA on flowering under LDs was clearly perceived as evident from the observations that GA treatment greatly accelerated flowering of co-1, co-2 and co-1 ga1 (Supplementary Table 1), and that gai-t6 rga-t2 rgl1-1 rgl2-1 or gai-t6 rga-t2 fully rescued the late-flowering phenotype of co-2 or the non-flowering phenotype of co-2 ga1-3 (Fig. 4a, Table 1). Furthermore, SOC1 and FT expression was upregulated in gai-t6 rga-t2 rgl1-1 rgl2 regardless of CO activity (Fig. 4b). These observations support that GA signalling directly modulates flowering regulators, such as SOC1, in parallel to CO.

**Figure 4: GA signalling promotes *SOC1* through enhancing NF-Y binding to the *SOC1* promoter.**

Although both CO and RGA interacted with NF-Y subunits (Fig. 1) and regulated SOC1 expression through NF-Y binding to SOC1 (Fig. 3b–e), the SOC1 promoter was only associated with CO and NF-Y, but not RGA (Fig. 3a, Supplementary Fig. 8). To understand how the interaction between RGA and NF-Y regulates SOC1 expression, we first examined whether treatment of GA or an inhibitor of GA biosynthesis, paclobutrazol (PAC), affects the protein levels of NF-YC9 that interacted in vivo with RGA (Fig. 1e). Neither GA treatment that degraded DELLA proteins including RGA nor PAC treatment that stabilized DELLA proteins affected the abundance of NF-YC9-FLAG in nf-yc9-1 pNF-YC9:NF-YC9-FLAG (Fig. 4c). However, ChIP assays of nf-yc9-1 pNF-YC9:NF-YC9-FLAG revealed that NF-YC9-FLAG binding to the SOC1 promoter was significantly enhanced or compromised by GA or PAC treatment, respectively (Fig. 4d). Thus, GA signalling regulates flowering through enhancing NF-Y binding to SOC1 rather than affecting the expression levels of NF-Y proteins.

NF-Y reduces H3K27me3 levels at SOC1

Although several studies have correlated NF-Y binding to histone marks at downstream genes^41,42,43, it is so far unclear whether NF-Y affects histone modifications in plants. To understand how NF-Y regulates SOC1 transcription, we compared the distribution profiles of several selected histone marks, including H3ac, H4ac, H3K4me2, H3K4me3, H3K9ac and H3K27me3, at the SOC1 locus between wild-type and nf-ya2-1 nf-yb2-1 nf-yb3-1 nf-yc9-1 plants by ChIP assays. While these histone marks were all enriched to different extents at SOC1, H3K27me3 exhibited the most dramatic change in its levels in nf-ya2-1 nf-yb2-1 nf-yb3-1 nf-yc9-1 as compared with wild-type plants (Fig. 5a, Supplementary Fig. 14). H3K27me3 is a histone mark associated with transcriptionally silent chromatin, which is specifically recognized by TERMINAL FLOWER 2 (TFL2), the only Arabidopsis homologue of HP1 (ref. 44). ChIP analysis using tfl2-1 35S:TFL2-3HA⁴⁵ showed the association of TFL2-3HA with SOC1 at the same region (SOC1-10) highly enriched for H3K27me3 (Fig. 5a,b), confirming the deposition of H3K27me3 at SOC1. The progressive increase of H3K27me3 levels at SOC1 in nf-yb2-1 nf-yb3-1 and nf-ya2-1 nf-yb2-1 nf-yb3-1 nf-yc9-1 (Fig. 5a) was consistent with decreased expression of SOC1 in these mutants (Fig. 2a), suggesting that NF-Y subunits might play a redundant role in affecting H3K27me3 levels at SOC1. In addition, H3K27me3 levels also greatly increased at SOC1 in co-1 and ga1 (Fig. 5c). These results indicate that the effect of CO and GA signalling on SOC1 mediated by NF-Y is associated with H3K27me3 levels at SOC1.

**Figure 5: NF-Y affects H3K27me3 dynamics at *SOC1* and interacts with REF6.**

To further examine the relationship between NF-Y and the H3K27me3 status at SOC1, we measured H3K27me3 levels at the native and mutated pSOC1:GUS transgene loci in the native and mutated pSOC1:GUS plants, respectively (Fig. 3d). H3K27me3 levels were much higher at the mutated locus (Supplementary Fig. 15), indicating that NFYBE bound by NF-Y is involved in mediating H3K27me3 levels at SOC1.

NF-Y mediates H3K27me3 demethylation at SOC1 partly via REF6

Because H3K27me3 is deposited by H3K27me3 methyltransferases of the polycomb repressive complex 2 and removed by H3K27 demethylases, we then examined whether NF-Y modulates H3K27me3 dynamics at SOC1 through its interaction with CURLY LEAF (CLF), a main H3K27me3 methyltransferase, or REF6, a H3K27 demethylase, both of which affect flowering in Arabidopsis (Table 1)^46,47,48,49.

As both clf and REF6-YFP-HA overexpression (REF6ox) plants exhibit global changes in H3K27me3 levels^46,47, we performed ChIP assays on these two types of plants and found that overexpression of REF6 dramatically reduced H3K27me3 levels at SOC1, whereas there was no significant change in H3K27me3 levels in clf (Fig. 5d). Although REF6 was also reported to act as an H3K4me3/H3K36me3 demethylase⁵⁰, we did not observe significant changes in H3K4me3/H3K36me3 levels at SOC1 in both REF6ox and ref6-1 compared with wild-type plants (Supplementary Fig. 16). In agreement with REF6 role in reducing H3K27me3 levels, SOC1 was upregulated in REF6ox and slightly downregulated in ref6-1 (Fig. 5e). In contrast, SOC1 was unexpectedly downregulated in clf (Supplementary Fig. 17), which could indirectly result from the effect of CLF on SOC1 upstream regulators, such as FLOWERING LOCUS C⁴⁶.

Having shown that both NF-Y and REF6 promoted SOC1 expression through reducing H3K27me3 levels (Fig. 5a,d,e), we crossed nf-yb2-1 nf-yb3-1 with REF6ox and ref6-1 to investigate whether NF-Y interacts with REF6 to affect SOC1 expression. Upregulation of SOC1 in REF6ox was attenuated by nf-yb2-1 nf-yb3-1 (Fig. 5e), which is consistent with the observation that nf-yb2-1 nf-yb3-1 partially suppressed the extremely early flowering phenotype of REF6ox (Fig. 5f, Table 1). Furthermore, SOC1 expression was lower in nf-yb2-1 nf-yb3-1 ref6-1 than in nf-yb2-1 nf-yb3-1 and ref6-1 (Fig. 5e). Consequently, the late-flowering phenotype of ref6-1 was enhanced by nf-yb2-1 nf-yb3-1 (Table 1). These results support that NF-Y partially mediates REF6 effect on SOC1 expression. As NF-Y did not affect mRNA expression of REF6 (Supplementary Fig. 18), we then tested the protein interaction between a NF-Y subunit, NF-YC9 and REF6. Yeast two-hybrid assay revealed the interaction between NF-YC9 and the amino terminus of REF6 in yeast (Fig. 5g), and co-immunoprecipitation analysis of nf-yc9-1 pNF-YC9:NF-YC9-FLAG REF6ox/− plants confirmed the interaction between NF-YC9 and REF6 in Arabidopsis (Fig. 5h). These findings suggest that NF-Y interacts with REF6 to regulate SOC1 expression. Consistently, reduction of H3K27me3 levels at SOC1 in REF6ox was suppressed by nf-yb2-1 nf-yb3-1, while H3K27me3 levels significantly increased in nf-yb2-1 nf-yb3-1 ref6-1 compared with those in ref6-1 and nf-yb2-1 nf-yb3-1 (Fig. 6a).

**Figure 6: Interaction between REF6 and NF-Y directly regulates *SOC1* expression downstream of photoperiod and GA pathways.**

ChIP assays using a previously established ref6-1 pREF6:REF6-HA transgenic line, in which a functional version of REF6-HA rescued the flowering defect of ref6-1 (ref. 47) showed that REF6-6HA was associated with SOC1, and that this association was significantly attenuated in nf-yb2-1 nf-yb3-1 and under SD conditions (Fig. 6b,d), but enhanced by GA treatment (Fig. 6c). These data, together with the observation that overexpression of REF6 dramatically suppressed late flowering of co-1 and ga1 (Fig. 6e, Table 1), substantiate that the interaction between REF6 and NF-Y directly regulates SOC1 downstream of CO and GA signalling.

Discussion

Combinatorial transcription factors, such as NF-Y complexes, play an essential role in regulating eukaryotic gene expression⁵¹. Unlike other eukaryotic organisms, the plant lineage has multiple genes that encode each subunit of a heterotrimeric NF-Y complex^32,34. Individual plant NF-Y subunits have been shown to affect various developmental processes and plant responses to environmental stresses^{28,29,30,52,53,54,55}. However, the precise function of complete NF-Y complexes in plants so far remains elusive.

Here we show a NF-Y complex composed of three different types of subunits, NF-YA2, NF-YB2 and NF-YC9, which controls flowering time through directly regulating SOC1 transcription in response to flowering signals from photoperiod and GA pathways in Arabidopsis (Fig. 7). nf-ya2-1 nf-yb2-1 nf-yb3-1 nf-yc9-1 exhibits much later flowering than nf-yb2-1 nf-yb3-1 (Table 1), indicating that these NF-Y subunits play redundant roles in promoting flowering. NF-YB and NF-YC subunits interact with CO in the photoperiod pathway as previously reported^28,29, and also with RGA in the GA pathway. Among the NF-Y subunits examined, NF-YC9 serves as a key component that not only mediates the interaction between NF-YA2 and NF-YB2 in the NF-Y heterotrimer (Fig. 1d), but also interacts in vivo with CO and RGA (Fig. 1e). A recent study has suggested that plant NF-YC enables translocation of NF-YB to the nucleus, and that the resulting NF-YB/NF-YC heterodimer is required for further recruitment of NF-YA in the final assembly of the heterotrimeric NF-Y complex as shown in mammals³⁵. Thus, our finding of NF-YC9 as a common partner of other NF-Y subunits, CO, and RGA implies that NF-YC9 could be a key subunit that coordinates the perception of flowering signals and the assembly of the specific combinatorial NF-Y transcription factor that triggers the downstream regulatory events in response to flowering signals.

**Figure 7: Photoperiod and GA pathways regulate *SOC1* expression via NF-Y-mediated H3K27me3 demethylation.**

Our results suggest that the CO-mediated photoperiod pathway and GA signalling function in parallel in promoting flowering under LDs. The integration of plant responses to these two pathways converges on the NF-Y complex. The genetic data showing the similar late-flowering phenotype of nf-yb2-1 nf-yb3-1 and nf-yb2-1 nf-yb3-1 co-1 and complete suppression of early flowering of SUC2:CO-6HA by nf-yb2-1 nf-yb3-1 suggest that CO regulates flowering in a NF-Y-dependent manner. Consistent with the protein interaction between NF-Y and CO, the SOC1 promoter region associated with NF-Y subunits overlaps with the region bound by CO. Furthermore, the NFYBE in the SOC1 promoter bound by NF-Y is required for the upregulation of SOC1 by CO. These observations all support that NF-Y is an important regulator that mediates CO effect on SOC1 transcription (Fig. 7).

In contrast to CO, RGA in the GA pathway represses SOC1 transcription. Although RGA interacts with NF-Y, RGA does not directly bind to the SOC1 promoter. GA treatment degrades RGA, which enhances NF-Y binding to SOC1. These results suggest that DELLA proteins, such as RGA, affect SOC1 expression through preventing NF-Y binding to SOC1 (Fig. 7). This is substantiated by the observations that the NFYBE in the SOC1 promoter is indispensable for upregulation of SOC1 by GA treatment (Fig. 3d,e), and that the flowering response to GA is partially compromised in nf-ya2-1 nf-yb2-1 nf-yb3-1 nf-yc9-1 (Supplementary Table 1). In addition to SOC1, the GA pathway also affects other components of the regulatory network that integrates flowering signals. For example, GA plays spatially distinct regulatory roles in promoting transcription of FT and SPL genes under LDs^21,56.

It has been demonstrated that NF-Y complexes bind to the CCAAT box, a cis-element present in nearly 25% of eukaryotic promoters, indicating that NF-Y complexes are involved in transcriptional regulation of a considerable number of eukaryotic genes^33,42,57. Several studies have tested in vitro binding sites of plant NF-Y subunits^55,58, but whether these sites are relevant to NF-Y endogenous function is unclear. Another recent study has proposed that NF-Y complexes may recognize a distal CCAAT site at the FT promoter based on ChIP assays of 35S:NF-YB2-YFP/HA plants⁵⁹, whereas we were unable to detect binding of NF-YB2-6Myc to FT using the nf-yb2-1 pNF-YB2:NF-YB2-6Myc tagging line (Supplementary Fig. 9).

We show here that the NF-YA2/NF-YB2/NF-YC9 complex binds to a unique NFYBE, 5′-TTCACAAACACCATT-3′, through NF-YA2. It is noteworthy that this NFYBE does not contain the exact NF-Y consensus binding site, CCAAT, as shown in yeast and animals, implying that the diversity in plant NF-Y subunits may evolve with the divergence of their corresponding cis-elements. The SOC1 genomic region containing the NFYBE demonstrates two unique features that are relevant to regulation of SOC1 by CO and GA signalling. First, it has been proposed that CO activates SOC1 through being recruited by a DNA-binding factor to a motif located between nt −372 and −248 in the SOC1 promoter¹⁸. This region exactly overlaps with the SOC1-5 fragment containing the newly identified NFYBE, implying that NF-Y could be the long-sought DNA-binding factor for CO. Furthermore, CO has been shown to bind a CO-responsive element containing a consensus TGTG(N2-3)ATG motif in the FT promoter⁶⁰. Notably, there are two similar motifs (TGTGTATG) located 199 bp upstream and 262 bp downstream from the NFYBE in the SOC1 promoter, respectively. The entire region containing these motifs is associated with CO in vivo (Fig. 3a). In concert, these results suggest that CO and NF-Y interact at the same region centred on the NFYBE to activate SOC1 transcription.

Second, the forepart of the NFYBE is similar to a GA-responsive cis-element (GARE), 5′-TAACAAA/G-3′, which has been identified from an α-amylase gene in barley aleurone layers⁶¹. This element is bound by GAMYB, a GA-inducible transcriptional factor, which activates the α-amylase gene expression in aleurone cells⁶². In Arabidopsis, the GARE is significantly enriched in the promoters of GA-responsive genes⁶³. AtMYB33, a GAMYB protein, has been suggested to promote flowering through binding to a GARE-like element in the LFY promoter⁶⁴. Thus, it is tempting to speculate that the interaction between DELLAs and NF-Y may also affect NF-Y interaction with other GARE-binding trans-acting partners at the NFYBE, thus mediating the downstream response of GA signalling in plants.

NF-YB and NF-YC subunits have conserved histone fold motifs that resemble H2B and H2A, respectively, both of which, together with H3 and H4, are core histones of the nucleosome, the fundamental unit of chromatin⁶⁵. In yeast and animals, NF-Y could modulate chromatin structure and function through replacement of H2A-H2B and/or covalent modifications of histones, such as methylation and acetylation³³. One of the most intriguing questions relevant to NF-Y function is how the interaction between NF-Y and other transcriptional regulators mediates spatial and temporal regulation of selected specific loci rather than bulk chromatin.

In this study, we show that a plant NF-Y complex plays an important role in integrating environmental and developmental signals to govern the selection of a specific key regulator to be targeted by a general histone demethylase. NF-Y mediates the effect of photoperiod and GA signalling on SOC1 expression partly through modulating H3K27me3 demethylation via a H3K27 demethylase REF6. Both CO and RGA interact with NF-Y that directly binds to the NFYBE in the SOC1 promoter, which in turn mediates REF6 to modulate H3K27me3 levels at SOC1. Our findings establish NF-Y as a key complex that orchestrates plant responses to seasonal changes in day length and the endogenous phytohormone GA to mediate epigenetic control of the floral transition in a locus-specific manner. As different combinations of multiple NF-Y subunits may result in the formation of various NF-Y complexes with different intrinsic properties and trans-acting partners, this flexibility could allow plants to respond and adapt to various stresses through controlling a large number of specific targets at the molecular level, although they cannot move away from stressful environments.

Methods

Plant materials and growth conditions

Arabidopsis plants were grown at 22 °C under LDs (16 h light/8 h dark) or short days (8 h light/16 h dark). The mutants ga1-3, rga-t2, gai-t6, rgl1-1, rgl2-1 and co-2 are in Ler background, while all the other mutants are in Col background. nf-yb2-1 (SALK_025666), nf-yb3-1 (SALK_062245), nf-yc9-1 (SALK_058903), nf-yc4-1 (SALK_032163) and clf (SALK_006658) seeds were obtained from the Arabidopsis Biological Resource Centre, while nf-ya2-1 (GK-440G05) and nf-yc3-2 (GK-051E10) were obtained from the European Arabidopsis Stock Centre. Seeds with ga1-3 or ga1 background were imbibed in 100 μM GA₃ at 4 °C for 7 days and rinsed thoroughly with water before sowing.

Plasmid construction and plant transformation

To construct 35S:NF-YC9-6HA, 35S:NF-YC3-6HA and 35S:NF-YA2-6HA, the cDNAs encoding NF-YC9, NF-YC3 and NF-YA2 were amplified and cloned into pGreen-35S-6HA, respectively⁵. To construct pNF-YC9: NF-YC9-FLAG and pNF-YB2:NF-YB2-6Myc, the genomic fragments of NF-YC9 and NF-YB2 were amplified and cloned into pHY105-6Myc and pPZPY122-FLAG²⁶, respectively. The SUC2 promoter fragment and the cDNA encoding CO were amplified and cloned into pHY105-6HA to obtain SUC2:CO-6HA. For the complementation test, a 6.6-kb SOC1 genomic fragment that includes 2.4 kb of the upstream sequence, 3.5 kb of the transcribed region, and 0.7 kb of the 3′-flanking region was amplified and cloned into pHY105 (ref. 5) to obtain the gSOC1 construct. Based on the native gSOC1 and the previously reported pSOC1:GUS constructs⁵, the derived constructs with the mutated NFYBE were produced using QuikChange II XL-Site-Directed Mutagenesis Kit (Stratagene). Primers used for plasmid construction are listed in Supplementary Table 2. Except for transgenic plants harbouring pNF-YC9:NF-YC9-FLAG that were selected on MS medium supplemented with gentamicin, transgenic plants with other constructs were selected by Basta on soil.

Yeast two-hybrid and three-hybrid assays

The coding regions of NF-YC1, NF-YC9, NF-YC3, NF-YB1, NF-YB2, NF-YB3, NF-YA1, NF-YA2, CO and DELLAs were amplified and cloned into either pGBKT7 or pGADT7 (Clontech). Yeast two-hybrid assays were performed using the Yeastmaker Yeast Transformation System 2 (Clontech). To screen an Arabidopsis cDNA library (CD4-30, from ABRC)⁶⁶, the fragment of RGAΔN (residues 200–587) was fused to the GAL4-BD as bait. To analyse the interaction between NF-YC9 and REF6, the carboxy- and N-terminal fragments of REF6 were amplified and cloned into pGBKT7 to obtain BD-REF6ΔN and BD-REF6ΔC, respectively. To analyse the formation of the trimeric NF-Y complex, the NF-YC9 coding region was amplified and cloned into pQH05, which was derived from pTH184 with a HIS3 selection marker. Primers used for generating constructs for yeast two-hybrid and three-hybrid assays are listed in Supplementary Table 2. Yeast AH109 cells were co-transformed with specific bait and prey constructs. For yeast three-hybrid assay, yeast AH109 cells were co-transformed with AD-NF-YB2/NF-YB3 and BD-NF-YA1/NF-YA2 in the presence of either pQH05 or pQH05-NF-YC9. All yeast transformants were grown on SD/-Trp/-Leu/-His/-Ade medium for selection or interaction test.

Expression analysis

Total RNA was extracted using the FavorPre Plant Total RNA Mini Kit (Favorgen) and reverse transcribed using the SuperScrip RT–PCR System (Invitrogen). Real-time PCR was performed in triplicates on CFX38 real-time system (Bio-Rad) with the iQ SYBR Green Supermix (Bio-Rad). The relative expression level was calculated as previously reported⁵. Primers used for gene expression analysis are listed in Supplementary Table 2. GUS staining and quantitative analysis of GUS activity were carried out as previously described⁶⁷.

In vitro pull-down assay

To produce GST-tagged proteins, the cDNAs encoding RGA and CO were cloned into pGEX-4T-1 vector (Pharmacia). To produce His-tagged proteins, the cDNAs encoding NF-YC9, NF-YB2 and NF-YA2 were cloned into pQE30 vector (Qiagen). Primers used for these constructs are listed in Supplementary Table 2. These constructs and the empty pGEX-4T-1 and pQE30 vectors were transformed into E. coli Rosetta (DE3) (Novagen), and protein expression was induced by IPTG. The soluble His fusion proteins were immobilized onto Ni-NTA agarose beads (30210, Qiagen), while the soluble GST fusion proteins were immobilized onto glutathione sepharose beads (17-0756-01, Amersham Biosciences). For pull-down assays, 2 μg His fusion proteins were incubated with the immobilized GST and GST fusion proteins at 4 °C for 1 h. Proteins retained on the beads were subsequently resolved by SDS–polyacrylamide gel electrophoresis and detected with anti-His antibody (Santa Cruz Biotechnology). Uncropped scans of western blot results are shown in Supplementary Fig. 19.

ChIP assay

Nine-day-old seedlings with various genetic backgrounds were fixed on ice for 45 min in 1% formaldehyde under vacuum. Fixed tissues were homogenized, and chromatin was isolated and sonicated as previously described⁵. The solubilized chromatin was immunoprecipitated by either a specific antibody or mouse IgG (15,381, Sigma) with Protein G PLUS agarose (sc-2002, Santa Cruz Biotechnology). NF-YC9-FLAG, NF-YB2-6Myc, NF-YA2-6HA and CO-6HA were immunoprecipitated by anti-FLAG (A2220, Sigma), anti-Myc (A7470, Sigma) and anti-HA agarose conjugates (A2095, Sigma), respectively. Various histone modifications were detected by anti-H3K27me3 (07-449, Millipore), anti-H3K4me2 (07-030, Millipore), anti-H3K4me3 (07-473, Millipore), anti-H3K36me3 (ab9050, Abcam), anti-H3K9ac (06-942, Millipore), anti-Acetyl-H3 (06-599, Millipore) and anti-Acetyl-H4 antibodies (06-866, Millipore). The co-immunoprecipitated DNA was recovered and analysed by quantitative real-time PCR in triplicates. For ChIP assays of H3K27me3 levels and binding of CO and various NF-Y subunits to SOC1, relative fold enrichment was calculated by normalizing the amount of a target DNA fragment against that of a genomic fragment of a reference gene, ACTIN7 (At5g09810), and then by normalizing the value for immunoprecipitation using a specific antibody against that of mouse IgG. The enrichment of a Tubulin (TUB8; At5g23860) genomic fragment was used as a negative control. For ChIP assays of H3ac, H4ac, H3K4me2, H3K4me3, H3K36me3 and H3K9ac levels, Ta2 transposon (At4g06488) served as a reference gene, while the enrichment of a Cinful-like (At4g03770) genomic fragment was used as a negative control⁶⁸.

Co-immunoprecipitation assay

Nine-day-old seedlings with various genetic backgrounds were collected, and nuclear proteins were extracted according to the ChIP protocol but without the tissue fixation step. NF-YC9-FLAG was immunoprecipitated by anti-FLAG agarose conjugate (A2220, Sigma), anti-Myc agarose conjugate (A7470, Sigma), or anti-GFP antibody (A11122, Invitrogen). Proteins bound to the beads were resolved by SDS–polyacrylamide gel electrophoresis and detected by anti-Myc (SAB4700447, Sigma), anti-FLAG (F3165, Sigma), anti-HA (H9658, Sigma), or anti-GFP antibody (A11122, Invitrogen). Uncropped scans of Western blot results are shown in Supplementary Fig. 19.

BiFC analysis

The cDNAs of the proteins tested were cloned into serial pSAT1 vectors containing either N- or C-terminal-enhanced yellow fluorescence protein fragments. The resulting cassettes were further subcloned into a pGreen binary vector HY105 for BiFC analysis as previously published⁶¹.

EMSA

EMSA assay was performed using the LightShift Chemiluminescent EMSA kit (Pierce). The SOC1-5 fragment or the double-stranded oligonucleotides of the native and mutated NF-YA2 binding element in the SOC1 promoter with 13 bp flanking sequences at both ends⁶⁹ were biotin end labelled and used as probes. Non-labelled probes were used as cold competitors.

Additional information

How to cite this article: Hou, X. et al. Nuclear factor Y-mediated H3K27me3 demethylation of the SOC1 locus orchestrates flowering responses of Arabidopsis. Nat. Commun. 5:4601 doi: 10.1038/ncomms5601 (2014).

References

Simpson, G. G. & Dean, C. Flowering–Arabidopsis, the rosetta stone of flowering time? Science 296, 285–289 (2002).
Article ADS CAS Google Scholar
Lee, H. et al. The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev. 14, 2366–2376 (2000).
Article CAS Google Scholar
Samach, A. et al. Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288, 1613–1616 (2000).
Article ADS CAS Google Scholar
Wang, J. W., Czech, B. & Weigel, D. miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana. Cell 138, 738–749 (2009).
Article CAS Google Scholar
Liu, C. et al. Direct interaction of AGL24 and SOC1 integrates flowering signals in Arabidopsis. Development 135, 1481–1491 (2008).
Article CAS Google Scholar
Putterill, J., Robson, F., Lee, K., Simon, R. & Coupland, G. The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors. Cell 80, 847–857 (1995).
Article CAS Google Scholar
Suarez-Lopez, P. et al. CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis. Nature 410, 1116–1120 (2001).
Article ADS CAS Google Scholar
Valverde, F. et al. Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303, 1003–1006 (2004).
Article ADS CAS Google Scholar
Kumar, S. V. et al. Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 484, 242–245 (2012).
Article ADS CAS Google Scholar
Wigge, P. A. et al. Integration of spatial and temporal information during floral induction in Arabidopsis. Science 309, 1056–1059 (2005).
Article ADS CAS Google Scholar
An, H. et al. CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis. Development 131, 3615–3626 (2004).
Article CAS Google Scholar
Corbesier, L. et al. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316, 1030–1033 (2007).
Article ADS CAS Google Scholar
Liu, L. et al. FTIP1 is an essential regulator required for florigen transport. PLoS Biol. 10, e1001313 (2012).
Article CAS Google Scholar
Li, D. et al. A repressor complex governs the integration of flowering signals in Arabidopsis. Dev. Cell 15, 110–120 (2008).
Article CAS Google Scholar
Moon, J. et al. The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis. Plant J. 35, 613–623 (2003).
Article CAS Google Scholar
Shen, L., Kang, Y. G., Liu, L. & Yu, H. The J-domain protein J3 mediates the integration of flowering signals in Arabidopsis. Plant Cell 23, 499–514 (2011).
Article CAS Google Scholar
Yoo, S. K. et al. CONSTANS activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis. Plant Physiol. 139, 770–778 (2005).
Article CAS Google Scholar
Hepworth, S. R., Valverde, F., Ravenscroft, D., Mouradov, A. & Coupland, G. Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs. EMBO J. 21, 4327–4337 (2002).
Article CAS Google Scholar
Wilson, R. N., Heckman, J. W. & Somerville, C. R. Gibberellin is required for flowering in Arabidopsis thaliana under short days. Plant Physiol. 100, 403–408 (1992).
Article CAS Google Scholar
Reeves, P. H. & Coupland, G. Analysis of flowering time control in Arabidopsis by comparison of double and triple mutants. Plant Physiol. 126, 1085–1091 (2001).
Article CAS Google Scholar
Porri, A., Torti, S., Romera-Branchat, M. & Coupland, G. Spatially distinct regulatory roles for gibberellins in the promotion of flowering of Arabidopsis under long photoperiods. Development 139, 2198–2209 (2012).
Article CAS Google Scholar
Dill, A. & Sun, T. Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana. Genetics 159, 777–785 (2001).
CAS PubMed PubMed Central Google Scholar
Fu, X. et al. The Arabidopsis mutant sleepy1 gar2-1 protein promotes plant growth by increasing the affinity of the SCFSLY1 E3 ubiquitin ligase for DELLA protein substrates. Plant Cell 16, 1406–1418 (2004).
Article CAS Google Scholar
de Lucas, M. et al. A molecular framework for light and gibberellin control of cell elongation. Nature 451, 480–484 (2008).
Article ADS CAS Google Scholar
Feng, S. et al. Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature 451, 475–479 (2008).
Article ADS CAS Google Scholar
Hou, X., Lee, L. Y., Xia, K., Yan, Y. & Yu, H. DELLAs modulate jasmonate signaling via competitive binding to JAZs. Dev. Cell 19, 884–894 (2010).
Article CAS Google Scholar
Yu, S. et al. Gibberellin regulates the Arabidopsis floral transition through miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE transcription factors. Plant Cell 24, 3320–3332 (2012).
Article CAS Google Scholar
Kumimoto, R. W., Zhang, Y., Siefers, N. & Holt, B. F. NF-YC3, NF-YC4 and NF-YC9 are required for CONSTANS-mediated, photoperiod-dependent flowering in Arabidopsis thaliana. Plant J. 63, 379–391 (2010).
Article CAS Google Scholar
Wenkel, S. et al. CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis. Plant Cell 18, 2971–2984 (2006).
Article CAS Google Scholar
Kumimoto, R. W. et al. The Nuclear Factor Y subunits NF-YB2 and NF-YB3 play additive roles in the promotion of flowering by inductive long-day photoperiods in Arabidopsis. Planta 228, 709–723 (2008).
Article CAS Google Scholar
Cai, X. N. et al. A putative CCAAT-binding transcription factor is a regulator of flowering timing in Arabidopsis. Plant Physiol. 145, 98–105 (2007).
Article CAS Google Scholar
Gusmaroli, G., Tonelli, C. & Mantovani, R. Regulation of novel members of the Arabidopsis thaliana CCAAT-binding nuclear factor Y subunits. Gene 283, 41–48 (2002).
Article CAS Google Scholar
Dolfini, D., Gatta, R. & Mantovani, R. NF-Y and the transcriptional activation of CCAAT promoters. Crit. Rev. Biochem. Mol. Biol. 47, 29–49 (2012).
Article CAS Google Scholar
Siefers, N. et al. Tissue-specific expression patterns of Arabidopsis NF-Y transcription factors suggest potential for extensive combinatorial complexity. Plant Physiol. 149, 625–641 (2009).
Article CAS Google Scholar
Hackenberg, D. et al. Studies on differential nuclear translocation mechanism and assembly of the three subunits of the Arabidopsis thaliana transcription factor NF-Y. Mol. Plant 5, 876–888 (2012).
Article CAS Google Scholar
Yu, H. et al. Floral homeotic genes are targets of gibberellin signaling in flower development. Proc. Natl Acad. Sci. USA 101, 7827–7832 (2004).
Article ADS CAS Google Scholar
Lee, L. Y. et al. STUNTED mediates the control of cell proliferation by GA in Arabidopsis. Development 139, 1568–1576 (2012).
Article CAS Google Scholar
Onouchi, H., Igeno, M. I., Perilleux, C., Graves, K. & Coupland, G. Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes. Plant Cell 12, 885–900 (2000).
Article CAS Google Scholar
Xing, Y., Fikes, J. D. & Guarente, L. Mutations in yeast HAP2/HAP3 define a hybrid CCAAT box binding domain. EMBO J. 12, 4647–4655 (1993).
Article CAS Google Scholar
Brazda, V., Laister, R. C., Jagelska, E. B. & Arrowsmith, C. Cruciform structures are a common DNA feature important for regulating biological processes. BMC Mol. Biol. 12, 33 (2011).
Article CAS Google Scholar
Gatta, R. & Mantovani, R. NF-Y affects histone acetylation and H2A.Z deposition in cell cycle promoters. Epigenetics 6, 526–534 (2011).
Article CAS Google Scholar
Donati, G. et al. An NF-Y-dependent switch of positive and negative histone methyl marks on CCAAT Promoters. PLoS ONE 3, e2066 (2008).
Article ADS Google Scholar
Gatta, R. & Mantovani, R. NF-Y substitutes H2A-H2B on active cell-cycle promoters: recruitment of CoREST-KDM1 and fine-tuning of H3 methylations. Nucleic Acids Res. 36, 6592–6607 (2008).
Article CAS Google Scholar
Zhang, X. et al. The Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation. Nat. Struct. Mol. Biol. 14, 869–871 (2007).
Article CAS Google Scholar
Liu, C., Xi, W., Shen, L., Tan, C. & Yu, H. Regulation of floral patterning by flowering time genes. Dev. Cell 16, 711–722 (2009).
Article CAS Google Scholar
Jiang, D. H., Wang, Y. Q., Wang, Y. Z. & He, Y. H. Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb Repressive Complex 2 components. PLoS ONE 3, e3404 (2008).
Article ADS Google Scholar
Lu, F. L., Cui, X., Zhang, S. B., Jenuwein, T. & Cao, X. F. Arabidopsis REF6 is a histone H3 lysine 27 demethylase. Nat. Genet. 43, 715–719 (2011).
Article CAS Google Scholar
Chanvivattana, Y. et al. Interaction of Polycomb-group proteins controlling flowering in Arabidopsis. Development 131, 5263–5276 (2004).
Article CAS Google Scholar
Hennig, L. & Derkacheva, M. Diversity of Polycomb group complexes in plants: same rules, different players? Trends Genet. 25, 414–423 (2009).
Article CAS Google Scholar
Ko, J. H. et al. Growth habit determination by the balance of histone methylation activities in Arabidopsis. EMBO J. 29, 3208–3215 (2010).
Article CAS Google Scholar
Singh, K. B. Transcriptional regulation in plants: the importance of combinatorial control. Plant Physiol. 118, 1111–1120 (1998).
Article CAS Google Scholar
Lotan, T. et al. Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell 93, 1195–1205 (1998).
Article CAS Google Scholar
Nelson, D. E. et al. Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres. Proc. Natl Acad. Sci. USA 104, 16450–16455 (2007).
Article ADS CAS Google Scholar
Li, W. X. et al. The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. Plant Cell 20, 2238–2251 (2008).
Article CAS Google Scholar
Liu, J. X. & Howell, S. H. bZIP28 and NF-Y Transcription factors are activated by ER stress and assemble into a transcriptional complex to regulate stress response genes in Arabidopsis. Plant Cell 22, 782–796 (2010).
Article CAS Google Scholar
Galvao, V. C., Horrer, D., Kuttner, F. & Schmid, M. Spatial control of flowering by DELLA proteins in Arabidopsis thaliana. Development 139, 4072–4082 (2012).
Article CAS Google Scholar
Mantovani, R. The molecular biology of the CCAAT-binding factor NF-Y. Gene 239, 15–27 (1999).
Article CAS Google Scholar
Kusnetsov, V., Landsberger, M., Meurer, J. & Oelmuller, R. The assembly of the CAAT-box binding complex at a photosynthesis gene promoter is regulated by light, cytokinin, and the stage of the plastids. J. Biol. Chem. 274, 36009–36014 (1999).
Article CAS Google Scholar
Cao, S. et al. A distal CCAAT/NUCLEAR FACTOR Y complex promotes chromatin looping at the FLOWERING LOCUS T promoter and regulates the timing of flowering in Arabidopsis. Plant Cell 26, 1009–1017 (2014).
Article CAS Google Scholar
Tiwari, S. B. et al. The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element. New Phytol. 187, 57–66 (2010).
Article CAS Google Scholar
Skriver, K., Olsen, F. L., Rogers, J. C. & Mundy, J. cis-acting DNA elements responsive to gibberellin and its antagonist abscisic acid. Proc. Natl Acad. Sci. USA 88, 7266–7270 (1991).
Article ADS CAS Google Scholar
Gubler, F., Kalla, R., Roberts, J. K. & Jacobsen, J. V. Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pI alpha-amylase gene promoter. Plant Cell 7, 1879–1891 (1995).
CAS PubMed PubMed Central Google Scholar
Bastian, R. et al. Gibberellic acid and cGMP-dependent transcriptional regulation in Arabidopsis thaliana. Plant Signal. Behav. 5, 224–232 (2010).
Article CAS Google Scholar
Gocal, G. F. W. et al. GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis. Plant Physiol. 127, 1682–1693 (2001).
Article CAS Google Scholar
Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8A resolution. Nature 389, 251–260 (1997).
Article ADS CAS Google Scholar
Fan, H. Y., Hu, Y., Tudor, M. & Ma, H. Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins. Plant J. 12, 999–1010 (1997).
Article CAS Google Scholar
Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907 (1987).
Article CAS Google Scholar
Schonrock, N. et al. Polycomb-group proteins repress the floral activator AGL19 in the FLC-independent vernalization pathway. Genes Dev. 20, 1667–1678 (2006).
Article CAS Google Scholar
Tang, W. & Perry, S. E. Binding site selection for the plant MADS domain protein AGL15: an in vitro and in vivo study. J. Biol. Chem. 278, 28154–28159 (2003).
Article CAS Google Scholar

Download references

Acknowledgements

We thank X.F. Cao for providing ref6-1, REF6ox and ref6-1 pREF6:REF6-HA seeds, the Arabidopsis Biological Resource Centre for nf-yb2-1, nf-yb3-1, nf-yc9-1, and nf-yc4-1 seeds, and the European Arabidopsis Stock Centre for nf-ya2-1 and nf-yc3-2 seeds. This work was supported by Academic Research Funds (MOE2011-T2-2-008 and R-154-000-506-112) from the Ministry of Education-Singapore, the Singapore National Research Foundation under its Competitive Research Programme (NRF2010NRF-CRP002-018), and the intramural resource support from National University of Singapore and Temasek Life Sciences Laboratory.

Author information

Authors and Affiliations

Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, Guangdong, China
Xingliang Hou
Department of Biological Sciences and Temasek Life Sciences Laboratory, National University of Singapore, Singapore, 117543, Singapore
Jiannan Zhou, Chang Liu, Lu Liu, Lisha Shen & Hao Yu

Authors

Xingliang Hou
View author publications
You can also search for this author in PubMed Google Scholar
Jiannan Zhou
View author publications
You can also search for this author in PubMed Google Scholar
Chang Liu
View author publications
You can also search for this author in PubMed Google Scholar
Lu Liu
View author publications
You can also search for this author in PubMed Google Scholar
Lisha Shen
View author publications
You can also search for this author in PubMed Google Scholar
Hao Yu
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

X.H. and H.Y. conceived and designed the research. X.H., J.Z., C.L., L.L., and L.S. performed the experiments. X.H., J.Z. and H.Y. analysed the data. X.H. and H.Y. wrote the paper.

Corresponding authors

Correspondence to Xingliang Hou or Hao Yu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-19, Supplementary Tables 1-2 (PDF 2411 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hou, X., Zhou, J., Liu, C. et al. Nuclear factor Y-mediated H3K27me3 demethylation of the SOC1 locus orchestrates flowering responses of Arabidopsis. Nat Commun 5, 4601 (2014). https://doi.org/10.1038/ncomms5601

Download citation

Received: 20 October 2013
Accepted: 07 July 2014
Published: 08 August 2014
DOI: https://doi.org/10.1038/ncomms5601

This article is cited by

Transcriptomic analysis reveals the molecular basis of photoperiod-regulated sex differentiation in tropical pumpkins (Cucurbita moschata Duch.)
- Shudan Xue
- Hexun Huang
- Yujuan Zhong
BMC Plant Biology (2024)
Identification of conserved miRNAs and their targets in Jatropha curcas: an in silico approach
- Foeaz Ahmed
- Md. Nazmul Islam Bappy
- Md. Shariful Islam
Journal of Genetic Engineering and Biotechnology (2023)
Epigenetic silencing of callose synthase by VIL1 promotes bud-growth transition in lily bulbs
- Wenqiang Pan
- Jingru Li
- Jian Wu
Nature Plants (2023)
Nuclear Factor-Y (NF-Y): Developmental and Stress-Responsive Roles in the Plant Lineage
- Polavarapu B. Kavi Kishor
- Showkat Ahmad Ganie
- Suprasanna Penna
Journal of Plant Growth Regulation (2023)
Characterization of sandalwood (E,E)-α-farnesene synthase whose overexpression enhances cold tolerance through jasmonic acid biosynthesis and signaling in Arabidopsis
- Xinhua Zhang
- Xiaohong Chen
- Guohua Ma
Planta (2023)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.