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The B3-Domain Transcription Factor VAL1 Regulates the Floral Transition by Repressing FLOWERING LOCUS T

Yanjun Jing, Qiang Guo, Rongcheng Lin

Published September 2019. DOI: https://doi.org/10.1104/pp.19.00642

Abstract

Many plants monitor changes in day length (or photoperiod) and adjust the timing of the floral transition accordingly to ensure reproductive success. In long-day plants, a long-day photoperiod triggers the production of florigen, which promotes the floral transition. FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana) encodes a major component of florigen, and FT expression is activated in leaf veins specifically at dusk through the photoperiod pathway. Repression of FT mediated by Polycomb group (PcG) proteins prevents precocious flowering and adds another layer to FT regulation. Here, we identified high-level trimethylation of histone H3 at Lys 27 (H3K27me3) in the high trimethylation region (HTR) of the FT locus from the second intron to the 3′ untranslated region. The HTR contains a cis-regulatory DNA element required for H3K27me3 enrichment that is recognized by the transcriptional repressor VIVIPAROUS1/ABSCISIC ACID INSENSITIVE3-LIKE1 (VAL1). VAL1 directly represses FT expression before dusk and at night, coinciding with the high abundance of both VAL1 mRNA and VAL1 homodimer. Furthermore, VAL1 recruits LIKE HETEROCHROMATIN PROTEIN1 and MULTICOPY SUPRESSOR OF IRA1 to FT chromatin, leading to an H3K27me3 peak at the HTR of FT. These findings reveal a mechanism for PcG repression of FT mediated by an intronic cis-silencing element and suggest a possible role for VAL1 in modulating PcG repression of FT during the flowering response.

Polycomb group (PcG) proteins play important roles in the transcriptional repression of developmental genes in multicellular eukaryotes ranging from plants to humans (Simon and Kingston, 2013; Mozgova and Hennig, 2015). PcG proteins form two main types of complexes: polycomb repressive complex 1 (PCR1) and PRC2. The evolutionarily conserved PRC2 catalyzes the trimethylation of histone H3 at Lys 27 (H3K27me3), whereas PRC1 mediates transcriptional repression via H2AK119 monoubiquitylation and chromatin compaction (Turck et al., 2007; Kim et al., 2012; Wang et al., 2014; Mozgova and Hennig, 2015; Li et al., 2018). Given that PcG complexes, per se, do not specifically bind to DNA, several mechanisms are involved in recruiting PcG proteins to their target chromatin. In fruit fly (Drosophila melanogaster), multiple DNA-binding proteins recognize cis-regulatory regions known as Polycomb response elements (PREs) and recruit PcG factors (Kassis and Brown, 2013). No consensus PRE sequences exist in mammals. Instead, CpG islands and long noncoding RNAs play important roles in recruiting PRC2 (Simon and Kingston, 2013; Mozgova and Hennig, 2015).

In Arabidopsis (Arabidopsis thaliana), several cis-elements were initially identified based on their PRE-like properties (Xiao and Wagner, 2015). A 50-bp cis-repression element in the promoter region of LEAFY COTYLEDON2, which harbors an RY motif, confers H3K27me3 deposition at transgenes (Berger et al., 2011). A PRE-like sequence in KNOX homeobox genes is bound by the ASYMMETRIC LEAVES complex, which recruits PRC2 to the target genes (Lodha et al., 2013). The PRE-like element in the upstream region of the floral meristem terminator KNUCKLES is required for its transcriptional repression in a cell division-dependent manner (Sun et al., 2014). Recent genome-wide studies have uncovered putative PRE sequences in Arabidopsis and specific cis-motifs as consensus PREs (e.g. GAGA motifs and telobox motifs) related to PcG recruitment, revealing an evolutionarily conserved model similar to that in Drosophila (Xiao et al., 2017; Zhou et al., 2018). In plants, long noncoding RNAs also bind to CURLY LEAF (CLF) or LIKE HETEROCHROMATIN PROTEIN1 (LHP1) to recruit PcG factors to their targets (Heo and Sung, 2011; Ariel et al., 2014). Nevertheless, it is unknown how changes in chromatin occur in response to environmental signals (He and Li, 2018).

The B3-domain–containing transcription factors VIVIPAROUS1/ABSCISIC ACID INSENSITIVE3-LIKE1 (VAL1) and VAL2 are thought to be widely involved in PRC1-mediated repression of specific target genes (Yang et al., 2013; Merini et al., 2017). VAL1/2 possess a plant-specific B3 DNA-binding domain that specifically recognizes Sph/RY elements (CATGCA) in the regulatory regions of target genes (Suzuki et al., 1997; Swaminathan et al., 2008; Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018). More than half of the genes that are derepressed >4-fold in val1 val2 double mutants possess at least one RY motif in their promoter regions or first introns (Suzuki et al., 2007). A PRE-like sequence comprising two to three nucleosomes around the first intron of FLOWERING LOCUS C (FLC) contains two RY motifs that are recognized by VAL1/2. VAL1 recruits LHP1 and the apoptosis- and splicing-associated protein complex to deposit H3K27me3 at the nucleation region, thereby silencing FLC after vernalization (Qüesta et al., 2016; Yuan et al., 2016). VAL1 directly interacts with MULTICOPY SUPRESSOR OF IRA1 (MSI1), the core PRC2 subunit, and specifically binds to two RY elements in the promoter of AGAMOUS-LIKE15 (AGL15), leading to the establishment of H3K27me3 and repressing seed maturation (Chen et al., 2018).

In rice (Oryza sativa), the binding of VAL1/2 homologs GERMINATION- DEFECTIVE1 and VAL2 to the intronic RY motif of ELONGATED UPPERMOST INTERNODE1 (Eui1) results in the recruitment of repressor complexes to down-regulate Eui1 expression via histone modification (Xie et al., 2018). Hence, the RY cis-element–mediated recruitment of chromatin-associated proteins by VAL1/2 is likely a conserved negative regulatory mechanism in plants. Structural analysis of VAL1-B3 indicated that it forms H-bonds with all six bp in the RY motif (Sasnauskas et al., 2018). In addition to the B3 DNA-binding domain, VALs contain three other domains, including the PHD-like (PHD-l) and cysteine and tryptophan residue-containing (CW) domains (which are associated with histone binding) and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) domain (Suzuki and McCarty, 2008; Jo et al., 2019). PHD-l reads the methylation state of histone H3K27, whereas the CW domain is responsible for the interaction between VAL1 and HISTONE DEACETYLASE19 (Zhou et al., 2013; Yuan et al., 2016).

The transition from vegetative to reproductive growth is a crucial developmental switch that ensures reproductive success in flowering plants. This transition is often synchronized with changes in seasonal cues, such as day length or photoperiod, through the photoperiod pathway (Andrés and Coupland, 2012). Photoperiod is perceived in leaves, leading to the transmission of a major component of florigen encoded by FLOWERING LOCUS T (FT) from the leaves to the shoot apical meristem (Corbesier et al., 2007; Turck et al., 2008). Under long-day (LD) conditions, FT is activated, primarily by the transcriptional regulator CONSTANS (CO; Andrés and Coupland, 2012; Song et al., 2015). Multiple transcription factors or regulators, including CO, directly bind to the proximal promoter of FT and regulate its expression (Andrés and Coupland, 2012). A chromatin loop brings the distal enhancer and proximal cis-elements of FT in close proximity, leading to FT activation (Cao et al., 2014; Liu et al., 2014). Moreover, the direct binding of transcription factors to the introns and 3′-untranslated region (3′-UTR) of FT has also been demonstrated (Searle et al., 2006; Mathieu et al., 2009).

Arabidopsis mutants of PcG factors, such as clf, embryonic flower1 (emf1), emf2, and lhp1, display extremely early flowering, largely due to increased expression of FT (Bratzel and Turck, 2015). CLF functions as an H3K27 methyltransferase that deposits H3K27me3 at FT, leading to its repression (Jiang et al., 2008). LHP1, SHORT LIFE (SHL), and EARLY BOLTING IN SHORT DAYS (EBS) are readers of the H3K27me3 marks of target genes; their binding to H3K27me3 on FT chromatin results in its silencing (Turck et al., 2007; Zhang et al., 2007a; López-González et al., 2014; Li et al., 2018). Both LHP1 and the bromo adjacent homology (BAH) domain-containing proteins SHL and EBS directly interact with EMBRYONIC FLOWER 1 (EMF1), forming two plant-specific complexes. Both of these complexes serve as PRC1 that directly represses FT expression and thus regulates flowering (Wang et al., 2014; Li et al., 2018). PcG proteins likely repress FT expression before dusk and at night, but it is unclear how the PcG complex silences FT expression.

Here, we show that LHP1 and the repressive histone mark H3K27me3 are more highly enriched at the Lys 27 (H3K27me3) region (HTR) within FT than in other regions. We identified a cis-regulatory DNA element (two RY motifs) in the HTR that is recognized by the B3 domain transcription factor VAL1. The levels of VAL1 homodimer accumulation, the binding of VAL1 to FT chromatin, and VAL1 repression of FT expression were higher at night than at dusk in LDs. Moreover, VAL1 recruits the PcG proteins LHP1 and MSI1 to FT chromatin, leading to the H3K27me3 peak at the HTR.

RESULTS

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

The deposition of H3K27me3 occurs throughout the various regions of FT locus, including the promoter region, gene body, and (particularly) downstream regions (Turck et al., 2007; Adrian et al., 2010). We examined whether a region in FT chromatin contains particularly high levels of H3K27me3. We produced amplicons across FT locus from −6.8 to 3.4 kb to further explore the contribution of PRC2 to the repression of FT (Fig. 1A). We compared the H3K27me3 profiles along FT in the leaves of wild-type Columbia (Col) and the mutant of the PRC2 core component CLF over a LD cycle. In Col, H3K27me3 was indeed enriched at various regions across the FT locus, with a high-level H3K27 HTR located from the second intron to the 3′UTR (Fig. 1, A and B). In clf-28, the levels of H3K27me3 across FT locus were strongly reduced, although substantial enrichment was still detected at the HTR (Fig. 1, A and B). The residual level of H3K27me3 at the FT locus in clf mutants is likely due to the H3K27 methyltransferase redundancy with SWINGER (Farrona et al., 2011).

Figure 1.
Figure 1.

Enrichment of H3K27me3 and LHP1 at the FT locus. A, Schematic diagram of the FT genomic region. Exons and the 5′UTR (or 3′UTR) are represented by black and gray boxes, respectively, whereas other genomic regions are represented by a black line. Numbers indicate the positions of amplicons for the chromatin immunoprecipitation (ChIP) experiments. B, ChIP experiments showing relative fold enrichments of H3K27me3 across the FT locus in wild-type (WT) and clf-28 seedlings at ZT8 in LD. C, ChIP analysis of LHP1-GFP enrichment on FT chromatin of wild-type (Col) and LHP1p:LHP1-GFP tfl2-1 seedlings grown in LDs at ZT2. The signals from Col samples were set to 1. In (B) and (C), error bars represent SDs of at least three independent biological replicates, and ACT2 (ACTIN 2) and/or Ta3 were used as controls.

LHP1 [also known as TERMINAL FLOWER2] binds to targets that colocalized with H3K27me3 (Turck et al., 2007; Zhang et al., 2007a). LHP1 is expressed in vascular tissue, and LHP1 is a component of PRC1 that binds to FT locus and represses its expression (Kotake et al., 2003; Adrian et al., 2010; Wang et al., 2014). H3K27me3 levels at FT locus in Col and tfl2-1/lhp1-3 were explored by ChIP–quantitative qPCR (qPCR); however, no significant changes were observed (Supplemental Fig. S1F), which is probably due to that LHP1 and EMF1 or the BAH-domain–containing proteins SHL and EBS act together in binding and maintaining H3K27me3 at FT locus (López-González et al., 2014; Wang et al., 2014; Li et al., 2018). We generated LHP1p:LHP1-GFP transgenic plants in the tfl2-1 background and found that the extremely early flowering phenotype of tfl2-1 was rescued in the homozygous lines, which showed similar FT expression levels to that of Col (Supplemental Fig. S1, A–C), implying that LHP1p:LHP1-GFP is biologically functional. LHP1-GFP fusion proteins produced fluorescent signals in the nucleus (Supplemental Fig. S1D). We then performed ChIP assays using LHP1p:LHP1-GFP tfl2-1, in which the LHP1-GFP fusion protein was enriched after immunoprecipitation (Supplemental Fig. S1E). LHP1-GFP was strongly enriched on FT chromatin, especially at the H3K27me3 HTR (Fig. 1C). Finally, ectopic FT activation is known to occur (but not ectopically expressed outside of the phloem) in plants in the absence of PRC2 and PRC1 function (Moon et al., 2003; Steinbach and Hennig, 2014; Wang et al., 2014). These observations indicate that PRC2 and PRC1 are required for proper FT expression.

Identification of Two RY Motifs as the Putative FT Repression Element

PcG proteins are recruited to their target chromatin and mediate the spread of H3K27me3 to the surrounding regions (Mozgova and Hennig, 2015; Xiao and Wagner, 2015). The role of PREs in the recruitment of PcG to specific genes is conserved in Arabidopsis and Drosophila (Kassis and Brown, 2013; Xiao et al., 2017; Zhou et al., 2018). We therefore attempted to identify the cis-regulatory element in the HTR required for FT expression. We generated a reporter construct in which GUS was located downstream of a cassette including FT enhancer and proximal promoter (hereafter referred to as EP-GUS; Fig. 2A; Liu et al., 2014). The putative PRE-containing fragments (PUTs) were inserted between the enhancer and proximal promoter to generate EP-PUT constructs (Fig. 2A). We then separated the HTR into intron 2 (PUT1), intron 3 (PUT2), and adjacent downstream PUT3 (similar length to PUT1) and examined their transcriptional activity in transgenic plants. The presence of PUT1, but not PUT2 or PUT3, was sufficient to repress GUS expression (Fig. 2, B and C).

Figure 2.
Figure 2.

Identification of the FT cis-silencing elements. A, Schematic representation of the sequence truncation series of the putative FT PREs (PUTs, blue boxes) fused to the GUS reporter gene. The PUT fragments were placed between the 548-bp enhancer and the 527-bp proximal promoter to generate various EP-PUT cassettes and drive GUS expression. The two RY (TGCATG; R, purine; Y, pyrimidine) motifs are marked by arrowheads. B and C, GUS expression analysis of T1 transgenic seedlings expressing the indicated EP-PUTs. The total number of plants of each transgenic line analyzed is indicated above the columns. S+M indicate lines with strong or medium GUS staining, whereas W+N indicate lines with weak or none GUS staining. The seventh set of leaves is shown. Scale bar = 5 mm. D, ChIP experiments showing H3K27me3 accumulation relative to H3 on the indicated amplicons in 10-d-old seedlings of the indicated genotype. Error bars are means ± sd of one biological replicate with three technical repeats for samples from at least three independent transgenic lines. Strong GUS staining lines were chosen for EP:GUS, EP-PUT7:GUS, EP-PUT6m:GUS, and EP-PUT6∆24:GUS, whereas weak GUS staining lines were used for EP-PUT1:GUS, EP-PUT4:GUS, and EP-PUT6:GUS. The signal from FT amplicon 15 was set to 1. The locations of the FT amplicons are shown in Figure 1.

Drosophila PREs are nucleosome depleted, perhaps to facilitate the assembly of the multiprotein PRC1 and PRC2 complexes (Mohd-Sarip et al., 2005, 2006). We then added additional amplicons around the HTR and performed ChIP and FAIRE (formaldehyde‐assisted isolation of regulatory elements) assays to narrow down the putative PRE-containing region (Supplemental Fig. S2A). The HRTs were located in similar regions in H3K27me3 ChIP normalized to either histone H3 or input (Supplemental Fig. S2, B and C). We identified a nucleosome-depleted region located in the HRT from the latter half of intron 2 (amplicon 14) to the 3′UTR (amplicon f), in which PUT4 overlaps with PUT1, as a possible PRE-containing fragment (Supplemental Fig. S2D). Indeed, the use of PUT4, but not PUT5, reduced GUS activity in the assay (Fig. 2, A and B). Using a sequence truncation approach, we found that the presence of a 183-bp PUT6 region was sufficient to repress GUS expression (Fig. 2, A–C). Two RY motifs shown to trigger the epigenetic repression of target genes were found within PUT6. The simultaneous mutation (EP-PUT6m) and deletion (EP-PUT6∆24) of these two RY motifs in PUT6 led to the release of GUS repression (Fig. 2C), suggesting that RY motifs might function as FT silencing elements.

PREs act as cis-motifs for PcG recruitment, leading to the deposition of H3K27me3 not only on genes containing these motifs, but also on adjacent genes (Simon and Kingston, 2013; Xiao et al., 2017). We conducted ChIP assays to explore whether the putative PRE-containing regions are involved in the enrichment of H3K27me3 at the GUS locus. In transgenic plants carrying RY motifs, the levels of H3K27me3 on GUS were higher than those of the wild type, in agreement with the notion that the GUS reporter genes were silenced. By contrast, in transgenic plants with mutated or no RY motifs, the levels of H3K27me3 at the GUS locus were lower than those of the wild type (Fig. 2D). Hence, the RY motifs are required for H3K27me3 binding and GUS silencing.

The Intronic RY Motifs of FT Confer Flowering Repression in LDs

The fusion of the FT enhancer and the proximal promoter sequence constitutes a short promoter (hereafter referred to as the EP promoter) that responds normally to day length (Liu et al., 2014). We investigated the biological effects of the RY motifs on the floral transition in LDs by generating various FT genomic fragments driven by the EP promoter and introducing them into ft-10 plants (Fig. 3A). All transgenic plants expressing FT fragments under the control of EP promoters flowered earlier than the parent line ft-10 (Fig. 3, A and B), suggesting that these constructs were biologically functional.

Figure 3.
Figure 3.

Phenotypic analysis of plants harboring FT transgenes driven by the recombinant EP promoter. A, Schematic diagram of the FT transgenes. The number 2800 indicates +2800 bp relative to the start codon. The exons and 3′UTR are represented by black and gray boxes, respectively; the deletion regions are represented by dashed lines; and the remaining genomic regions are represented by black lines. The FT transgenes were driven by the EP promoter to which the FT enhancer and proximal promoter were serially fused. B, Total number of leaves at flowering for the indicated plants (in the ft-10 background) grown under LD conditions. Each red triangle represents a single plant (n ≥ 23); means (black horizontal lines) ± sd (error bars) are shown. Statistical significance was determined by one-way ANOVA and multiple comparison by the Duncan method (P = 0.05). Letters above the bars indicate significant differences. C, Distribution of flowering time in T1 transgenic plants carrying the indicated FT genomic fragments in the ft-10 background grown in LDs. In (B) and (C), ft-10 served as a control.

Plants with a deletion of intron 1 or intron 2 (EP:FTg2800∆ intron 1 ft-10 and EP:FTg2800∆intron 2 ft-10) showed earlier flowering than EP:FTg2800 ft-10, whereas plants harboring a deletion of intron 3 (EP:FTg2800∆ intron 3 ft-10) exhibited a flowering phenotype similar to that of EP:FTg2800 ft-10, suggesting that both intron 1 and intron 2 might harbor FT cis-repression elements.

We further examined the role of RY motifs by introducing an FTg2800 transgene with these two RY motifs deleted (FTg2800∆24) into the ft-10 background and found that these plants exhibited earlier flowering than those harboring the wild-type FTg2800 transgene (EP:FTg2800 ft-10; Fig. 3). Taken together, these results indicate that the RY-motif–containing region serves as an FT-repressing element.

The Transcriptional Repressor VAL1 Binds to FT-Repressing RY Motifs

Plant-specific B3-domain transcription factors bind to RY motifs (Reidt et al., 2000; Braybrook et al., 2006; Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018; Sasnauskas et al., 2018). The B3 domain of VAL1 is necessary and sufficient for its binding to RY motifs (Suzuki et al., 1997; Qüesta et al., 2016; Sasnauskas et al., 2018). We therefore performed electrophoretic mobility shift assays (EMSAs) using recombinant VAL1-B3 fragments tagged with glutathione S-transferase (GST). GST-VAL1-B3 caused an up-shift of the biotinylated wild-type RY1/2 probe but not the RY1/2 mutant probe. We also detected binding of VAL1 B3 to single RY sites RY1 and RY2 (Crick strand of and just downstream of RY1; Fig. 4, A–D). Moreover, the amounts of shifted bands were substantially attenuated by the addition of excess unlabeled wild-type DNA but not by competitors with mutated RY motifs (Fig. 4, B and C). Interestingly, the presence of double RY motifs facilitated the formation of nonspecific complexes (multiple protein copies bound to a single DNA, indicated by arrowheads in Fig. 4, A and B).

Figure 4.
Figure 4.

VAL1 binds to the intronic RY motifs and represses FT expression. A to D, EMSA showing that GST-VAL1-B3 protein, but not GST alone, specifically binds to the double (RY1/2-wt; A andB) or single (RY1-wt and RY2-wt) RY wild-type probes (C and D) but not the mutant probes (RY1/2-m, RY1-m, and RY/2-m; B to D). Arrows indicate the specific complex, arrowheads indicate the nonspecific complex, and square brackets indicate free probe. E, GAD-VAL1, but not GAD-VAL2, GAD-RAV1, GAD-RAV2, or GAD alone, activates the expression of the LacZ reporter gene driven by the PUT4 fragment (shown in Fig. 2) in yeast. F, GAD-VAL1 activates the LacZ reporter gene driven by the wild-type PUT6 fragment (shown in Fig. 2) in yeast. Single mutations (RY1m and RY2m) and simultaneous mutation (RY1/2m) of the RY motifs attenuate or abolish the activation of LacZ gene expression. G, Evolutionary conservation of the RY motifs in FT intronic sequences from different Brassicaceae species. RY motifs (CATGCA, Crick strand TGCATG) are shown in red, and the Crick strand is underlined. H, Transcript levels of FT in VAL1p:VAL1-GR val1-2 transgenic seedlings in LDs. The 10-d-old seedlings were harvested at ZT4 after treatment for 4 h with or without DEX (20 µM) in the presence or absence of CHX (50 µM). Expression levels were normalized to IPP2. Means ± sd from three biological replicates are shown (*P < 0.05, **P < 0.01, Student’s t test). I, GUS staining of the indicated transgenic GUS lines (as depicted in Fig. 2A) in the 35S:VAL1-GR background. Seedlings were grown in MS medium with or without 20 µM DEX (Mock, 0.1% [v/v] ethanol) for 6 d in LDs. At least two independent transgenic GUS lines were examined with similar results. Scale bar = 1 mm.

We performed yeast one-hybrid assays to explore the two RY motifs recognized by VAL1. Fusion proteins comprising the GAL4 transcriptional activation domain fused to VAL1 (GAD-VAL1), but not GAD-VAL2, GAD-RAV1, GAD-RAV2, or GAD alone, activated the LacZ reporter gene driven by the RY-motif–containing PUT4 and PUT6 fragments (Fig. 4, E and F). Mutation of any of the two RY motifs (RY1-m or RY2-m) in PUT6 attenuated the binding of VAL1, whereas both RY mutations (PUT6-RY1/2-m) and deletions (PUT6∆24) abolished the activation of the reporter gene by GAD-VAL1 (Fig. 4F). These results indicate that VAL1 recognizes and binds to the RY motifs of the FT repression element. We aligned the intronic sequences of FT from Arabidopsis and FT homologs from other plants of the Brassicaceae family, including Arabidopsis lyrata, Brassica rapa, and Brassica oleracea. This alignment revealed that both RY motifs are highly conserved in species of the Brassicaceae family (Fig. 4G), suggesting they play important roles in regulating FT expression.

To investigate whether VAL1 directly regulates FT expression, we generated VAL1p:VAL1-glucocorticoid receptor (GR) val1-2 transgenic plants, in which VAL1 was fused with the GR and driven by the 3.4-kb native VAL1 promoter. In the presence of dexamethasone (DEX), the flowering phenotype of VAL1p:VAL1-GR val1-2 was rescued (Supplemental Fig. S3, A and B). AGAMOUS LIKE15 (AGL15) is a direct target of VAL1 (Chen et al., 2018). Both val1-2 and VAL1p:VAL1-GR val1-2 plants exhibited higher levels of AGL15 than the wild type, whereas only VAL1p:VAL1-GR val1-2 was rescued by DEX induction (Supplemental Fig. S3C), demonstrating that VAL1-GR is functional. DEX treatment supplemented with or without cycloheximide, a protein synthesis inhibitor, led to reduced FT expression (Fig. 4H), suggesting that the repression of FT requires nucleus-localized VAL1. However, upon VAL1 mutation, FT expression was down-regulated and the plants flowered late (Supplemental Fig. S3, D and E), arguing against the notion that VAL1 represses FT expression.

MADS domain transcription factors FLC and AGL15, whose encoding genes are directly repressed by VAL1, act as FT transcriptional repressors to prevent flowering (Searle et al., 2006; Adamczyk et al., 2007; Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018). We reasoned that the defect in FT repression in the val1 mutants might be due to the secondary effects of VAL1 on the regulation of flowering. This notion is supported by the finding that FT expression and flowering time were largely rescued in val flc (Supplemental Fig. S3, D and E). GUS transcript levels and GUS staining activity were explored in the randomly chosen T1 EP:GUS and EP-PUTs:GUS reporter lines. The attitudes of GUS staining reflect GUS mRNA levels (Supplemental Fig. S3F), supporting that these lines were effective. We then crossed EP:GUS and EP-PUTs:GUS reporter lines to val1-2 and to plants harboring functional 35S:VAL1-GR (Fig. 4I; Supplemental Fig. S3, G and H). Indeed, GUS expression driven by RY-motif-containing fragments (EP-PUT6:GUS) was derepressed in the val1-2 background, whereas GUS expression driven by fragments lacking RY motifs (EP-PUT6∆24:GUS val1-2) showed almost no effect upon VAL1 mutation (Supplemental Fig. S3H). When introduced into 35S:VAL1-GR plants, the weak GUS activity of EP-PUT6:GUS was further reduced in response to DEX treatment compared with mock treatment, whereas little difference in GUS staining was observed in the other reporter lines (bearing a mutated or no RY motif, i.e. EP-PUT6m:GUS 35S:VAL1-GR and EP-PUT6∆24:GUS 35S:VAL1-GR) with or without DEX treatment (Fig. 4I). Furthermore, we conducted luciferase-based transient assay. The Nicotiana benthamiana cells coexpressing 35S:GFP with PUT4:LUC (luciferase) or PUT4m:LUC displayed comparable signals, whereas the cells coexpressing 35S:VAL1-GFP/PUT4:LUC displayed attenuated signals compared with those coexpressing 35S:VAL1-GFP/PUT4m:LUC (Supplemental Fig. S3, I and J), indicating that VAL1 repressed the expression of FT RY-motif–containing fragment of PUT4:LUC. Collectively, these results support the notion that the transcription factor VAL1 represses FT expression via its RY motifs.

VAL1 Directly Represses FT Expression before Dusk and at Night in LDs

The binding of the PcG factors CLF, LHP1, and EMF1 and the histone mark H3K27me3 to FT locus is reduced at dusk compared with night and midday (Luo et al., 2018). We explored whether VAL1 is involved in the photoperiodic regulation of flowering time. We examined the diurnal expression patterns of VAL1 in LDs and found that VAL1 mRNA levels peaked at ZT0, declined during the day, reached a trough at ZT16, and subsequently increased (Fig. 5A). VAL2 showed a similar expression pattern, but with much weaker fluctuations, whereas VAL3 was expressed at very low levels throughout the period (Fig. 5A). Consistent with this notion, microarray data from the eFP browser showed that the expression of VAL1, but not VAL2 or VAL3, oscillates and reaches a trough at the end of the day (Supplemental Fig. S4; Michael et al., 2008). VAL1 was expressed in leaf veins of both cotyledons and true leaves (Fig. 5B).

Figure 5.
Figure 5.

Analysis of VAL1 expression patterns and its binding to FT chromatin. A, VAL1, VAL2, and VAL3 mRNA levels in 10-d-old (wild type [WT]) seedlings over a 24-h LD cycle. The mRNA levels were normalized to IPP2. Error bars indicate SD of three independent biological replicates. White and black bars indicate light and dark periods, respectively. B, Spatial expression patterns of VAL1p:GUS plants grown in LDs determined by histochemical staining for GUS activity. Scale bars = 1 mm (the cotyledon) and 2 mm (the fifth set of true leaf), respectively. C, ChIP analysis of VAL1-GFP enrichment at the FT locus in 10-d-old wild type (Col) and VAL1p:VAL1-GFP val1-2 seedlings grown in LDs. The levels of immunoprecipitated genomic fragments were measured by qPCR; the signals from Col samples were set to 1. Ta3 was used as a negative control. Error bars represent SD from three independent biological replicates (*P < 0.05, Student’s t test). The locations of the FT amplicons are shown in Figure 1. D, FT mRNA levels in VAL1p:VAL1-GR val1-2 seedlings grown for 10 d in LDs. Seedlings were harvested at the indicated ZTs after treatment for 2 h with DEX (20 µM) in the presence or absence of CHX (50 µM). Expression levels were normalized to IPP2. Values are means ± sd of two biological replicates for samples from three independent transgenic lines. The signals from CHX treatments were set to 1.E, Coimmunoprecipitation assay showing the homodimerization of VAL1 in 10-d-old seedlings expressing the VAL1p:VAL1-GFP and 35S:Myc-VAL1 constructs. The panel at bottom shows higher intensity coimmunoprecipitation signals detected by Myc antibody. F, Quantification of the homodimerization level of VAL1 after normalization to immunoprecipitated GFP using ImageJ software. The level of VAL1 homodimer at ZT16 was set to 1. Means ± sd from three biological replicates are shown. In (D) and (F) Significance of differences from ZT16 was calculated using Student’s t test (**P < 0.01).

To explore whether VAL1 binds to FT chromatin, we generated VAL1p:VAL1-GFP val1-2 transgenic plants in which VAL1-GFP was driven by the VAL1 native promoter. The representative lines exhibited similar flowering times and FT expression levels as those of the wild type (Supplemental Fig. S5, A–C) and produced fluorescent signals in the nucleus (Supplemental Fig. S5D), implying that the VAL1-GFP fusion protein is biologically functional. We conducted ChIP using representative VAL1p:VAL1-GFP val1-2 seedlings harvested at ZT16 and ZT24. At ZT24, VAL1-GFP was strongly enriched on amplicon 14 (containing RY motifs) and on amplicon 15 to a lesser extent, whereas at ZT16, the enrichment of VAL1-GFP at FT was significantly reduced (Fig. 5C). We measured the protein level of VAL1-GFP using VAL1p:VAL1-GFP val1-2 seedlings harvested at ZT4, ZT16, and ZT24 in LDs and found that the protein levels did not change obviously (Supplemental Fig. S5E), indicating that the reduced binding ability of VAL1-GFP at FT locus at ZT16 might not be due to the VAL1-GFP protein abundance.

The MADS domain transcription factors FLC and AGL15, whose encoding genes are directly repressed by VAL1, act as FT transcriptional repressors to prevent flowering (Searle et al., 2006; Adamczyk et al., 2007; Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018). To identify the particular time in LDs when VAL1 represses FT expression and to avoid the effects of FLC and AGL15 (Searle et al., 2006; Adamczyk et al., 2007; Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018), we measured FT transcript levels using RNA extracted from dissected leaves of VAL1p:VAL1-GR val1-2 transgenic plants simultaneously treated with DEX and cycloheximide (CHX; to reduce secondary transcriptional effects after VAL1 moves into the nucleus). The repression of FT by VAL1 was much stronger when VAL1 activity was induced at midday (ZT6) and at the end of the night (ZT24) versus dusk (ZT16; Fig. 5D). These results indicate that the ability of VAL1 to bind to and repress FT depends on day length, with maximum activity at night, which coincides well with the finding that VAL1 is strongly expressed at this time.

VAL1 likely forms homodimers that bind to the RY motifs in its target loci (Qüesta et al., 2016; Yuan et al., 2016). We therefore generated VAL1p:VAL1-GFP 35S:Myc-VAL1 val1-2 transgenic lines and performed coimmunoprecipitation assays. VAL1 indeed formed homodimers in seedlings harvested at ZT16 and ZT24 (Fig. 5E), as previously reported (Chhun et al., 2016). Intriguingly, the homodimerization of VAL1 was significantly higher at ZT24 compared with ZT16 (Fig. 5, E and F). This finding is consistent with the observation that higher levels of VAL1 binding to FT chromatin and stronger repression of FT by VAL1 occur at night versus the end of LDs.

VAL1 Recruits PcG Proteins LHP1 and MSI1 to the FT Locus

LHP1 and MSI1 interact with VAL1 to repress the expression of target genes involved in seed maturation and the control of flowering time (Yuan et al., 2016; Chen et al., 2018). MSI1 regulates the photoperiodic floral transition (Steinbach and Hennig, 2014). We therefore investigated whether the enrichment of LHP1 and MSI1 on FT chromatin is dependent on VAL1. We generated 35S:Myc-MSI1 transgenic plants in which Myc-MSI1 fusion protein was enriched after immunoprecipitation (Supplemental Fig. S6, A and B). We introduced the LHP1p:LHP1-GFP and 35S:Myc-MSI1 constructs into 35S:VAL1-GR transgenic lines and measured the association of LHP1-GFP and Myc-MSI1 with FT chromatin by ChIP assays. Both LHP1-GFP and Myc-MSI1 were enriched at the HTR of FT after 35S:VAL1-GR was induced by DEX treatment compared with the mock control (Fig. 6, A and B). Thus, VAL1 binding to the RY motif in the HTR increased recruitment of both LHP1 and MSI1 to FT chromatin.

Figure 6.
Figure 6.

VAL1-mediated PcG repression of FT expression. A to C, Association of LHP1-GFP (A), Myc-MSI1 (B), and H3K27me3 (C) with the FT locus revealed by ChIP-qPCR. The 10-d-old seedlings were treated with or without 20 µM DEX for 4 h and harvested at ZT4. qPCR signals from DEX-treated samples were normalized to input (A and (B) and H3 (C), respectively. The signals from mock-treated samples (Mock) were set to 1. TA3 was used as a negative control. Values in (A) to (C) are means ± sd of three biological replicates. The locations of the FT amplicons are shown in Figure 1.

MSI1 is a component of PRC2 that catalyzes H3K27me3 (Köhler et al., 2003). In addition, LHP1 reads the H3K27me3 marks deposited by PRC2 (Turck et al., 2007; Zhang et al., 2007a). MSI1 interacts with LHP1; this interaction links LHP1 to PRC2 and facilitates the recruitment of PRC2 to chromatin bearing H3K27me3 (Derkacheva et al., 2013). We examined whether VAL1 recruits LHP1 and MSI1 to promote H3K27me3 deposition at the FT locus by measuring H3K27me3 levels on FT chromatin in 35S:VAL1-GR val1 seedlings. Upon DEX treatment, H3K27me3 was enriched at the FT locus at the HTR compared with the uninduced control (Fig. 6C). FT transcript levels in 35S:VAL1-GR versus LHP1p:LHP1-GFP 35S:VAL1-GR or 35S:Myc-MSI1 35S:VAL1-GR were also examined by qPCR; however, no significant changes upon DEX treatment were observed (Supplemental Fig. S7), probably due to that the encoding genes of FT repressors, such as FLC and AGL15, were also down-regulated upon DEX treatment in LHP1p:LHP1-GFP 35S:VAL1-GR and 35S:Myc-MSI1 35S:VAL1-GR (Supplemental Fig. S7).

val1/2 strong mutants exhibit embryonic traits as those in the mutants severely compromised of PRC2 or PRC1 function (Supplemental Fig. S8A; Yang et al., 2013; Jo et al., 2019). We further examined the role of VAL1/2 proteins in H3K27 trimethylation at FT locus. Double mutation of VAL1 and VAL2 caused a reduction of H3K27me3 levels, but did reach the levels as those in clf-28, indicating VAL1/2 proteins mediate H3K27me3 deposition on FT chromatin (Supplemental Fig. S8B). Together, these findings indicate that VAL1 represses FT transcription via facilitating the recruitment of LHP1 and MSI1 and mediating H3K27 trimethylation at the FT HTR.

DISCUSSION

In this study, we demonstrated that the plant-specific transcription factor VAL1 contributes to periodic PcG repression of the florigen gene FT in Arabidopsis under LDs. VAL1 binds specifically to the two intronic RY motifs at the HTR of the FT locus, likely as a homodimer, which accumulates to higher levels at night than at dusk in LDs. Thus, LHP1 and MSI1 recruitment to the HTR is facilitated by interacting with VAL1, leading to increased levels of H3K27me3 on FT and its repression at night.

The FT locus is widely bound by LHP1 and the repressive mark H3K27me3, especially at the downstream region (Turck et al., 2007; Zhang et al., 2007b; Adrian et al., 2010). The H3K27me3 and LHP1 profiles across FT in our ChIP assays largely overlap with those obtained in previous studies, with an HTR covering the second intron and the 3′UTR (Fig. 1). PcG-induced silencing of FLC is best characterized in Arabidopsis. During vernalization, H3K27me3 levels increase locally at the nucleation region of FLC in response to cold and spread over the entire FLC gene body following transfer to warm conditions (Qüesta et al., 2016; Yang et al., 2017). The nucleation of H3K27me3 occurs in a region of two or three nucleosomes around the first exon, which confers metastable epigenetic silencing of FLC in the cold. The spreading of H3K27me3 across the FLC locus results in the stable silencing of FLC after transfer to warm conditions (Yang et al., 2017). Both the PcG factors LHP1 and CLF and H3K27me3 are enriched on FT chromatin (Luo et al., 2018). Consistently, we found that both LHP1 and H3K27me3 highly enriched at the HTR of the FT locus (Fig. 1, B and C). We inferred that the HTR may confer metastable epigenetic repression of FT during the photoperiodic response to rapid light/dark cycles.

We narrowed down a putative PRE-containing region based on molecular dissection of the HTR via GUS staining and colocalization analysis of the HTR and nucleosome-depleted region revealed by ChIP and FAIRE (Fig. 2, A–C; Supplemental Fig. S2). RY motifs were shown to be involved in the epigenetic silencing of target genes. Further experiments (i.e. RY motif deletion and mutagenesis approaches) confirmed that RY motifs are necessary to confer the repressive activity of H3K27me3 deposition (Fig. 2D). The FAIRE assay revealed four nucleosome-depleted regions across the FT locus. The proximal promoter region (amplicons 9 and 10) carries two CO-responsive elements (COREs; containing CCACA) and is bound by the output of the photoperiod pathway (CO; Adrian et al., 2010; Tiwari et al., 2010; Song et al., 2012), suggesting this region might facilitate CO-dependent FT expression, consistent with our previous report (Jing et al., 2019). A FAIRE signal peak covered the latter half of intron 2 to the 3′UTR and overlapped with the HTR, harboring two RY motifs bound by VAL1. These findings suggest that this region is accessible to the transcriptional repressor VAL1, which recognizes the RY motif, thereby enabling the recruitment of PcG proteins LHP1 and MSI1. Further experiments are needed to investigate whether this region favors the assembly of the multiprotein complexes PRC2, as shown in Drosophila (Mohd-Sarip et al., 2005, 2006). The first FAIRE peak (amplicon 6) appears to be bound by chromatin-remodeling factor PICKLE (Jing et al., 2019), whereas in the region of the third FAIRE peak (amplicon 12), no candidate transcription factors have been reported that recognize the target DNA sequences.

An experiment using the short fusion promoter (EP promoter) driving FT expression revealed that the two RY motifs are required for the proper flowering response in LDs (Fig. 3A). Interestingly, plants harboring constructs with a deletion of intron 1 or intron 2 of the FT genomic sequence (EP:FTg2800∆intron1 ft-10 and EP:FTg2800∆intron2 ft-10) exhibited earlier flowering than those harboring a construct lacking a deletion (EP:FTg2800 ft-10), indicating that the polymorphisms in the cis-regulatory regions within intron 1 or intron 2 negatively modulate its function.

Several proteins, including FLC, can bind to the first intron in FT to repress its expression (Golembeski and Imaizumi, 2015). We cannot exclude the possibility that intron 2 carries other cis-silencing elements besides RY motifs. Similar to our results, introducing an FLC transgene with a deleted RY motif (FLCΔ10) into the flc mutant background led to the up-regulation of FLC and resulted in late flowering (Yuan et al., 2016). The response of flowering to the disruption of the intronic native RY motifs of FT remains to be examined. A recently discovered dominant rice mutant, dwarf Eui1 (dEui1), exhibits a severe enclosed-panicle phenotype. The phenotypic defects in dEui1 are caused by increased expression of Eui1 in which T-DNA replaces the 135-bp fragment containing a cis-silencing element harboring an RY motif in the Eui1 intron (Xie et al., 2018).

Unlike the repressive effect of VAL1 on FT expression, the val1 mutant exhibited late flowering and decreased FT expression (Supplemental Fig. S3, D and E). One possible explanation is that two proteins of VAL1 silencing targets, FLC and AGL15, are both direct repressors of FT (Searle et al., 2006; Adamczyk et al., 2007; Qüesta et al., 2016; Yuan et al., 2016; Chen et al., 2018). This study provides several lines of evidence supporting this hypothesis. First, introducing the flc mutation into val1-2 strongly attenuated the late-flowering phenotype of val1-2 and derepressed FT transcription (Supplemental Fig. S3, D and E). Second, GUS activity in the transgenic line EP-PUT6:GUS (harboring the GUS reporter gene driven by the RY-motif-containing fragment PUT6) increased upon VAL1 mutation (Supplemental Fig. S3G). Third, FT expression was much more strongly repressed in VAL1p:VAL1-GR val1-2 plants after DEX and CHX treatment compared with DEX treatment alone (Fig. 4H), indicating that the secondary effect of VAL1 on the transcriptional regulation of FT was attenuated by DEX/CHX treatment.

H3K27me3 was largely reduced in the val1/2 double mutant, but not to the level of that in clf-28 (Supplemental Fig. S8B), indicating that there may exist other proteins other than VAL1/2 to mediate PcG repression of FT. FLC was previously shown to directly bind to the first intron of FT and directly interacts with EMF1, and this interaction is required for FT repression (Searle et al., 2006; Wang et al., 2014), suggesting that other than VAL1-PcG, FLC-PcG is also required for FT repression. Further work would be needed to determine how FLC-PcG and VAL1-PcG function in concert to repress FT expression.

VAL1 recruits repressive factors or complexes to its target loci. Both VAL1 and LHP1 can read H3K27me3 marks and interact with the PRC2 component MSI1 (Turck et al., 2007; Zhang et al., 2007a; Derkacheva et al., 2013; Yuan et al., 2016). Our results suggest that VAL1 (and possibly VAL2) binds to RY motifs and recruits LHP1 to FT chromatin, which may favor the recruitment of a PRC2 complex (including the indispensable subunit MSI1) to FT chromatin to deposit H3K27me3, resulting in the H3K27me3 peak at the HTR. The abundance of VAL1 mRNA and VAL1 homodimers (and possibly heterodimers with VAL2), the binding of VAL1 and H3K27me3 to the HTR, and the repression of FT expression by VAL1 exhibited a periodic pattern, with higher levels at night than at dusk. We also examined the enrichment of LHP1-GFP, Myc-MSI1, and the repressive histone mark H3K27me3 at FT locus over a LD cycle using ChIP-qPCR. When compared with those at ZT4 and ZT24, the levels of LHP1-GFP and H3K27me3, but not Myc-MSI1, at the HTR fragment (amplicon 15) were reduced at dusk (ZT16; Supplemental Fig. S8, C–E). These results, together with observations that PcG factors repress FT expression at night and from dawn to late afternoon under LDs and that PcG factors (CLF, EMF1, and LHP1) moderately associate with FT chromatin at dusk (Wang et al., 2014; Luo et al., 2018), suggest that VAL1 functions in PcG repression of FT at night and from dawn to late afternoon in LDs. When CO accumulates from late afternoon through dusk, VAL1-mediated PcG repression of FT is relieved to some extent; thus, FT is derepressed. Together, these findings indicate that the transcriptional repressor VAL1 recognizes RY motifs and triggers epigenetic repression of the florigen gene FT, conferring the photoperiodic control of flowering in angiosperms.

METHODS

Plant Material and Growth Conditions

All Arabidopsis (Arabidopsis thaliana) plants used in this study are of the Columbia (Col) ecotype, and all stable transgenic lines were generated by floral dip using Agrobacterium tumefaciens strain GV3101 (Clough and Bent, 1998). The mutants ft-10 (GK-290E08; Yoo et al., 2005), flc-6 (SALK_041126; Schönrock et al., 2006), val1-2 (SALK_088606; Suzuki et al., 2007), val2-3 (SALK_059568C; Yang et al., 2013), clf-28 (Doyle and Amasino, 2009), and tfl2-1 (Larsson et al., 1998) were obtained from the Arabidopsis Biological Resource Center (ABRC; The Ohio State University, Columbus). Double mutants/transgenic plants were generated by genetic crossing, and homozygous lines were used. Surface-sterilized seeds were plated on half-strength Murashige and Skoog medium containing 0.6% (w/v) phytoagar, 3% (w/v) Suc, and 0.05% (w/v) MES (pH 5.7) and kept for 3 d at 4°C in the dark before being transferred to the growth room. Plants were grown pots of soil in controlled conditions at 22°C, under LD (16-h light/8-h dark).

Plasmid Construction and Plant Transformation

The distal enhancer and proximal promoter of FT reported previously (Adrian et al., 2010; Liu et al., 2014) were fused and inserted into the HindIII and SalI restriction sites of a pBI101.1-derived plasmid to make the construct EP-GUS4-pBI. A 2.8-kb FT genomic fragment (FTg2800), DNA fragments containing a deletion mutant (FTg2800∆24), and FT complementary DNA (cDNA) were amplified and inserted into the SalI and SmaI restriction sites of EP-GUS4-pBI in which GUS fragment was deleted, to make the constructs EP-FTg2800, EP-FTg2800∆24, and EP-FTcDNA, respectively. These plasmids were transformed into the ft-10 mutant.

The distal enhancer and proximal promoter of FT were amplified and inserted into the SalI-EcoRI and EcoRI-SalI sites of pBI101.1-derived plasmid EP-GUS3-pBI, respectively, to make the construct EP-GUS3-pBI. Candidate fragments (PUT1 ∼ 7, PUT6m, and PUT∆24) were amplified and inserted into the EcoRI-XhoI site of EP-GUS3-pBI. The resulting plasmids were transformed into Col.

The 3.4-kb VAL1 promoter fragment and the cDNA encoding VAL1 were amplified and inserted in sequence into the HindIII-SalI and EcoR1-SalI sites of pBI101.1-derived plasmids pBI-GFP and pGI-GR, to make the constructs VAL1p:VAL1-GFP and VAL1p:VAL1-GR, respectively. The same VAL1 promoter fragment was also inserted into the HindIII-SalI site of pBI101.1 to create the construct VAL1p-GFP. The VAL1 cDNA fragment was also inserted into the EcoRI-SalI sites of pRI-Myc and pRI-GR, to give rise to the constructs Myc-VAL1 and VAL1-GR, respectively. The 1.4-kb LHP1 promoter fragment and the LHP1 cDNA fragment were amplified and cloned into the HindIII-EcoRI and EcoRI-SalI sites of pBI-GFP to obtain LHP1p:LHP1-GFP. The MSI1 cDNA fragment was amplified and cloned into the EcoRI-XhoI sites of the pVIP-Myc vector to obtain Myc-MSI1. These plasmids were transformed into Col or val1-2 mutants indicated in the text.

To prepare constructs for the yeast one-hybrid assay, the RAV1, RAV2, VAL1, and VAL2 full-length coding sequence fragments were ligated into EcoRI- and SalI-digested pJG4-5 vector (Clontech), to generate pGAD-RAV1, pGAD-RAV2, pGAD-VAL1, and pGAD-VAL2, respectively. PUT4 and PUT7 fragments of FT were amplified and then cloned into the EcoRI-XhoI sites of the pLacZi2µ vector (Jing et al., 2013), to generate the FT-PUT4:LacZ and FT-PUT7:LacZ constructs. The PUT7 fragment containing the deletion (PUT7∆24) and mutated RY-motif (PUT7-RY1-m, PUT7-RY2-m, PUT7-RY1/2-m) were also inserted into the EcoRI-XhoI sites of the pLacZi2µ vector to make the constructs PUT7∆24:LacZ, PUT7-RY1-m:LacZ, PUT7-RY2-m:LacZ, and PUT7-RY1/2-m:LacZ, respectively.

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

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

The primers are listed in Supplemental Table S1.

Yeast One-Hybrid Analysis

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

Gene Expression Analysis

Total RNA was extracted from 10-d-old seedlings at various time points using an RNA Extraction Kit (Tiangen) according to the manufacture’s instruction. Total RNAs were used to prepare first-strand cDNAs by Moloney murine leukemia virus reverse transcriptase (Invitrogen). Reverse transcription-qPCR was carried out in a LightCycler 480 (Roche) using a SYBR Premix ExTaq Kit (Takara) following the manufacturers’ instructions. Expression analysis was performed with three technical repeats. The expression level was normalized to that of IPP2 or UBQ10 (internal controls). Primers used for gene expression analysis are listed in Supplemental Table S1.

GUS Histochemical Analysis

The rosette leaves or seedlings were incubated in 0.1 m sodium phosphate buffer containing 50 mm K3Fe(CN)6, 50 mm K4Fe(CN)6, and 1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronide at 37°C in the dark for 20 h. The staining reactions were terminated by replacing the buffer with ethanol. The samples were subjected to various conditions as indicated in the text, and GUS staining images were captured by a digital camera (Olympus).

Luciferase-Based Transient Assays

Agrobacterium-mediated transient assay was carried out as described previously (Chen et al., 2008). The Agrobacterium strains GV3101 carrying the indicated constructs were incubated in Luria–Bertani medium at 28°C overnight. The culture was pelleted, washed twice, and resuspended in 10 mm MgCl2 containing 0.2 mm acetosyringone to a final concentration of OD600 = 1.5. The bacteria were kept at 28°C for 3–5 h without shaking. The Agrobacterium suspensions were coinfiltrated into fully expanded young Nicotiana benthamiana leaves with a needleless syringe. The plants were then grown at 22°C for 2 d under LD conditions before LUC activity was examined. LUC images were captured using a NightSHADE LB985 plant imaging apparatus equipped with a CCD camera (Berthold Technologies). The bioluminescence intensities were calculated using Indigo software (v2.0.3.0, Berthold Technologies). Three independent replicates were used for the data analysis.

EMSAs

VAL1-B3-GST and the empty pGEX-4T-1 were transformed into E. coli BL21 strain (DE3), and protein expression was induced by isopropylthio-β-galactoside. The soluble GST fusion proteins were purified using Glutathione Sepharose 4B beads (GE Healthcare; for GST fusions). EMSAs were performed using the LightShift Chemiluminescent EMSA kit (Thermo). Double-stranded DNA was generated by annealing sense and antisense oligonucleotides. Binding reaction conditions were as following: 1× Binding buffer (10 mm Tris, 0.1 mm EDTA, and 5 mm B-mercaptoethanol, [pH7.5]), 5% (v/v) glycerol, 5 mm MgCl2, 50 ng/μl Poly (dIdC), and 0.05% (w/v) Nonidet P-40, in a final volume of 20 μL. Binding reactions were incubated at 25°C for 20 min. Free DNA and protein-DNA complexes were separated on 5% (w/v) native polyacrylamide gels in 30 mm MES/30 mm His (pH 6.3) as reported (Sasnauskas et al., 2018), for 90 min. Detection of biotin-labeled DNA by chemiluminescence was performed according to the manufacturer’s instructions.

ChIP Assay

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

FAIRE Assay

The FAIRE assay was performed as previously described (Omidbakhshfard et al., 2014; Jing et al., 2019). In short, 0.5 g 10-d-old seedlings were fixed in 1% (v/v) formaldehyde at room temperature under a vacuum for 8 min, followed by sonication, resulting in the production of 0.3–1 kb DNA fragments. After four rounds of phenol–chloroform extraction, the purified DNA was eluted in 200 mL Tris-EDTA buffer. Relative enrichment in the FAIRE-treated DNA was calculated with DNA from uncrosslinked DNA samples serving as the control. The enrichment of fragmented genomic DNA was normalized to that of the Ta3 retrotransposon (internal control; Wu et al., 2015). All primer sequences are listed in Supplemental Table S1.

Statistical Analyses

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

Accession Numbers

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

Supplemental Data

The following supplemental materials are available.

Acknowledgments

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

Footnotes

  • Received May 28, 2019.
  • Accepted June 25, 2019.
  • Published July 9, 2019.

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The B3-Domain Transcription Factor VAL1 Regulates the Floral Transition by Repressing FLOWERING LOCUS T
Yanjun Jing, Qiang Guo, Rongcheng Lin
Plant Physiology Sep 2019, 181 (1) 236-248; DOI: 10.1104/pp.19.00642
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