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. 2018 Dec 4;179(2):477–490. doi: 10.1104/pp.18.00865

ABI5-BINDING PROTEIN2 Coordinates CONSTANS to Delay Flowering by Recruiting the Transcriptional Corepressor TPR21

Guanxiao Chang a, Wenjuan Yang b, Qili Zhang b, Jinling Huang c,d, Yongping Yang a,2, Xiangyang Hu a,b,2,3
PMCID: PMC6426417  PMID: 30514725

ABI5-BINDING PROTEIN2 interacts with TPR2 and CONSTANS to delay flowering time

Abstract

ABI5-BINDING PROTEIN2 (AFP2) negatively regulates the abscisic acid signal by accelerating ABI5 degradation during seed germination in Arabidopsis (Arabidopsis thaliana). The abscisic acid signal is reported to delay flowering by up-regulating Flowering Locus C expression, but the role of AFP2 in regulating flowering time is unknown. Here, we found that flowering time was markedly delayed and CONSTANS (CO) expression was reduced in a transgenic Arabidopsis line overexpressing AFP2 under LD conditions. Conversely, the loss-of-function afp2 mutant showed slightly earlier flowering, with higher CO expression. These data suggest that AFP2 negatively regulates photoperiod-dependent flowering time by modulating the CO signal. We then found that AFP2 exhibited circadian expression rhythms that peaked during the night. Furthermore, the C-terminus of AFP2 interacted with CO, while its N-terminal ethylene response factor–associated amphiphilic repression motif interacted with the transcriptional corepressor TOPLESS-related protein2 (TPR2). Thus, AFP2 bridges CO and TPR2 to form the CO-AFP2-TPR2 complex. Biochemical and genetic analyses showed that AFP2 mediated CO degradation during the night. AFP2 also recruited histone deacetylase activity at Flowering Locus T chromatin through its interaction with TPR2. Taken together, our results reveal an elaborate mechanism by which AFP2 modulates flowering time through coordinating the activity and stability of CO.

Flowering is a critical phase in the life cycle of plants and heralds the transition from vegetative to reproductive growth. Plants have evolved complex mechanisms to ensure that flowering occurs at an appropriate time in response to environmental cues (such as photoperiod) and endogenous signals (Andrés and Coupland, 2012; Johansson and Staiger, 2015; Song et al., 2015). In Arabidopsis (Arabidopsis thaliana), environmental photoperiod information is processed by the core factor CONSTANS (CO), which is transcribed at the plant apex or in the leaf vasculature (Putterill et al., 1995; Suárez-López et al., 2001).

In leaves, CO activates the expression of florigen Flowering Locus T (FT). Loss-of-function mutants of FT are late flowering, whereas overexpression of FT promotes flowering (Corbesier et al., 2007; Jaeger and Wigge, 2007; Mathieu et al., 2007). The expression level of CO is controlled by the circadian clock and light signaling pathway (Suárez-López et al., 2001; Valverde et al., 2004; Turck et al., 2008). The CO transcriptional level oscillates rhythmically, peaking in the afternoon in long days but at dusk in short days (Hayama and Coupland, 2003). The Cycling DNA binding with one finger (DOF) Factor (CDF) transcription factors, including CDF1, CDF3, CDF4, and CDF5, synergistically bind to the DOF binding sites in the CO promoter to suppress FT expression in the morning, while the transcription of CDF is regulated by the circadian genes CIRCADIAN CLOCK ASSOCIATED1 and LATEELONGATED HYPOCOTYL in the morning and suppressed by PSEUDO RESPONSE REGULATOR5 (PRR5), PRR7, and PRR9 in the afternoon (Nakamichi et al., 2007; Fornara et al., 2009). The blue light receptor Flavin Binding, Kelch Repeat, F-box acts together with GIGANTEA to degrade CDF under long-day (LD) conditions (Sawa et al., 2007; Song et al., 2014). Once CDF has been removed from the CO promoter, the basic helix-loop-helix transcriptional activators FBH1, FBH2, FBH3, and FBH4 recognize the E-box cis-elements in the CO promoter to activate its transcription (Ito et al., 2012; Nagel et al., 2014).

In addition to regulation at the transcriptional level, CO protein stability is tightly controlled by an elaborate mechanism. CO is degraded by the ring finger E3 ligase CONSTITUTIVE PHOTOMORPHORGENIC1 and its partner SUPPRESSOR OF PHYA1 in the night (Jang et al., 2008; Liu et al., 2008; Zuo et al., 2011), or by another E3 ligase, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1, in the morning (Lazaro et al., 2012; Joon Seo et al., 2013). Recently, Goralogia et al. reported that CDF recruits the transcriptional corepressor TOPLESS (TPL) to reduce CO expression and that two small B-box microProteins named microProtein 1a (miP1a) and miP1b form a bridge between CO and TPL, thereby suppressing the FT signal (Goralogia et al., 2017). These findings suggest that TPL has an essential function in photoperiod-mediated flowering time. Normally, TPL and its homolog TOPLESS-RELATED PROTEINS are associated with the histone deacetylase complex (HDAC) during plant development and immunity (Long et al., 2006; Wang et al., 2013a; Oh et al., 2014; Ryu et al., 2014), and CO is required for periodic modification of the histone acetylation level at FT chromatin through HDAC activity (Gu et al., 2013). Nevertheless, the mechanism by which CO recruits TPL/TPRs to epigenetically modulate FT expression remains largely unknown.

The phytohormone signal also affects flowering time (Davis, 2009; de Wit et al., 2016); for example, mutation of the jasmonic acid signal receptor CORONATINE INSENSITIVE1 promotes flowering, while overexpression of COI-targeted JASMONATE-ZIM-DOMAIN PROTEIN1 delays flowering (Zhai et al., 2015). Further investigation has shown that JASMONATE-ZIM-DOMAIN PROTEIN1 orchestrates the activity of the APETALA2 family members Target of Eat1 (TOE1) and TOE2, which repress the transcription of FT. TOE1 and TOE2 also form a complex with CO in the morning (Zhang et al., 2015) or with FKF1 in the afternoon (Song et al., 2012; Zhang et al., 2015) to suppress CO activity. Gibberellic acid-mediated activation of the FT signal, which results in early flowering, depends on CO, and the GA signal repressor DELLA interacts with CO to suppress its activity (Wang et al., 2016; Xu et al., 2016). The bZIP transcription factor ABSCISIC ACID-INSENSITIVE MUTANT5, which plays an essential role in ABA-inhibited seed germination, also binds to the ABRE/G-box promoter elements of the Flowering Locus C (FLC) promoter to up-regulate FLC expression and delay flowering (Wang et al., 2013b). Arabidopsis ABI5-BINDING PROTEIN1 (AFP1) was first reported as a negative regulator of ABA signaling that promotes ABI5 degradation during seed germination. Overexpression of AFP1 enhances tolerance to ABA and reduces ABI5 accumulation, whereas afp1 mutants are sensitive to ABA and have high levels of ABI5 (Lopez-Molina et al., 2003). There are three AFP1 homologs (i.e. AFP2, AFP3, and AFP4) in the Arabidopsis genome, and their expression is differentially induced by environmental stress (Garcia et al., 2008). AFP2, similar to AFP1, functions epistatically to ABI5, and mutation of AFP2 leads to increased sensitivity to ABA stress (Lopez-Molina et al., 2003). AFP2/3 also interact with the corepressor TPL/TPRs to impair ABA-regulated gene expression (Pauwels et al., 2010; Lynch et al., 2017). We have recently reported that AFP2 interacts with ABI5 to enhance seed germination under high temperature stress (Chang et al., 2018), but other physiological functions mediated by AFPs and their TPL/TPR partner remain to be investigated.

Although it has been established that ABI5 delays flowering by up-regulating FLC (Wang et al., 2013b), it is unknown whether and how AFPs affect flowering time. In this study, we sought to determine the potential role of AFP2 in controlling flowering time. We showed that overexpression of AFP2 (AFP2-ox) substantially delayed flowering under LD conditions by inactivating the CO signal; conversely, the afp2 mutant exhibited early flowering at a higher CO level. Additional biochemical analysis demonstrated that AFP2 bridged CO and TPR2 to form a complex, and AFP2 not only interacted with TPR2 to dampen FT expression through HDAC activity at the transcriptional level but also interacted with CO to promote ubiquitin-mediated proteolysis of CO at the post-transcriptional level. Thus, our findings unravel an elaborate mechanism by which AFP2 recruits the corepressor TPR2 to coordinate the CO signal and thereby ensure that flowering occurs at an appropriate time.

RESULTS

AFP2 and AFP3 Modulate Flowering Time

To investigate the functions of AFP genes in regulating flowering time, we obtained several T-DNA insertion mutants from the Arabidopsis Biological Resource Center, including afp1-1 (Salk-020158), afp1-2 (Salk-005054), afp2-1 (Salk-131676), afp2-2 (Salk-145086), afp3-1 (Salk-037555), afp3-2 (Salk-052114), afp4-1 (GABI-019E07), and afp4-2 (Salk-208284). For each mutant, the T-DNA transposon was inserted into the exons of the corresponding gene (Supplemental Fig. S1, A and B), and the functional transcript was nearly undetectable by RT-qPCR analysis (Supplemental Fig. S1C). We then generated several individual transgenic lines in which the full-length AFPs were fused to a Flag tag and driven by the cauliflower mosaic virus 35S promoter (named AFP1-ox, AFP2-ox, AFP3-ox, and AFP4-ox, respectively; Supplemental Fig. S2). Immunoblot analysis using anti-Flag antibody revealed strong immunoblot bands in these transgenic lines, but not in the Col wild type, confirming their overexpression (Supplemental Fig. S2).

We then compared the flowering times of the different lines under LD conditions (16 h light/8 h darkness). As shown in Figure 1, A and B, and Supplemental Figure S3, the afp4-1 and afp4-2 mutants and AFP4-ox transgenic lines had similar flowering times to the wild type, suggesting that AFP4 does not affect the flowering time (Supplemental Fig. S3). The flowering time of the afp1-1 and afp1-2 mutants was not different from that of the wild type, whereas the afp2-1 (hereafter named afp2), afp2-2, afp3-1, and afp3-2 mutants showed earlier flowering times than the wild type, with the afp1-1/afp2-1/afp3-1 triple mutant flowering even earlier (Supplemental Fig. S3), suggesting a functional redundancy of AFP2 and AFP3 in flowering time regulation. Correspondingly, AFP2-ox or AFP3-ox markedly delayed flowering time, whereas overexpression of AFP1 was only slightly later than that of wild-type Col (Supplemental Fig. S3), suggesting that AFP2 and AFP3 are key regulators of flowering time under LD conditions.

Figure 1.

Figure 1.

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AFP2 regulates flowering time under LD conditions. A, Flowering phenotype of the wild-type (Col), afp2 mutant, and AFP2-ox lines. The photograph was taken 25 d after germination under LD conditions. Bar = 2 cm. B, Flowering time, as indicated by the total rosette leaf number, under LD conditions. Data are means ± sd of three biological replicates; for each line, 20 plants were scored. Bars with different letters are significantly different at P < 0.05 (Tukey’s test). C, RT-qPCR analysis of AFP2 transcriptional levels in the wild-type Col line grown under LD conditions for 14 d. IPP2 was used as an internal control. Data are means ± sd of three biological replicates. White and dark bars above the x axis indicate light and dark periods, respectively.

We also examined their flowering times under short-day (SD) conditions (8 h light/16 h darkness), and both afp2 and AFP2-ox showed a similar late flowering time to the wild-type Col line (Supplemental Fig. S4, A and B). Similarly, the flowering time was not significantly different among the afp3-1, afp1-1/afp2/afp3-1 triple mutant, AFP3-ox transgenic, and Col lines under SD conditions (Supplemental Fig. S4B), indicating that AFP2 and AFP3 mainly delay flowering time through modulating the photoperiodic pathway. We also measured the transcriptional level of AFP2 during the day and night in 2-week-old wild-type seedlings under LD conditions (16L/8D) and found that the transcriptional level of AFP2 presented a circadian rhythm, with a gradual increase during the day and peak during the night at zeitgeber time 20 (ZT20), followed by a rapid decline to a very low level at the next dawn (ZT0; Fig. 1C).

AFP2 Interacts with TPR2

To investigate the role of AFP2 in regulating flowering time, we used AFP2 as the bait to screen an Arabidopsis-normalized cDNA library by a yeast two-hybrid (Y2H) assay. After two rounds of screening, we obtained several positive clones that interacted with AFP2 in yeast. After sequencing these clones, we identified a gene encoding TPR2, a member of the TPL/TPR transcriptional corepressor family. To confirm their interaction, we fused full-length TPR2 into the prey vector (AD-TPR2) and AFP2 into the Gal4 DNA-binding domain of the bait vector (BD-AFP2). As shown in Supplemental Figure S5, A and B, we observed a strong interaction between AFP2 and TPR2 in yeast. As the control, AFP2 did not interact with the empty AD vector in yeast cells.

Bioinformatics analysis using SMART software (http://smart.embl-heidelberg.de/) showed that AFP2 contained three functional motifs: EAR, NINJA, and JAS (Supplemental Fig. S5A). We then expressed a truncated version of AFP2 that lacked the EAR motif (AFP2∆E), NINJA motif (AFP2∆N), or JAS motif (AFP2∆J), respectively, and tested the interaction with TPR2 in yeast. We found that TPR2 interacted with AFP2∆N or AFP2∆J, but not with AFP2∆E (Supplemental Fig. S5, A and B), suggesting that the EAR motif is required for the interaction between AFP2 and TPR2.

Next, we determined the cellular localization of AFP2 and TPR2 by fusing AFP2 with a cyan fluorescence protein (AFP2-CFP) and by fusing TPR2 to a yellow fluorescence protein (TPR2-YFP). The fusions were then transiently expressed in Nicotiana benthamiana leaves. The strong CFP or YFP fluorescence was observed in the nucleus (Fig. 2A). We then examined the interaction between AFP2 and TPR2 in planta via bimolecular fluorescence complementation (BiFC). Full-length AFP2 and AFP2 lacking the EAR, NINJA, or JAS motif (AFP2∆E, AFP2∆N, AFP2∆J) were fused to the N-terminal half of YFP (named AFP2-nYFP, AFP2∆E-nYFP, AFP2∆N-nYFP, and AFP2∆J-nYFP), while full-length TPR2 was fused to the C-terminal half of YFP (cYFP-TPR2). Coexpression of AFP2-nYFP, AFP2∆N-nYFP, or AFP2∆J-nYFP with cYFP-TPR2 caused strong YFP fluorescence, whereas coexpression of AFP2∆E-nYFP with cYFP-TPR2 did not. As a control, we coexpressed empty nYFP with cYFP-TPR2. As expected, this did not yield any YFP fluorescence (Fig. 2B; Supplemental Fig. S5C), suggesting that AFP2, specifically the EAR motif, interacts with TPR2 in planta.

Figure 2.

Figure 2.

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AFP2 interacts with TPR2 in vitro and in vivo. A, Colocalization of AFP2 and TPR2 in the nucleus. AFP2-CFP and TPR2-YFP were transiently cotransformed into N. benthamiana leaf epidermal cells, and the localization of AFP2 and TPR2 were observed based on CFP and YFP fluorescence, respectively. From top to bottom, CFP fluorescence, YFP fluorescence, merged image of CFP and YFP. Bar = 10 μm. B, BiFC analysis of the interaction of AFP2 and TPR2 in planta. Full-length or truncated AFP2 was fused to nYFP (AFP2-nYFP or AFP2∆E-nYFP) and full-length TPR2 was fused to cYFP (cYFP-TPR2), and both constructs were cotransformed into N. benthamiana epidermal cells and YFP fluorescence was monitored. Bar = 10 μm. C and D, Co-IP analysis of the AFP2 and TPR2 interaction. Flag-tagged full-length AFP2 or truncated AFP2 and GFP-tagged TPR2 were cotransformed into N. benthamiana epidermal leaves. TPR2-GFP was immunoprecipitated with GFP-TRAP beads and detected with anti-Flag antibody. TPR2-GFP in the immunoprecipitates was detected using an anti-GFP antibody.

In agreement with this finding, the co-immunoprecipitation (Co-IP) results showed that full-length AFP2, or the truncated AFP2 without the NINJA or JAS motif (AFP2∆N or AFP2∆J) fused to a Flag tag, could be coimmunoprecipitated by anti-GFP resin when coexpressed with TPR2 fused to GFP (TPR2-GFP), while no Co-IP signal could be detected when AFP2∆E fused to Flag tag was coexpressed with TPR2-GFP (Fig. 2, C and D). These data indicate that the EAR motif of AFP2 interacts with TPR2 in living cells.

AFP2 Depends on TPR2 To Delay Flowering Time

Given that AFP2 interacts with TPR2 in vitro and in vivo and that AFP2-ox delays flowering time, we next evaluated the role of TPR2 in flowering time regulation. We identified two T-DNA insertion mutants, tpr2-1 (Salk_112730) and tpr2-2 (Salk_079848), in which the T-DNA insertions were located in the 13th and 20th exons, respectively (Supplemental Fig. S6A). The corresponding upstream or downstream primers for the 13th (F1/R1 primers pair) exon and the 20th (F2/R2 primers pair) exon could successfully amplify the correct bands using wild-type Col DNA as template, whereas they failed to amplify the bands with mutant DNA templates. In contrast, the primer pair of LB/R1 and LB/R2 successfully amplified the mutant band from the Salk_112730 and Salk_079848 lines, respectively. LB was a primer located on T-DNA (Supplemental Fig. S6B). RT-qPCR analysis also showed that the transcript of TPR2 was significantly reduced in these two mutants (Supplemental Fig. S6C), confirming that tpr2-1 (hereafter named tpr2) and tpr2-2 were loss-of-function mutants. The flowering time of tpr2 or tpr2-2 was substantially earlier than that of the wild-type Col line under LD conditions (Fig. 3, A and B; Supplemental Fig. S6, D and E).

Figure 3.

Figure 3.

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The tpr2 mutation reduces flowering time in the later-flowering AFP2-ox background. A, The flowering phenotype of wild type (Col), tpr2, AFP2-ox, and AFP2-ox/tpr2. Bar = 2.5 cm. The photos were taken at 18 d after seeds germination. B, The different flowering times and FT transcriptional levels in Col, tpr2, AFP2-ox, and AFP2-ox/tpr2. The flowering times of these lines are indicated by the total rosette leaf number under LD conditions. The FT transcriptional level was measured by RT-qPCR analysis in 10-d-old seedlings under LD conditions. Data are the means ± sd of three biological replicates. For each line, 20 plants per line were observed. Bars with different letters are significantly different at P < 0.05 (Tukey’s test). C, RT-qPCR analysis of CO, FT, and SOC1 expression in 10-d-old wild-type, afp2, and AFP2-ox lines under LD conditions, at a 4-h resolution. Actin was used as an internal control. Data are means ± se of three biological replicates. Bars with different letters are significantly different at P < 0.05 (two-way ANOVA with Tukey’s test).

To investigate the function of TPR2, we expressed TPR2 in Escherichia coli and used the purified TPR2 as antigen to prepare an antibody against TPR2. As shown in Supplemental Figure S7A, our TPR2 antibody specifically recognized endogenous TPR2 in wild-type Col, but not in the tpr2 mutant, demonstrating the specificity of our TPR2 antibody. We then crossed the early flowering tpr2 mutant with the late flowering AFP2-ox line to obtain AFP2-ox/tpr2 and confirmed the genotype of AFP2-ox/tpr2 by immunoblot analysis (Supplemental Fig. S7B). In contrast to the late flowering phenotype of AFP2-ox, knockout of TPR2 in the AFP2-ox/tpr2 line markedly promoted flowering (Fig. 3, A and B), suggesting that TPR2 is required for the late flowering phenotype of the AFP2-ox line.

AFP2 Represses the CO-Mediated Early Flowering Phenotype

Because the flowering time of AFP2-ox was late under LD conditions but could not be distinguished from wild-type Col under SD conditions (Supplemental Fig. S4, A and B), we hypothesized that the AFP2-regulated flowering time was dependent on the photoperiodic pathway. We thus compared the transcriptional level of CO and its downstream targets FT and SOC1 in the Col wild-type, afp2, and AFP2-ox lines under LD conditions. We observed the circadian expression of CO in Col under LD conditions (Fig. 3C). CO also presented similar circadian expression in the afp2 and AFP2-ox lines, but the CO circadian altitude was slightly higher in afp2 and strikingly lower in AFP2-ox (Fig. 3C). Consistent with these findings, FT and SOC1 expression were clearly reduced in the AFP2-ox line but higher in afp2 than in the wild type. These data suggest that AFP2 represses the transcription of CO and its downstream targets FT and SOC1 to delay flowering under LD conditions. Because both tpr2 and AFP2-ox/tpr2 presented early flowering but AFP2-ox showed a late flowering time, we also compared the FT expression patterns among AFP2-ox, tpr2, AFP2-ox/tpr2, and wild-type Col as the control. The expression levels of FT at ZT16 in tpr2 or AFP2-ox/tpr2 were significantly higher than in AFP2-ox and Col (Fig. 3B, bottom panel), suggesting that the inhibitory effect of AFP2 on FT expression required TPR2.

TPL interacts with CO and microprotein miP1a/1b as a trimer complex to regulate flowering time (Graeff et al., 2016), and our above result suggested that AFP2 interacts with TPR2. It is possible that AFP2 interacts with CO to delay flowering time. To test this possibility, we examined whether there was an interaction between CO protein lacking activation domains (deleted N-terminal amino acids 1–175, termed “CO-CT”) and AFP2 using Y2H analysis. As shown in Figure 4A, we found that CO-CT interacted with the full-length AFP2, as well as with the truncated AFP2 lacking the EAR or NINJA domain, but not with the truncated AFP2 lacking the JAS domain, suggesting that the JAS domain in AFP2 is required for the interaction between AFP2 and CO. Consistent with this finding, a Co-IP assay using transiently transformed tobacco leaves revealed the strong interaction between CO and full-length AFP2, or truncated AFP2 lacking the EAR or NINJA motif, but not between CO and truncated AFP2 lacking the JAS motif (Fig. 4B), demonstrating that the JAS motif is required for the interaction between AFP2 and CO in vitro and in vivo.

Figure 4.

Figure 4.

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AFP2 influences the ability of CO to activate FT expression. A, AFP2 interacts with CO-CT in yeast. Full-length or truncated AFP2 was fused to GAL4-AD, and CO-CT, in which the N-terminal 1-175 amino acid of CO was deleted, was fused to GAL4-BD, and both constructs were transformed into yeast cells. The interaction of AFP2 or truncated AFP2 with CO-CT was assessed on selective media lacking Leu, Trp, His, or Ade. Gal4-BD was used as negative controls. Photographs were obtained 3 d after incubation at 28°C. B, Co-IP analysis of the interaction between AFP2 and CO. Flag-fused AFP2 and GFP-fused CO were transiently cotransformed into N. benthamiana leaves, and CO-GFP was immunoprecipitated with GFP-TRAP and detected with anti-Flag antibody. The protein input was detected with anti-GFP and anti-Flag, respectively. C, The flowering phenotype of Col, AFP2-ox, CO-HA, and AFP2-ox/CO-HA. Bar = 3 cm. The photos were taken at 18 d after seeds germination. D, Flowering times of Col, AFP2-ox, CO-HA, and AFP2-ox/CO-HA based on the rosette leaf number under LD conditions. Data are the means ± sd of three biological replicates. For each line, 20 plants were observed. Bars with different letters are significantly different at P < 0.05 (Tukey’s test). E and F, Circadian accumulation of CO-HA in the CO-HA and AFP2-ox/CO-HA lines. The CO-HA and AFP2-ox/CO-HA lines were grown under LD conditions for 2 weeks, and samples were collected every 4 h from ZT0. Total proteins were extracted to detect CO-HA accumulation using anti-HA antibody. Antitubulin was used as the loading control. The intensity ratio of the CO-HA signal to that of tubulin is shown in (F). Data are the means ± sd of three biological replicates. G, Flowering times of CO-HA, CO-HA/AFP2-ox, cop1, and CO-HA/AFP2-ox/cop1 indicated by the total rosette leaf number under LD conditions. Data are the means ± sd of three biological replicates. For each line, 20 plants were observed. Bars with different letters are significantly different at P < 0.05 (Tukey’s test).

ABI5 was previously reported as the target protein of AFP2 during seed germination (Lopez-Molina et al., 2003), and overexpression of ABI5 activates the flowering negative factor FLC at the transcriptional level to repress flowering (Wang et al., 2013b). Thus, we compared the transcription of FLC in Col, afp2, and AFP2-ox under LD conditions. We did not observe any obvious differences among the FLC transcript levels in these lines (Supplemental Fig. S8); this suggests that AFP2-mediated late flowering occurs independently of FLC.

Furthermore, we measured the effects of AFP2 on CO activity and flowering time by genetic analysis. The SUC2::CO-HA line (expressing CO fused to an HA tag and driven by the SUC2 promoter, hereafter termed CO-HA) showed earlier flowering than the wild-type Col line. We crossed the CO-HA line with AFP2-ox to generate the CO-HA/AFP2-ox line and confirmed these lines by immunoblot analysis using anti-HA and anti-GFP antibodies (Supplemental Fig. S9). In comparison to the early flowering CO-HA line, the flowering time of the CO-HA/AFP2-ox lines was markedly later, similar to that of AFP2-ox (Fig. 4, C and D). In agreement with this finding, the FT transcriptional level in CO-HA/AFP2-ox was also lower than that of CO-HA (Supplemental Fig. S10A). The co mutant showing a later flowering time was crossed with the earlier flowering afp2 mutant to obtain the afp2/co line. Similar to the later flowering co mutant, the afp2/co line presented a late flowering phenotype (Supplemental Fig. S10B). These data suggest that CO is epistatic to AFP2, and AFP2 represses the early flowering phenotype of CO-HA by negatively regulating the CO signal.

AFP2 has been reported to accelerate ABI5 degradation (Lopez-Molina et al., 2003), and AFP2-ox suppressed the activating activity of CO in CO-HA/AFP2-ox lines, suggesting that AFP2 not only suppresses the transcription of CO (Fig. 3C), but also possibly affects the stability of the CO protein itself. To test this possibility, we compared CO protein accumulation dynamics between the CO-HA and CO-HA/AFP2-ox lines during the day and night under LD conditions. As shown in Figure 4, E and F, the protein accumulation level of CO in both CO-HA and CO-HA/AFP2-ox oscillated with the same rhythm, peaking at ZT16, but the CO protein content during the evening and night, especially at ZT16 and ZT20, was higher in the transgenic CO-HA line than in the crossed CO-HA/AFP2-ox line, supporting the notion that AFP2 promotes the degradation of CO during the night.

We then found that AFP2-induced CO degradation was partially inhibited by pretreatment with the proteasome inhibitor MG132 (Supplemental Fig. S11), suggesting that CO degradation potentially depends on the ubiquitin proteasome system pathway. A previous study has shown that COP1 interacts with AFP2 in the nucleus (Lopez-Molina et al., 2003), and COP1 as the E3 ligase mediates the degradation of CO during the night (Jang et al., 2008). It is possible that AFP2-promoted degradation of CO requires COP1, and therefore we crossed the cop1-4 (termed cop1 hereafter) mutant with CO-HA/AFP2-ox to generate the CO-HA/AFP2-ox/cop1 line. Similar to the earlier flowering CO-HA, the flowering time of the CO-HA/AFP2-ox/cop1 line was earlier than that of CO-HA/AFP2-ox under LD conditions (Fig. 4E). Consistent with this finding, the CO protein content at ZT16 and ZT20 was higher in CO-HA/AFP2-ox/cop1 than in CO-HA/AFP2-ox (Supplemental Fig. S12). These findings indicate that AFP2-dependent CO degradation and the late flowering phenotype require COP1.

AFP2 Recruits TPR2 To Suppress CO Expression and Delay Flowering

Microproteins miP1a/1b bridge TPL and CO to form the complex (Graeff et al., 2016). Because AFP2 interacts with CO or TPR2, it is possible that AFP2 bridges TPR2 and CO to form the CO-AFP2-TPR2 complex. To test this possibility, we conducted a yeast three-hybrid (Y3H) analysis to examine the interactions among CO, AFP2, and TPR2. Cotransformation of empty pDR plasmid with AD-CO and BD-TPL did not enable growth on selection medium, but in the presence of the AFP2 plasmid, the cotransformed yeast grew well on selective medium (Fig. 5A). Yeast cotransformed with truncated AFP2 lacking either EAR or JAS did not grow well, supporting the idea that full-length AFP2 is required to reinforce the interaction between CO and TPR2 (Fig. 5A).

Figure 5.

Figure 5.

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AFP2 reinforces CO and TPR2 to coordinate flowering time. A, Y3H analysis to detect the formation of the CO-AFP2-TPR2 complex. Yeast cotransformed with these three constructs could grow well on the nonselective medium lacking Leu, Trp, and uracil (−L/−W/−U), but only yeast harboring constructs that had positive interactions were able to grow on restrictive growth medium supplemented with 10 mm 3-aminotriazole plus 2% (w/v) Gal and lacking His/Leu/Trp/Ura (−H/−L/−W/−U). B, In vitro pull-down analysis of the interactions among CO, AFP2, and TPR2. Recombinant GST-TPR2 and MBP-CO proteins were produced in E. coli. After cell lysis, cell extracts of GST-TPR2 and MBP-CO were mixed with HIS-AFP2, HIS-AFP∆E, or HIS-AFP2∆J, respectively, and then incubated with magnetic anti-His-coupled magnetic beads. His-tagged full-length or truncated AFP2 was precipitated and washed using a magnetic stand, eluted by boiling in SDS loading buffer, and separated by SDS-PAGE. GST-TPR2 and MBP-CO were detected by immunoblotting. C, Co-IP analysis of the CO-AFP2-TPR2 complex in vivo. The CO-HA/afp2 transgenic line was crossed with AFP-ox/afp2 to obtain CO-HA/AFP2-ox/afp2, which was subjected to Co-IP analysis. Total proteins were extracted from CO-HA/AFP2-ox/afp2 and immunoprecipitated with anti-Flag agarose beads, and the immunoprecipitated proteins were detected with anti-TPR2 antibody. D, Flowering phenotype of the afp2 mutant and the indicated transgenic lines in the afp2 mutant background. The photos were taken at 18 d after seeds germination. Bar = 3 cm. E, Flowering times based on the total rosette leaf number under LD conditions. Data are means ± sd of three biological replicates. For each line, 20 plants were observed. Bars with different letters are significantly different at P < 0.05 (Tukey’s test). F, RT-qPCR analysis of CO transcript levels in the afp2 mutant and different transgenic lines in the afp2 background. IPP2 was used as an internal control. Data are means ± sd of three biological replicates. Bars with different letters are significantly different at P < 0.05 (Tukey’s test). 3AT, 3-aminotriazole.

The presence of the CO-AFP2-TPR2 complex was further supported by an in vitro pull-down assay. As shown in Figure 5B, MBP-CO and GST-TPR2 did not coprecipitate when incubated in the absence of HIS-AFP2, but they did coprecipitate in the presence of HIS-AFP2, suggesting formation of the CO-AFP2-TPR2 complex. We also tested whether CO and TPR2 could interact with each other in the presence of truncated AFP2 and found that deletion of the EAR or JAS domain blocked the interaction between CO and TPR2 (Fig. 5B). These findings suggest that these two domains are required for the formation of the CO-AFP2-TPR2 complex in vitro.

To test whether the CO-AFP2-TPR2 complex exists in vivo, we first generated the AFP2-ox/afp2 (overexpressing full-length AFP2-ox in the afp2 mutant background), AFP2∆E-ox/afp2 (overexpressing AFP2∆EN-ox in the afp2 mutant background), and AFP2∆J-ox/afp2 (overexpressing AFP2∆J-ox in the afp2 mutant background) lines, and then crossed them to obtain AFP2-ox/CO-HA/afp2, AFP2∆E-ox/CO-HA/afp2, AFP2∆N-ox/CO-HA/afp2, and AFP2∆J-ox/CO-HA/afp2. Using the TPR2 antibody, we found that CO-HA coprecipitated with TPR2 in the crossed AFP2-ox/CO-HA/afp2 line, but not in the crossed AFP2∆E-ox/CO-HA/afp2 or AFP2∆E-ox/CO-HA/afp2 line (Fig. 5C). These data suggest that full-length AFP2 reinforces the interaction between CO and TPR2 in vivo through the EAR and JAS domains.

We further evaluated the function of the CO-AFP2-TPR2 complex in modulating flowering time. Both afp2 and CO-HA/afp2 flowered early, while AFP2-ox/afp2 flowered late. The AFP2-ox/afp2 and CO-HA/AFP2-ox/afp2 lines showed later flowering than the afp2 mutant (Fig. 5, D and E), suggesting that AFP2 overexpression suppresses the early flowering of CO-HA. However, CO-HA/AFP2∆E-ox/afp2 or CO-HA/AFP2∆J-ox/afp2 flowered early, similar to CO-HA/afp2, but CO-HA/AFP2∆N-ox/afp2 showed a similar flowering time to CO-HA/AFP2-ox/afp2 (Fig. 5, D and E), suggesting that the EAR and JAS domains are required for AFP2-mediated inhibition of CO activity. In agreement with this notion, FT transcript levels were higher in CO-HA/afp2, CO-HA/AFP2∆E/afp2, and CO-HA/AFP2∆J/afp2 and lower in CO-HA/AFP2-ox/afp2 and CO-HA/AFP2∆N/afp2 (Fig. 5F).

AFP2 and TPR2 Suppress CO Transcriptional Activity By Reducing the Chromatin Acetylation Level at the FT Locus

Having shown that AFP2 reinforces the interaction between CO and TPR2, we next investigated the effects of AFP2 and TPR2 on the transcriptional activity of CO using an Arabidopsis mesophyll protoplast analysis. Transient transformation of CO-HA/afp2 protoplasts with the FT::LUC reporter construct resulted in high bioluminescence emission with a high Luc/Ren ratio, because CO-HA activates FT transcription (Fig. 6, A and B). The CO-HA-induced increase in the Luc/Ren ratio could be limited by cotransformation with full-length AFP2-ox or AFP2∆N-ox, but not by AFP2∆E-ox or AFP2∆J-ox, suggesting that AFP2, mainly the EAR and JAS domains, suppresses the transcriptional activation effect of CO on FT expression. Cotransformation of AFP2-ox or AFP2∆N-ox with TPR2-ox further suppressed the bioluminescence and Luc/Ren ratio, but cotransformation of AFP2∆E-ox or AFP2∆J-ox with TPR2-ox did not suppress the CO-induced increase of bioluminescence, suggesting that functional AFP2 is required for the corepressive activity of TPR2 on the transcriptional activation activity of CO on FT.

Figure 6.

Figure 6.

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AFP2 and TPR2 repress the transcriptional activation activity of CO on FT. A, Schematic of the reporter and effectors used in the transient protoplast transformation assay. B, Transient dual-luciferase reporter analysis of the inhibitory effect of AFP2 and TPR2 on CO-induced FT expression. Error bars indicate sd from three biological replicates. C, ChIP-qPCR analysis of the histone H3 acetylation levels in the indicated region (denoted as FT-P, FT-E, and FT-L locus) of FT chromatin in Col, CO-HA, AFP2-ox, and the various crossed lines. An antiacetyl-histone H3 antibody was used for immunoprecipitation. TUB2 was used as an internal control, and the relative histone H3 acetylation levels of FT were normalized to those of TUB2. The detailed position of FT-P, FT-E, and FT-L are indicated by bars below the FT gene. Exons are shown as solid boxes and introns as solid lines. Data are means ± sd of triplicate experiments. For (B) and (C), bars with different letters are significantly different at P < 0.05 (Tukey’s test).

Members of the TPL/TPR family recruit HDAC to suppress target gene transcription through deacetylation (Long et al., 2006; Zhu et al., 2010; Oh et al., 2014; Ryu et al., 2014). Our above results showed that TPR2 suppressed CO-dependent transcription, possibly by recruiting HDAC to FT chromatin and reducing histone H3 acetylation at this region. Thus, we examined the histone acetylation level at the promoter region of FT chromatin. The histone H3 acetylation level in the proximal region of the FT promoter in the CO-HA/AFP2-ox and CO-HA/AFP2∆N-ox lines was lower than that in the control wild-type Col line, as well as in the CO-HA/AFP2∆E-ox and CO-HA/AFP2∆J-ox lines, in agreement with the lower FT transcriptional level and late flowering time of the CO-HA/AFP2-ox and CO-HA/AFP2∆N-ox lines (Fig. 6C). The histone H3 acetylation level in CO-HA/AFP2-ox/tpr2 was higher than that in CO-HA/AFP2-ox, suggesting that the HDAC activity-mediated low H3 acetylation level in CO-HA/AFP2-ox requires TPR2. Thus, these data indicate that the AFP2-CO-TPR2 complex suppresses flowering time through HDAC-mediated reduction of the H3 acetylation level at the FT promoter.

DISCUSSION

AFP2 Delays Flowering Time via CO

In this study, we report that AFP2 is a regulator of flowering time. By comparing the flowering times among various afp mutants and AFP overexpression lines, we determined that AFP2 and AFP3 mainly affect flowering time, as the flowering time of afp1 was only slightly earlier, but knocking out or overexpressing AFP4 did not markedly affect flowering time (Fig. 1; Supplemental Figs. S3 and S4). These phenotypes suggest that the different AFP isoforms have evolved diverse functions to differentially regulate Arabidopsis growth and development, or the response to environmental stress. For example, only AFP1 and AFP2 were substantially induced by salt and ABA stress (Garcia et al., 2008). A previous study has shown that AFP1 promotes the degradation of ABI5 during seed germination and that ABI5 binds to the FLC promoter to up-regulate FLC expression and thereby delay flowering (Wang et al., 2013a, 2013b). However, here we found that FLC transcript levels in AFP2-ox were similar to those in the wild type (Supplemental Fig. S8), which excludes the possibility that AFP2 delays flowering through an ABI5-dependent FLC signal.

Furthermore, the delaying effect on flowering time in the AFP2-ox line was obvious under LD but not SD conditions, suggesting that the later flowering phenotype of the AFP2-ox lines was dependent on the photoperiod time. In support of this proposal, our Y2H, in vitro pull-down, and in vivo Co-IP experiments confirmed the interaction between CO and AFP2 (Supplemental Fig. S5); AFP2-ox also reduced the expression level of CO and of its downstream targets FT and SOC1, while the afp2 mutant showed slightly higher transcription of CO, FT, and SOC1 (Fig. 3C). These results support the notion that AFP2 interacts with CO to repress CO activity and the expression of FT and SOC1, ultimately delaying flowering time.

Arabidopsis AFP1 has been reported to colocalize with the E3 ligase COP1 in the nucleus (Lopez-Molina et al., 2003), and COP1 induces proteasome-mediated CO degradation in the dark (Jang et al., 2008). OsbZIP46 is the rice (Oryza sativa) homolog of Arabidopsis ABI5, while OsMODD is the rice homolog of Arabidopsis AFP2. The transcriptional activity of OsbZIP46 is suppressed by OsMODD by recruiting HADC to OsbZIP46 chromatin to reduce its acetylation level. The protein stability of OsbZIP46 is also regulated by OsMODD through recruitment of the U-box-type ubiquitin E3 ligase OsPUB7 (Tang et al., 2016). Consistent with this finding, we detected a lower CO protein level in the CO-HA/AFP2-ox line than in the CO-HA line during the night (Fig. 4E), and the flowering time of the CO-HA/AFP2-ox line was later than that of the CO-HA line (Fig. 4, C and D). It is possible that AFP2 recruits the E3 ligase COP1 and thereby destabilizes CO to delay flowering. The progeny of CO-HA/AFP2-ox/cop1-4 flowered earlier than CO-HA/AFP2-ox (Fig. 4E), and the CO protein content at ZT20 was higher in CO-HA/AFP2-ox/cop1-4 than in CO-HA/AFP2-ox (Supplemental Fig. S12), supporting the notion that COP1 is required for AFP2-mediated CO protein stability during the night. Thus, the detailed mechanism by which AFP2 combines with COP1 to trigger CO degradation or CO stabilization merits further investigation.

AFP2 Recruits TPR2 To Suppress CO Activity, Resulting in Delayed Flowering

The EAR motif characterized by the consensus sequence of LxLxL or DLNxxP is a principal transcriptional repression motif in plants (Causier et al., 2012a, 2012b). Several studies have demonstrated that EAR motif-containing proteins interact with the corepressor TPL/TPR, which in turn recruits histone deacetylases (HDAC), including HDA6 or HDA19, to promote chromatin compaction and transcriptional inactivation (Long et al., 2006; Zhu et al., 2010; Tao et al., 2013; Oh et al., 2014; Ryu et al., 2014). The interactions of AFP2 and AFP3 with TPL was reported previously. Y2H analysis showed that the EAR motif of AFP2 and AFP3 is required for the observed interaction between AFPs and TPL (Pauwels et al., 2010; Causier et al., 2012a). AFP2 also interacts with all four TPR proteins, but AFP3 interacts with all except TPR4. However, unlike AFP2 and AFP3, AFP1 only shows weak interaction with TPL or TPR1/2/3 (Lynch et al., 2017). In agreement with these earlier findings, we found that AFP2 interacts with TPR2 in yeast and in planta, mainly through the EAR motif (Supplemental Fig. S5, A and B; Fig. 2, C and D).

Furthermore, we found that the late flowering phenotype of AFP2-ox could be reversed by the TPR2 mutation (Fig. 3, A and B). Transient protoplast transformation experiments showed that AFP2-ox inhibited CO-mediated FT transcription, while overexpression of TPR2 enhanced the inhibitory effect of AFP2 on FT transcription (Fig. 6, A and B). The acetylation level at the FT locus of CO-HA/AFP2-ox was lower than in CO-HA (Fig. 6C), in agreement with the lower FT transcript levels in CO-HA/AFP2-ox (Fig. 6B). Thus, AFP2 may recruit the TPR2-HADC complex to increase deacetylation levels at the FT locus and thereby delay flowering. This result supports the critical role of TPR2 in controlling histone acetylation levels at FT chromatin through the AFP2-CO complex. Gu et al. reported that S​A​P30 F​U​N​C​T​I​O​N-R​E​L​A​T​E​D1 (AFR1) and AFR2 are the components of HDAC complexes that modulate the periodic histone acetylation level of FT chromatin (Gu et al., 2013). It remains to be determined whether TPR2 recruits AFR1/2 to delay flowering in the AFP2-ox line.

AFP2 Reinforces CO and TPR2 To Form a Complex that Represses the FT Signal

Although TPL suppresses CO activity, TPL does not interact directly with CO. Previous studies have shown that a microprotein called miP1a/b can bridge CO and TPL and recruit TPL, which suppresses CO activity (Graeff et al., 2016). In this study, AFP2 was found to interact with CO through the C terminus JAS motif (Fig. 4, A and B), as well as with TPR2 through the EAR motifs (Fig. 2; Supplemental Fig. S5); however, we did not detect a direct interaction between CO and TPR2 (Fig. 5A). Similar to miP1a/b, Y3H analysis showed that AFP2 mediated the interaction between CO and TPR2. Furthermore, pull-down and Co-IP experiments showed that both the EAR and JAS motifs were required for the formation of the CO-AFP2-TPR2 complex (Fig. 5, B and C). Consistently, the flowering time of the CO-HA/AFP2-ox/afp2 or CO-HA/AFP2∆N-ox/afp2 line was later than that of the CO-HA/afp2, CO-HA/AFP2∆E-ox/afp2, or CO-HA/AFP2∆J-ox/afp2 line (Fig. 5, D and E), but the flowering time of the AFP2-ox/tpr2 line was earlier than that of the AFP2-ox line (Fig. 3A).

CO activity has been reported to be required for AFR2 recruitment to FT chromatin, thereby dampening FT expression by reducing histone acetylation levels at the FT locus (Gu et al., 2013). Here, we found that the CO-AFP2-TPR2 complex recruited HDAC activity to reduce the histone acetylation level at the FT promoter, which led to lower FT expression and a delayed flowering time in the CO-HA/AFP2-ox line (Fig. 5F). Conversely, histone acetylation was restored to normal levels in the CO-HA/AFP2-ox/tpr2 line (Fig. 6C), confirming that AFP2 delays flowering through TPR2-recruited HDAC activity and that AFP2 mediates the indirect interaction between CO and TPR2 and subsequently suppresses CO-dependent activation of FT expression.

Consistent with our findings, a previous study has shown that HDAC activity participates in the AFP2-mediated inhibition of ABA-responsive gene expression and ABA-induced seed dormancy (Lopez-Molina et al., 2003; Wang et al., 2013a; Tang et al., 2016; Lynch et al., 2017). AFP2 may not be the sole component linking CO and TPL/TPR. For example, TPL interacts with TOE1/TOE2, overexpression of TOE1/TOE2 reduces FT expression and delays flowering, and TPL/TPR is required for TOE1/2-mediated repression of FT expression (Causier et al., 2012a). TOE1/TOE2 also interact with CO to induce its degradation at the posttranslational level (Zhang et al., 2015); thus, similar to AFP2, it is possible that TOE1/2 bridges the interaction between CO and TPL to repress FT expression. Further studies should investigate how AFP2 and TOE1/2 coordinate to recruit TPL/TPR and tightly regulate the FT signal and flowering time. Similarly, although CO activity is required for AGL18- or AFR1/2-mediated periodic histone deacetylation at FT chromatin (Gu et al., 2013), CO did not interact directly with AGL18 and AFR1/2. Thus, whether or how AFP2 integrates CO and AFR1/2 for FT expression requires further investigation.

In conclusion, our findings demonstrate that AFP2 controls flowering time by recruiting the corepressor TPR2 to silence CO activity and the downstream FT signal. Based on our data, we propose a working model to illustrate the regulatory mechanism by which AFP2 fine-tunes CO activity and flowering time (Fig. 7). In the early morning, there is insufficient CO to efficiently activate FT transcription. CO protein levels gradually accumulate and peak in the afternoon, at which point CO efficiently activates FT transcription. Some other protein, such as FKF, also stabilizes CO during this period. However, upon arrival of the evening or night, more AFP2 accumulates and interacts with CO. On the one hand, AFP2 induces protein degradation of CO, possibly coordinating with COP1; on the other, AFP2 recruits TPR2 and HDAC to reduce the acetylation level at the promoter of the FT locus, subsequently suppressing FT transcription during the evening or night. Therefore, the CO-AFP2-TPR module is a major regulator that fine-tunes photoperiodic flowering time by temporally modulating histone deacetylation at a region of chromatin underlying FT expression.

Figure 7.

Figure 7.

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Proposed model to illustrate the role of AFP2 in regulating the circadian transcription of FT. In this model, AFP2 restrains CO-induced FT transcription by degrading CO and recruits TPR2 to remove histone acetylation at the FT chromatin, leading to lower FT transcript levels during the evening hours.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

All the Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants, including afp1-1 (Salk-020158), afp1-2 (Salk-005054), afp2-1 (Salk-131676), afp2-2 (Salk-145086), afp3-1 (Salk-037555), afp3-2 (Salk-052114), afp4-1(GK-019E07), afp4-2 (Salk-208284), TPR2 (Salk_112730), and TPR2-2 (Salk_079848), were obtained from the Arabidopsis Biological Resource Center. The SUC2::CO-HA seeds were kindly provided by Dr. Brigitte Koop and Prof. George Coupland (Max Planck Institute for Plant Breeding Research) and by Prof. Takato Imaizumi (University of WA, Seattle). The plants were grown under a white light photoperiod at 22°C under LD (16-h light [100 μmol−2 s−1]/8-h darkness) or SD (8-h light [100 μmol−2 s−1]/16-h darkness) conditions. Flowering time was determined by counting the number of rosette leaves at the time when the inflorescence had grown to a height of 3–5 cm.

Generation of Transgenic Plants

To generate the transgenic lines overexpressing full-length or truncated AFP2, the full-length CDS sequence or truncated versions of AFP2 lacking the EAR, NINJA, or JAS domains were amplified using the primers listed in Supplemental Table S1. The amplified fragments were cloned into the SalI/BamHI (New England Biolabs) sites of the pRI101-6Flag vector [6xFlag tag inserted in the EcoRI/SacI site of the pRI101-AN vector (3262; Takara)] using the In-fusion HD Cloning Kit (638911, Clontech), upstream of the 6xFlag and driven by the 35S constitutive promoter. The constructs were named pRI101-AFP2-6Flag, pRI101-AFP2∆E-6Flag, pRI101∆N-AFP2-6Flag, and pRI101-AFP2∆J-6Flag, respectively. Similarly, the full-length sequences of AFP1, AFP3, and AFP4 were amplified and cloned into pRI101-6Flag to obtain pRI101-AFP1-6Flag, pRI101-AFP3-6Flag, and pRI101-AFP4-6Flag, and full-length TPR2 was cloned into the SalI/BamHI sites of the pRI101-GFP vector (GFP tag inserted in the NdeI/SalI site of the pRI101-AN vector [3262; Takara]) to obtain the pRI101-GFP-TPR2 constructs. These constructs were transformed into Arabidopsis plants using the Agrobacterium tumefaciens-mediated floral dip method (Clough and Bent, 1998).

Y2H and Y3H Assays

To generate full-length AFP2 and TPR2, or truncated TPR2, the primers listed in Supplemental Table S1 were used to amplify the coding sequences from Arabidopsis cDNA template. These PCR products were then cloned into the prey vector pGADT7 (630442, Clontech) and bait vector pGBKT7 (630443, Clontech) using In-fusion Cloning Technology (Clontech). Yeast strains Y187 and AH109 were transformed with the prey and bait vectors, respectively, using polyethylene glycol-mediated yeast transformation as described in Hu et al. (2014). After screening on −Trp medium (630413, Clontech) or −Leu medium (630414, Clontech), three independent clones were mated and grown on −Trp/−Leu medium (630417, Clontech) for 3 d to confirm mating success, and the corresponding clones were transferred to −TPR/−Leu/−His/−Ade medium (630428, Clontech) to measure their growth status.

For the Y3H assay, the TPR2 coding sequence was recombined into a pGBKT7 vector (pGBKT7-TPR2) and CO into the pGADT7 vector (pGADT7-CO). The full-length or truncated AFP2 were cloned into the pYES2 vector (Invitrogen). Yeast strains Y187 and AH109 were cotransformed with pGBKT7-TPR2 and the empty pYES2 or pYES2 with full-length (or truncated) AFP2 as described above, and positive clones were screened on −Trp/−Ura medium (630427, Clontech). pGADT7-CO was transformed into the yeast strain AH109 and selected on −Leu medium. The selected yeast strains Y187 and AH109 were then mated for 2 d at 28°C and then selected on −Trp/−Leu/−Ura medium (630426, Clontech). The interaction of the TPR2-AFP2-CO complex was determined by screening the mated yeast cell on −Trp/−Leu/−Ura/−His selective medium (630425, Clontech) supplemented with 2% (w/v) Gal and 10 mm 3-aminotriazole.

BiFC Analysis

BiFC experiments were performed using 3-week-old Nicotiana benthamiana plants according to a method reported in both Lee et al. (2008) and Hu et al. (2014). The full-length AFP2 and TPR2 sequences and truncated AFP2 sequence were recombined into the binary BIFC vector pSPYNE or pSPYCE, respectively (Walter et al., 2004), so that AFP2 was fused to the N-terminal vector and TPR2 was fused to the C-terminal vector. The constructs were transformed into A. tumefaciens–competent LBA4404 (9115, Clontech), and the cultures were incubated in a rotator at 300 rpm for 6 h at 28°C. After culture, Agrobacteria harboring nYFP or cYFP were mixed together and centrifuged at 300 rpm for 5 min at 4°C, and the pellets were dissolved in injection solution (10 mm Tri-HCl buffer, 25 mm MgCl2, pH 5.6) to an OD600 of 0.1. Then, the Agrobacteria solution was injected into N. benthamiana leaves, and YFP fluorescence was observed 3 d after injection using a confocal microscope (LSM710; Zeiss).

In Vitro Pull-Down Analysis

In vitro pull-down analysis was carried out as described in Hu et al. (2014). In brief, full-length AFP2 or the truncated AFP2 coding sequence was cloned into the EcoRI/XhoI sites of pET28a to generate the HIS-AFP2, HIS-AFP∆E, HIS-AFP2∆N, and HIS-AFP2∆J constructs. The full-length coding region of CO was cloned into the EcoR1/XhoI site of pMAL-C2 to generate MBP-CO, and the full-length coding region of TPR2 was cloned into pGEX-4T-1 to generate GST-TPR2. All the constructs were manipulated using the In-Fusion Cloning technique (Clontech), and primer information is provided in Supplemental Table S1. The Escherichia coli BL21 (DE3) strain harboring an expression construct was incubated at 37°C for 2 h, shifted to 22°C, and then incubated for an additional 12 h after induction with 1 mm isopropyl β-d-1-thio-galactopyranoside (V900917; Sigma-Aldrich). HIS-, GST-, or MBP-tagged recombinant proteins were purified using glutathione- or MBP-Sepharose according to the manufacturers’ protocols (GE Healthcare for GST-tagged protein, New England Biolabs for MBP-tagged protein).

For the in vitro pull-down assays, 100–300 µg of MBP-CO and GST-TPR2 proteins were incubated with 100 µg of HIS-AFP2 (or its truncated protein) and 2 mg of glutathione-Sepharose 4B beads (50 μL) in PBS buffer (1× PBS, 1× protease inhibitor cocktail; Roche) with 0.1% (v/v) NP-40 for 2 h at 4°C. The pulled-down proteins were extensively washed with buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, and 0.6% [v/v] Triton X-100) before the samples were resolved on 8% (w/v) SDS-PAGE gels and analyzed by protein gel blot analysis using anti-MBP (1:5000, New England Biolabs), anti-HIS (1:300, Qiagen), and anti-GST (1:3000, Invitrogen), followed by a mouse secondary antibody (1:5000, Promega) and the ECL system (Invitrogen).

Antibody Production and Immunoblot Analysis

The TPR2 antibody was produced commercially (YouKe Biotech). A region encoding a polypeptide from amino acids 152–353 of TPR2 was subcloned into the prokaryotic expression vector pET28a. The polypeptide was expressed, purified, and used as antigen to raise polyclonal antibodies in rabbits. To obtain a purified antibody against TPR2, the immunity serum was further applied to His-TPR2-Sepharose, and the bound antibodies were eluted with 0.2 m Gly-HCl buffer, pH 2.2. The washed antibodies were then preserved in 10 mm PBS buffer, pH 7.4 with 0.01% (w/v) NaN3 and 20% (v/v) glycerol for further immunoblot analysis.

For immunoblot analysis, 2-week-old seedlings were rapidly frozen in liquid N and ground in extraction buffer (50 mm sodium P pH 7.0, 100 mm NaCl, 5 mm EDTA, 0.1% [v/v] Triton X-100, 0.1% [w/v]sodium deoxycholate, and protease inhibitor tablet; Roche). The supernatant was collected after centrifugation at 21,000g for 5 min at 4°C. Approximately 10 μg of total protein was separated on a 12% (w/v) SDS-polyacrylamide electrophoresis gel and transferred to a nitrocellulose membrane, which was then probed with the appropriate primary antibody (anti-GFP, 1:3000, Clontech; anti-Flag, 1:3000, Sigma-Aldrich; anti-HA, 1:3000, Roche) and horseradish-conjugated goat anti-mouse secondary antibody (1:3000, Promega). Signals were detected using an ECL Kit (Invitrogen).

Co-IP Analysis

Co-IP was performed as described in Hu et al. (2014). The leaves of three-week-old N. benthamiana seedlings were injected with Agrobacterium LBA4404 harboring 35S:AFP2-6Flag and 35S:TPR2-GFP, or the truncated 35S:AFP2∆E-6xFlag or 35S:AFP2∆J-6xFlag with 35S:TPR2-GFP, respectively, for 3 d. Total proteins were extracted using extraction buffer (50 mm sodium P pH 7.0, 100 mm NaCl, 5 mm EDTA, 0.1% [v/v] Triton X-100, 0.1% [w/v] sodium deoxycholate, 20 μg mL−1 MG132, and a protease inhibitor tablet) and incubated with GFP-Trap-A (Chromotek) for 4 h at 4°C. The beads were then washed three times using washing buffer (50 mm sodium P pH 7.0, 100 mm NaCl, 5 mm EDTA, 0.1% [v/v] Triton X-100, 20 μg mL−1 MG132 and a protease inhibitor tablet). The immunoprecipitates were separated on a 12% (w/v) SDS-polyacrylamide gel and detected by immunoblot analysis with anti-GFP (Clontech) or anti-Flag (Sigma-Aldrich) antibodies, and the immunoblot signals were detected using an ECL Kit (Invitrogen).

RNA Isolation and RT-qPCR

Total RNA was extracted from 2-week-old seedlings using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1.5 μg DNase-treated RNA in a 20-μL reaction volume using M-MuLV reverse transcriptase (Takara) with oligo(dT)18 primers. The relative transcript level of each gene was quantified by qPCR using SYBR Green I Master Mix (04 707 516 001, Roche) and a Light Cycler 480 Real-Time PCR machine (Roche) according to a method described in Hu et al. (2014). At least three biological replicates for each sample were performed to confirm the gene expression pattern. IPP2 was used as an internal gene expression control. The gene-specific primers used to detect the transcripts are listed in Supplemental Table S1.

Transient Transactivation Assay

The fragment containing the 2,675-bp region upstream of the start codon of FT was amplified and cloned into the pGreenII 0800-LUC vector to generate the reporter construct. The full-length AFP2 or TPR2, or the truncated AFP2 region, was amplified and cloned into pGreenII-SK to generate different effector constructs. All primers used to generate these constructs are listed in Supplemental Table S1. Preparation of Arabidopsis mesophyll protoplasts from CO-HA/afp2 leaves and subsequent transfections were performed as described in Yoo et al. (2007) with minor modifications. A dual-luciferase reporter assay system (Promega) was used to measure firefly Luc and Renilla luciferase (REN) activities. The REN gene under the control of the cauliflower mosaic virus 35S promoter and the LUC gene were in the pGreenII 0800-LUC vector. Relative REN activity was used as an internal control, and LUC/REN ratios were calculated.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) experiments were carried out as described in Gu et al. (2013) with minor modifications. Briefly, 5 g of 2-week-old seedlings grown in LD conditions was fixed in fixation buffer (10 mm Tris-HCl, pH 8.0, 0.4 m Suc, 1 mm EDTA, 1 mm PMSF, 0.25% [v/v] Triton X-100, and 1% [v/v] formaldehyde) for 10–15 min under a vacuum. The fixation procedure was then stopped by the addition of 0.125 m Gly for 10 min under a vacuum. After washing three times with 50 mL cold water, the fixed seedlings were ground to a fine powder in liquid N and suspended in extraction buffer I (10 mm Tris-HCl, pH 8.0, 0.4 m Suc, 5 mm EDTA, 5 mm β-mercaptoethanol, 1 mm PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 5 μg/mL antipain, 1 μg/mL pepstatin, and 50 μm MG-132). After filtration through Miracloth (EMD Millipore), the cells were centrifuged at 1000g for 20 min and washed with extraction buffer II (10 mm Tris-HCl, pH 8.0, 0.25 m Suc, 10 mm MgCl2, 1% [v/v] Triton X-100, 5 mm EDTA, 5 mm β-mercaptoethanol, 1 mm PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 5 μg/mL antipain, 1 μg/mL pepstatin, and 50 μm MG-132) to purify the nuclei. The nuclei were then resuspended in 1 mL of nuclei lysis buffer (10 mm Tris-HCl, pH 8.0, 10 mm EDTA, 1% [w/v] SDS, 5 mm β-mercaptoethanol, 1 mm PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 5 μg/mL antipain, 1 μg/mL pepstatin, and 50 μm MG-132) and sonicated for 5 min (30 s on, 30 s off, low intensity) with a sonicator (Bioruptor; Diagenode). Approximately 20 μg of chromatin was then diluted in 1 mL of ChIP dilution buffer (15 mm Tris-HCl, pH 8.0, 167 mm NaCl, 1 mm EDTA, 1% [v/v] Triton X-100, 1 mm PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 5 μg/mL antipain, 1 μg/mL pepstatin, and 50 μm MG-132) and incubated with rabbit polyclonal antiacetylated histone H3 (with acetyl K9+K14+K18+K23+K27, catalog no. ab47915; Abcam) overnight at 4°C. The protein-DNA complexes were collected by incubation with 50 μL of equilibrated Protein G beads (Dynabeads; Invitrogen) for 2–3 h. The beads were sequentially washed with low-salt buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% [v/v] Triton X-100, 1 mm PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 5 μg/mL antipain, 1 μg/mL pepstatin, and 50 μm MG-132), high-salt buffer (low-salt buffer replacing 150 mm NaCl with 500 mm NaCl), LiCl buffer (low-salt buffer replacing 150 mm NaCl with 250 mm LiCl), and TE buffer (10 mm Tris-HCl, pH 8.0, and 1 mm EDTA). Protein-DNA complexes were released by incubation with 300 μL of ChIP elution buffer (20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 50 mm NaCl, 1% [w/v] SDS, and 50 μg/mL proteinase K) for 1 h at 65°C. Immunoprecipitated DNA was purified using a PCR Purification Kit (New England Biolabs), and qPCR was conducted to measure the amounts of FT fragment on a Light Cycler 480 Real-Time PCR machine (Roche) using SYBR Green PCR Master Mix. TUB2 was used to normalize the qPCR results in each ChIP sample. The primers used are specified in Supplemental Table S1.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis TAIR database under the following accession numbers: AFP1, At1g69260; AFP2, At1G13740; AFP3, At3g29575; AFP4, At3g02140; TPL, At1g15750; TPR2, At1g04130; CONSTANS, At5g15840; FT, At1g65480.

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Professors George Coupland (Max Planck Institute for Plant Breeding Research, Cologne, Germany), Takato Imaizumi (University of Washington, Seattle), and Ruth Finkelstein (University of California at Santa Barbara) for contributing related seeds. We also thank all the former colleagues and students for their work at the beginning of this project.

Footnotes

1

This work was supported by start-up funding from Shanghai University, the National Natural Science Foundation of China (grant nos. 31470348 and 31570279), the Applied Basic Research Project of Yunnan Province (grant no. 2016FA015), and the 13th Five-Year Informatization Plan of the Chinese Academy of Sciences (grant no. XXH13506).

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