MicroRNA319-regulated TCPs interact with FBHs and PFT1 to activate CO transcription and control flowering time in Arabidopsis

The transcription factor CONSTANS (CO) is a central component that promotes Arabidopsis flowering under long-day conditions (LDs). Here, we show that the microRNA319-regulated TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factors promote photoperiodic flowering through binding to the CO promoter and activating its transcription. Meanwhile, these TCPs directly interact with the flowering activators FLOWERING BHLH (FBHs), but not the flowering repressors CYCLING DOF FACTORs (CDFs), to additively activate CO expression. Furthermore, both the TCPs and FBHs physically interact with the flowering time regulator PHYTOCHROME AND FLOWERING TIME 1 (PFT1) to facilitate CO transcription. Our findings provide evidence that a set of transcriptional activators act directly and additively at the CO promoter to promote CO transcription, and establish a molecular mechanism underlying the regulation of photoperiodic flowering time in Arabidopsis.


Introduction
Flowering is a transition from the vegetative to the reproductive phase in the plant life cycle, which is crucial for successful reproduction. Genetic approaches in the model plant Arabidopsis, in which flowering is often promoted under long-day (LD) but is delayed during short-day (SD) conditions, reveal that CONSTANS (CO) plays crucial roles in photoperiod monitoring and flowering time determination [1][2][3].
In Arabidopsis, CO encodes a B-box-type zinc finger transcriptional activator [4]. The co mutant lines flower late under LDs, whereas the plants overexpressing CO display early flowering phenotype in both LDs and SDs [4,5]. Under LDs, CO displays a biphasic diurnal expression pattern that its transcript levels first rise at the late afternoon to form a small peak in the light period, and a second peak appears during the midnight [5]. Several studies have revealed that the CO protein stabilization is tightly controlled in a light-dependent manner by a number of factors, such as phytochrome A (PHYA), cryptochrome 2 (CRY2) and FKF1 (FLAVIN-BINDING, KELCHREPEAT, F-BOX1) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) [6][7][8][9][10]. Therefore, the induction of CO mRNA levels at dusk under LDs but not the peak expression at night is essential for the CO protein accumulation and subsequent photoperiodic flowering promotion.
To date, several components have been identified to precisely regulate the diurnal transcription of CO in Arabidopsis. The transcription factors CYCLING DOF FACTORs (CDF1-5) are the well characterized repressors of CO transcription [11,12]. However, as the repressors, CDFs could not fully explain the remarkable up-regulation of CO transcript levels at dusk. The four basic helix-loop-helix-type (bHLH) transcription factors FLOWERING BHLH 1 (FBH1), FBH2, FBH3, and FBH4 have been identified as the CO transcriptional activators that preferentially bind to the E-box cis-elements of the CO promoter in the afternoon to induce the expression of CO [13], proposing a complicated temporal interplay among repressors and activators in restricting the CO transcription. However, unlike CDFs, FBHs do not show robust daily oscillation at either mRNA or protein levels, implying that their time-dependent binding preference on CO promoter is potentially affected by some other unidentified regulators or coactivators [13].
In addition to the transcription factors, PHYTOCHROME AND FLOWERING TIME 1 (PFT1), encoding the Mediator complex subunit 25 (MED25) in Arabidopsis, was reported to genetically act upstream of CO and promote flowering [14,15]. However, the molecular mechanisms about how PFT1 relies on the information from light signals to control flowering time through affecting CO transcript levels remain obscure.
The plant-specific TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family transcription factors contain a conserved non-canonical bHLH domain, which mediates DNA binding or interactions with other proteins [16]. In Arabidopsis, the jaw-D mutants, in which micro-RNA319 (miR319) is over accumulated and five class II TCP genes including TCP2, TCP3, TCP4, TCP10, and TCP24 are down-regulated, show delayed flowering phenotype [17][18][19]. However, the functional mode and action mechanism of these TCPs transcription factors in regulation of Arabidopsis flowering time remain unclear. In this study, we demonstrate that the miR319-regulated TCPs function as direct transcriptional activators of the photoperiodic flowering regulator CO to promote Arabidopsis flowering under the inductive photoperiod. Furthermore, these TCPs transcription factors physically interact with the flowering activators FBHs. Meanwhile, we found that these TCPs and FBHs transcription factors directly interact with the flowering time regulator PFT1 to facilitate CO transcription, and this conclusion is further supported by the observation that PFT1 proteins are exclusively enriched in the TCPand FBH-binding regions of CO promoter under LDs. Thus, we uncover a transcriptional activation complex for direct activation of CO transcription to promote Arabidopsis photoperiodic flowering.

Results
The miR319-regulated TCPs promote Arabidopsis photoperiodic flowering Previous studies have shown that the Arabidopsis tcp4 and jaw-D mutants displayed delayed flowering under LDs [17][18][19]. To further evaluate the potential role of the miR319-regulated TCPs transcription factors in the photoperiodic flowering pathway, we examined the flowering time phenotype of jaw-D mutant lines under both LD (16 h light/8 h dark) and SD (8 h light/ 16 h dark) conditions. As expected, the jaw-D plants displayed an obvious late-flowering phenotype compared with wild type (WT) Columbia-0 (Col-0) under inductive LDs (Fig 1A  and 1B), but flowered normally under the non-inductive SDs (S1 Fig). Next, we analyzed the expression patterns of miR319 together with the five miR319-regulated TCPs, including TCP2, TCP3, TCP4, TCP10 and TCP24, in both wild type and jaw-D mutant plants. In consistent with previous reports [17,19], we also showed that the five miR319-regulated TCPs were all significantly down-regulated in jaw-D plants compared with WT, coupling with the elevated miR319 levels ( Fig 1C). Intriguingly, the mRNA levels of TCP2, TCP4 and TCP24 exhibited similar diurnal expression patterns ( Fig 1C). Accordingly, we assumed that miR319-regulated TCPs may be involved in the Arabidopsis photoperiodic flowering pathway. To further confirm this hypothesis, we generated transgenic plants carrying a 35S:TCP4 construct [17] [12][13][14][15][16], as compared with Col-0 seedlings (Fig 2A, left panel). On the contrary, the CO mRNA levels in the 35S:TCP4 line were obviously increased compared with those in WT (Fig 2A, right panel). Notably, we observed a significant up-regulation of CO in the 35S:TCP4 seedlings at dusk during the light phase (ZT 12-16) (Fig 2A, right panel). As is well known, CO directly activates the expression of its downstream targeting flowering-time gene FLOWERING LOCUS T (FT). Thus, we assumed that the FT transcription might be also affected in the jaw-D and 35S:TCP4 plants. As expected, FT expression was partially compromised in jaw-D mutant, but significantly up-regulated in the 35S:TCP4 transgenic line at different time points (S3 Fig), consistent with the altered CO levels in these lines. Together, these results imply that the miR319-regulated TCPs might play positive regulatory roles in activating CO transcription.
The miR319-regulated TCPs act genetically upstream of CO in regulating flowering To investigate the functional relationship between TCPs and CO in vivo, we tested for genetic interactions between these genes. We crossed the CO-overexpressing transgenic line 35S:CO into the jaw-D background to generate the jaw-D/35S:CO plant and examined its flowering time phenotype in LDs. As expected, the 35S:CO and jaw-D plants displayed early-flowering and late-flowering phenotypes, respectively (Fig 2B and 2C). However, all of the jaw-D/35S:CO seedlings flowered early resembling the flowering time phenotype of 35S:CO plants (Fig 2B  and 2C), indicating that overexpression of CO can rescue the late-flowering phenotype of jaw-D. Meanwhile, we introduced 35S:TCP4 into the co-9 mutant background [20] to generate the co-9/35S:TCP4 line. The flowering time analysis in LDs revealed that, similar to co-9, all the co-9/35S:TCP4 seedlings exhibited late-flowering phenotype compared to Col-0 ( Fig 2D and 2E), suggesting that the early flowering phenotype caused by 35S:TCP4 is largely dependent on the function of CO. Together, these data suggest that the miR319-regulated TCPs may function genetically upstream of CO to promote photoperiodic flowering.
TCP4 directly binds to the CO promoter and activate CO transcription As plant-specific transcription factors, the miR319-regulated TCPs predominantly bind to the common TCP-binding motifs [TBM, GGACC(A/C)] to regulate the expression of target genes [19]. We screened the CO promoter sequence (2-kb), and identified five putative TBM sequences. Two of the TBMs are adjacent to the CO transcriptional start site (named as TBM 1 and TBM 2) with positions of -263/-257 and -324/-318, while the other three located at -1341/-1335 (named as TBM 3), -1371/-1365 (named as TBM 4) and -1484/-1478 (named as TBM 5), respectively ( Fig 3A, upper panel). To investigate the association of TCP4 with the CO promoter in vivo, we performed chromatin immunoprecipitation (ChIP) assay. Considering that  [21,22] for the ChIP assay. Here, we designed eight specific amplicons (represented by P1-P8 in Fig 3A, upper panel) which covered the 2-kb region of CO promoter. As a result, the TCP4-GFP enrichments were specifically observed at the P8 and P2/P3/P4 regions of CO promoter in the rTCP4-GFP ChIP samples (rTCP4-GFP + αGFP in Fig 3A), compared with the negative controls (WT + αGFP and rTCP4-GFP -αGFP in Fig 3A), with the highest level at the P8 region ( Fig 3A). Consistently, P8 and P3 span the CO promoter regions where the TCP-binding motifs are located (TBM 1/2 in P8, and TBM 3/4/5 in P3 in Fig 3A), indicating that TCP4 is specifically associated with the TBM cis-elements on the CO promoter region in vivo. This binding specificity was further supported by the yeast one-hybrid (Y1H) and electrophoretic mobility shift (EMSA) assays, in which TCP4 exclusively associated with the P8 and P3 fragments, but not the TBM cis- Error bars denote ± SD, and three independent biological replicates were performed (n = 3). (B) Yeast one-hybrid (Y1H) assays showing the direct association of TCP4 proteins with CO promoter fragments in vitro. mP8 and mP3, the fragments which contain mutated TBM cis-elements; SD, synthetic dextrose medium; L, Leu; W, Trp; H, His; 3-AT, 3-amino-1,2,4-triazole. 10 −1 , 10 −2 and 10 −3 denote the different dilution series. (C) EMSA revealing the binding of TCP4 to the TBM ciselements in CO promoter. Competition for TCP4 binding was performed with 125× cold probes containing wild type TBM (Competitor wt) or mutated TBM cis-elements (Competitor mut). (D and E) Transcriptional activity assays in Nicotiana benthamiana illustrating the activation of CO transcription by miR319-targeted TCPs. A representative leaf image was shown in (D), and the quantification of the relative luminescence intensities was done in (E) by using n = 18 independent leaves. Error bars denote SDs. (F) Activation of CO transcription in pERGW-rTCP4 transgenic Arabidopsis plants after βestradiol application. pERGW-rTCP4 transgenic line was separately treated with DMSO (Mock) or 10 μM β-estradiol at ZT 3, and collected 24 h later for the quantification of the transcript levels of TCP4 and CO (mean ± SD, n = 3). Asterisks in (E) and (F) denote significant differences against the control sample values at P < 0.01 (**, Student's t test).
https://doi.org/10.1371/journal.pgen.1006833.g003 elements mutated mP8 and mP3 [the TBM cis-elements GGACC(C/A) were replaced by AAAAAA] (Fig 3B and 3C; S1 and S2 Tables). Together, these data strongly demonstrate that TCP4 directly binds to the TBM cis-elements in the CO promoter region both in vitro and in vivo.
Next, to evaluate the direct regulation of TCP4 as well as other miR319-regulated TCPs on CO expression, we performed transient transcriptional activity assays in Nicotiana benthamiana leaves using the CO promoter (2-kb) fused with the LUC gene that encodes firefly luciferase as a reporter [23]. Results showed that the LUC signals were significantly elevated by coexpression of the rTCPs, including rTCP2, rTCP3, rTCP4, rTCP10 and rTCP24 (Fig 3D and 3E; S5 Fig), supporting the hypothesis that the miR319-regulated TCPs are direct transcriptional activators of CO transcription. This conclusion was further confirmed in Arabidopsis by generating the β-estradiol-inducible pERGW-rTCP4 transgenic plants ( Fig 3F). In the presence of the chemical inducer β-estradiol, the pERGW-rTCP4 seedlings showed efficient induced expression of TCP4 (Fig 3F, left panel). Most importantly, the CO transcript levels were also significantly up-regulated following the β-estradiol treatment ( Fig 3F, right panel), confirming the direct activation of CO expression by TCP4.

The miR319-regulated TCPs physically interact with the flowering activators FBHs but not the flowering repressors CDFs
Because the FBHs and CDFs transcription factors have been shown to separately act as activators and repressors of CO transcription [11][12][13]24], we asked whether the miR319-regulated TCPs physically interact with these transcription factors. Indeed, yeast two-hybrid (Y2H) assays revealed an obvious interaction between TCP4 and FBH1 (Fig 4A), and this interaction was further confirmed by LUC complementation imaging (LCI) assay in N. benthamiana ( Fig  4B, upper panel). However, no interaction was detected between TCP4 and CDF1 ( Fig 4A; Fig  4B, lower panel), suggesting that the interaction between TCP4 and FBH1 may be specifically occurred with biological significance. To further confirm the interaction between TCP4 and FBH1 in vivo, we crossed FBH1-GFP with TCP4-Myc to generate the FBH1-GFP/TCP4-Myc double transgenic Arabidopsis plant, and conducted co-immunoprecipitation (Co-IP) assay. Confidently, the interaction signal was exclusively observed in FBH1-GFP/TCP4-Myc plant, but not in FBH1-GFP or TCP4-Myc control samples (Fig 4C), further supporting the physical interaction between TCP4 and FBH1. Moreover, we conducted Förster resonance energy transfer (FRET) assays using Arabidopsis protoplast cells. Here, we employed a quantitative non-invasive fluorescence lifetime imaging (FLIM) approach to detect FRET efficiency [25].
In this assay, two tested proteins are separately fused with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to generate the donors and acceptors, and the lifetime (described as τ) of the donor fluorescence (CFP) is measured in the presence or absence of the acceptor proteins (represented by τ DA and τ D , respectively). If there is a physical interaction between the donor and acceptor, the lifetime τ DA will be considerably shorter than τ D . First, we fused TCP4 with CFP to generate the donor, and FBH1 and CDF1 with YFP as the acceptors (Fig 4D-4F). The combination of TCP4-CFP/YFP was also designed to represent the negative control in the absence of acceptor (τ D , Fig 4D-4F). Confocal microscope detection suggested that all the indicated proteins were properly accumulated in the protoplast cells, and the fluorescent signals of TCP4-CFP, FBH1-YFP and CDF1-YFP fusion proteins were all exclusively observed and perfectly merged in the nuclei (Fig 4D), indicating similar subcellular localization of TCP4, FBH1 and CDF1. Subsequently, we measured the CFP lifetime in the combination samples. As expected, the average lifetime of CFP in the TCP4-CFP/FBH1-YFP co-expression cells was 0.99 ± 0.19 ns (τ DA ; mean ± SD, n = 17 nuclei), which was remarkably (P < 0.01; Student's t test) shorter than the 2.59 ± 0.39 ns (τ D ; mean ± SD, n = 7 nuclei) determined in the negative control TCP4-CFP/YFP (Fig 4E and 4F), suggesting a strong physical interaction between TCP4 and FBH1. However, in the TCP4-CFP/CDF1-YFP co-expression samples, the average lifetime of CFP was 2.48 ± 0.16 ns (τ DA ; mean ± SD, n = 15 nuclei), very similar to that of the negative control, illustrating that TCP4 does not interact with CDF1 ( Fig  4E and 4F).
Further assays by Y2H in yeast and LCI in N. benthamiana revealed that TCP4 also interacts with FBH2, FBH3 and FBH4 (S6A and S6B Fig); meanwhile, FBH1 could also interact with other miR319-regulated TCPs such as TCP2, TCP3, TCP10 and TCP24 (S6C and S6D Fig). These results strongly suggest the functional conservation and redundancy among the miR319-regulated TCPs as well as the FBH homologs. Our simultaneous analyses using the CDF homologs CDF1, CDF2 and CDF3 revealed that these CDFs failed to interact with all the tested TCPs (TCP2, TCP3  The above conclusion led us to further evaluate the biological significance of the physical interaction between TCP4 and FBH1 on their transcriptional activation activities. To this end, we carried out transient transcriptional activity assays in N. benthamiana. As the activators of CO transcription, both TCP4 and FBH1 significantly led to obvious induction in the luminescence intensity of CO pro :LUC by about 6-to 10-fold changes compared with the empty vector control sample (Fig 4G and 4H; combinations 2 and 3). Most importantly, we observed a more significant up-regulation of LUC reporter activity in the TCP4 and FBH1 co-expressed sample (Fig 4G and 4H; combination 4), in which the luminescence intensity was almost 20 times higher than that in the negative control (Fig 4G and 4H; combination 1), confidently suggesting an additive effect of TCP4 and FBH1 on CO transcription activation. Meanwhile, our qRT-PCR assays revealed that TCP4 and FBH1 were similarly expressed in different infiltrated samples (Fig 4I). Together, these data imply that TCP4 and FBH1 might function additively to regulate the expression of CO in Arabidopsis.
Both the miR319-regulated TCPs and FBHs interact with the flowering time regulator PFT1 PFT1, encoding the Arabidopsis Mediator complex subunit 25 (MED25) which usually acts as a transcriptional co-activator, was previously described as an essential regulator of flowering time [14,15]. These observations promoted us to test whether there may be a functional relationship between PFT1 and the CO activators TCPs and/or FBHs. Indeed, we found that both TCP4 and FBH1 could interact with PFT1, according to the Y2H assay in yeast as well as the LCI and Co-IP assays in N. benthamiana (Fig 5A-5C). Next, we conducted FLIM-FRET to further confirm these physical interactions in Arabidopsis protoplast cells. Similarly, confocal microscope detection confirmed that the TCP4-CFP, FBH1-YFP and PFT1-YFP/CFP fusion proteins were all properly accumulated, and the fluorescent signals of these fusion proteins were exclusively merged in the nuclei (Fig 5D). Subsequently, an average lifetime of 0.90 ± 0.08 ns (τ DA ; mean ± SD, n = 18 nuclei) was determined in the TCP4-CFP/PFT1-YFP co-expression samples (Fig 5E and 5F), which was significantly (P < 0.01; Student's t test) shorter than that of 2.59 ± 0.39 ns (τ D ) in the negative control (TCP4-CFP/YFP as shown in Fig 4E and 4F); meanwhile, the average lifetime of CFP in the PFT1-CFP/FBH1-YFP samples was only 0.95 ± 0.28 ns (τ DA ; mean ± SD, n = 9 nuclei), remarkably (P < 0.01; Student's t test) shorter than the 2.80 ± 0.28 ns (τ D ; mean ± SD, n = 6 nuclei) in the negative control PFT1-CFP/YFP (Fig 5E and 5F). These data strongly demonstrate that PFT1 directly interacts with TCP4 and FBH1 in Arabidopsis. Certainly, our extended assays further confirmed that PFT1 also interacts with other miR319-regulated TCPs (including TCP2, TCP3, TCP10 and TCP24) and FBH1 homologs (FBH2, FBH3 and FBH4) (S9 Fig). PFT1 Is genetically required for the role of TCP4 in promoting flowering The above finding that PFT1 physically interacts with TCP4 promoted us to ask whether PFT1 is functionally involved in the TCP4-regulated photoperiodic flowering pathway. To address this question, we crossed the 35S:TCP4 transgenic plants with the pft1-2 mutant line to generate the 35S:TCP4/pft1-2 plant, and examined its flowering time phenotype in LDs. Our results showed that pft1-2 exhibited a late-flowering phenotype compared to the WT Col-0 ( Fig 5G  and 5H), which is consistent with the previous studies [14,15,26]. More importantly, even though 35S:TCP4 could trigger early flowering (Fig 1A and 1B; Fig 5G and 5H), its promotional effect on flowering was largely compromised in the pft1-2 background (Fig 5G and 5H). These results imply that PFT1 is genetically required for the role of TCP4 in promoting flowering.
PFT1 Acts as co-activator of miR319-regulated TCPs and FBHs to facilitate CO transcription We further hypothesized that PFT1 might be required for the full functions of miR319-regulated TCPs in the activation of CO transcription. To test this idea, we performed the transient activation activity assays in N. benthamiana (Fig 6A). Consistent with the above results, the expression of TCP4 alone led to about 10-fold up-regulation of the CO pro :LUC reporter activity (Fig 3D and 3E, combination 4; Fig 6A, combination 3); whereas, PFT1 failed to elevate the CO pro :LUC reporter activity (Fig 6A, combination 2), indicating that PFT1 alone is not able to activate CO transcription. Interestingly, when we co-expressed PFT1 and TCP4, an obvious additive effect was observed as the luminescence intensities increased by almost 30 folds (Fig 6A, combination 4), compared with the empty vector control (Fig 6A, combination 1), significantly higher than that in the TCP4 single-expression samples (Fig 6A, combination 3), suggesting that PFT1 potentially facilitates the transcriptional activation activity of TCP4 on CO transcription. Parallel experiments showed that PFT1 could also dramatically enhance the transcriptional activation activity of FBH1 in promoting CO transcription (Fig 6A, combinations 5-8). Our qRT-PCR assays revealed that TCP4, FBH1 and PFT1 were all similarly expressed in different infiltrated samples (Fig 6B). Thus, we concluded that PFT1 may function as co-activator of both TCPs and FBHs in the activation of CO transcription. mRNA levels in the afternoon (ZT 8-12) and an obvious peak at the midnight (ZT 20-24) ( Fig  6C). In consistence with the previous report [14], our data confirmed that the CO expression in the pft1-2 mutant line was significantly compromised compared with that in the WT Col-0 control (Fig 6D), indicating an essential role of PFT1 in facilitating CO transcription.
Based on our findings that PFT1 physically interacts with the CO activators TCPs and FBHs (Fig 4), we were interested to test whether PFT1 proteins are enriched in the CO promoter regions in vivo. To this end, we conducted the ChIP assays using the 35S:PFT1-GFP transgenic Arabidopsis plants [15]. As expected, the PFT1-GFP enrichments were remarkably detected at the P8 and P3 regions of CO promoter with a maximum enrichment at P8 (PFT1-GFP + αGFP in Fig 6E), compared with the negative controls (WT + αGFP and PFT1-GFP -αGFP in Fig  6E). Significantly, the enrichment tendency of PFT1 at the different CO promoter regions well correlated with that of TCP4 (Fig 3A), and was also very similar to that of FBH1 as shown in a previous study [13]. Taken together, we propose that PFT1 is probably enriched in the CO promoter regions to act synergistically with TCPs and FBHs to facilitate CO transcription.

Discussion
In this study, we showed that the miR319-regulated TCPs interact with the flowering time regulators FBHs and PFT1 to activate CO transcription and promote Arabidopsis photoperiodic flowering.
Previous observations suggested that the miR319-regulated TCPs transcription factors may be involved in regulation of Arabidopsis flowering time [17][18][19]. However, the functional mode and action mechanism of these TCPs transcription factors in regulation of Arabidopsis flowering time remain unclear. In this study, we show that down-regulation of the miR319-regulated TCPs in the jaw-D mutant plants causes late flowering phenotype in LDs, but not in SDs (Fig 1A and 1B; S1 Fig), demonstrating that the miR319-regulated TCPs modulate flowering time through regulating the photoperiodic flowering pathway in Arabidopsis. In support of this view, the expression of CO, a central component of the photoperiodic flowering pathway in Arabidopsis, were significantly reduced in the jaw-D mutant plants in amplitude under LDs (Fig 2A), while up-regulated by both constitutive and inducible overexpression of TCP4 (Figs  2A and 3F). Further, we showed that TCP4 can bind to the TBM cis-elements of the CO promoter and all the miR319-regulated TCPs directly activate CO transcription (Fig 3A-3E). Based on these findings, we conclude that the miR319-regulated TCPs may act as positive regulators of photoperiodic flowering through direct activation of CO transcription in Arabidopsis. Nevertheless, the in planta interplay between the miR319-regulated TCP transcription factors and CO promoter still needs to be intensively analyzed in the future, considering that the non-native promoter used for driving TCP4 expression in this study might cause a nonphysiological effect for TCP4. Therefore, it should be intriguing to uncover the dynamic enrichment pattern of each member of these TCP proteins on the CO promoter, which will be useful for better understanding the contribution of these TCPs to the daily CO oscillation.
FBHs act as CO transcription activators in regulating flowering time [13]. Our findings that TCPs physically interact with FBHs provide a novel mechanism for the regulation of CO transcription in the photoperiodic flowering pathway (Fig 4; S6 and S8 Figs). The previous study showed that the E-box cis-elements contained in the -509/-196 region of CO promoter are essential for FBH1 binding as well as FBH1-dependent gene activation [13]. Coincidently, our ChIP assays revealed a preferred binding fragment of CO promoter by TCP4 containing the TBM cis-elements in the -348/-155 region (P8 in Fig 3A), which is adjacent to and partially overlapped with the FBH1 binding region. The spatial proximity of the DNA-binding sites to some extent causes the possibility of direct interaction between TCP4 and FBH1. However, our further assays revealed that TCP4 and FBH1 interact with each other through their transcriptional activation domains (S8 Fig), not through their DNA-binding domains (i.e. the bHLH domains that located in the N-or C-terminals of TCP4 and FBH1, respectively, as shown in S8A Fig), suggesting the physical interaction between TCP4 and FBH1 might facilitate their transcriptional activation activities on CO transcription. Indeed, an additive effect of TCP4 and FBH1 in activating CO transcription was obviously observed in our analyses ( Fig  4G and 4H), implying a potential interplay among the TCPs and FBHs transcription factors. However, it should be noticed that TCP4 and FBH1 themselves could, at least in part, activate the transcription of CO (Fig 4G and 4H). Thus, the additive effect of TCP4 and FBH1 might be attributed to more abundant activators enriched on the CO promoter and/or their cooperation upon the co-expression of these two transcription factors. Here, we assume that the miR319-regulated TCPs and FBHs might function cooperatively and/or independently to activate the CO expression in certain situations. However, it is eagerly needed to explore the genetic interaction between the TCPs and FBHs regarding the regulation of CO expression in vivo in the future.
In this study, we confirmed that both TCPs and FBHs physically interact with the transcriptional co-activator PFT1 (Fig 5 and S9 Fig). Although PFT1, encoding the Mediator subunit 25 in Arabidopsis, was initially identified as a positive regulator of flowering time more than ten years ago [14], the molecular mechanisms of its action in regulation of flowering time remain obscure to date. Mediator is a multiprotein complex that promotes transcription by recruiting the RNA polymerase II (RNAPII) to the promoter regions upon the physical interaction with specific DNA-bound transcription factors [28,29]. Our observations reinforce that co-expression of PFT1/MED25 with TCP4 or FBH1 additively elevated the CO transcription levels (Fig 6A), while the loss-of-function of PFT1 leads to an obvious reduction of CO mRNA levels ( Fig 6D). It is noteworthy, in our assays, that PFT1 failed to promote CO transcription in the absence of TCP4 or FBH1 (Fig 6A), implying the essential roles of TCPs and FBHs for the function of PFT1 in activating CO transcription. This hypothesis was further supported by our ChIP assay results that the PFT1 proteins were enriched with the peaks in the CO promoter regions near the TCP4-and FBH1-binding sites (Figs 3A and 6E). Collectively, our results suggest that PFT1 potentially acts as positive regulator of CO transcription.
Based on our findings, we proposed a working model on the control of photoperiodic flowering time (Fig 6F). Briefly, the miR319-regulated TCPs and FBHs directly bind to the adjacent regions of CO promoter in the wild-type Arabidopsis plants; they physically interact with each other through their transcriptional activation domains to activate CO transcription through direct interaction with PFT1, and consequently promote flowering under LDs (Fig 6F, upper  panel). By contrast, in the jaw-D mutant plants, the association of TCPs with CO promoter is drastically blocked due to the overdose of miR319 and consequent decrease of TCP proteins, leading to down-regulation of CO transcription during the peak expression time, which as a result causes delayed flowering (Fig 6F, lower panel).
Arabidopsis thaliana were grown under LD (16-h-light/8-h-dark) or SD (8-h-light/16-hdark) conditions at 22˚C. Time-course analyses were performed on 12-d-old seedlings grown on half-strength Murashige and Skoog medium. Nicotiana benthamiana was grown in a greenhouse at 22˚C with a 16-h-light/8-h-dark cycle.

Analyses of flowering time phenotype
Analyses of flowering time were performed as previously described [15]. Flowering time was recorded from at least 15 plants per genotype that were grown in soil under either LDs or SDs, and was scored as the number of days from germination to the first appearance of buds at the apex (days to bolting). The rosette leaf number was counted after the main stem has bolted 1 cm.

DNA constructs and generation of transgenic/hybrid plants
For Gateway cloning, all the gene sequences were cloned into the pQBV3 or pENTRY vectors (Gateway) and subsequently introduced into certain destination vectors following the Gateway technology (Invitrogen). For ligase dependent cloning, the endonuclease digested vectors and PCR fragments were separately purified by PCR cleanup kit (Axygen, AP-PCR-250), and ligated at 16˚C with T4 DNA ligase (New England Biolabs, M2020). For ligase-independent ligation, the ligation free cloning mastermix (abm) was used following the application handbook.
For generation of miR319-cleavage-resistant forms of TCPs (rTCPs) as well as the TBM ciselements mutated CO promoter fragments, the one-step site-directed mutagenesis strategy was performed. The wild type TCP (TCP2, TCP3, TCP4, TCP10 and TCP24) sequences or the CO promoter fragments were first connected into the pQBV3 entry vector, and then the mutations on miR319-target sites or TBM cis-elements were introduced by the specifically designed primers, as described previously [31]. The PCR amplifications were carried out by pre-heating at 94˚C for 3 min, 16 cycles of 94˚C for 1 min, 55˚C for 1 min and 68˚C for 7 min, followed by incubation at 68˚C for 1 h. The PCR products were purified by PCR cleanup kit (Axygen, AP-PCR-250), and digested by 1 μl of DpnI (New England Biolabs, R0176L). The obtained products were transformed into Escherichia coli competent cells for sequencing. The primer sequences used for site-directed mutagenesis are listed in S5 Table. For the generation of 35S:TCP4 lines, the expression vector 35S:TCP4 [17] was transformed into the Agrobacterium strain GV3101 (pMP90). For the construction of TCP4 pro :GUS transgenic lines, the 5' upstream region of the TCP4 sequence (-2000/-1) was amplified from Col-0 genomic DNA, and cloned into the binary vector pMDC162 vector to generate TCP4 pro :GUS expression construct. For the generation of β-estradiol-inducible pERGW-rTCP4 transgenic plants, the construct pQBV3-rTCP4 containing the miR319-cleavage-resistant TCP4 was introduced into the destination vector pERGW to fuse with a β-estradiol-inducible promoter following the Gateway cloning strategy. All of the binary vectors were introduced into the wild type Col-0 plants by Agrobacterium-mediated transformation to generate transgenic plants [20]. More details of the DNA constructs are listed in S6 Table. jaw-D/35S:CO, co-9/35S:TCP4, FBH1-GFP/TCP4-Myc and 35S:TCP4/pft1-2 were generated by genetic crossing.

RNA extraction and gene expression analysis
The 12-d-old Arabidopsis seedlings grown on half-strength Murashige and Skoog medium were collected at the indicated time after the onset of light. Total RNA was extracted using Trizol (Invitrogen) reagent. About 2 μg of total RNA and Moloney murine leukemia virus reverse transcriptase (M-MLV; Promega) were further used for reverse transcription. The cDNA was diluted to 100 μL with water in a 1:5 ratio, and 2 μL of the diluted cDNA was used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with the SYBR Premix Ex Taq (Perfect Real Time; TaKaRa). The reverse transcription of mature miRNAs was performed as described previously [32], by using specifically designed stem-loop primers. qRT-PCR was performed using the following program: 120 sec at 95˚C, 45 cycles of 10 sec at 95˚C, and 1 min at 60˚C. ACTIN7 and U6 expression levels were used as the internal controls for coding genes and miRNAs, respectively. All the experiments were performed independently three times. All the primers used for real time quantification are listed in S3 Table. Yeast experiments For yeast one-hybrid assay, the pHIS2 derivatives were co-transformed with GAL4-AD constructs harboring the P8 and P3 sequences, or mP8 and mP3 fragments which contain mutated TBM cis-elements [GGACC(C/A) were replaced by AAAAAA] (see S1 Table) into the yeast (Saccharomyces cerevisiae) strain AH109. The transformed cells were first grown on synthetic dextrose medium lacking Leu and Trp (SD-L/W) and then were transferred to the synthetic dextrose medium lacking Leu, Trp and His (SD-L/W/H) medium supplemented with 3-amino-1,2,4-triazole (3-AT) for selection. For yeast two-hybrid assay, the GAL4-AD and GAL4-BD derivatives were co-transformed into the yeast strain AH109, and grown on SD-L/W. Further, the yeast cells were screened on the SD-L/W/H or SD-L/W/H/ A media with 3-AT. For transcriptional activation activity analysis, the GAL4-BD derivatives were separately transformed into yeast strain AH109, and the transformed yeast cells were grown and selected on SD-L and SD-L/H/A media, respectively. Each experiment was independently repeated for three times with similar results.

LCI assays
The LCI assays for the protein interaction detection was performed in N. benthamiana leaves as described previously [33]. Briefly, the full-length or truncated forms of the genes were separately fused with the N-and C-terminal parts of the luciferase reporter gene LUC. Agrobacteria cells harboring the nLUC and cLUC derivative constructs were co-infiltrated into N. benthamiana leaves, and the LUC activities were analyzed 48 h after infiltration using NightSHADE LB 985 (Berthold). In each analysis, five independent N. benthamiana leaves were infiltrated and analyzed, and totally three biological replications were performed with similar results.

Transcriptional activity assays in N. benthamiana
The transcriptional activity assays were performed in N. benthamiana leaves as previously described [23]. The 2-kb CO promoter sequence was amplified from Col-0 genome DNA, and fused with the luciferase reporter gene LUC through Gateway reactions (Invitrogen) into the plant binary vector pGWB35 [34] to generate the reporter construct CO pro :LUC. For the construction of the effectors, the full-length coding sequences of indicated genes were amplified and cloned into the plant binary vector pGWB17 [34]. The reporter and effector constructs were separately introduced into Agrobacterium strain GV3101 (pMP90), to carry out the coinfiltration in N. benthamiana leaves. LUC activities were observed and quantified 48 h after infiltration using NightSHADE LB 985 (Berthold). In each experiment, 10 independent N. benthamiana leaves were infiltrated and analyzed, and totally three biological replications were performed with quantification.

FLIM-FRET assays
For FLIM-FRET assays, the indicated proteins were separately fused with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to generate the donors and receptors. The FLIM-FRET experiments were performed as described previously [25] with some modifications. Briefly, the donors and receptors were co-expressed in Arabidopsis protoplast cells. After 24 h incubation, the CFP and YFP fluorescence signals were imaged with the confocal microscope (Carl Zeiss, LSM880). Förster resonance energy transfer was measured by fluorescence lifetime imaging using the PicoHarp 300 time-correlated single-photon counting (TCSPC) module (PicoQuant, Germany). A pulsed laser (405 nm) tuned at 84MHz was used to excite the CFP. The emission from 450 to 490 nm was collected by the detector in 512×512 pixel format. The acquired FLIM decay curve from regions of interest (ROI) was fitted by two-exponential theoretical models using SymPhoTime 64 software, and the mean CFP lifetimes were calculated as the mean values of the fit function and analyzed using SymPhoTime 64 software. In each analysis, 6 independent nuclei were quantitatively analyzed, and totally three independent replications were performed with similar results.

ChIP assays
Arabidopsis seedlings were grown on half-strength Murashige and Skoog medium in 16-hlight/8-h-dark LDs for 14 days, and the samples (2 to 3 grams) were collected at ZT 14. The ChIP assays were carried out following the procedure described previously [23]. The ChIP assays were separately performed with (+ αGFP) or without (-αGFP) the anti-GFP antibody. Finally, the GFP-specific enrichments of the fragments from CO promoter were analyzed by qPCR, and the enrichment fold of a certain fragment was calculated by normalizing to the amount of ACTIN7 promoter enriched in the same sample. Anti-GFP-CHIP grade antibody (Abcam, ab290) and protein G plus agarose (Santa cruz, sc-2002) were used for the immunoprecipitation. The enrichment of DNA fragments was determined by qPCR with specific primers, as shown in S4 Table. Three biological replicates were performed.

EMSA
The maltose binding protein (MBP) tagged TCP4 protein was expressed in E. coli strain Transetta-DE3 (Transgen biotech, CD801), and purified using the amylose resin (New England Biolabs, E8021V) following the manual. The CO promoter probes containing the TBM cis-element were synthesized and labeled with digoxigenin-11-ddUTP at the 3' end by using DIG gel shift kit (Roche, 03353591910). Unlabeled dimerized oligo-nucleotides of CO promoter fragments containing the wild type or mutated TBM cis-elements were generated as the competitors. EMSAs were performed as previously described [33]. Competition for TCP4 binding was performed with 125× cold probes containing TBM cis-elements [GGACC(C/A)] or mutated TBM cis-elements (AAAAAA). Sequences of probes and competitors are shown in S2 Table. Three biological replicates were performed.

Accession numbers
Sequence data from this study can be found in the Arabidopsis Genome Initiative database under the following accession numbers: CO (At5g15840), TCP2 (At4g18390), TCP3 (At1g532