Research ArticleResearch Article

BBX19 Interacts with CONSTANS to Repress FLOWERING LOCUS T Transcription, Defining a Flowering Time Checkpoint in Arabidopsis

Chang-Quan Wang, Cade Guthrie, Mostafa Khoshhal Sarmast, Katayoon Dehesh

Published September 2014. DOI: https://doi.org/10.1105/tpc.114.130252

Abstract

The timely transition of vegetative to reproductive development is coordinated through quantitative regulation of floral pathway genes in response to physiological and environmental cues. Here, we show that the circadian-controlled expression of the Arabidopsis thaliana floral transition regulators FLOWERING LOCUS T (FT) and CONSTANS (CO) is antiphasic to that of BBX19, a transcription factor with two B-Box motifs. Diminished expression of BBX19 by RNA interference accelerates flowering, and constitutive expression of BBX19 delays flowering under inductive photoperiods. This delay is not accompanied by the alteration of CO expression levels but rather by a reduction of transcript levels of FT and the FT-regulated genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1, LEAFY, and FRUITFUL. Similar to CO, BBX19 is expressed in vasculature. BBX19 and CO colocalize in the nucleus and interact physically in vivo. In transient assays, coinfiltration of 10-fold more CO overcomes the BBX19-mediated repression of FT activation. Substitution of the conserved Cys-25 to Ser in the BBX19 Box1 motif abolishes the BBX19–CO interaction and eliminates the negative regulation of flowering time, while the analogous C76S substitution in the Box2 motif is ineffective. Together, these results implicate BBX19 as a circadian clock output that depletes the active CO pool to accurately monitor daylength and precisely time FT expression.

INTRODUCTION

The B-box (BBX) proteins are a functionally diverse class of zinc-finger transcription factors encoded by a highly conserved group of genes across multicellular species (Yamawaki et al., 2011; Huang et al., 2012; Crocco and Botto, 2013). These proteins are characterized by the presence of one or two B-Box motifs in the N-terminal domain, alone or in combination with a C-terminal CCT (for CONSTANS, CO like, and TOC) domain (Khanna et al., 2009; Huang et al., 2012). In Arabidopsis thaliana, the BBX family consists of 32 proteins arranged into five structural groups based on the number of B-Box motifs and the presence or absence of the CCT domain (Robson et al., 2001; Kumagai et al., 2008; Khanna et al., 2009; Gangappa and Botto, 2014).

The CCT domain has weak similarity to histone proteins and as such is implicated in transcriptional regulation and in some instances in nuclear protein transport (Robson et al., 2001; Laubinger et al., 2006; Jang et al., 2008; Yan et al., 2011; Gendron et al., 2012).

The two B-Box motifs differ in their consensus sequences and the interspacing of the zinc binding residues, potentially caused by segmented duplication and internal deletion events (Massiah et al., 2007; Crocco and Botto, 2013). The B-Box motif is well established as being critical for both transcriptional regulation and also heterodimeric formation with other BBX family members as well as other nonfamily member proteins (Datta et al., 2007, 2008; Qi et al., 2012; Gangappa et al., 2013; Gangappa and Botto, 2014). Specifically, the conserved Cys and His residues in B-Box motifs are known to be involved in protein–protein zinc ligation (Gangappa and Botto, 2014).

CONSTANS (CO)/BBX1 was the first member of the BBX (BBX1) family identified in Arabidopsis; it contains two B-Box motifs and one CCT domain. CO induces flowering in response to the long-day (LD) inductive photoperiod via activating the expression of floral inducers such as FLOWERING LOCUS T (FT) and SUPPRESSOR OF CONSTANS1 (SOC1) (Samach et al., 2000; Hayama et al., 2003; Abe et al., 2005; Wigge et al., 2005; Yoo et al., 2005; Searle et al., 2006). CO activates FT transcription through direct binding to CO-responsive elements and CCAAT-box elements in the FT promoter (Wenkel et al., 2006; Tiwari et al., 2010; Cao et al., 2014).

Control of flowering time is not limited to CO/BBX1, since other BBX family members, such as BBX4, BBX6, BBX7 (COL9), BBX24 (STO), and BBX32 (EILP6), also regulate flowering through distinct and overlapping as well as antagonistic functions (Cheng and Wang, 2005; Datta et al., 2006; Hassidim et al., 2009; Park et al., 2011; Li et al., 2014). Specifically, bbx4 mutants flower early under both short-day (SD) and LD conditions, suggesting an antagonist function to that of CO (Datta et al., 2006), while overexpression of BBX7 delays flowering under inductive LD conditions by reducing the expression levels of CO and in turn decreasing FT expression (Cheng and Wang, 2005). By contrast, bbx6 mutant plants flower normally under SD conditions, but BBX6 overexpression induces early flowering by promoting FT expression (Hassidim et al., 2009). On the other hand, bbx24 (sto-1) mutants display delayed flowering under SD conditions, while the overexpression lines show accelerated flowering time under both the SD and LD regimes. BBX24 functions by competing with FLOWERING LOCUS C (FLC) to regulate downstream flowering genes (Li et al., 2014). Furthermore, overexpression of BBX32 delays flowering time, potentially through heterodimer formation with EMBRYONIC FLOWER1, a necessary regulator of vegetative development (Park et al., 2011).

In addition to their roles in altering the expression levels of flowering time genes, the BBX proteins can also modulate transcript levels of several genes in hormonal signaling networks. For example, BBX18 regulates the gibberellin (GA) signaling pathway by increasing the expression of GA-oxidase (GA3ox1 and GA20ox1) metabolic genes and suppressing the expression of GA2ox1 and GA2ox8 catabolic genes in response to light (Wang et al., 2011). Additional studies suggest that BBX18 may act as an integrator of GA and light signaling pathways (Weller et al., 2009).

Studies of how plants constantly sense and rapidly respond to environmental challenges have uncovered a host of stress signaling agents, among them a small metabolite, methylerythritol cyclodiphosphate (MEcPP), that is responsible for the communication of environmental perturbations sensed by plastids back to the nucleus (Xiao et al., 2012). MEcPP is a precursor of isoprenoids produced by the plastidial methylerythritol phosphate pathway, and its stress-induced accumulation elicits the expression of selected stress-responsive genes (Xiao et al., 2012, 2013). Specifically, the constitutively active HPL (ceh1) mutants accumulate high levels of MEcPP and display robustly altered expression levels of selected genes, among them BBX19, encoding a member of the BBX family of proteins with two B-Box motifs. The altered levels of BBX19 transcript in ceh1 mutants, together with the established regulatory role of some BBX proteins in stress-induced signaling pathways (Nagaoka and Takano, 2003; Laubinger et al., 2006; Wang et al., 2013), led us to explore the potential function of BBX19 in plant stress responses. These studies, however, unexpectedly led to the discovery of BBX19 as a regulator of flowering time.

Here, we show that BBX19 functions as a negative regulator of flowering time under the inductive photoperiod. The mode of BBX19 action is through physical interaction with CO, leading to depletion of the active CO pool required for the transcription of FT. The integrity of the Box1 motif is essential for this protein–protein interaction and is thus essential for the BBX19-associated regulation of flowering time. This mode of action distinguishes BBX19 from the other previously reported regulators of flowering time.

RESULTS

BBX19 Is a Negative Regulator of Flowering Time under the Inductive Photoperiod

We have previously reported on the robust alteration in expression levels of a selected group of stress-responsive genes in response to an accumulation of the retrograde signal MEcPP in the ceh1 mutant plant (Xiao et al., 2012). The initial microarray data identified BBX19 as a transcription factor whose transcript levels in ceh1 mutants are reduced to 50% of the wild-type levels. Here, we verified the initial microarray data by reverse transcription and quantitative PCR (RT-qPCR) analyses (Supplemental Figure 1A).

To begin to dissect the potential function of BBX19 in plant stress responses, we incorporated a BBX19 overexpression construct into ceh1 by crossing the mutant plants to the line constitutively expressing CaMV35S:BBX19-GFP in the Columbia-0 (Col-0) background. The constitutive expression of BBX19 resulted in >2 weeks (equivalent to ∼15 additional leaves per plant to flowering) delayed flowering time in all overexpression genotypes, namely in Col-0 (OE44 and OE56) as well as in the ceh1 mutant background, under the inductive LD photoperiod (Figures 1A and 1B; Supplemental Figure 1B). These results also revealed a relationship between the expression levels of BBX19 and the length of delay in flowering time, since the OE56 line, which expresses BBX19 at a higher level relative to the OE44 line, flowered ∼3 d (equivalent to 2 additional leaves per plant to flowering) later than OE44 (Figures 1A and 1B; Supplemental Figure 2).

Figure 1.
Figure 1.

BBX19 Is a Negative Regulator of Flowering Time under the Inductive Photoperiod.

(A) Flowering phenotypes of 45-d-old wild-type Col-0, T-DNA insertion lines (bbx19-1 and bbx19-2), and BBX19 overexpression lines (OE44 and OE56).

(B) Leaf number per plant at flowering time of the aforementioned genotypes.

(C) Flowering phenotypes of 28-d-old wild-type Col-0 and BBX19 RNAi lines (Ri323 and Ri513).

(D) Leaf number per plant at flowering time of the wild type and RNAi lines (Ri323 and Ri513).

In (B) and (D), data are means ± sd (n ≥ 30). Asterisks denote significant differences in flowering time between control (Col-0) and overexpression or RNAi lines (P < 0.05).

[See online article for color version of this figure.]

Next, we examined the flowering time of the T-DNA insertion lines, here designated bbx19-1 and bbx19-2. The bbx19-1 line has an insertion in the first intron, and bbx19-2 has an insertion in the end of the fourth exon at the C terminus (Supplemental Figure 3A). However, 30 cycles of PCR showed easily detectable full-length and truncated BBX19 transcript levels in the bbx19-1 and bbx19-2 plants, respectively, and as such, no changes in the respective flowering time as compared with the wild type (Figures 1A and 1B; Supplemental Figures 3B and 3C). Importantly, similar flowering time between the wild-type and bbx19-2 plants suggests the full functionality of the encoded truncated polypeptide lacking the C-terminal portion of the coding region (Figures 1A and 1B; Supplemental Figures 3A and 3C).

Since BBX19 function was not lost in the T-DNA insertion lines, we generated RNA interference (RNAi) lines displaying strongly reduced levels of BBX19 transcript detected upon several rounds of amplifications. The RT-qPCR–based quantitation of the transcript levels showed 70 and 85% reduction in BBX19 expression levels in the two independent RNAi lines, Ri323 and Ri513, respectively, as compared with the wild type (Supplemental Figure 3D).

Based on protein sequence analyses and the presence of two B-boxes in their N termini, BBX19 together with seven other BBXs are placed within the group IV subfamily (BBX18 to BBX25) (Khanna et al., 2009; Crocco and Botto, 2013; Gangappa and Botto, 2014). In fact, the conserved residues in these B-box motifs have been shown to be crucial in mediating protein–protein interactions and transcriptional regulation (Gangappa and Botto, 2014). Based on these structural and functional similarities, we questioned whether the RNAi lines are specific to BBX19 and whether the expression levels of the other seven members of group IV might be altered. To address this concern, we performed RT-qPCR on the group IV family members and determined that the RNAi lines are indeed specific to BBX19, as the expression levels of the other seven genes remained unaltered (Supplemental Figures 3D and 4). These data confirmed the suitability of these RNAi lines for functional analyses of BBX19, justifying their subsequent utilization for additional studies. Further analyses showed accelerated flowering time (3 d = two additional leaves per plant to flowering) of the RNAi lines as compared with the wild type under the inductive photoperiod (Figures 1C and 1D).

Collectively, these data provide strong support for the role of BBX19 as a negative regulator of flowering time in Arabidopsis.

Next, we questioned whether BBX19 functions independently of the photoperiodic flowering by comparing the flowering time of various genotypes, including the wild type (Col-0), OE44 and OE56 lines, bbx19-b, and Ri323 and Ri513, under noninductive SD conditions (Supplemental Figures 5A to 5D). These data, analyzed based on plant leaf numbers at flowering, clearly display similar flowering time among these genotypes, thus illustrating the exclusive involvement of BBX19 protein in flowering time regulation under inductive photoperiods.

BBX19 Overexpression Reduces FT mRNA Levels but Does Not Alter Expression Levels of the Known Activator or Suppressors of FT Expression

To begin to examine the mechanism of action of BBX19 in the regulation of flowering time, we used RT-qPCR and measured the expression levels of CO, FT, and SOC1, genes key in the photoperiod pathway regulation of flowering, as well as the floral identity genes LEAFY (LFY) and FRUITFUL (FUL) (Blázquez and Weigel, 2000; Onouchi et al., 2000; Samach et al., 2000; Michaels et al., 2003; Wang et al., 2009; Yamaguchi et al., 2009). These data show that, with the exception of CO, whose expression levels remained unchanged, the transcript levels of all examined floral regulators were notably reduced in OE44 and OE56 overexpressing lines as compared with the wild type (Col-0) plant, with FT being the most significantly reduced, followed by LFY, FUL, and SOC1 (Figures 2A to 2E).

Figure 2.
Figure 2.

BBX19 Suppresses the Expression of FT but Not That of CO, Independently of Known FT Transcriptional Repressors.

Total RNA was extracted from wild-type Col-0 and OE44 and OE56 lines and subjected to real-time quantitative PCR analysis. The transcript levels of each gene (CO, FT, SOC1, LFY, FUL, TEM1, FLC, and SVP) were normalized to At4g34270 (T1P41-like family protein) and At4g26410 (M3E9) measured in the same samples. Data are mean fold differences ± sd of three biological replicates each with three technical repeats. Asterisks denote significant differences (P < 0.05).

Next, we examined whether the overexpression of BBX19 shifted the balance in favor of the expression of FT transcriptional repressors, in particular TEMPRANILLO1 (TEM1), FLC, and SHORT VEGETATIVE PHASE (SVP) (Searle et al., 2006; Castillejo and Pelaz, 2008; Li et al., 2008). As shown (Figures 2F to 2H), the expression levels of these three FT transcriptional repressors in the OE44 and OE56 lines are comparable to those of the wild-type Col plants.

Collectively, the data confirmed that the BBX19-mediated repression of FT is not through modulation of the expression levels of the activator (CO) or the known suppressors of FT.

BBX19 Represses CO Activation of FT Transcription

To differentiate between transcriptional and posttranscriptional modifications, we performed Agrobacterium tumefaciens infiltration–based transient assays in Nicotiana benthamiana. Specifically, a construct containing the FT promoter region fused to firefly luciferase (PFT:LUC) was agroinfiltrated into N. benthamiana leaves alone or together with either 35S:CO or 35S:BBX19. Additionally, PFT:LUC was also coinfiltrated with the truncated BBX19 (35S:BBX19-T), which lacks the C terminus of the coding region mirroring the bbx19-2–encoded polypeptide (Figures 3A, 3C, and 3E; Supplemental Figure 3A). These data clearly demonstrate a notable induction above background LUC bioluminescence in leaves coinfiltrated with PFT:LUC and 35S:CO, in contrast with the highly reduced intensity of bioluminescence observed in leaves infiltrated with PFT:LUC together with either the full-length 35S:BBX19 or the truncated 35S:BBX19-T. This finding supports the antagonistic function of the two BBX family members, CO and BBX19, in regard to the regulation of FT expression and further verifies the functionality of the truncated polypeptide in suppressing FT promoter activity.

Figure 3.
Figure 3.

BBX19 Represses FT Transcription by Depleting the Pool of Active CO Required for the Activation of FT Transcription.

(A) and (C) Representative images of transient expression assays in N. benthamiana displayed by bright field (A) and dark field (C) of leaves expressing PFT:LUC alone, or together with 35S:CO, or with full-length (35S:BBX19), or with truncated BBX19 (35S:BBX19-T).

(B) and (D) Representative images of transient expression assays in N. benthamiana displayed by bright field (B) and dark field (D) of leaves expressing PFT:LUC either alone or together with 35S:CO or with both 35S:CO and 35S:BBX19 in a 1:1:1 or 1:10:1 ratio, respectively.

The color-coded bar displays the intensity of LUC activity.

(E) and (F) Intensities of the LUC bioluminescence presented in (C) and (D), respectively, using Andor Solis image-analysis software. Data are means ± se (n = 22). Asterisks denote significant differences (P < 0.05).

To examine the potential competition between BBX19 and CO transcription factors in modulating FT expression, we coinfiltrated PFT:LUC together with the 35S:CO and 35S:BBX19 constructs in a 1:1:1 or 1:10:1 ratio (Figures 3B, 3D, and 3F). These data clearly demonstrate the suppression of LUC bioluminescence in the presence of equal ratios of BBX19 and CO and the full reversion of this suppression with CO coinfiltrated at 10-fold higher levels than that of BBX19 (Figures 3D and 3F). This implies an antagonistic function of, as well as a potential direct competition between, these two BBX transcription factors. This competition may be the result of the binding of these two transcription factors to the same or adjacent cis-elements, or it may be caused by their physical interaction leading to depletion of the active CO pool required for the induction of FT expression.

The Box1 Motif Is Indispensable for the BBX19–CO Interaction and BBX19-Mediated Regulation of Flowering Time

Because of the established function of B-Box motifs in heterodimer formation between BBX family members and other nonfamily member proteins (Datta et al., 2007, 2008; Qi et al., 2012; Gangappa et al., 2013; Gangappa and Botto, 2014), we questioned whether BBX19 could physically interact with CO (BBX1). Thus, we exploited split luciferase complementation assays by generating fusion constructs of BBX19, CO, and PHYTOCHROME INTERACTING FACTOR5 (PIF5) as a control to N- and C-terminal fragments of luciferase. Subsequently, these constructs were used in varying combinations in agroinfiltration-based transient assays in N. benthamiana (Figures 4A and 4B). The outcome clearly shows reconstitution of luciferase activity in the leaves coinfiltrated with BBX19 and CO fusion constructs and not in the leaves coinfiltrated with the control (PIF5 and BBX19). These data were further verified using a yeast two-hybrid assay (Supplemental Figure 6). Next, we examined the role that individual B-Box motifs might play in the BBX19/CO protein–protein interaction by performing luciferase activity reconstitution assays with two independently mutated BBX19 constructs. One mutant construct contains a substituted Cys-25 to Ser in Box1 (BBX19-M1) and the other contains the analogous substitution Cys-76 to Ser in Box2 (BBX19-M2) (Figures 4A, 4B, and 5A). These data clearly show that the CO interaction with the BBX19 protein is eliminated in leaves coinfiltrated with CO and BBX19 mutated in Box1, whereas this protein–protein interaction remains unaffected by mutation in Box2.

Figure 4.
Figure 4.

BBX19 and CO Physically Interact.

Representative images of split luciferase complementation assays in N. benthamiana displayed by bright field (A) and dark field (B) of leaves expressing BBX19, CO, or PIF5 fused to N- and C-terminal fragments of luciferase.

Figure 5.
Figure 5.

Box1 Is Indispensable for BBX19-Mediated Regulation of Flowering Time.

(A) Schematic representation of B-Box motif amino acid sequences and the sites of the Cys-25 to Ser mutation in Box1 and the Cys-76 to Ser substitution in Box2.

(B) Flowering phenotypes of a line overexpressing intact BBX19 (OE44) and two lines overexpressing mutated Box1 (OEM1) and mutated Box2 (OEM2).

(C) Leaf number at flowering. Data are means ± sd (n ≥ 30). Asterisks denote significant differences (P < 0.05) as determined by Student’s t test.

[See online article for color version of this figure.]

Because of the critical role of Box1 in the BBX19–CO interaction, we next examined the role of this motif in the regulation of flowering time. Thus, we generated independent transgenic lines overexpressing BBX19 mutated in Box1 (BBX19-M1) or Box2 (BBX19-M2) with the same amino acid substitution as those used in protein–protein interaction assays (Figures 4 and 5A). Comparison of flowering time under the LD inductive photoperiod shows that while the BBX19 overexpression lines expressing intact motifs and the BBX19-M2 plants display a delay in flowering time of >2 weeks (equivalent to 15 additional leaves per plant to flowering), BBX19-M1 flowers at the same time as the wild-type plants (Figures 5B and 5C). This finding extends the indispensable role of Box1 in BBX19 protein–protein interaction to the functionality of BBX19 as a negative regulator of flowering time.

BBX19 Is Expressed in Vasculature and Colocalizes with CO in the Nucleus

To interact, BBX19 and CO must colocalize. To examine the probability of this colocalization, we employed two different approaches. In one approach, we analyzed the spatial expression pattern of BBX19 using Arabidopsis plants expressing the PBBX19:GUS construct. These data show a distinctive expression pattern of BBX19 in the vasculature of leaves, presented here at two different developmental stages (3- and 10-d-old leaves) (Figures 6A and 6B). This pattern fully coincides with the established expression patterns of not only CO but also FT in the vasculature (An et al., 2004; Kumar et al., 2012).

Figure 6.
Figure 6.

BBX19 Is Expressed in the Vasculature and Is Nuclear Colocalized with CO.

(A) and (B) Histochemical GUS staining of 3-d-old (A) and 10-d-old (B) leaves from transgenic seedlings expressing PBBX19:GUS in the Col-0 background.

(C) Representative transient assays in N. benthamiana expressing 35S:CO-YFP or 35S:BBX19-GFP, individually or in combination, displaying nuclear colocalization of these two transcription factors in the same cell.

In a second approach, we exploited colocalization assays in N. benthamiana leaves using CO-YFP (for yellow fluorescent protein) and BBX19-GFP (for green fluorescent protein) constructs (Figure 6C). The vascular expression of both CO (An et al., 2004) and BBX19 together with the nuclear colocalization of CO-YFP and BBX19-GFP provide compelling evidence for their presence in the same tissue and the same cell in planta, thus allowing their physical interaction as the mechanism by which BBX19 negatively controls flowering time.

BBX19-Mediated Delayed Flowering Is DELLA Independent

Because of the well-established role of elevated GA levels in the termination of vegetative phase and the increasing expression of flower-promoting transcription factors such as SPLs and LFY (Yamaguchi et al., 2014), we asked whether this pathway might also play a role in the BBX19 negative regulation of flowering time under LD conditions. Thus, we exogenously treated various genotypes with GA and determined that there is a partial recovery of delayed flowering in OE44 and OE56 lines in response to the GA treatment (Figures 7A and 7B). This result led us to question whether BBX19, either directly or indirectly, decelerates the GA-dependent degradation of DELLA proteins. To address this, we transformed the global-DELLA (gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4) mutant, which lacks all five DELLA proteins (Fuentes et al., 2012), and the corresponding Landsberg erecta (Ler) background with the 35S:BBX19 construct. Subsequent analyses of the various genotypes showed the anticipated early flowering of the global mutant line as compared with the corresponding Ler background (Figures 7C and 7D). By contrast, the BBX19-overexpressing Ler (Ler-OE) and global mutant (glob-OE) genotypes exhibited delayed flowering time (Figures 7C and 7D), supporting a DELLA-independent mechanism of BBX19 action.

Figure 7.
Figure 7.

BBX19-Mediated Delayed Flowering Is DELLA-Independent.

(A) Flowering phenotypes of wild-type Col-0 and BBX19 overexpression lines (OE44 and OE56) sprayed with 0.001% Triton X-100 (−GA) or with 100 µM GA3 in 0.001% Triton X-100 (+GA).

(B) Leaf number at flowering time of the mock-treated (gray bars) and GA-treated (black bars) representatives of the various genotypes.

(C) Flowering phenotypes of Ler, the global mutant, which lacks all five DELLA proteins, and the respective BBX19-overexpressing homozygous lines, Ler-OE and glob-OE.

(D) Leaf number to flowering.

In (B) and (D), data are means ± sd (n ≥ 30). Asterisks denote significant differences (P < 0.05) as determined by Student’s t test.

[See online article for color version of this figure.]

To gain further insight into the molecular basis of the partial recovery of flowering in GA-treated BBX19 overexpression lines, we next examined the expression levels of the SPL transcription factors, which activate the floral integrator genes SOC1, LFY, and FUL and thus promote flowering (Wang et al., 2009; Yamaguchi et al., 2009). We specifically focused on SPL3 and SPL9, which are well-established direct activators of floral integrator genes, as well as SPL4, which appears to control flowering time (Wu and Poethig, 2006; Wang et al., 2009). The RT-qPCR analyses showed higher basal transcript levels of SPL3 and SPL9 in the overexpression lines as compared with the wild type (Col-0), and the GA application increased these levels relative to mock treatment in all genotypes (Figures 8A and 8C). By contrast, GA application reduced the SPL4 transcript in the wild type to similar levels found in mock- and GA-treated overexpression plants (Figure 8B).

Figure 8.
Figure 8.

GA Partially Recovers the Delayed Flowering in BBX19 Overexpression Lines by Inducing LFY and FUL.

Total RNA was extracted from wild-type Col-0 and OE44 and OE56 lines mock-treated (gray bars) or GA-treated (black bars) and subjected to real-time quantitative PCR analysis. The transcript levels of the SPL transcription factors (SPL4, SPL3, and SPL9) and floral integrator genes (LFY, SOC1, and FUL) were normalized as described in Figure 2. Data are mean fold differences ± sd of three biological replicates each with three technical repeats. Asterisks denote significant differences (P < 0.05).

The overall differences in the relative expression levels of SPL genes in mock- and GA-treated Arabidopsis was verified by transient expression assays in N. benthamiana leaves agroinfiltrated with the individual SPL promoters fused to LUC (PSPL:LUC). These data, in concordance with the RT-qPCR results, showed a similar ranking of the SPL expression levels, as determined by bioluminescence intensity measurements, as a marker for the activity of their respective promoters (Supplemental Figures 7A to 7C). Specifically, we show that SPL4 displays the least basal and GA-treated bioluminescence intensity in N. benthamiana leaves, while GA induces high levels of bioluminescence intensity from the promoters of SPL3 and SPL9.

Next, we tested the expression levels of the floral identity genes LFY, SOC1, and FUL in mock- and GA-treated wild-type and overexpression lines. These data verified the earlier RT-qPCR analyses showing reduced transcript levels for all three genes in mock-treated overexpression lines as compared with the wild type (Figures 8D and 8F). However, in response to GA application, the expression levels of these genes are modified in the overexpression lines and not in the wild type. Specifically, GA application reduced the expression levels of SOC1 but induced those of LFY and FUL. The partial recovery of flowering time in GA-treated overexpression lines in spite of lower LFY and FUL transcripts, as compared with the corresponding wild-type levels, suggests that these GA-induced levels are at a threshold critical for partial recovery of the delayed flowering in overexpression lines. Furthermore, the data establish that GA application does not fully substitute for the BBX19 suppression of FT expression required for the full recovery of flowering time.

Circadian-Controlled Expression of BBX19 Is Antiphasic to the FT and CO Rhythms

The circadian regulation of CO transcript levels in conjunction with the light-induced stabilization of CO protein is an established basis for monitoring daylength and a guarantee for the induction of FT under LD inductive photoperiods (Suárez-López et al., 2001; Valverde, 2011). In view of the BBX19 physical interaction with CO, and thus the repression of FT, we questioned whether BBX19 might be circadian-regulated and how such potential rhythms might compare with those of CO and FT. In addition, we tested how reduced expression levels of BBX19 in RNAi lines might influence the levels and/or the rhythms of FT and CO transcript accumulation. Thus, we measured the transcript levels of BBX19, CO, and FT every 4 h in a 24-h cycle starting at the onset of light at 6 am in Col-0, OE44, and Ri323 lines. This high-resolution examination of the transcriptional rhythms clearly showed the expected highly reduced expression levels of BBX19 in Ri323 as opposed to the notably induced levels in OE44 lines (Figure 9A). The data further demonstrate that, in contrast with the Ri323 and OE44 lines, the wild-type Col-0 plants well display circadian regulation of BBX19 expression, peaking at noon and declining to hardly detectable levels at 6 pm, a time point coinciding with the onset of CO expression and FT transcript accumulation (Figures 9A to 9C). In addition, neither the rhythm nor the levels of CO transcript are altered in any of the three genotypes (Figure 9B). By contrast, FT expression at all time points is notably induced in Ri323 while the levels in OE44 lines are reduced, particularly at the peak time (22 pm), as compared with the Col-0 plants (Figure 9C). Moreover, in agreement with previous reports (Yanovsky and Kay, 2002; Galvão et al., 2012), high-resolution measurements showed two peaks for the accumulation of FT transcript, one at the expected time coinciding with accumulation of CO at 22 pm and the other independent of CO expression levels at 10 am. The CO-independent peak is at the lowest in the BBX19 overexpression lines, followed by a higher level in the wild-type Col-0, and reaching to notable height in the Ri323 plants. This result raises an intriguing scenario, expanding the regulatory function of BBX19 in flowering time from just depleting the active CO pool to sequestering/suppressing other as yet unknown regulator(s) of FT. Further studies using a BBX19/CO double mutant will be instrumental in furthering our understanding of the regulatory roles of BBX19 and CO in mediating the FT morning peak. Regardless, this BBX19-dependent alteration of FT transcript levels validates the functional role of BBX19 in mediating the regulation of FT expression.

Figure 9.
Figure 9.

Circadian-Controlled Expression of BBX19 Is Antiphasic to the FT and CO Rhythms.

Total RNA was extracted from the leaves of Col-0 plants collected at 4-h intervals and subjected to real-time quantitative PCR analysis. The transcript levels of BBX19, FT, and CO were normalized as described in Figure 2. The bar above the graphs indicates the light conditions, with day and night denoted in white and black, respectively. Data are mean fold differences ± sd of three biological replicates each with three technical repeats.

This finding establishes BBX19 as a negative regulator of flowering time that depletes the active CO pool during the day via its physical interaction with this activator of FT and, in turn, prevents the untimely CO activation of FT expression. Collectively, these results further provide compelling evidence for the role of BBX19 as a key circadian output to ensure a tight control over flowering time under the inductive photoperiod.

DISCUSSION

Plants have evolved a tightly controlled quantitative balance between repressors and activators to provide a robust regulation of the transition from vegetative to reproductive growth. Research in the last decade has led to the identification of a number of repressors with distinct and overlapping modes of action. The most studied repressor is FLC, which regulates flowering by quantitatively repressing the floral pathway integrators FT, SOC1, and LFY (Michaels and Amasino, 1999; Blázquez and Weigel, 2000; Samach et al., 2000). Other known repressors include TERMINAL FLOWER1, which extends the vegetative growth phase via delaying the induction of LFY and APETALA1 (Ratcliffe et al., 1998); SVP, which delays flowering but not by affecting the photoperiod or vernalization network (Hartmann et al., 2000); TARGET OF EAT1/2, proposed to control flowering time via interaction with miRNA172 (Aukerman and Sakai, 2003); TEM1 and TEM2, which are direct repressors of FT (Castillejo and Pelaz, 2008); and BBX7 (COL9), an indirect repressor of FT that suppresses CO under the inductive LD condition (Cheng and Wang, 2005).

Here, we have identified a member of the BBX family, BBX19, as a negative regulator of flowering time, which interacts with CO protein and potentially with other CO-independent regulators of FT to deplete their active pool required for FT activation and thus the promotion of flowering. This mode of function differentiates BBX19 from the two coiled-coil domain–containing proteins, SUPPRESSOR OF PHYA1 and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), which bind to, and promote, the degradation of the CO protein and hence cause a delay in flowering (Laubinger et al., 2006; Jang et al., 2008). Furthermore, the BBX19 mode of action is also distinct from that of BBX7, which reduces FT through the suppression of CO expression rather than via physical interaction with CO protein (Cheng and Wang, 2005). Indeed, the inverse relationship between FT expression levels and the transcript levels of BBX19, in contrast with unaltered CO expression levels regardless of BBX19 (Figure 9), provides additional ground for the distinct mechanisms of action of BBX19 and BBX7.

Moreover, while a truncated form of BBX19 missing the C-terminal portion of the coding region downstream of the Box1 and Box2 motifs is dispensable in repressing FT, the integrity of Box1 but not Box2 is essential for the BBX19-associated repression of FT. This validates the crucial role of Box1 in the BBX19 and CO physical interaction, and thus in the indirect repression of FT expression. The recovery of FT:LUC activity in the presence of 10-fold higher levels of CO compared with those of BBX19 is additional evidence supporting the function of BBX19 in reducing the active CO pool via physical interaction, easily permissible by the colocalization of CO and BBX19.

Collectively, these data show the crucial role of the Box1 motif in the functionality of BBX19 and the importance of the BBX19/CO balance in flowering time.

The BBX19-mediated delayed flowering is not through the suppression of GA-dependent degradation of DELLA proteins. However, GA application recovers the delayed flowering, albeit only partially, through the induction of LFY and FUL to quantitative thresholds required to trigger flowering. The lower LFY and FUL expression in BBX19 overexpression lines as compared with the corresponding wild-type levels, despite the notably elevated SPL3 and SPL9 transcript levels in overexpression plants, supports the dominant role of FT in the regulation of these genes. Moreover, the reduced level of SOC1 in the overexpression lines is not surprising and in fact supports the earlier reports indicating that SOC1 activation requires FT (Moon et al., 2005; Yoo et al., 2005; Lee and Lee, 2010).

Collectively, these data led to a simplified model by which the BBX19-mediated control of flowering time is through the depletion of CO and ultimately the repression of FT as the dominant pathway regulating SOC1 and, in turn, full induction of LFY and FUL (Figure 10).

Figure 10.
Figure 10.

Simplified Schematic Model of BBX19 Binding to CO and Indirect Suppression of FT and the Downstream Flower-Promoting Genes.

Previously, BBX19 was reported to be a circadian-regulated gene with a function in the output pathway downstream of the clock (Kumagai et al., 2008; Dally et al., 2014). Our data corroborate the circadian regulation of BBX19 and further demonstrate its antiphasic circadian rhythm compared with those of FT and CO. This suggests that BBX19 functions as a checkpoint to prevent the expression of FT soon after the onset of CO expression and the light-induced stabilization of its protein necessary for binding to the FT promoter. Intriguingly, the B2 flowering time locus of the biennial root crop sugar beet (Beta vulgaris) was recently identified as Bv-BBX19, a close homolog of Arabidopsis BBX19 (Dally et al., 2014). The authors reported that induced mutations within the second B-box of Bv-BBX19 caused the up-regulation of FLOWERING LOCUS1 (Bv-FT1), and they further proposed a model of action by which Bv-BBX19 and BOLTING TIME CONTROL1 (BTC1) complement each other and thereby acquire a CO function to regulate the downstream Bv-FT1 and Bv-FT2 genes. The alteration of Bv-FT1 levels in response to a functional mutation in Bv-BBX19 is reminiscent of the BBX19-mediated regulation of FT expression levels. Additional future analyses of the mode of action of BBX19 in sugar beet may help to determine the overlapping and distinct molecular functions of these two homologs in regulating the flowering time of annual and biennial plants.

In summary, this report expands the repertoire of FT repressors safeguarding the optimal timing of the vegetative-to-floral phase transition by avoiding precocious flowering, albeit through distinct modes of action. We specifically propose that the balance of BBX19/CO superimposed on their opposing expression rhythms functions as an additional mechanism by which plants accurately monitor the daylength and precisely time the expression of FT.

METHODS

Plant Growth Conditions and Treatment

Arabidopsis thaliana ecotype Col-0 and Ler plants were grown in LD (16/8-h photoperiod) or SD (10/14-h photoperiod) conditions when identified at 22°C. Time-course analyses were performed on 2-week-old seedlings grown on half-strength Murashige and Skoog medium.

The BBX19 T-DNA insertion lines were obtained from the ABRC (SALK_087493 and SALK_088902), and the homozygous plants were identified through PCR-based genotyping using the primers listed in Supplemental Table 1.

Exogenous application of GA was conducted by spraying 2-week-old soil-grown plants with either 100 µM GA3 (Sigma) in 0.001% Triton X-100, or Triton X-100 alone as the control, every 2 d until flowering. The number of rosette leaves and flowering days were recorded throughout the experiments.

Plasmid Construction and Generation of Transgenic Plants

The overexpression construct was prepared by amplifying BBX19 (At4g38960) from the cDNA of Col-0 using the listed primers (Supplemental Table 1) followed by cloning the DNA into pENTR-D-TOPO vector (Invitrogen) and subsequent subcloning into the Gateway vector pFAST-R05, which contains a GFP fusion at the C-terminal end and a seed-specific red fluorescent protein marker for selection (http://www.psb.ugent.be/).

For the generation of BBX19 RNAi lines, we amplified BBX19 from an Arabidopsis (Col-0) cDNA library using the primers listed in Supplemental Table 1 and subcloned the DNA fragment into the Gateway vector pFAST-R03 to obtain an RNAi construct that can form a hairpin RNA.

Substitutions of Cys-25 and Cys-76 to Ser in Box1 and Box2, respectively, were obtained using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies), with the primers listed in Supplemental Table 1.

The resulting binary vectors were used to transform various genotypes (Col-0, Ler, and glob) by the floral dip method (Clough and Bent, 1998).

Constructs used in transient assays contained 2 kb upstream of the translational start site of FT, SPL3, SPL4, and SPL9 amplified from Col-0 genomic DNA and subsequently were cloned into the pBGWL7 destination vector.

For the expression pattern analysis, the promoter of BBX19 characterized by 1.6 kb upstream of the translational start site was amplified from the genomic DNA of Arabidopsis (Col-0), followed by construction of the pBBX19:GUS construct into the pBGWFS7 destination vector.

Primer sequences used for vector construction are presented in Supplemental Table 1.

Protein–Protein Interaction and Nuclear Localization Assays

To perform protein-protein interaction analysis, p35S was cloned from pEN-L4-p35S-R1, the coding regions of BBX19, CO, and PIF5 were cloned from Arabidopsis (Col-0) cDNA, mutated BBX19 was reamplified using the mutated template described above, and the firefly luciferase fragments amino acids 1 to 416 (NLuc) and amino acids 398 to 550 (CLuc) were cloned from the pBGWL7 vector using the primers listed in Supplemental Table 1. We designed PCR primers to include 22- and 25-bp attB and attBr sites followed by at least 18 to 25 bp of gene-specific sequences. The BP reactions were subsequently performed with PCR products and corresponding donor vector pDONR221 P1-P4, pDONR221 P4r-P3r, and pDONR221 P3-P2 to generate pENTR vectors L1-35S-L4, R4-BBX19-R3, R4-BBX19-M1-R3, R4-BBX19-M2-R3, R4-CO-R3, R4-PIF5-R3, L3-NLuc-L2, and L3-NLuc-L2. Multiple LR reactions were performed to construct the binary vectors 35S:BBX19:CLuc, 35S:BBX19-M1:CLuc, 35S:BBX19-M2:CLuc, 35S:CO:NLuc, and 35S:PIF5:NLuc.

Localization studies were performed with constructs containing the BBX19 or CO coding region lacking the stop codon, which was inserted into pENTR-D-TOPO vector flanked by a pair of attL1 and attL2 combination sites. Subsequent recombination with pEN-L4-p35S-R1 and pEN-R2-YFP-L3 using multiple LR reactions together with pB7m34GW as a destination vector was performed according to MultiSite Gateway Pro (Invitrogen; http://www.psb.ugent.be/). The binary vectors generated contained 35S:CO-YFP and 35S:BBX19-GFP sequences.

Expression Analyses

Expression analyses using RT-qPCR were conducted as described previously (Walley et al., 2008). Briefly, total RNA from rosette leaves was isolated by TRIzol extraction (Life Technologies) and further purified using the Qiagen RNeasy kit with on-column DNase treatment (Qiagen) to eliminate DNA contamination. One microgram of RNA was reverse transcribed using SuperScript III (Invitrogen). RT-qPCR was performed using At4g34270 and At4g26410 as reference genes for the internal controls as described previously for transcript normalization (Walley et al., 2008). The primers used are listed in Supplemental Table 1.

The BBX19 transcript levels in the T-DNA insertion lines (bbx19-1 [SALK_088902] and bbx19-2 [SALK_087493]) were tested by 30 cycles of RT-PCR using one pair of primers to amplify the full length and the second set of primers designed to amplify the truncated fragment (BBX19-T) from the cDNA (Supplemental Figure 4). The primers used are listed in Supplemental Table 1.

GUS Histochemical Assay

For GUS staining, plants were grown on half-strength Murashige and Skoog plates in long days (16/8-h photoperiods) for 10 d. Plants were stained in buffer containing 100 mM phosphate buffer, pH 7, 10 mM EDTA, 0.1% Triton X-100, 0.5 mM potassium ferrocyanide, and 1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid at 37°C for 24 h before destaining in ethanol.

Transient Expression Assays in Nicotiana benthamiana

Overnight cultures of Agrobacterium tumefaciens GV3101 carrying various promoter constructs (FT, SPL3, SPL4, and SPL9) fused to LUC, 35S-BBX19:GFP, 35S-BBX19-T:GFP, or 35S-CO:YFP were prepared in infiltration medium (2 mM Na3PO4, 50 mM MES, 0.5% Glc, and 100 μM acetosyringone) at OD600 = 0.2. The cultures were spot-infiltrated into 6- to 7-week-old N. benthamiana leaves. The competition assays were performed with cultures of PFT:LUC plus 35S:BBX19-GFP and 35S:CO-YFP at a ratio of either 1:1:1 or 1:1:10, respectively.

Yeast Two-Hybrid Assay

The yeast mating-based two-hybrid system was provided kindly by Siobhan Brady (University of California, Davis). Full-length cDNAs of BBX19, BBX19-M, and CO were cloned into the pENTR/D-TOPO vector (Invitrogen), and upon sequence verification, they were subcloned into the Gateway vector Y-AD or X-DB using LR reaction to generate the bait and prey plasmids. The plasmids were then transformed into MaV103 and MaV203 yeast strains. Yeast mating and selection were performed as described previously, in the absence of Leu, Trp, His, and uracil, in X-Gal–containing solid medium (Walhout and Vidal, 2001).

Luciferase Imaging

Luciferase imaging was performed as described previously (Walley et al., 2007) using a CCD camera (Andor Technology) 48 to 96 h after infiltration. Images were acquired every 10 min for 90 min, and luciferase activity was quantified as mean counts per pixel per exposure time using Andor Solis image-analysis software (Andor Technology).

Statistical Analyses

These analyses were performed by two-tailed Student’s t tests. The difference was considered significant at P < 0.050.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: SPL3 (AT2G33810), SPL4 (AT1G53160), SPL9 (AT2G42200), CO (BBX1; AT5G15840), FT (AT1G65480), SOC1 (AT2G45660), LFY (AT5G61850), FUL (AT5G60910), and BBX19 (AT4G38960).

Supplemental Data

The following materials are available in the online version of this article.

Acknowledgments

We thank Andrew Groover, Jinzheng Wang, Marta Bjornson, and Geoff Benn for their critical review of the article and Lars Ostergaard for providing the DELLA global mutant seeds. This work was supported by the National Institutes of Health (Grant R01GM107311 to K.D.) and the National Science Foundation (Grants IOS-1036491and IOS-1352478 to K.D.).

AUTHOR CONTRIBUTIONS

C.-Q.W. planned and executed the experimental procedures. C.G. and M.K.S. executed the experiments. K.D. provided guidance and prepared the article.

Footnotes

  • www.plantcell.org/cgi/doi/10.1105/tpc.114.130252

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Katayoon Dehesh (kdehesh{at}ucdavis.edu).

  • ↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.

  • ↵[W] Online version contains Web-only data.

Glossary

LD
long-day
SD
short-day
MEcPP
methylerythritol cyclodiphosphate
RT-qPCR
reverse transcription and quantitative PCR
Col-0
Columbia-0
RNAi
RNA interference
Ler
Landsberg erecta
  • Received July 19, 2014.
  • Revised August 25, 2014.
  • Accepted September 3, 2014.
  • Published September 16, 2014.

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BBX19 Interacts with CONSTANS to Repress FLOWERING LOCUS T Transcription, Defining a Flowering Time Checkpoint in Arabidopsis
Chang-Quan Wang, Cade Guthrie, Mostafa Khoshhal Sarmast, Katayoon Dehesh
The Plant Cell Sep 2014, 26 (9) 3589-3602; DOI: 10.1105/tpc.114.130252
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