FLOWERING LOCUS T Promoter Research Articles

Two B-box domain proteins, BBX28 and BBX29, regulate flowering time at low ambient temperature in Arabidopsis

This paper demonstrates that BBX28 and BBX29 proteins in Arabidopsis promote flowering in association with the CO-FT regulatory module at low ambient temperature under LD conditions. Flowering plants integrate internal developmental signals with external environmental stimuli for precise flowering time control. The expression of BBX29 is up-regulated by low temperature treatment, but the biological function of BBX29 in low temperature response is unknown. In the current study, we examined the biological role of BBX29 and its close-related protein BBX28 in flowering time control under long-day conditions. Although neither BBX28 single mutant nor BBX29 single mutant has a flowering-associated phenotype, the bbx28 bbx29 double mutant plants have an obvious delayed flowering phenotype grown at low ambient temperature (16°C) compared to the wild-type (WT) plants. The expression of FT and TSF was lower in bbx28 bbx29 double mutant plants than in wild-type plants at 16°C. Both BBX28 and BBX29 interact with CONSTANS (CO), an important flowering integrator that directly binds to the FLOWERING LOCUS T (FT) promoter. In the effector-reporter assays, transcriptional activation activity of CO on the FT promoter was reduced in bbx28 bbx29 double mutant plants compared to that in WT plants. Taken together, our results reveal that BBX28 and BBX29 are promoters of flowering in Arabidopsis, especially at low ambient temperature.

Structural insights into the multivalent binding of the Arabidopsis FLOWERING LOCUS T promoter by the CO-NF-Y master transcription factor complex.

Flowering plants sense various environmental and endogenous signals to trigger the floral transition and start the reproductive growth cycle. CONSTANS (CO) is a master transcription factor in the photoperiod floral pathway that integrates upstream signals and activates the florigen gene FLOWERING LOCUS T (FT). Here, we performed comprehensive structural and biochemical analyses to study the molecular mechanism underlying the regulation of FT by CO in Arabidopsis thaliana. We show that the four previously characterized cis-elements in the FT promoter proximal region, CORE1, CORE2, P1, and P2, are all direct CO binding sites. Structural analysis of CO in complex with NUCLEAR FACTOR-YB/YC (NF-YB/YC) and the CORE2 or CORE1 elements revealed the molecular basis for the specific recognition of the shared TGTG motifs. Biochemical analysis suggested that CO might form a homomultimeric assembly via its N-terminal B-Box domain and simultaneously occupy multiple cis-elements within the FT promoter. We suggest that this multivalent binding gives the CO-NF-Y complex high affinity and specificity for FT promoter binding. Overall, our data provide a detailed molecular model for the regulation of FT by the master transcription factor complex CO-NF-Y during the floral transition.

Arabidopsis CIB3 regulates photoperiodic flowering in an FKF1-dependent way.

Arabidopsis cryptochrome 2 (CRY2) and FLAVIN-BINDING, KELCH REPEAT, and F-BOX 1 (FKF1) are blue light receptors mediating light regulation of growth and development, such as photoperiodic flowering. CRY2 interacts with a basic helix-loop-helix transcription factor CIB1 in response to blue light to activate the transcription of the flowering integrator gene FLOWERING LOCUS T (FT). CIB1, CIB2, CIB4, and CIB5 function redundantly to promote flowering in a CRY2-dependent way and form various heterodimers to bind to the noncanonical E-box sequence in the FT promoter. However, the function of CIB3 has not been described. We discovered that CIB3 promotes photoperiodic flowering independently of CRY2. Moreover, CIB3 does not interact with CRY2 but interacts with CIB1 and functions synergistically with CIB1 to promote the transcription of the GI gene. FKF1 is required for CIB3 to promote flowering and enhances the CIB1-CIB3 interaction in response to blue light.

Characterization and Activity Analyses of the FLOWERING LOCUS T Promoter in Gossypium Hirsutum.

Flowering transition is a crucial development process in cotton (Gossypium hirsutum L.), and the flowering time is closely correlated with the timing of FLOWERING LOCUS T (FT) expression. However, the mechanism underlying the coordination of various cis-regulatory elements in the FT promoter of cotton has not been determined. In this study, a 5.9-kb promoter of FT was identified from cotton. A bioinformatics analysis showed that multiple insertion–deletion sites existed in the 5.9-kb promoter. Different expression levels of a reporter gene, and the induction by sequential deletions in GhFT promoter, demonstrated that 1.8-kb of the GhFT promoter was stronger than 4.2-, 4.8-, and 5.9-kb promoter fragments. The binding sites of the CONSTANS (CO) and NUCLEAR FACTOR Y transcription factors were located within the 1.0-kb sequence upstream of the FT transcription start site. A large number of repeat segments were identified in proximal promoter regions (−1.1 to −1.4 kb). A complementation analysis of deletion constructs between 1.0 and 1.8 kb of G. hirsutum, Gossypium arboretum, and Gossypium raimondii FT promoters revealed that the 1.0-kb fragment significantly rescued the late-flowering phenotype of the Arabidopsis FT loss-of-function mutant ft-10, whereas the 1.8-kb promoter only slightly rescued the late-flowering phenotype. Furthermore, the conserved CORE motif in the cotton FT promoter is an atypical TGTG(N2-3)ATG, but the number of arbitrary bases between TGTG and ATG is uncertain. Thus, the proximal FT promoter region might play an important role affecting the activity levels of FT promoters in cotton flowering.

Targeted DNA methylation represses two enhancers of FLOWERING LOCUS T in Arabidopsis thaliana.

FLOWERING LOCUS T (FT) plays a major role in regulating the floral transition in response to an inductive long day photoperiod in Arabidopsis thaliana. Expression of FT in leaves is dependent on the distal transcriptional enhancer Block C, located 5-kilobases (kb) upstream of the transcriptional start site (TSS). We expressed an inverted repeat of Block C to induce local DNA methylation and heterochromatin formation, which lead to FT downregulation in an inductive photoperiod. Using targeted DNA methylation as a tool to uncover further regulatory regions at the FT locus, we identified Block E, located 1 kb downstream of the gene, as a novel enhancer of FT. As Block C, Block E is conserved across Brassicaceae and located in accessible chromatin. Block C and E act as additive transcriptional enhancers that, in combination with the proximal FT promoter, control expression of FT in response to photoperiod in the leaf phloem.

CIB1 and CO interact to mediate CRY2-dependent regulation of flowering.

Cryptochromes are photolyase-like photoreceptors. Arabidopsis CRY2 (cryptochrome 2) primarily mediates the photoperiodic regulation of floral initiation. CRY2 has been shown to promote FT (FLOWERING LOCUS T) mRNA expression in response to blue light by suppressing the degradation of the CO (CONSTANS) protein and activating CIB1 (CRY2-interacting bHLH1). Although CIB1 and CO are both transcriptional activators of FT, their relationship is unknown. Here, we show that CIB1 physically interacts with CO and promotes FT transcription in a CO-dependent manner. CRY2, CIB1, and CO form a protein complex in response to blue light to activate FT transcription, and the complex is regulated by the photoperiod and peaks at dusk along with higher FT expression. We also determined that CRY2 was recruited to the FT chromatin by CIB1 and CO and that all three proteins are bound to the same region within the FT promoter. Therefore, there is crosstalk between the CRY2-CO and CRY2-CIBs pathways, and CIB1 and CO act together to regulate FT transcription and flowering.

The NUCLEAR FACTOR-CONSTANS complex antagonizes Polycomb repression to de-repress FLOWERING LOCUS T expression in response to inductive long days in Arabidopsis.

Many plants sense the seasonal cues, day length or photoperiod changes, to align the timing of the developmental transition to flowering with changing seasons for reproductive success. Inductive day lengths through the photoperiod pathway induce the expression of FLOWERING LOCUS T (FT) or FT relatives that encode a major mobile florigen to promote flowering. In Arabidopsis thaliana, under inductive long days the photoperiod pathway output CONSTANS (CO) accumulates toward the end of the day, and associates with the B and C subunits of Nuclear Factor Y (NF-Y) to form the NF-CO complex that acts to promote FT expression near dusk, whereas Polycomb group (PcG) proteins function to silence FT expression. How NF-CO acts to antagonize the function of PcG proteins to regulate FT expression remains unclear. Here, we show that the NF-CO complex bound to the proximal FT promoter, through chromatin looping, acts in concert with an NF-Y complex bound to a distal enhancer to reduce the levels of PcG proteins, including both Polycomb repressive complex 1 (PRC1) and PRC2 at the FT promoter, leading to a relieving of Polycomb silencing and thus FT de-repression near dusk. Thus, our study provides molecular insights on how the 'active' photoperiod pathway and the 'repressive' Polycomb silencing system interact to control temporal FT expression, conferring the long-day induction of flowering in Arabidopsis.

FE Controls the Transcription of Downstream Flowering Regulators Through Two Distinct Mechanisms in Leaf Phloem Companion Cells.

In the facultative long-day plant Arabidopsis thaliana, FLOWERING LOCUS T (FT), encoding the mobile hormone florigen, plays an essential role in modulating the optimal timing of flowering to ensure reproductive success. Under inductive long-day conditions, the transcription of FT is activated by the CONSTANS (CO)/NUCLEAR FACTOR-Y (NF-Y) protein complex in leaf phloem companion cells. FT is transported to the shoot apical meristem through interaction with florigen transporters, such as SODIUM POTASSIUM ROOT DEFECTIVE 1 (NaKR1). Some regulators involved in photoperiod-dependent FT function have been reported previously; however, the molecular mechanism that coordinates FT protein synthesis and transport efficiently needs to be investigated. The present study examined the role of an Myb-related transcription factor, FE, in the activation of FT gene transcription and FT protein transport. Expression analysis using FE-inducible systems and chromatin immunoprecipitation assays showed that FE directly bound to the FT and NaKR1 promoters and activated the transcription of downstream target genes. FE failed to activate FT expression without CO function, whereas FE-mediated NaKR1 induction was not affected by CO function. Taken together, our data indicate that FE regulates the transcription of FT and florigen transporter genes via different mechanisms.

CYCLING DOF FACTOR 1 represses transcription through the TOPLESS co-repressor to control photoperiodic flowering in Arabidopsis.

CYCLING DOF FACTOR 1 (CDF1) and its homologs play an important role in the floral transition by repressing the expression of floral activator genes such as CONSTANS (CO) and FLOWERING LOCUS T (FT) in Arabidopsis. The day-length-specific removal of CDF1-dependent repression is a critical mechanism in photoperiodic flowering. However, the mechanism by which CDF1 represses CO and FT transcription remained elusive. Here we demonstrate that Arabidopsis CDF proteins contain non-EAR motif-like conserved domains required for interaction with the TOPLESS (TPL) co-repressor protein. This TPL interaction confers a repressive function on CDF1, as mutations of the N-terminal TPL binding domain largely impair the ability of CDF1 protein to repress its targets. TPL proteins are present on specific regions of the CO and FT promoters where CDF1 binds during the morning. In addition, TPL binding increases when CDF1 expression is elevated, suggesting that TPL is recruited to these promoters in a time-dependent fashion by CDFs. Moreover, reduction of TPL activity induced by expressing a dominant negative version of TPL (tpl-1) in phloem companion cells results in early flowering and a decreased sensitivity to photoperiod in a manner similar to a cdf loss-of-function mutant. Our results indicate that the mechanism of CDF1 repression is through the formation of a CDF-TPL transcriptional complex, which reduces the expression levels of CO and FT during the morning for seasonal flowering.

In silico Functional Analysis of FLC and FT-Genes Responsible for Postponingand Accelerating the Onset of Flowering

In silico functional analyses of FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT) genes were investigated herein. The recognition of cis-regulatory modules and their organization are a prerequisite to information on the regulation of gene expression. Accordingly, this study carried out a promoter analysis on FT and FLC genes in Arabidopsis and its orthologs in Prunus persica by PlantPan and PLANCARE servers in which the integrating transcription factor binding site (TFBs) was involved in hormonal and stress signals. Light responsive elements were also studied. Among the responsive elements identified in FT and FLC, Arabidopsis had the highest number of responsive elements. This is partly because P. persica is day-neutral, whereas Arabidopsis is a facultative long day plant. The results of motif recognition in FT and FLC proteins with the MEME server showed that FT is highly conserved, but the number of motifs suggests that the FLC protein had far more numerous motifs, which is likely due to its higher amino acid length. Some specified TFs like AGAMOUS (AG), AGAMOUS LIKE (AGL), LEAFY (LFY) and APETALA1 (AP1) were found by the PlantPan web server in FT and FLC promoters’ analysis. They were also present in the protein-protein interaction network analysis which raises the question whether or not FT or FLC can be positive or negative regulators of their own expression in a feedback loop.

NUCLEAR FACTOR Y, Subunit A (NF-YA) Proteins Positively Regulate Flowering and Act Through FLOWERING LOCUS T.

Photoperiod dependent flowering is one of several mechanisms used by plants to initiate the developmental transition from vegetative growth to reproductive growth. The NUCLEAR FACTOR Y (NF-Y) transcription factors are heterotrimeric complexes composed of NF-YA and histone-fold domain (HFD) containing NF-YB/NF-YC, that initiate photoperiod-dependent flowering by cooperatively interacting with CONSTANS (CO) to drive the expression of FLOWERING LOCUS T (FT). This involves NF-Y and CO binding at distal CCAAT and proximal “CORE” elements, respectively, in the FT promoter. While this is well established for the HFD subunits, there remains some question over the potential role of NF-YA as either positive or negative regulators of this process. Here we provide strong support, in the form of genetic and biochemical analyses, that NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box in the FT promoter and are positive regulators of flowering in an FT-dependent manner.

WRKY71 accelerates flowering via the direct activation of FLOWERING LOCUS T and LEAFY in Arabidopsis thaliana.

Flowering is crucial for achieving reproductive success. A large number of well-delineated factors affecting flowering are involved in complex genetic networks in Arabidopsis thaliana. However, the underlying part played by the WRKY transcription factors in this process is not yet clear. Here, we report that WRKY71 is able to accelerate flowering in Arabidopsis. An activation-tagged mutant WRKY71-1D and a constitutive over-expresser of WRKY71 both flowered earlier than the wild type (WT). In contrast, both the RNA interference-based multiple WRKY knock-out mutant (w71w8 + 28RNAi) and the dominant repression line (W71-SRDX) flowered later. Gene expression analysis showed that the transcript abundance of the flowering time integrator gene FLOWERING LOCUS T (FT) and the floral meristem identity genes LEAFY (LFY), APETALA1 (AP1) and FRUITFULL (FUL) were greater in WRKY71-1D than in the WT, but lower in w71w8 + 28RNAi and W71-SRDX. Further, WRKY71 was shown to bind to the W-boxes in the FT and LFY promoters in vitro and in vivo. The suggestion is that WRKY71 activity hastens flowering via the direct activation of FT and LFY.

FLOWERING LOCUS T has higher protein mobility than TWIN SISTER OF FT.

In plants, successful reproduction requires the proper timing of flowering under changing environmental conditions. Arabidopsis FLOWERING LOCUS T (FT), which encodes a proposed phloem-mobile florigen, has a close homologue, TWIN SISTER OF FT (TSF). During the vegetative phase, TSF shows high levels of expression in the hypocotyl before FT induction, but the tsf mutation does not have an apparent flowering-time phenotype on its own under long-day conditions. This study compared the protein mobility of FT and TSF. With TSF-overexpressing plants as the rootstock, the flowering time of ft tsf scion plants was only slightly accelerated. Previous work has shown that FT is graft-transmissible; by contrast, this study did not detect movement of TSF from the roots into the shoot of the scion plants. This study used plants overexpressing FT/TSF chimeric proteins to map a region responsible for FT movement. A chimeric TSF with region II of FT (L28 to G98) expressed in the rootstock caused early flowering in ft tsf scion plants; movement of the chimeric protein from the rootstocks into the shoot apical region of the ft tsf scion plants was also detected. Misexpression of TSF in the leaf under the control of the FT promoter or grafting of 35S::TSF cotyledons accelerated flowering of ft-10 plants. FT was more stable than TSF. Taking these results together, we propose that protein mobility of FT is higher than that of TSF, possibly due to a protein domain that confers mobility and/or protein stability.

Induced and natural variation of promoter length modulates the photoperiodic response of FLOWERING LOCUS T.

FLOWERING LOCUS T (FT) regulates the floral transition in many plant species by integrating environmental seasonal signals and internal cues. Here we show that two interdependent regulatory regions are necessary and sufficient to convey photoperiod responsiveness to FT. While a minimal distance between the regulatory regions is required to fully suppress FT expression under short days, increased distance reduces promoter response to long days. Natural variation at FT creating promoter length differences is widespread, correlates with longitudinal and latitudinal clines and affects a promoter region physically interacting with both photoperiod control regions. Three major FT promoter variants correlate with differences in FT allele usage in F1 hybrids. We propose that FT variation in cis could be adaptive by conferring differences in FT transcriptional control ultimately translating to increased fitness.

A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis

For many plant species, reproductive success relies on the proper timing of flowering, and photoperiod provides a key environmental input. Photoperiod-dependent flowering depends on timely expression of FLOWERING LOCUS T (FT); however, the coordination of various cis-regulatory elements in the FT promoter is not well understood. Here, we provide evidence that long-distance chromatin loops bring distal enhancer elements into close association with the proximal promoter elements bound by CONSTANS (CO). Additionally, we show that NUCLEAR FACTOR Y (NF-Y) binds a CCAAT box in the distal enhancer element and that CCAAT disruption dramatically reduces FT promoter activity. Thus, we propose the recruitment model of photoperiod-dependent flowering where NF-Y complexes, bound at the FT distal enhancer element, help recruit CO to proximal cis-regulatory elements and initiate the transition to reproductive growth.

Elevated Levels of MYB30 in the Phloem Accelerate Flowering in Arabidopsis through the Regulation of FLOWERING LOCUS T

In Arabidopsis thaliana, the R2R3 MYB-like transcription factor MYB30 is a positive regulator of the pathogen-induced hypersensitive response and of brassinosteroid and abscisic acid signaling. Here, we show that MYB30 expressed under the control of the strong phloem-specific SUC2 promoter accelerates flowering both in long and short days. Early flowering is mediated by elevated expression of FLOWERING LOCUS T (FT), which can be observed in the absence and presence of CONSTANS (CO), the main activator of FT. CO-independent activation by high MYB30 expression results in FT levels that remain below those observed in the wild-type plants, which show an additive CO-dependent activation. In contrast, TWIN SISTER OF FT (TSF) is repressed in plants expressing high levels of MYB30 in the phloem. In transient assays, MYB30 and CO additively increase the activity of a reporter construct driven by a 1 kb FT promoter. Acceleration of flowering by MYB30 does not require the presence of salicylic acid and is independent of FLC. Taken together, increased levels of MYB30, which was reported to be induced in response to the perception of pathogens, can accelerate flowering and MYB30 may thus be a candidate to mediate cross-talk between gene networks involved in biotic stress perception and flowering time.

Multiple bHLH Proteins form Heterodimers to Mediate CRY2-Dependent Regulation of Flowering-Time in Arabidopsis

Arabidopsis thaliana cryptochrome 2 (CRY2) mediates light control of flowering time. CIB1 (CRY2-interacting bHLH 1) specifically interacts with CRY2 in response to blue light to activate the transcription of FT (Flowering Locus T). In vitro, CIB1 binds to the canonical E-box (CACGTG, also referred to as G-box) with much higher affinity than its interaction with non-canonical E-box (CANNTG) DNA sequences. However, in vivo, CIB1 binds to the chromatin region of the FT promoter, which only contains the non-canonical E-box sequences. Here, we show that CRY2 also interacts with at least CIB5, in response to blue light, but not in darkness or in response to other wavelengths of light. Our genetic analysis demonstrates that CIB1, CIB2, CIB4, and CIB5 act redundantly to activate the transcription of FT and that they are positive regulators of CRY2 mediated flowering. More importantly, CIB1 and other CIBs proteins form heterodimers, and some of the heterodimers have a higher binding affinity than the CIB homodimers to the non-canonical E-box in the in vitro DNA-binding assays. This result explains why in vitro CIB1 and other CIBs bind to the canonical E-box (G-box) with a higher affinity, whereas they are all associated with the non-canonical E-boxes at the FT promoter in vivo. Consistent with the hypothesis that different CIB proteins play similar roles in the CRY2-midiated blue light signaling, the expression of CIB proteins is regulated specifically by blue light. Our study demonstrates that CIBs function redundantly in regulating CRY2-dependent flowering, and that different CIBs form heterodimers to interact with the non-canonical E-box DNA in vivo.

BAC Library Development and Clone Characterization for Dormancy-Responsive DREB4A, DAM, and FT from Leafy Spurge (Euphorbia esula) Identifies Differential Splicing and Conserved Promoter Motifs

We developed two leafy spurge bacterial artificial chromosome (BAC) libraries that together represent approximately 5× coverage of the leafy spurge genome. The BAC libraries have an average insert size of approximately 143 kb, and copies of the library and filters for hybridization-based screening are publicly available through the Arizona Genomics Institute. These libraries were used to clone full-length genomic copies of an AP2/ERF transcription factor of the A4 subfamily of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEINS (DREB) known to be differentially expressed in crown buds of leafy spurge during endodormancy, a DORMANCY ASSOCIATED MADS-BOX (DAM) gene, and several FLOWERING LOCUS T (FT) genes. Sequencing of these BAC clones revealed the presence of multiple FT genes in leafy spurge. Sequencing also provided evidence that two different DAM transcripts expressed in crown buds of leafy spurge during endo- and eco-dormancy result from alternate splicing of a single DAM gene. Sequence data from the FT promoters was used to identify several conserved elements previously recognized in Arabidopsis, as well as potential novel transcription factor binding sites that may regulate FT. These leafy spurge BAC libraries represent a new genomics-based tool that complements existing genomics resources for the study of plant growth and development in this model perennial weed. Furthermore, phylogenetic footprinting using genes identified with this resource demonstrate the usefulness of studying weedy species to further our general knowledge of agriculturally important genes.

ThemicroRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3Module Regulates Ambient Temperature-Responsive Flowering viaFLOWERING LOCUS Tin Arabidopsis

The flowering time of plants is affected by modest changes in ambient temperature. However, little is known about the regulation of ambient temperature-responsive flowering by small RNAs. In this study, we show that the microRNA156 (miR156)-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) module directly regulates FLOWERING LOCUS T (FT) expression in the leaf to control ambient temperature-responsive flowering. Overexpression of miR156 led to more delayed flowering at a lower ambient temperature (16°C), which was associated with down-regulation of FT and FRUITFULL expression. Among miR156 target genes, SPL3 mRNA levels were mainly reduced, probably because miR156-mediated cleavage of SPL3 mRNA was higher at 16°C. Overexpression of miR156-resistant SPL3 [SPL3(-)] caused early flowering, regardless of the ambient temperature, which was associated with up-regulation of FT and FRUITFULL expression. Reduction of miR156 activity by target mimicry led to a phenotype similar to that of SUC2::rSPL3 plants. FT up-regulation was observed after dexamethasone treatment in GVG-rSPL3 plants. Misexpression and artificial microRNA-mediated suppression of FT in the leaf dramatically altered the ambient temperature-responsive flowering of plants overexpressing miR156 and SPL3(-). Chromatin immunoprecipitation assay showed that the SPL3 protein directly binds to GTAC motifs within the FT promoter. Lesions in TERMINAL FLOWER1, SHORT VEGETATIVE PHASE, and EARLY FLOWERING3 did not alter the expression of miR156 and SPL3. Taken together, our data suggest that the interaction between the miR156-SPL3 module and FT is part of the regulatory mechanism controlling flowering time in response to ambient temperature.

CONSTANS and ASYMMETRIC LEAVES 1 complex is involved in the induction ofFLOWERING LOCUS Tin photoperiodic flowering in Arabidopsis

Plants monitor changes in day length to coordinate flowering with favorable seasons to increase their fitness. The day-length specific induction of FLOWERING LOCUS T (FT) regulated by CONSTANS (CO) is the crucial aspect of photoperiodic flowering in Arabidopsis thaliana. Recent studies have elucidated some mechanisms of CO-dependent FT induction. Here, we demonstrate another mechanism of CO-dependent FT regulation. Our results indicate that CO protein partially regulates FT transcription by forming a complex with ASYMMETRIC LEAVES 1 (AS1) protein, which regulates leaf development partly by controlling gibberellin (GA) levels. We identified AS1 as a CO-interacting protein in yeast and verified their interaction in vitro and in planta. We also showed that the temporal and spatial expression pattern of AS1 overlapped with that of CO. In addition, as1 mutants showed GA-independent delayed flowering under different light/dark conditions. FT expression levels in the as1 mutants and the SUC2:CO-HA/as1 line under long-day and 12-h light/12-h dark conditions were reduced compared with wild-type plants and the SUC2:HA-CO line, respectively. Moreover, AS1 bound directly to the specific regions of the FT promoter in vivo. These results indicate that CO forms a functional complex with AS1 to regulate FT expression and that AS1 plays different roles in two regulatory pathways, both of which concomitantly regulate the precise timing of flowering.