J3-AFP2 Antagonism as a Novel Layer of CO/FT Regulation in Flowering Time Control

Plants perceive daylength through photoreceptors and relay this environmental cue into flowering signals via the photoperiodic pathway (Rahul et al. 2024). CONSTANS (CO), a phloem-specific B-box transcription factor, serves as a central hub integrating signals from the photoperiodic and other pathways to activate flowering (Qu et al. 2025). CO directly binds to the promoter of FLOWERING LOCUS T (FT), the florigen gene expressed in the phloem, to promote its transcription (Lv et al. 2021). Once synthesised, the FT protein moves from the source leaf to the shoot apical meristem (SAM), where it interacts with the bZIP transcription factor FD and activates flowering (Chen et al. 2018). CO activity is tightly regulated at multiple levels including transcription, protein stability, and interactions with partner proteins, to ensure FT induction only under permissive long-day (LD) conditions (Takagi et al. 2023). Another important gene regulating flowering is the TOPLESS-related protein2 (TPR2). TPR2 is a transcriptional repressor from the TOPLESS family (Plant et al. 2021). It interacts with the promoter region of the FT protein and suppresses the transcription of the gene (Chang et al. 2019).

Abscisic acid (ABA), a key hormone in abiotic stress responses, has also been implicated in flowering regulation. ABA-responsive transcription factors such as ABI4/5 upregulate FLOWERING LOCUS C (FLC), a floral repressor (Wang et al. 2013). Members of the ABI5-BINDING PROTEIN (AFP) family, particularly AFP2, directly recruit the TPR2 co-repressor, interact with CO, and inhibit FT transcription through histone deacetylation (Chang et al. 2019). AFP2 also cooperates with COP1 to promote CO degradation during the night. These dual transcriptional and posttranslational mechanisms integrate ABA signalling into the photoperiodic flowering pathway.

Recently, Zhi et al. (2025) used a yeast two-hybrid (Y2H) system to screen potential AFP2-interacting proteins, and J3 was identified (Zhi et al. 2025). To investigate the role of J3, the flowering phenotypes of j3 loss-of-function mutants and J3 overexpression lines were examined. Under both LD and short-day (SD) conditions, j3 mutants exhibited significantly delayed flowering, while J3 overexpression lines flowered earlier compared to the wild-type controls. These results indicate that J3 functions as a positive regulator of flowering time.

Previous studies showed that J3 interacts with the MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) to promote the expression of SOC1 and FT, thus accelerating flowering (Shen et al. 2011). Beyond this transcriptional activation via SVP, the current study reveals that J3 also influences flowering by antagonising the function of AFP2. AFP2 is known to promote CO degradation, leading to delayed flowering. Overexpression of AFP2 delayed flowering, whereas co-overexpression of J3 with AFP2 partially rescued this late-flowering phenotype. Conversely, j3 mutation strengthened the late-flowering phenotype caused by AFP2 overexpression. Immunoblot analyses showed that CO protein levels were significantly higher in plants overexpressing both AFP2 and J3 compared to plants overexpressing AFP2 alone. Similarly, in the afp2 mutant background, CO protein levels were higher than in the afp2 j3 double mutant. These results demonstrate that J3 counteracts AFP2-mediated CO degradation, contributing to positive regulation of flowering time.

To further investigate the mechanism by which J3 antagonises AFP2, a yeast three-hybrid assay was performed. The results showed that J3 interferes with the interaction between AFP2 and CO. In the absence of J3, AFP2 efficiently interacted with CO, promoting CO degradation. In the presence of J3, the AFP2-CO interaction was disrupted, thereby stabilising CO protein (Figure 1). This suggests that J3 stabilises CO not only by directly binding AFP2 but also by competitively inhibiting AFP2-CO complex formation, thus promoting flowering through maintenance of CO protein levels.

AFP2 has also been reported to repress FT expression by reducing histone H3 acetylation at the FT locus (Chang et al. 2019). To determine whether J3 affects this epigenetic regulation, chromatin immunoprecipitation (ChIP) assays were conducted. Compared to the wild-type control, CO-HA plants exhibited elevated histone H3 acetylation at the proximal FT promoter region, while AFP2-Flag and j3-1 mutants showed reduced acetylation. Notably, J3-GFP and J3-GFP AFP2-Flag plants displayed higher acetylation levels than AFP2-Flag plants alone, correlating with increased FT transcription and earlier flowering. These results suggest that J3 mitigates AFP2-mediated repression of FT by enhancing histone H3 acetylation at the FT promoter.

Together, this study reveals new mechanisms by which J3 regulates flowering time: stabilising CO protein by antagonising AFP2-mediated degradation and promoting FT expression through modulation of histone H3 acetylation (Figure 1). Given that AFP2 recruits TPR2 to regulate CO transcription and cooperates with COP1 to mediate CO degradation, similar approaches could be used to explore potential interactions among J3, TPR2, and COP1. Such studies could further elucidate the broader regulatory role of J3 in flowering. It also remains intriguing to investigate whether the physical interaction between J3 and AFP2 and their competition at the FT promoter represent two distinct yet synergistic processes in regulating FT expression and flowering.

The authors declare no conflicts of interest.