CmCOL4-CmAFL1-CmSEP3 Integrates Photoperiod Signals to Regulate Chrysanthemum Flowering

Chrysanthemum morifolium is an important ornamental crop whose flowering is strictly regulated by photoperiod. However, the molecular mechanisms linking photoperiod signals to floral meristem identity and organ development in Asteraceae remain unclear. In this study, we identified and characterized CmAFL1, a FUL-like MADS-box gene specific to the Asteraceae family, in the short-day cultivar ‘Jinba’. CmAFL1 expression is upregulated under short-day conditions, primarily expressed in the shoot apical meristem during flower bud differentiation and predominantly in the receptacle during organ development. RNA interference-mediated silencing of CmAFL1 resulted in delayed flowering, arrested inflorescence development, malformed tubular florets, plant dwarfism, and excessive lateral branching, indicating its pleiotropic roles in flowering time, plant architecture, and floral organ patterning. At the molecular level, yeast one-hybrid assays revealed that the photoperiod pathway transcription factor CmCOL4 directly binds to the promoter region of CmAFL1. Furthermore, yeast one-hybrid and dual-luciferase assays confirmed that CmAFL1 directly interacts with and activates the promoter of the E-class floral organ identity gene CmSEP3. Together, our findings identify a novel transcriptional regulatory module CmCOL4-CmAFL1-CmSEP3 that potentially links photoperiod signaling to floral meristem identity and organ specification in Chrysanthemums. While further in planta validation is warranted, these findings provide novel insights into photoperiodic flowering regulation in Asteraceae and may serve as a theoretical foundation for molecular breeding strategies targeting flowering time manipulation in ornamental Chrysanthemums.