The photoperiod is a key environmental factor regulating flowering in the short-day plant chrysanthemum, and the molecular mechanisms of key genes responding to short-day conditions remain incompletely elucidated. This study focused on CmELF4-LIKE 3, a chrysanthemum homolog of the core photoperiod-sensing gene EARLY FLOWERING 4 (ELF4). CmELF4-LIKE 3 displayed circadian expression patterns under both long-day (LD) and short-day (SD) conditions, peaking 8 h after dawn, with significantly higher expression under SD than LD. The gene was highly expressed in stems and leaves, and subcellular localization confirmed its nuclear localization. Genetic transformation experiments showed that overexpression of CmELF4-LIKE 3 accelerated flower bud appearance by 3-5 days compared to the wild type, whereas RNAi lines delayed bud emergence by 4-8 days, demonstrating that CmELF4-LIKE 3 promotes flowering in chrysanthemum. Yeast two-hybrid (Y2H) and tobacco luciferase complementation (LUC) assays both verified a physical interaction between CmELF4-LIKE 3 and CmPIF7, a key component of the light signaling pathway. Quantitative real-time PCR (qRT-PCR) analysis revealed significant changes in the expression of downstream flowering-related genes CmCOL5 and CmCO in CmELF4-LIKE 3 transgenic lines. Additionally, tobacco transient LUC assays demonstrated that CmPIF7 activates the CmCOL5 promoter. In summary, this study established that CmELF4-LIKE 3 interacts with CmPIF7 to cooperatively regulate short-day-induced flowering in chrysanthemum. These findings not only provide key genetic resources for precise flowering time manipulation in chrysanthemum, but also offer new insights into the crosstalk between the circadian clock and light signaling pathways in plants.
