CiBBX19 negatively regulates the flowering time of Chrysanthemum indicum by recruiting CiBBX5 to inhibit the expression of CiFTL3.

Flowering is a critical stage in plant growth and reproduction. Chrysanthemum indicum, a species with both ornamental and medicinal value, has its flowering time directly affecting the quality of inflorescences. This study elucidates the molecular mechanism by which the BBX transcription factor family members CiBBX19 and CiBBX5 coordinately regulate the flowering time of Chrysanthemum indicum. Phylogenetic analysis reveals that CiBBX19 is the ortholog of AtBBX19, with its expression significantly downregulated during the vegetative-to-reproductive transition in leaves. Functional validation demonstrates that overexpression of CiBBX19 delays flowering, while its silencing accelerates this developmental transition, confirming its biological role in maintaining vegetative growth. Through qPCR time-course analysis and genetic transformation experiments, we identified the short-day responsive florigen gene CiFTL3 in Chrysanthemum indicum. The expression of CiFTL3 surges during the flower bud differentiation stage, and its overexpression advances the flowering time. The expression of CiFTL3 significantly decreases in CiBBX19-overexpressing lines, but yeast one-hybrid and LUC assays confirm that CiBBX19 does not directly bind to the CiFTL3 promoter. Protein interaction analysis reveals that CiBBX19 forms a heterodimer with CiBBX5. Although CiBBX5 exhibits stable expression during the developmental transition period, it specifically binds to the G-box cis-element (TACGTG) of the CiFTL3 promoter, thereby suppressing the expression of CiFTL3 and delaying the flowering in Chrysanthemum indicum. Through dual-luciferase reporter assays, we established that CiBBX19-CiBBX5 heterodimerization synergistically enhances transcriptional repression of CiFTL3, exceeding the inhibitory effect mediated by CiBBX5 alone. Our experiment showed that CiBBX19 can interact with CiBBX5 to indirectly inhibit the transcription of CiFTL3. This synergistic regulatory mechanism provides new insights into unraveling the dynamic balance between vegetative growth and reproductive development in Asteraceae plants and lays a molecular foundation for precise regulation of Chrysanthemum indicum flowering time.