The Cm14-3-3μ protein and CCT transcription factor CmNRRa delay flowering in chrysanthemum.

Floral transition from vegetative to reproductive growth is pivotal in the plant life cycle. NUTRITION RESPONSE AND ROOT GROWTH (OsNRRa) as a CONSTANS, CONSTANS-LIKE, TOC1 (CCT) domain protein delays flowering in rice and an orthologous gene CmNRRa inhibits flowering in chrysanthemum; however, the mechanism remains unknown. In this study, using yeast two-hybrid screening, we identified the 14-3-3 family member Cm14-3-3µ as a CmNRRa-interacting protein. Biochemical assays using a combination of bimolecular fluorescence complementation (BiFC), pull-down, and Co-immunoprecipitation (Co-IP) were performed to confirm the physical interaction between CmNRRa and Cm14-3-3µ in chrysanthemum. In addition, expression analysis showed that CmNRRa, but not Cm14-3-3µ, responded to the diurnal rhythm, whereas both genes were highly expressed in the leaves. Moreover, the function in flowering time regulation of Cm14-3-3µ is similar to that of CmNRRa. Furthermore, CmNRRa repressed chrysanthemum FLOWERING LOCUS T-like 3 (CmFTL3) and APETALA 1 (AP1)/FRUITFULL (FUL)-like gene (CmAFL1), but induced TERMINAL FLOWER1 (CmTFL1) directly by binding to their promoters. Cm14-3-3µ enhanced the ability of CmNRRa to regulate the expression of these genes. These findings suggest that there is a synergistic relationship between CmNRRa and Cm14-3-3µ in flowering repression in chrysanthemum.