Molecular basis of CONSTANS oligomerization in FLOWERING LOCUS T activation

The transcription factor CONSTANS (CO) integrates day-length information to induce the expression of florigen FLOWERING LOCUS T (FT) in Arabidopsis. We recently reported that the C-terminal CCT domain of CO forms a complex with NUCLEAR FACTOR-YB/YC to recognize multiple cis-elements in the FT promoter, and the N-terminal tandem B-box domains form a homomultimeric assembly. However, the mechanism and biological function of CO multimerization remained unclear. Here, we report that CO takes on a head-to-tail oligomeric configuration via its B-boxes to mediate FT activation in long days. The crystal structure of B-boxes reveals a closely connected tandem B-box fold forming a continuous head-to-tail assembly through unique CDHH zinc fingers. Mutating the key residues involved in CO oligomerization resulted in a non-functional CO, as evidenced by the inability to rescue co mutants. By contrast, a transgene encoding a human p53-derived tetrameric peptide in place of the B-boxes rescued co mutant, emphasizing the essential role of B-boxes-mediated oligomerization. Furthermore, we found that the four TGTG-bearing cis-elements in FT proximal promoter are required for FT activation in long days. Our results suggest that CO forms a multimer to bind to the four TGTG motifs in the FT promoter to mediate FT activation.