Fig. 1

CO forms homodimers via B-box motifs. A, C. Schematic representations of truncated CO proteins. B, D, F. Yeast two-hybrid assays mapping minimum binding motifs required for CO homodimerization. Various combinations of indicated truncated CO proteins were expressed in yeast. LacZ (β-galactosidase) activity was quantified and represented interaction strength. Similar means were obtained for all three independent assays. E. Yeast cells were grown on SD agar media (1) lacking Trp (T), Leu (L), and His (H) in the presence of 5 mM 3-AT (denoted as SD -TLH + 5 mM 3AT) or (2) lacking T, L, H, and Ade (A) (denoted as SD -TLAH). G. GST pull-down assays confirming interactions in yeast. GST-fused full-length CO (CO 1–373) and truncated CO proteins were pulled down along with 6XHis-fused CO 1–373. Proteins were visualized by western blotting against anti-GST and anti-6XHis antibodies. H In vivo CO 1–132 variant homodimerization to validate interactions observed in vitro and in yeast. The YFP N-terminal fragment (N-YFP) and the YFP C-terminal fragment (C-YFP) were fused with CO 1–132 variant. N-YFP and C-YFP fusion constructs constitutively expressing N-YFP-CO 1–132 and C-YFP-CO 1–132 were transfected into Arabidopsis mesophyll protoplasts. DAPI (4′,6-diamidino-2-phenylindole) was used for nuclear staining