Plants flower in an appropriate season to allow sufficient vegetative development and to position flower development in favorable environments.The Arabidopsis CO and FKF1 genes promote flowering by inducing FT expression in the long-day afternoon.The CO protein is present in the morning but could not activate FT expression due to unknown negative mechanisms,referred to as the "morning gate",which prevent premature flowering.The AP2/EREBP genes participate in development and stress responses,including inhibition of flowering by the TOE genes.To probe the conservation and divergence of AP2/EREBP genes,we analyzed the duplication patterns of this family in Brassicaceae.Some AP2/EREBP duplicates generated early in Brassicaceae history were quickly lost,while others were retained in all tested Brassicaceae species,suggesting early functional divergence followed by persistent conservation.Furthermore,we used 16 representative Arabidopsis AP2/EREBP proteins as baits and identifies 1,970 potential AP2/EREBP-interacting proteins,with a small subset of interactions verified in planta.The putative AP2-interacting proteins participate in many functions in development and stress responses,including photomorphogenesis,flower development,drought and cold responses,abscisic acid and auxin signaling.In particular,TOE1 and related proteins interact with the activation domain of CO and COLs and inhibit CO activity.TOE1 binds to the FT promoter near the CO-binding site and reducing TOE function results in a morning peak of the FT mRNA.In addition,TOE1 interacts with FKF1 and likely interferes with the FKF1-CO interaction,resulting in degradation of the CO protein in afternoon to prevent premature flowering.Our results uncovered that TOE proteins are the molecular keepers of the "morning gate" and indicate that positive and negative regulators coordinate to precisely regulate flowering time,thereby ensuring maximum reproduction in response to environmental signals.