B-box (BBX) zinc finger proteins play critical roles in both vegetative and reproductive development in plants. Many BBX proteins have been identified in Arabidopsis thaliana as floral transition regulatory factors, such as CO, BBX7 (COL9), BBX19, and BBX32. BBX32 is involved in flowering time control through repression of COL3 in Arabidopsis thaliana, but it is still elusive that whether and how BBX32 directly interacts with flowering signal integrators of AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) in Chinese cabbage (Brassica rapa L. ssp. pekinensis) or other plants. In this study, B-box-32(BBX32), a transcription factor in this family with one B-box motif was cloned from B. rapa, acted as a circadian clock protein, showing expression changes during the circadian period. Additional experiments using GST pull-down and yeast two-hybrid assays indicated that BrBBX32 interacts with BrAGL24 and does not interact with BrSOC1, while BrAGL24 does interact with BrSOC1. To investigate the domains involved in these protein-protein interactions, we tested three regions of BrBBX32. Only the N-terminus interacted with BrAGL24, indicating that the B-box domain may be the key region for protein interaction. Based on these data, we propose that BrBBX32 may act in the circadian clock pathway and relate to the mechanism of flowering time regulation by binding to BrAGL24 through the B-box domain. This study will provide valuable information for unraveling the molecular regulatory mechanisms of BrBBX32 in flowering time of B. rapa.
Newly Described Components and Regulatory Mechanisms of Circadian Clock Function in Arabidopsis thaliana