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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 .

Many MCM1-AGAMOUS-DEFICIENS-SRF (MADS) genes have been proved to play an important role in the flowering time regulation of plants. The flowering-inhibiting factor AGAMOUS-LIKE 18 (AGL18) integrates into the two flowering-activating factors SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and AGAMOUS-LIKE 24 (AGL24), which play an important role during the plant developmental stages of the flowering pathway. However, it remains unknown whether and how the AGL18 protein directly interacts with SOC1 and/or AGL24 genes to regulate flowering time in Brassica juncea. In this study, three members (AGL18-1 in florescence, AGL18-2 and AGL18-3 in young seedlings) of the AGL18 family, and SOC1 and AGL24 in florescence were cloned in Brassica juncea. Yeast One-Hybrid assays and Dual-Glo® Luciferase assays showed that the SOC1 and AGL24 promoters interacted only with AGL18-1 protein, not AGL18-2 and AGL18-3. The typical conserved structure of the M-domain of AGL18-1 was the key region that mediated the interaction between the AGL18-1 protein and SOC1 promoter, and the I-domain, K-domain and C-domain did not regulate the interaction of AGL18-1/SOC1. In contrast, the K-domain and M-domain in AGL18-1 could mediate the interaction between the AGL18-1 protein and AGL24 promoter. This indicated that the AGL18-1 protein must have its unique functions that differed from AGL18-2 and AGL18-3. This work provides valuable information for in-depth studies into the molecular mechanisms of the AGL18 protein with SOC1 and AGL24 for flowering time control of Brassica juncea.