An inappropriate flowering time and drought stress resistance in Brassica juncea (mustard) will decrease its yield and quality. B-box (BBX) transcription factors play an important role in the regulation of flowering and drought tolerance, but the biological functions and molecular mechanisms of the BBX family—especially BjuBBX6—remain largely unknown in Brassica juncea. In this study, we cloned the BjuBBX6-1 gene from Brassica juncea and found that it was expressed mainly in flowers and leaves. BjuBBX6-1 was localized in the nucleus and had transcriptional activation activity. Overexpressing BjuBBX6-1 caused earlier flowering compared with the wild type, while BjuBBX6-1 silencing in RNAi lines led to later flowering. BjuBBX6-1 interacted with flowering factors BjuNF-YB2 and BjuNF-YB3 and subsequently bound to the promoters of its downstream genes to promote BjuABI2 expression. However, it repressed BjuFLC and BjuGLK expression, resulting in earlier flowering. Under drought stress, overexpressing BjuBBX6-1 plants reduced chlorophyll content and antioxidant enzyme activities, and increased malondialdehyde (MDA) content, while silencing BjuBBX6-1 plants exhibited the opposite trend. The results indicate that BjuBBX6-1 negatively regulates drought tolerance in Brassica juncea. This study lays a theoretical foundation for in-depth research on the molecular mechanism of the BBX family in flowering regulation and drought tolerance breeding of Brassica juncea.

Photoperiod and temperature are crucial factors that trigger flowering in Brassica juncea (B. juncea). However, the underlying regulatory mechanisms remain poorly understood. The MADS-box transcription factor AGL18 acts as a pivotal repressor of floral transition and functions redundantly with AGL15. In this study, we isolated BjuAGL18-1 from B. juncea and identified two unique transcripts, resulting in two distinct proteins: a full-length protein, BjuAGL18-1L, and a truncated protein, BjuAGL18-1S. Further investigation showed that the two isoforms had similar subcellular localizations but different expression patterns in various plant tissues. Notably, BjuAGL18-1L and BjuAGL18-1S were abundantly induced under short- and long-day photoperiods, respectively. BjuAGL18-1L overexpression in B. juncea and Arabidopsis thaliana (A. thaliana) led to late flowering, whereas BjuAGL18-1S overexpression resulted in early flowering. Yeast two-hybrid, bimolecular fluorescent complementation, and luciferase complementation assays showed that BjuAGL18-1L, but not BjuAGL18-1S (which lacked the EAR motif), interacted with the co-repressor BjuAFR2 and the histone deacetylase BjuHDA9 to form a multiprotein complex. Further analysis indicated that BjuAGL18-1L could also form a complex with BjuAGL15 and bind to the BjuFUL promoter, thus inhibiting its expression. However, BjuAGL18-1S interacted with BjuAGL18-1L to form heterodimers, which attenuated their activities, likely by disrupting their binding to target genes, resulting in accelerated flowering progression. These results suggest that BjuAGL18-1 is involved in photoperiod-induced flowering via different regulatory mechanisms in B. juncea.