Orphan Gene BR2 connects vernalization to the floral integrator BrSOC1c to delay flowering in Chinese cabbage

Orphan genes ( OGs ) are usually species specific and lack recognizable domains, posing challenges for their functional analysis. Flowering time is a key agronomic trait affecting the yield and adaptability of Chinese cabbage ( Brassica rapa ). However, the molecular mechanism by which Chinese cabbage OGs regulate flowering remains unclear. Here, we verified that OG BOLTING RESISTANCE 2 ( BR2 ) is a key inhibitory factor for flowering in Chinese cabbage. The BR2 loss-of-function mutant ‘ br2-M ’, exhibited a significantly earlier flowering time. In contrast, the BR2 overexpression line ‘ BR2OE ’ delayed flowering. Further analysis showed that the expression of BR2 was directly repressed by vernalization treatment. To clarify its mechanism, we used yeast two-hybrid library screening and protein interaction experiments and found that BR2 specifically bound to the MADS-box domain of flowering integrator BrSOC1c. Molecular mechanism studies showed that BrSOC1c directly bound to the promoter of downstream flowering gene BrLFYb and activated its transcription, whereas BR2 significantly inhibited its transcriptional activation ability by interacting with BrSOC1c. Vernalization treatment relieved the inhibitory effect of BR2 on the BrSOC1c– BrLFYb pathway, promoting flowering. In conclusion, this study identified BR2 as a negative regulatory factor induced by vernalization that delays the flowering process by directly inhibiting the activity of BrSOC1c . These results uncover the molecular mechanism underlying vernalization-mediated flowering regulation in Chinese cabbage.