Flowering is critical to the success of plant propagation. The MYB family transcription factor CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) is an essential component of the core loop of the circadian clock and plays a crucial role in regulating plant flowering time. In this study, we found that photoperiod affects the expression pattern and expression level of BcCCA1, which is delayed flowering time under short-day conditions in Pak-choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis]. We detected overexpression and silencing of BcCCA1 in Pak-choi, resulting in delayed and promoted flowering time, respectively. Furthermore, we also discovered that FLOWERING LOCUS C (BcFLC) and SUPPRESSOR OF CONSTANS1 (BcSOC1) were expressed significantly differently in BcCCA1 overexpression and silencing plants compared with control plants. Therefore, we further investigated the interaction relationship between BcCCA1, BcFLC, and BcSOC1, and the results showed that BcCCA1 and BcFLC as a complex interacted with each other. Moreover, both BcCCA1 and BcFLC can directly bind to the promoter of BcSOC1 and repress its transcription, and BcCCA1 can form a complex with BcFLC to enhance the transcriptional inhibition of BcSOC1 by BcFLC. This study reveals a new mechanism by which the circadian clock regulates flowering time.

Brassinosteroids (BRs) play critical roles in plant growth by promoting cell elongation and division, leading to increased leaf inclination angles. BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMS-SUPPRESSOR 1 (BES1) act as transcription factors in the brassinosteroid signaling pathway and are involved in several physiological activities regulated by BRs. In this study, we identified and cloned BcBZR1 from the heitacai non-heading Chinese cabbage (NHCC) cultivar. The sequence analysis showed that the coding sequence length of BcBZR1 is 996 bp, encoding 331 amino acid residues. Subcellular localization assays showed that BcBZR1 is localized in the nucleus and cytoplasm and that BcBZR1 protein is transported to the nucleus after receiving BR signals. Compared with Col-0, the leaf inclination angle was smaller in BcBZR1-OX. The EBR treatment experiment indicated that BRs regulate the differential expression of paclobutrazol resistance1 (PRE1) and ILI1 binding bHLH1 (IBH1) in the adaxial and abaxial cells of the petiole through BZR1, thus regulating the leaf inclination angle. The bimolecular fluorescence complementation (BiFC) assay indicated that BcBZR1 interacts with C-repeat Binding Factor2 (BcCBF2) and CBF3. Taken together, our findings not only validate the function of BcBZR1 in leaf inclination angle distribution in non-heading Chinese cabbage, but also contribute to the mechanism of leaf inclination angle regulation in this species under cold stress.

The primary domain/leucine zipper (bZIP) transcription factor, Elongated Hypocotyl (HY5), is crucial for the photomorphogenesis of seedlings. Here, we identified BcHY5 as a regulator of hypocotyl length from the non-heading Chinese cabbage (NHCC) variety ‘Suzhouqing’, which encoded a protein comprised 164 amino acid residues. Ectopic expression of BcHY5 in Arabidopsis shortens the length of the hypocotyl. Additionally, we discovered a protein called BcBBX24 containing the B-BOX (BBX) domain, which is the interacting partner of BcHY5. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and GST pull-down assays revealed that BcHY5 interacted with BcBBX24. Additionally, by physically binding to the promoter of BcHY5, BcBBX24 inhibited the transcriptional activity of BcHY5. Therefore, our findings reveal a transcriptional mechanism through which light response occurs in NHCC seedlings, where BcHY5 can interact with BcBBX24 and BcBBX24 can prevent BcHY5 transcription.

Leaves are the primary organs for plant photosynthesis, and their flat, symmetric morphology is crucial for plant growth and development. The LBD family transcription factor ASYMMETRIC LEAVES 2 (AS2) plays a central role in the establishment of leaf polarity. In this study, we cloned the BcAS2 gene from the non-heading Chinese cabbage cultivar “NHCC001” and successfully generated overexpression strains through genetic transformation. Phenotypic analysis revealed that overexpression of BcAS2 led to significant upward curling of leaves in non-heading Chinese cabbage. Additionally, we found that the expression of BcPHB, a gene associated with leaf adaxial polarity development, was significantly up-regulated in BcAS2-overexpressing plants compared to controls. This interaction was further confirmed through yeast one-hybridization (Y1H), dual-luciferase reporter assays, and electrophoretic mobility shift assay (EMSA), all of which demonstrated that BcAS2 directly binds to the GATA-motif site of the BcPHB promoter and promotes its transcription. Functional validation via overexpression and silencing of BcPHB confirmed its role in regulating adaxial polarity development in non-heading Chinese cabbage leaves. This study elucidates the molecular mechanism of the BcAS2-BcPHB pathway in regulating leaf polarity in non-heading Chinese cabbage, providing a theoretical foundation for morphological improvement breeding.