The purpose of this study was to explore the regulation of BcMYB44 on anthocyanin synthesis and drought tolerance of non-heading Chinese cabbage. The BcMYB44 gene was cloned from the purple inbred line ‘NJZX1-3’ and its green mutant ‘NJZX1-0’. Sequence analysis confirmed that BcMYB44 belongs to the R2R3-MYB family and has the highest homology with BnMYB44. Subcellular localization revealed that BcMYB44 is a nuclear protein. Yeast two-hybrid (Y2H) and Bimolecular Fluorescent Complimentary (BiFC) experiments showed that BcMYB44 interacts with BcPAP1 and BcEGL3. Pigment detection of BcPAP1 and BcMYB44 protein activity in N. benthamiana indicates that BcMYB44 plays a negative regulatory role by inhibiting the expression of key structural genes (F3H, DFR, etc.) in anthocyanin synthesis. Virus-induced gene silencing (VIGS) further confirmed this inhibition. Analysis of drought tolerance of non-heading Chinese cabbage based on VIGS showed that pTY-S plants are more resistant to drought than pTY-BcMYB44 plants. The results indicate that BcMYB44 has a positive regulatory role in drought stress, which most likely is achieved by inhibiting anthocyanin accumulation, regulating stomatal movement, and improving osmotic regulation and homeostasis of reactive oxygen species (ROS).

Non-heading Chinese cabbage (Brassica campestris ssp. chinensis) is an important vegetative crop in the south of China. As an antioxidant, anthocyanin is the major quality trait for vegetables with purple leaves or petioles. However, the molecular biosynthetic mechanism of anthocyanin in non-heading Chinese cabbage has not been explained exclusively. In this study, two non-heading Chinese cabbage with contrasting colors in the leaves were used as the materials for RNA-seq. A total of 906 DEGs were detected, and we found that the anthocyanin and flavonoid biosynthetic pathways are significantly enriched in the purple NHCC. The transcriptome result was verified by RT-qPCR. Though bioinformatics analysis, BcTT8 was selected as the candidate gene for the regulation of anthocyanin synthesis, and the characterization of BcTT8 was elucidated by the functional analyses. The results proved that BcTT8 is a nucleus protein and phylogenetically close to the TT8 protein from Brassica. After silencing BcTT8, the total anthocyanin content of pTY-BcTT8 plants decreased by 42.5%, and the relative expression levels of anthocyanin pathway genes BcDFR, BcLODX and BcUF3GT-1 were significantly downregulated, while the transcription level of BcFLS was significantly upregulated. Compared with the wild type, the transgenic Arabidopsis showed obvious violet in the cotyledons part, and the anthocyanin biosynthetic genes such as AtDFR and AtLODX were significantly upregulated. In conclusion, BcTT8 is critical in the anthocyanin synthesis process of non-heading Chinese cabbage. Our findings illustrated the molecular mechanism of anthocyanin biosynthesis in non-heading Chinese cabbage.

Pak choi exhibits a diverse color range and serves as a rich source of flavonoids and terpenoids. However, the mechanisms underlying the heterosis and coordinated regulation of these compounds—particularly isorhamnetin—remain unclear. This study involved three hybrid combinations and the detection of 528 metabolites from all combinations, including 26 flavonoids and 88 terpenoids, through untargeted metabolomics. Analysis of differential metabolites indicated that the heterosis for the flavonoid and terpenoid contents was parent-dependent, and positive heterosis was observed for isorhamnetin in the two hybrid combinations (SZQ, 002 and HMG, ZMG). Moreover, there was a high transcription level of flavone 3′-O-methyltransferase, which is involved in isorhamnetin biosynthesis. The third group was considered the ideal hybrid combination for investigating the heterosis of flavonoid and terpenoid contents. Transcriptome analysis identified a total of 12,652 DEGs (TPM > 1) in various groups that were used for comparison, and DEGs encoding enzymes involved in various categories, including “carotenoid bio-synthesis” and “anthocyanin biosynthesis”, were enriched in the hybrid combination (SZQ, 002). Moreover, the category of anthocyanin biosynthesis also was enriched in the hybrid combination (HMG, ZMG). The flavonoid pathway demonstrated more differential metabolites than the terpenoid pathway did. The WGCNA demonstrated notable positive correlations between the dark-green modules and many flavonoids and terpenoids. Moreover, there were 23 ERF genes in the co-expression network (r ≥ 0.90 and p < 0.05). Thus, ERF genes may play a significant role in regulating flavonoid and terpenoid biosynthesis. These findings enhance our understanding of the heterosis and coordinated regulation of flavonoid and terpenoid biosynthesis in pak choi, offering insights for genomics-based breeding improvements.

Polyploid plants generally demonstrate enhanced photosynthetic capacity compared to their diploid ancestors; however, the mechanisms underlying this phenomenon remain largely unexplored. This study utilizes pak choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis] as the material to investigate the molecular mechanisms underlying enhanced photosynthesis in autotetraploid. Our findings indicate that autotetraploid pak choi leaves exhibit increased thickness, enlarged intercellular spaces, and cell dimensions, as well as augmented accumulation of thylakoids and grana. Photosynthesis data indicate that the net photosynthesis (Pn) of autotetraploid plants is significantly greater than that of diploid plants. Transcriptome and miRNA sequencing revealed that the differentially expressed genes were significantly enriched in the photosynthesis thylakoid pathway. Joint analysis revealed that novel-miRNA117 regulates BcLhcb1 and BcLhcb4.2, whereas Bc-miR403-5p regulates BcLhcb4.2. The downregulation of BcLhcb1, BcLhcb2.1, and BcLhcb4.2 resulted in reduced Pn in pak choi, whereas their overexpression increased the growth of Arabidopsis and increased plant weight, which was attributed to the tight stacking of thylakoid granules in the overexpressing plants. Additionally, we observed weakened growth in Arabidopsis overexpressing novel-miRNA117 and Bc-miR403-5p, which was consistent with the phenotype resulting from the silencing of BcLhcbs. These findings further demonstrated that miRNA117 regulates BcLhcb1 and BcLhcb4.2, whereas Bc-miR403-5p regulates BcLhcb2.1. This study enhances our understanding of the molecular mechanisms underlying photosynthesis in homogenous autotetraploid pak choi. Furthermore, it provides novel insights into the regulatory roles of miRNAs in polyploid photosynthesis and contributes to the molecular breeding of pak choi.