Copper is a mineral element, which is necessary for the normal growth and development of plants, but high levels of copper will seriously damage plants. Studies have shown that AtGR1 improves the tolerance of Arabidopsis to aluminum and cadmium stress. However, the role of GR in the copper stress response of plants is still unclear. Here, we identified four genes (named BcGR1.1, BcGR1.2, BcGR2.1 and BcGR2.2, respectively) encoding glutathione reductase (GR) in non-heading Chinese cabbage (Brassica campestris (syn. Brassica rapa) ssp. chinensis), which could be divided into two types based on the subcellular localization. Among them, BcGR1.1, which belonged to the cytoplasmic localization type, was significantly upregulated under copper stress. Compared to WT (the wild type), Arabidopsis thaliana heterologously overexpressed BcGR1.1 had longer roots, higher fresh weight, higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and accumulated more superoxide dismutase and peroxidase under copper stress. However, in the AsA-GSH cycle under copper stress, the contents of AsA and AsA/DHA were significantly downregulated, and the contents of DHA and T-AsA (total AsA) were upregulated, in the BcGR1.1-overexpressing Arabidopsis. Therefore, BcGR1.1 could improve the scavenging ability of reactive oxygen species (ROS) by increasing the activity of GR, antioxidant enzymes and the utilization of AsA, and then enhance the copper stress tolerance of plants.

Flavonols have been shown to respond to a variety of abiotic stresses in plants, including cold stress. Higher total flavonoid content was found in non-heading Chinese cabbage (NHCC, Brassica campestris (syn. Brassica rapa) ssp. chinensis) after cold stress. A non-targeted metabolome analysis showed a significant increase in flavonol content, including that of quercetin and kaempferol. Here, we found that an R2R3–MYB transcription factor, BcMYB111, may play a role in this process. BcMYB111 was up-regulated in response to cold treatment, with an accompanying accumulation of flavonols. Then, it was found that BcMYB111 could regulate the synthesis of flavonols by directly binding to the promoters of BcF3H and BcFLS1. In the transgenic hairy roots of NHCC or stable transgenic Arabidopsis, overexpression of BcMYB111 increased flavonol synthesis and accumulation, while these were reduced in virus-induced gene silencing lines in NHCC. After cold stress, the higher proline content and lower malondialdehyde (MDA) content showed that there was less damage in transgenic Arabidopsis than in the wild-type (WT). The BcMYB111 transgenic lines performed better in terms of antioxidant capacity because of their lower H2O2 content and higher superoxide dismutase (SOD) and peroxidase (POD) enzyme activities. In addition, a key cold signaling gene, BcCBF2, could specifically bind to the DRE element and activate the expression of BcMYB111 in vitro and in vivo. The results suggested that BcMYB111 played a positive role in enhancing the flavonol synthesis and cold tolerance of NHCC. Taken together, these findings reveal that cold stress induces the accumulation of flavonols to increase tolerance via the pathway of BcCBF2–BcMYB111–BcF3H/BcFLS1 in NHCC.

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.