The SWEET family, which includes transcripts of a cohort of plant hexose and sucrose transporters, is considered key to improving crop stress tolerance and yield through its role in manipulating the carbohydrate partitioning process. The functions and regulatory roles of this gene family are variable among different species; thus, to determine these roles, more species-specific information is needed. Brassica rapa displays complicated regulation after a whole-genome triplication (WGT) event, which provides enormous advantages for use in genetic studies, thus it is an ideal model for exploring the functional and regulatory roles of SWEETs from a genetic perspective. In this study, the results of a homology search and phylogenetic relationship analysis revealed the evolutionary footprint of SWEETs among different plant taxa, which showed that plant SWEETs may have originated from Clade II and then expanded from vascular plants. The amino acid sequence characteristics and an analysis of the exon-intron structure of BrSWEETs duplicates clarified that SWEETs retention occurred after a WGT event in B. rapa. An analysis of the transcriptional levels of BrSWEETs in different tissues identified the expression differences among duplicated co-orthologs. In addition, qRT-PCR indicated that the BrSWEETs’ co-orthologs were varied in their stress responses. This study greatly enriches our knowledge of SWEETs in the B. rapa species, which will contribute to future studies on the Brassica-specific regulatory pathways and to creating genetic innovations.

As a reducing substance, ascorbic acid functioned well in abiotic and biotic stress. However, the regulatory mechanism of drought resistance is rarely known in pak choi. Here we found a gene BcSRC2 containing a C2 domain that responds to ABA signal and drought regulation in pak choi. Silencing of BcSRC2 reduces ascorbic acid content and drought resistance of pak choi. In Arabidopsis, BcSRC2 overexpression promotes ascorbic acid accumulation and increases drought tolerance. Meanwhile, transcriptome analysis between WT and BcSRC2-overexpressing pak choi suggests that ascorbic acid-related genes are regulated. BcSRC2 interacts with BcAPX4 and inhibit APX activity in vitro and in vivo, increasing the ascorbic acid content. We also found that drought stress increases ABA content, which reduces the expression of BcMYB30. BcMYB30 bound to the promoter of BcSRC2 and reduced its expression. Overall, our results suggest that a regulatory module, BcMYB30-BcSRC2-BcAPX4, plays a central role in increasing ascorbic acid content for responding ABA-mediated drought regulation in pak choi.

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.

As the storage organ of Chinese cabbage, the leafy head was harvested as a commercial product due to its edible value. In this study, the bulked segregant analysis (BSA) and bulked segregant RNA-Seq (BSR) were performed with F₂ separation progeny to study the molecular mechanism of leafy head formation in Chinese cabbage. BSA-Seq analysis located four candidate regions containing 40 candidate genes, while BSR-Seq analysis revealed eight candidate regions containing 607 candidate genes. The conjunctive analyses of these two methods identified that Casein kinase gene BrCKL8 (Bra035974) is the common candidate gene related with leafy head formation in Chinese cabbage, and it showed high expression levels at the three segments of heading type plant leaves. The differentially expressed genes (DEGs) between two set pairs of cDNA sequencing bulks were divided into two categories: one category was related with five hormone pathways (Auxin, Ethylene, Abscisic acid, Jasmonic acid and Gibberellin), the other category was composed of genes that associate with the calcium signaling pathway. Moreover, a series of upregulated transcriptional factors (TFs) were also identified by the association analysis of BSR-Seq analysis. The leafy head development was regulated by various biological processes and effected by diverse external environment factors, so our research will contribute to the breeding of perfect leaf-heading types of Chinese cabbage.

The transcription factor WRKY33 is a vital regulator of the biological process of the necrotrophic fungus Botrytis cinerea (B. cinerea). However, its specific regulatory mechanism remains to be further investigated. In non-heading Chinese cabbage (NHCC, Brassica campestris (syn. Brassica rapa) ssp. Chinensis), our previous study showed that BcWRKY33A is induced not only by salt stress, but also by B. cinerea infection. Here, we noticed that BcWRKY33A is expressed in trichomes and confer plant defense resistance. Disease symptoms and qRT-PCR analyses revealed that BcWRKY33A-overexpressing and -silencing lines were less and more severely impaired, respectively, than wild type upon B. cinerea treatment. Meanwhile, the transcripts’ abundance of indolic glucosinolates’ (IGSs) biosynthetic genes is consistent with plants’ B. cinerea tolerance. Identification and expression pattern analysis of BcMYB51s showed that BcMYB51-3 has a similar trend to BcWRKY33A upon B. cinerea infection. Moreover, BcWRKY33A directly binds to the BcMYB51-3 promoter, which was jointly confirmed by Y1H, dual-LUC, and EMSA assays. The importance of MYB51, the homolog of BcMYB51-3, in the BcWRKY33A-mediated B. cinerea resistance was also verified using the TRV-based VIGS system. Overall, our data concludes that BcWRKY33A directly activates the expression of BcMYB51-3 and downstream IGSs’ biosynthetic genes, thereby improving the B. cinerea tolerance of NHCC plants.

Heterosis plays a significant role in enhancing variety, boosting yield, and raising economic value in crops, but the molecular mechanism is still unclear. We analyzed the transcriptomes and 3D genomes of a hybrid (F₁) and its parents (w30 and 082). The analysis of the expression revealed a total of 485 specially expressed genes (SEGs), 173 differentially expressed genes (DEGs) above the parental expression level, more actively expressed genes, and up-regulated DEGs in the F₁. Further study revealed that the DEGs detected in the F₁ and its parents were mainly involved in the response to auxin, plant hormone signal transduction, DNA metabolic process, purine metabolism, starch, and sucrose metabolism, which suggested that these biological processes may play a crucial role in the heterosis of Brassica rapa. The analysis of 3D genome data revealed that hybrid F₁ plants tend to contain more transcriptionally active A chromatin compartments after hybridization. Supplementaryly, the F₁ had a smaller TAD (topologically associated domain) genome length, but the number was the highest, and the expression change in activated TAD was higher than that of repressed TAD. More specific TAD boundaries were detected between the parents and F₁. Subsequently, 140 DEGs with genomic structural variants were selected as potential candidate genes. We found two DEGs with consistent expression changes in A/B compartments and TADs. Our findings suggested that genomic structural variants, such as TADs and A/B chromatin compartments, may affect gene expression and contribute to heterosis in Brassica rapa. This study provides further insight into the molecular mechanism of heterosis in Brassica rapa.