OCTOPUS regulates BIN2 to control leaf curvature in Chinese cabbage.

Heading is one of the most important agronomic traits for Chinese cabbage crops. During the heading stage, leaf axial growth is an essential process. In the past, most genes predicted to be involved in the heading process have been based on leaf development studies in Arabidopsis. No genes that control leaf axial growth have been mapped and cloned via forward genetics in Chinese cabbage. In this study, we characterize the inward curling mutant ic1 in Brassica rapa ssp. pekinensis and identify a mutation in the OCTOPUS (BrOPS) gene by map-based cloning. OPS is involved in phloem differentiation in Arabidopsis, a functionalization of regulating leaf curvature that is differentiated in Chinese cabbage. In the presence of brassinosteroid (BR) at the early heading stage in ic1, the mutation of BrOPS fails to sequester brassinosteroid insensitive 2 (BrBIN2) from the nucleus, allowing BrBIN2 to phosphorylate and inactivate BrBES1, which in turn relieves the repression of BrAS1 and results in leaf inward curving. Taken together, the results of our findings indicate that BrOPS positively regulates BR signaling by antagonizing BrBIN2 to promote leaf epinastic growth at the early heading stage in Chinese cabbage.

Transcriptional Regulation and Gene Mapping of Internode Elongation and Late Budding in the Chinese Cabbage Mutant lcc.

Two important traits of Chinese cabbage, internode length and budding time, destroy the maintenance of rosette leaves in the vegetative growth stage and affect flowering in the reproductive growth stage. Internodes have received much attention and research in rice due to their effect on lodging resistance, but they are rarely studied in Chinese cabbage. In Chinese cabbage, internode elongation affects not only the maintenance of rosette leaves but also bolting and yield. Budding is also an important characteristic of Chinese cabbage entering reproductive growth. Although many studies have reported on flowering and bolting, studies on bud emergence and the timing of budding are scarce. In this study, the mutant lcc induced by EMS (Ethyl Methane Sulfonate) was used to study internode elongation in the seedling stage and late budding in the budding stage. By comparing the gene expression patterns of mutant lcc and wild-type A03, 2280 differentially expressed genes were identified in the seedling stage, 714 differentially expressed genes were identified in the early budding stage, and 1052 differentially expressed genes were identified in the budding stage. Here, the transcript expression patterns of genes in the plant hormone signaling and clock rhythm pathways were investigated in relation to the regulation of internode elongation and budding in Chinese cabbage. In addition, an F2 population was constructed with the mutants lcc and R500. A high-density genetic map with 1602 marker loci was created, and QTLs for internode length and budding time were identified. Specifically, five QTLs for internode length and five QTLs for budding time were obtained. According to transcriptome data analysis, the internode length candidate gene BraA02g005840.3C (PIN8) and budding time candidate genes BraA02g003870.3C (HY5-1) and BraA02g005190.3C (CHS-1) were identified. These findings provide insight into the regulation of internode length and budding time in Chinese cabbage.

Hormones and carbohydrates synergistically regulate the formation of swollen roots in a Chinese cabbage translocation line.

The genus Brassica contains a rich diversity of species and morphological types, including leaf, root, and oil crops, all of which show substantial phenotypic variation. Both Chinese cabbage and cabbage are typical leaf-type crops with normal roots. We created translocation lines based on interspecific crosses between Chinese cabbage and cabbage and identified qdh225, which exhibited a swollen-root phenotype. The swollen root of qdh225 contained a large number of granular substances, and the formation of its irregular morphological tissue was caused by a thickening of the phloem. Transcriptomic and metabolomic data suggested that differential expression of genes encoding nine types of enzymes involved in starch and sucrose metabolism caused changes in starch synthesis and degradation in the swollen root. These genes jointly regulated sucrose and starch levels, leading to significant enrichment of starch and soluble proteins in the swollen root and a reduction in the content of soluble sugars such as d-glucose and trehalose 6-phosphate. A significant increase in auxin (IAA) and abscisic acid (ABA) contents and a decrease in gibberellin (GA) content in the swollen root likely promoted the differential expression of genes associated with hormone signal transduction, thereby regulating the development of the swollen root. Taken together, our data suggest that accumulation of IAA and ABA and reduction in GA promote swollen root formation by regulating hormone-mediated signaling, leading to a thickening of phloem, root enlargement, and substantial accumulation of starch and soluble proteins. The latter provide materials, energy, and nutrient sources for the development of swollen roots.

HSP70 Gene Family in Brassica rapa: Genome-Wide Identification, Characterization, and Expression Patterns in Response to Heat and Cold Stress.

Heat shock proteins protect plants from abiotic stress, such as salt, drought, heat, and cold stress. HSP70 is one of the major members of the heat shock protein family. To explore the mechanism of HSP70 in Brassica rapa, we identified 28 putative HSP70 gene family members using state-of-the-art bioinformatics-based tools and methods. Based on chromosomal mapping, HSP70 genes were the most differentially distributed on chromosome A03 and the least distributed on chromosome A05. Ka/Ks analysis revealed that B. rapa evolution was subjected to intense purifying selection of the HSP70 gene family. RNA-sequencing data and expression profiling showed that heat and cold stress induced HSP70 genes. The qRT-PCR results verified that the HSP70 genes in Chinese cabbage (Brassica rapa ssp. pekinensis) are stress-inducible under both cold and heat stress. The upregulated expression pattern of these genes indicated the potential of HSP70 to mitigate environmental stress. These findings further explain the molecular mechanism underlying the responses of HSP70 to heat and cold stress.

Single-cell transcriptome reveals dominant subgenome expression and transcriptional response to heat stress in Chinese cabbage.

BACKGROUND: Chinese cabbage (Brassica rapa ssp. pekinensis) experienced a whole-genome triplication event and thus has three subgenomes: least fractioned, medium fractioned, and most fractioned subgenome. Environmental changes affect leaf development, which in turn influence the yield. To improve the yield and resistance to different climate scenarios, a comprehensive understanding of leaf development is required including insights into the full diversity of cell types and transcriptional networks underlying their specificity. RESULTS: Here, we generate the transcriptional landscape of Chinese cabbage leaf at single-cell resolution by performing single-cell RNA sequencing of 30,000 individual cells. We characterize seven major cell types with 19 transcriptionally distinct cell clusters based on the expression of the reported marker genes. We find that genes in the least fractioned subgenome are predominantly expressed compared with those in the medium and most fractioned subgenomes in different cell types. Moreover, we generate a single-cell transcriptional map of leaves in response to high temperature. We find that heat stress not only affects gene expression in a cell type-specific manner but also impacts subgenome dominance. CONCLUSIONS: Our study highlights the transcriptional networks in different cell types and provides a better understanding of transcriptional regulation during leaf development and transcriptional response to heat stress in Chinese cabbage.

Construction of a high-density mutant population of Chinese cabbage facilitates the genetic dissection of agronomic traits.

Chinese cabbage (Brassica rapa ssp. pekinensis) is an economically important vegetable crop throughout the world, especially in Asia. High-quality genome sequences are available for Chinese cabbage, but gene functional studies remain challenging. To promote functional genomic studies of Chinese cabbage, we generated an ethyl methane sulfonate (EMS) mutant population of ∼8000 M2 plants using the double haploid inbred line A03 as the parent. The genome of A03 was sequenced and used as a reference for high-throughput functional characterization of gene mutations at the whole-genome level. A total of 300 M2 to M5 EMS mutants were phenotypically screened and then sequenced, revealing 750 629 SNPs and 46 272 InDel mutations that cover 98.27% of all predicted genes in the A03 genome. A forward-genetics approach was successfully used to identify two genes with chloroplast-related functions that are responsible for the yellow leaf mutant trait. A reverse-genetics approach was also used to identify associations between mutations in five genes of the glucosinolate biosynthetic pathway and variations in glucosinolate content of the mutant plants. In addition, we built the Chinese cabbage EMS mutation database (CCEMD, www.bioinformaticslab.cn/EMSmutation/home) to increase the usability of this mutant population resource. In summary, we performed large-scale screening of a heading Chinese cabbage EMS mutant collection at the phenotypic and genotypic levels, which will facilitate gene mining of Chinese cabbage and might also be useful for the study of other Brassica crops.

Mutation in a chlorophyll-binding motif of Brassica ferrochelatase enhances both heme and chlorophyll biosynthesis.

The heme branch of tetrapyrrole biosynthesis contributes to the regulation of chlorophyll levels. However, the mechanism underlying the balance between chlorophyll and heme synthesis remains elusive. Here, we identify a dark green leaf mutant, dg, from an ethyl methanesulfonate (EMS)-induced mutant library of Chinese cabbage. The dg phenotype is caused by an amino acid substitution in the conserved chlorophyll a/b-binding motif (CAB) of ferrochelatase 2 (BrFC2). This mutation increases the formation of BrFC2 homodimer to promote heme production. Moreover, wild-type BrFC2 and dBrFC2 interact with protochlorophyllide (Pchlide) oxidoreductase B1 and B2 (BrPORB1 and BrPORB2), and dBrFC2 exhibits higher binding ability to substrate Pchlide, thereby promoting BrPORBs-catalyzed production of chlorophyllide (Chlide), which can be directly converted into chlorophyll. Our results show that dBrFC2 is a gain-of-function mutation contributing to balancing heme and chlorophyll synthesis via a regulatory mechanism in which dBrFC2 promotes BrPORB enzymatic reaction to enhance chlorophyll synthesis.

Genetic analysis of the "head top shape" quality trait of Chinese cabbage and its association with rosette leaf variation.

The agricultural and consumer quality of Chinese cabbage is determined by its shape. The shape is defined by the folding of the heading leaves, which defines the head top shape (HTS). The overlapping HTS, in which the heading leaves curve inward and overlap at the top, is the shape preferred by consumers. To understand the genetic regulation of HTS, we generated a large segregating F2 population from a cross between pak choi and Chinese cabbage, with phenotypes ranging from nonheading to heading with either outward curving or inward curving overlapping heading leaves. HTS was correlated with plant height, outer/rosette leaf length, and petiole length. A high-density genetic map was constructed. Quantitative trait locus (QTL) analysis resulted in the identification of 22 QTLs for leafy head-related traits, which included five HTS QTLs. Bulked segregant analysis (BSA) was used to confirm HTS QTLs and identify candidate genes based on informative single-nucleotide polymorphisms. Interestingly, the HTS QTLs colocalized with QTLs for plant height, outer/rosette leaf, and petiole length, consistent with the observed phenotypic correlations. Combined QTL analysis and BSA laid a foundation for molecular marker-assisted breeding of Chinese cabbage HTS and directions for further research on the genetic regulation of this trait.

Development and Application of SSR Markers Related to Genes Involved in Leaf Adaxial-Abaxial Polarity Establishment in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

In Chinese cabbage (Brassica rapa L. ssp. pekinensis), leaf adaxial-abaxial (ad-ab) polarity is tightly related to leaf incurvature, an essential factor for the formation of leafy heads. Therefore, identification of the genes responsible for leaf ad-ab polarity and studying their genetic variation may clarify the mechanism of leafy head formation. By comparing the sequences of the genes regulating leaf ad-ab polarity development in Arabidopsis thaliana (A. thaliana), 41 candidate genes distributed on 10 chromosomes were found to be responsible for the establishment of ad-ab polarity in Chinese cabbage. Orthologous genes, including 10 single copies, 14 double copies, and one triple copies, were detected in the Chinese cabbage. The gene structure and conserved domain analyses showed that the number of exons of the 41 candidate genes range from one to 25, and that most genes share the conserved motifs 1, 6, and 10. Based on the 41 candidate genes, 341 simple sequence repeats (SSRs) were detected, including five replicated types: single, double, triple, quintuple, and sextuple nucleotide replications. Among these sequence repeat (SSR) loci, 323 loci were used to design 969 specific primers, and 362 primer pairs were selected randomly and evaluated using 12 Chinese cabbage accessions with different heading types. 23 primer pairs resulting with clear, polymorphic bands, combined with other 127 markers, was used to construct a linkage map by using an F2 population containing 214 lines derived from the hybrid of the overlapping heading Chinese cabbage "14Q-141" and the outward curling heading Chinese cabbage "14Q-279." The result showed that the sequences of markers in the genetic linkage map and the physical map was consistent in general. Our study could help to accelerate the breeding process of leafy head quality in Chinese cabbage.

Characterization of Non-heading Mutation in Heading Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Heading is a key agronomic trait of Chinese cabbage. A non-heading mutant with flat growth of heading leaves (fg-1) was isolated from an EMS-induced mutant population of the heading Chinese cabbage inbred line A03. In fg-1 mutant plants, the heading leaves are flat similar to rosette leaves. The epidermal cells on the adaxial surface of these leaves are significantly smaller, while those on the abaxial surface are much larger than in A03 plants. The segregation of the heading phenotype in the F2 and BC1 population suggests that the mutant trait is controlled by a pair of recessive alleles. Phytohormone analysis at the early heading stage showed significant decreases in IAA, ABA, JA and SA, with increases in methyl IAA and trans-Zeatin levels, suggesting they may coordinate leaf adaxial-abaxial polarity, development and morphology in fg-1. RNA-sequencing analysis at the early heading stage showed a decrease in expression levels of several auxin transport (BrAUX1, BrLAXs, and BrPINs) and responsive genes. Transcript levels of important ABA responsive genes, including BrABF3, were up-regulated in mid-leaf sections suggesting that both auxin and ABA signaling pathways play important roles in regulating leaf heading. In addition, a significant reduction in BrIAMT1 transcripts in fg-1 might contribute to leaf epinastic growth. The expression profiles of 19 genes with known roles in leaf polarity were significantly different in fg-1 leaves compared to wild type, suggesting that these genes might also regulate leaf heading in Chinese cabbage. In conclusion, leaf heading in Chinese cabbage is controlled through a complex network of hormone signaling and abaxial-adaxial patterning pathways. These findings increase our understanding of the molecular basis of head formation in Chinese cabbage.