Shifan Zhang

Leaf-color variation in plants should be associated with chlorophyll metabolism and chloroplast development. Here, we characterized a low-temperature-sensitive pakchoi DH line, 1197, which exhibited green leaves at 25 °C, but showed yellowing at 4 °C. Low temperature significantly reduced chlorophyll accumulation and disrupted chloroplast ultrastructure. After transfer from 4 °C to 25 °C for 7 days, yellow leaves partially regreened, and chlorophyll a content increased by 366.67%. RNA-seq analysis identified 3058 core DEGs associated with the yellowing–regreening transition, which were significantly enriched in photosynthesis–antenna proteins, photosynthesis, and porphyrin metabolism pathways. Leaf yellowing was characterized by repression of chlorophyll biosynthesis genes (e.g., CHLD, CHLM, PORC) and induction of degradation genes (SGR1, SGR2, NYC1, PAO), together with widespread downregulation of chloroplast function-related genes. In addition, GLK2, HBI1, NAC047, and NAC029 were identified as candidate regulators of temperature-dependent leaf-color conversion. This study provides candidate molecular insights into low-temperature-induced yellowing and regreening in pakchoi and offers candidate genes for future functional validation and Brassica breeding.

Disease resistance breeding is an important direction for the genetic improvement of Chinese cabbage. The traditional elite variety ‘Yutian Baojian’ Chinese cabbage is highly regarded for its tall cylindrical head with a pointed tip, tightly twisted wrapper leaves, and sweet taste. However, long-term cultivation has led to a significant decline in its resistance to clubroot caused by Plasmodiophora brassicae. To restore clubroot resistance while maintaining its desirable horticultural traits, this study used the clubroot-susceptible ‘Yutian Baojian’ as the recurrent parent and the resistant donor ‘Shaocai’, which carries the CRd resistance gene, to develop backcross populations. Using marker-assisted selection (MAS), plants were comprehensively screened based on foreground selection with markers tightly linked to the CRd gene, background selection with 73 genome-wide polymorphic markers, and phenotypic evaluation of horticultural traits, including plant height, plant spread, head shape, and soluble solids content. In the BC₁ population, three individuals showing high genetic similarity were selected. From the BC₂ population, four elite individuals were obtained, exhibiting 99.32% genetic similarity, stable clubroot resistance, and typical horticultural characteristics. Furthermore, three homozygous resistant inbred lines (BC₂S₂) with the ‘Yutian Baojian’ phenotype were developed. These results enrich the clubroot-resistant germplasm resources of Chinese cabbage and provide an effective MAS-based strategy for the precise improvement and germplasm innovation of local cultivars.

Bolting and flowering time are critical agronomic traits affecting the commercial value and breeding efficiency of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Although vernalization is a key environmental signal promoting flowering, its regulatory mechanisms remain poorly understood in this crop. Here, we identify the flowering repressor gene BrMAF5 and its antisense long non-coding RNA BrMAF5L as negative regulators of vernalization-induced flowering. During vernalization, transcript levels of both genes showed a decreasing trend as the vernalization period extended. Functional assays in Arabidopsis thaliana demonstrated that ectopic expression of BrMAF5 or BrMAF5L significantly delayed flowering, accompanied by increased expression of floral repressors (AtFLC, AtTEM1) and reduced expression of floral activators (AtFT, AtSOC1). Moreover, protein interaction analysis revealed that BrMAF5 associates with BrACP4 and BrRAB1A, linking it to fatty acid metabolism and membrane trafficking pathways. Collectively, our results reveal a novel regulatory module in vernalization-mediated flowering. These findings pave the way for developing bolting-resistant Brassicaceae crops by identifying promising molecular targets.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a key vegetable crop in Asia, but its commercial value is often reduced by premature flowering triggered by vernalization. The molecular mechanisms behind this process are not fully understood. MADS-box genes, as crucial transcriptional regulators, play vital roles in plant development, including flowering. In this study, 102 MADS-box genes were identified in Chinese cabbage through bioinformatics analyses, covering phylogeny, chromosomal localization, and gene structure. Real-time quantitative PCR and RNA-seq data analysis revealed that the expression level of AGL27 declined as vernalization time increased. To determine BrAGL27′s functions, we obtained BrAGL27-overexpressed (OE) Arabidopsis thaliana lines that showed significantly later flowering compared with the wild type (WT). The expression levels of flowering suppressor genes AtFLC and AtTEM1 were significantly high-regulated in the BrAGL27-OE lines compared to WT plants, while the expression levels of the floral genes AtSPL15, AtSOC1, AtFT, and AtAP3 were significantly lower in the BrAGL27-overexpressed lines than in the wild type. These findings enhance understanding of MADS-box genes in vernalization and flowering regulation, offering a basis for further research on bolting resistance and flowering control in Chinese cabbage.

Among the Brassica species in China, including Chinese cabbage, pak-choi, caixin, zicaitai, and wucai, Brassica rapa plays an important role in vegetable production. Purple resources from the species itself are scarce. It is worth noting that the tissue positions expressing a purple phenotype vary greatly between purple pak-choi and zicaitai. In this study, the genetic patterns of the purple phenotype were analyzed in purple pak-choi and zicaitai, and the F₁ showed purple leaves and green stems, which indicated that purple traits in the leaves and stem were inherited independently. In conjunction with field identification, RNA-seq was used to sequence the transcriptomes of the purple expression sites of purple pak-choi, zicaitai, and their F₁. The high expression of the regulatory genes Dark_pur and BrTT8 affected the purple color of pak-choi and caused the high expression of structural genes of the anthocyanin metabolism pathway and the accumulation of anthocyanins in leaves. The regulatory genes BrPAP₂ and BrTT8 affected the purple color of zicaitai stems, were significantly upregulated, and caused high expression of related structural genes, leading to the accumulation of anthocyanins in the stem epidermis. This suggested that BrPAP₂ and Dark_pur were both R2R3-MYB transcription factors, which were tissue-specific for the regulation of purple color traits in B. rapa. They also had a gene epistatic effect, which influenced the expression of purple traits in the F₁. The gene MYBL2 was highly expressed in all purple tissue sites. The present study on the regulatory genes of the purple phenotype of zicaitai and purple pak-choi provides a theoretical basis for revealing the influence of purple traits on B. rapa leaves and stems, and it may lay the foundation for the selection and breeding of purple vegetables of B. rapa.

The trichomes of mustard leaves have significance due to their ability to combat unfavorable external conditions and enhance disease resistance. It was demonstrated that the MYB-bHLH-WD40 (MBW) ternary complex consists of MYB, basic Helix-Loop-Helix (bHLH), and WD40-repeat (WD40) family proteins and plays a key role in regulating trichome formation and density. The bHLH gene family, particularly the Myelocytomatosis (MYC) proteins that possess the structural bHLH domain (termed bHLH-MYC), are crucial to the formation and development of leaf trichomes in plants. bHLH constitutes one of the largest families of transcription factors in eukaryotes, of which MYC is a subfamily member. However, studies on bHLH-MYC transcription factors in mustard have yet to be reported. In this study, a total of 45 bHLH-MYC transcription factors were identified within the Brassica juncea genome, and a comprehensive series of bioinformatic analyses were conducted on their structures and properties: an examination of protein physicochemical properties, an exploration of conserved structural domains, an assessment of chromosomal positional distributions, an analysis of the conserved motifs, an evaluation of the gene structures, microsynteny analyses, three-dimensional structure prediction, and an analysis of sequence signatures. Finally, transcriptome analyses and a subcellular localization examination were performed. The results revealed that these transcription factors were unevenly distributed across 18 chromosomes, showing relatively consistent conserved motifs and gene structures and high homology. The final results of the transcriptome analysis and gene annotation showed a high degree of variability in the expression of bHLH-MYC transcription factors. Five genes that may be associated with trichome development (BjuVA09G22490, BjuVA09G13750, BjuVB04G14560, BjuVA05G24810, and BjuVA06G44820) were identified. The subcellular localization results indicated that the transcription and translation products of these five genes were expressed in the same organelle: the nucleus. This finding provides a basis for elucidating the roles of bHLH-MYC family members in plant growth and development, and the molecular mechanisms underlying trichome development in mustard leaves.

Turnip, a subspecies of Brassica rapa, is one of the important root vegetable crops in China. Its roots, as significant product organs, possess values for consumption, feed, and medicinal purposes. A previous study localized a gene associated with root swelling traits. In this study, we cloned the turnip root swelling-related gene BraTSD2 (TUMOROUS SHOOT DEVELOPMENT 2) and performed a preliminary functional analysis. The bioinformatics analysis of BraTSD2 indicated that the protein is likely to possess a transmembrane feature. Additionally, predictions of its secondary and tertiary structures suggested that BraTSD2 shares similarities with the structure of a pectin methyltransferase. Temporal and spatial expression analysis of BraTSD2 using qPCR techniques indicated that the BraTSD2 gene had the highest expression levels in roots and hypocotyls during the swelling stage. Analysis of the promoter cis-acting elements of the BraTSD2 gene revealed that the promoter is regulated by signals associated with light response, abscisic acid, stress, salicylic acid, and MYBHI. The promoter of the turnip BraTSD2 gene was expressed throughout the whole plant in Arabidopsis thaliana. Phenotypic observation and analysis of A. thaliana overexpression BraTSD2 transgenic lines showed that plants overexpressing BraTSD2 had fewer primary and lateral roots compared to the wild type, with lighter leaf color, and delayed development and flowering. A previously reported protein CGR3, associated with pectin methyltransferase function, interacts with protein BraTSD2. When the BraTSD2 gene is overexpressed in A. thaliana, it can affect root morphology, which may be the basic structural condition for the generation of turnip root swelling traits.

In Chinese cabbage development the interplay between shoot apex activity and vernalization is pivotal for flowering timing. The intricate relationship between various cell types in the shoot apex meristem and their roles in regulating flowering gene expression in Chinese cabbage is not yet fully understood. A thorough analysis of single-cell types in the Chinese cabbage shoot apex and their influence on flowering genes and vernalization is essential for deeper insight. Our study first established a single-cell transcriptomic atlas of Chinese cabbage after 25 days of non-vernalization. Analyzing 19 602 single cells, we differentiated them into 15 distinct cell clusters using established marker genes. We found that key genes in shoot apex development and flowering were primarily present in shoot meristematic cells (SMCs), companion cells (CCs), and mesophyll cells (MCs). MADS-box protein FLOWERING LOCUS C 2 (BrFLC2), a gene suppressing flowering, was observed in CCs, mirroring patterns found in Arabidopsis. By mapping developmental trajectories of SMCs, CCs, and MCs, we elucidated the evolutionary pathways of crucial genes in shoot apex development and flowering. The creation of a single-cell transcriptional atlas of the Chinese cabbage shoot apex under vernalization revealed distinct alterations in the expression of known flowering genes, such as VERNALIZATION INSENSITIVE 3 (VIN3), VERNALIZATION 1 (VRN1), VERNALIZATION 2 (VRN2), BrFLC, and FLOWERING LOCUS T (FT), which varied by cell type. Our study underscores the transformative impact of single-cell RNA sequencing (scRNA-seq) for unraveling the complex differentiation and vernalization processes in the Chinese cabbage shoot apex. These insights are pivotal for enhancing breeding strategies and cultivation management of this vital vegetable.

The purple-leaf phenotype in pak choi is due to the accumulation of anthocyanin. The main regulated genes are unclear. The gene controlling the purple-leaf phenotype was mapped on A03 using BSA-seq, but the candidate interval was not further narrowed with InDel markers. Based on our previous study, we hypothesized that the candidate gene that regulates purple leaves in pak choi may also be the Dark_Pur gene from B. juncea. Using the Dark_Pur-1 marker to identify P₁, P₂, F₁, and F₂, it was confirmed that the purple trait in purple-leaf pak choi was controlled by the Dark_Pur gene from B. juncea through distant hybridization. A DNA segment of approximately 514 Kb containing the Dark_Pur gene was reintroduced into pak choi from B. juncea. Meanwhile, a new purple pak choi germplasm line was created with green pak choi × purple B. juncea via distant hybridization, which proved that distant hybridization is an effective method for creating new germplasms. Furthermore, the purple-leaf phenotypes of 20 pak choi varieties were identified, and the purple-leaf traits of all lines were derived from B. juncea via distant hybridization. At present, few studies have focused on the background of the purple trait in pak choi; however, in this study, our results suggest that there is a high probability that the purple trait in pak choi may be completely derived from purple B. juncea via distant hybridization. This study also lays a good foundation for research on the creation of new germplasms through distant hybridization among the Brassica species.

The soil-borne pathogen Plasmodiophora brassicae is the causal agent of clubroot, a major disease in Chinese cabbage (Brassica rapa ssp. pekinensis). The host’s resistance genes often confer immunity to only specific pathotypes and may be rapidly overcome. Identification of novel clubroot resistance (CR) from germplasm sources is necessary. In this study, Bap246 was tested by being crossed with different highly susceptible B. rapa materials and showed recessive resistance to clubroot. An F₂ population derived from Bap246 × Bac1344 was used to locate the resistance Quantitative Trait Loci (QTL) by Bulk Segregant Analysis Sequencing (BSA-Seq) and QTL mapping methods. Two QTL on chromosomes A01 (4.67–6.06 Mb) and A08 (10.42–11.43 Mb) were found and named Cr4Ba1.1 and Cr4Ba8.1, respectively. Fifteen and eleven SNP/InDel markers were used to narrow the target regions in the larger F₂ population to 4.67–5.17 Mb (A01) and 10.70–10.84 Mb (A08), with 85 and 19 candidate genes, respectively. The phenotypic variation explained (PVE) of the two QTL were 30.97% and 8.65%, respectively. Combined with gene annotation, mutation site analysis, and real-time quantitative polymerase chain reaction (qRT-PCR) analysis, one candidate gene in A08 was identified, namely Bra020861. And an insertion and deletion (InDel) marker (co-segregated) named Crr1-196 was developed based on the gene sequence. Bra013275, Bra013299, Bra013336, Bra013339, Bra013341, and Bra013357 in A01 were the candidate genes that may confer clubroot resistance in Chinese cabbage. The resistance resource and the developed marker will be helpful in Brassica breeding programs.