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Main conclusion: BcERF98 is induced by ethylene signaling and inhibits the expression of BcFT by interacting with BcNF-YA2 and BcEIP9, thereby inhibiting plant flowering. Several stresses trigger the accumulation of ethylene, which then transmits the signal to ethylene response factors (ERFs) to participate in the regulation of plant development to adapt to the environment. This study clarifies the function of BcERF98, a homolog of AtERF98, in the regulation of plant flowering time mediated by high concentrations of ethylene. Results indicate that BcERF98 is a nuclear and the cell membrane-localized transcription factor and highly responsive to ethylene signaling. BcERF98 inhibits the expression of BcFT by interacting with BcEIP9 and BcNF-YA2, which are related to flowering time regulation, thereby participating in ethylene-mediated plant late flowering regulation. The results have enriched the theoretical knowledge of flowering regulation in non-heading Chinese cabbage (NHCC), providing the scientific basis and gene reserves for cultivating new varieties of NHCC with different flowering times. [ABSTRACT FROM AUTHOR]

This article discusses effective strategies for creating self-compatible Brassica rapa (Chinese cabbage) plants by introducing mutated SRK and SCR/SP11 genes from Brassica napus (rapeseed). Self-incompatibility (SI) is a common trait in Brassica vegetable crops, but it hinders the development and propagation of inbred lines. The manipulation of SI genes, such as SRK and SCR/SP11, can lead to the creation of self-compatible materials. The researchers used CRISPR/Cas9 technology to mutate the BnSRK-1 gene in B. napus and then employed distant hybridization to introduce the mutated gene into B. rapa, resulting in the generation of self-compatible materials. This research has important implications for improving the breeding process of Brassica vegetable crops. [Extracted from the article]

Abstract Background Purple non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis] has become popular because of its richness in anthocyanin. However, anthocyanin only accumulates in the upper epidermis of leaves. Further studies are needed to investigate the molecular mechanisms underlying the specific accumulation of it. Results In this study, we used the laser capture frozen section method (LCM) to divide purple (ZBC) and green (LBC) non-heading Chinese cabbage leaves into upper and lower epidermis parts (Pup represents the purple upper epidermis, Plow represents the purple lower epidermis, Gup represents the green upper epidermis, Glow represents the green lower epidermis). Through transcriptome sequencing, we found that the DIHYDROFLAVONOL 4-REDUCTASE-encoding gene BcDFR, is strongly expressed in Pup but hardly in others (Plow, Gup, Glow). Further, a deletion and insertion in the promoter of BcDFR in LBC were found, which may interfere with BcDFR expression. Subsequent analysis of gene structure and conserved structural domains showed that BcDFR is highly conserved in Brassica species. The predicted protein-protein interaction network of BcDFR suggests that it interacts with almost all functional proteins in the anthocyanin biosynthesis pathway. Finally, the results of the tobacco transient expression also demonstrated that BcDFR promotes the synthesis and accumulation of anthocyanin. Conclusions BcDFR is specifically highly expressed on the upper epidermis of purple non-heading Chinese cabbage leaves and regulates anthocyanin biosynthesis and accumulation. Our study provides new insights into the functional analysis and transcriptional regulatory network of anthocyanin-related genes in purple non-heading Chinese cabbage.

The application of a male-sterile line is an ideal approach for hybrid seed production in non-heading Chinese cabbage (Brassica rapa ssp. chinensis). However, the molecular mechanisms underlying male sterility in B. rapa are still largely unclear. We previously obtained the natural male sterile line WS24-3 of non-heading Chinese cabbage and located the male sterile locus, Bra2Ms, on the A2 chromosome. Cytological observations revealed that the male sterility of WS24-3 resulted from disruption of the meiosis process during pollen formation. Fine mapping of Bra2Ms delimited the locus within a physical distance of about 129 kb on the A2 chromosome of B. rapa. The Bra039753 gene encodes a plant homeodomain (PHD)-finger protein and is considered a potential candidate gene for Bra2Ms. Bra039753 was significantly downregulated in sterile line WS24-3 compared to the fertile line at the meiotic anther stage. Sequence analysis of Bra039753 identified a 369 bp fragment insertion in the first exon in male sterile plants, which led to an amino acid insertion in the Bra039753 protein. In addition, the 369 bp fragment insertion was found to cosegregate with the male sterility trait. This study identified a novel locus related to male sterility in non-heading Chinese cabbage, and the molecular marker obtained in this study will be beneficial for the marker-assisted selection of excellent sterile lines in non-heading Chinese cabbage and other Brassica crops.

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However, the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated (ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches (one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings (Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%–51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin. The one-step method outperformed the two-step method; the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily, quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Leaves are the predominant photosynthetic and edible organs in non-heading Chinese cabbage (Brassica campestris ssp. chinensis, NHCC), contributing significantly to yield, appearance, and desirability to consumers. However, the genetic basis of leaf shape and size in non-heading Chinese cabbage remains unclear. In this study, we developed a RIL population using 'Maertou', with slender leaves and narrow petioles, and 'Suzhouqing', with oval leaves and wide petioles, to construct a genetic linkage map and detect QTLs. To obtain stable and reliable QTLs, the 11 leaf-related traits, including the leaf length, leaf width, and fresh weight of the lamina and petiole and the thickness of petiole was observed on two locations—while the leaf shape, petiole shape, index of lamina/petiole length, and index of petiole fresh weight were calculated based on 7 leaf-related traits. QTL mapping illustrated that a total of 27 QTLs for leaf-related traits were preliminarily detected. The candidate genes were annotated and several genes involved in leaf development and leaf shape appeared in the overlapping regions of multiple loci, such as KRP2, GRF4, ARGOS, and SAUR9. This study lays the foundation for further exploration of the genetic mechanisms and development of effective molecular markers for leaf shape and size in NHCC. [ABSTRACT FROM AUTHOR]

Background: Purple non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis] has become popular because of its richness in anthocyanin. However, anthocyanin only accumulates in the upper epidermis of leaves. Further studies are needed to investigate the molecular mechanisms underlying the specific accumulation of it. Results: In this study, we used the laser capture frozen section method (LCM) to divide purple (ZBC) and green (LBC) non-heading Chinese cabbage leaves into upper and lower epidermis parts (Pup represents the purple upper epidermis, Plow represents the purple lower epidermis, Gup represents the green upper epidermis, Glow represents the green lower epidermis). Through transcriptome sequencing, we found that the DIHYDROFLAVONOL 4-REDUCTASE-encoding gene BcDFR, is strongly expressed in Pup but hardly in others (Plow, Gup, Glow). Further, a deletion and insertion in the promoter of BcDFR in LBC were found, which may interfere with BcDFR expression. Subsequent analysis of gene structure and conserved structural domains showed that BcDFR is highly conserved in Brassica species. The predicted protein-protein interaction network of BcDFR suggests that it interacts with almost all functional proteins in the anthocyanin biosynthesis pathway. Finally, the results of the tobacco transient expression also demonstrated that BcDFR promotes the synthesis and accumulation of anthocyanin. Conclusions: BcDFR is specifically highly expressed on the upper epidermis of purple non-heading Chinese cabbage leaves and regulates anthocyanin biosynthesis and accumulation. Our study provides new insights into the functional analysis and transcriptional regulatory network of anthocyanin-related genes in purple non-heading Chinese cabbage. [ABSTRACT FROM AUTHOR]

To clarify the impact of different washing and cooking approaches on the dietary exposure risks of two fat-soluble pesticides (chlorfenapyr and beta-cypermethrin) in mustard green (Brassica juncea (L.) Czern.) and non-heading Chinese cabbage (Brassica chinensis L.), five common cleaning approaches (edible salt water, edible baking soda water, rice-washing water, tap water, edible vinegar aqueous solution) were used to clean the mustard green and non-heading Chinese cabbage in a simulated Chinese kitchen, and four cooking approaches (boiling, steaming, frying, and pickling) were used to process the samples. The residues of chlorfenapyr and beta-cypermethrin in samples before and after kitchen processing were determined using QuEChERS sample preparation and GC-MS/MS and UPLC-MS/MS detection. By estimating the processing factors (PF) and the changes in exposure doses and the contributions to long-term dietary exposure risk (ADIi%) and short-term dietary exposure risk (%ARfD) to three representative Chinese populations (children, women of childbearing age, and the general population), the role of washing and cooking in reducing dietary exposure risk and in reducing uncertainty in dietary exposure risk assessment were estimated. The results showed that, edible baking soda water and the edible salt water had the best removal rates for mustard green and non-heading Chinese cabbage, and it was significantly better than the washing with tap water. After washing and cooking, the processing factors (PFTotal) in mustard green were 0.06-0.24 for chlorfenapyr and 0.04-0.11 for beta-cypermethrin, and those in non-heading Chinese cabbage were 0.05-0.21 for chlorfenapyr and 0.06-0.11 for beta-cypermethrin. The estimated exposure doses of these 2 pesticide residues were reduced by 76%-96% using washing and cooking processing. The study showed that routine kitchen processing can differentially reduce the risk of long-term and short-term dietary exposure of both chlorfenapyr and beta-cypermethrin residues to representative populations in China. Specifically for mustard green and non-heading Chinese cabbage, kitchen processing resulted in %ARfD < 100% for the residue of beta-cypermethrin, but not for the residue of chlorfenapyr [ABSTRACT FROM AUTHOR]

Brassinosteroids (BRs) play critical roles in plant growth by promoting cell elongation and division, leading to increased leaf inclination angles. BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMS-SUPPRESSOR 1 (BES1) act as transcription factors in the brassinosteroid signaling pathway and are involved in several physiological activities regulated by BRs. In this study, we identified and cloned BcBZR1 from the heitacai non-heading Chinese cabbage (NHCC) cultivar. The sequence analysis showed that the coding sequence length of BcBZR1 is 996 bp, encoding 331 amino acid residues. Subcellular localization assays showed that BcBZR1 is localized in the nucleus and cytoplasm and that BcBZR1 protein is transported to the nucleus after receiving BR signals. Compared with Col-0, the leaf inclination angle was smaller in BcBZR1-OX. The EBR treatment experiment indicated that BRs regulate the differential expression of paclobutrazol resistance1 (PRE1) and ILI1 binding bHLH1 (IBH1) in the adaxial and abaxial cells of the petiole through BZR1, thus regulating the leaf inclination angle. The bimolecular fluorescence complementation (BiFC) assay indicated that BcBZR1 interacts with C-repeat Binding Factor2 (BcCBF2) and CBF3. Taken together, our findings not only validate the function of BcBZR1 in leaf inclination angle distribution in non-heading Chinese cabbage, but also contribute to the mechanism of leaf inclination angle regulation in this species under cold stress. [ABSTRACT FROM AUTHOR]

Cold stress is a serious threat to global crop production and food security, but plant cold resistance is accompanied by reductions in growth and yield. In this study, we determined that the novel gene BcGSTF10 in non-heading Chinese cabbage [NHCC; Brassica campestris (syn. Brassica rapa) ssp. chinensis ] is implicated in resistance to cold stress. Biochemical and genetic analyses demonstrated that BcGSTF10 interacts with BcICE1 to induce C-REPEAT BINDING FACTOR (CBF) genes that enhance freezing tolerance in NHCC and in Arabidopsis. However, BcCBF2 represses BcGSTF10 and the latter promotes growth in NHCC and Arabidopsis. This dual function of BcGSTF10 indicates its pivotal role in balancing cold stress and growth, and this important understanding has the potential to inform the future development of strategies to breed crops that are both climate-resilient and high-yielding. [ABSTRACT FROM AUTHOR]

Leaf trichome is an important trait for stress-resistance breeding in Brassica crops, but its scientific evaluation and identification are rarely reported. To investigate the inheritance of leaf trichome in non-heading Chinese cabbage, two inbred lines, '14XS23W' (glabrous) and '14XS23Y' (hirsute), were used to generate F1 and F2 populations for joint analysis using a mixed major genes plus polygene inheritance model. The density of adaxial leaf trichomes was controlled by two pairs of additive-dominance-epistasis major genes plus additive-dominance polygenes. The broad-sense heritabilities of the major genes were 93.8% and 85.51%, respectively, for greenhouse and open-field conditions. However, the density of the abaxial leaf trichome was controlled by two pairs of additive-dominance-epistasis major genes plus additive dominance-epistasis polygenes. The broad-sense heritabilities of the major genes were 91.45% and 72.20%, while the polygenes were 0% and 14.5%, respectively, for greenhouse and open field environments. These results indicated that trichome density in non-heading Chinese cabbage was primarily controlled by two pairs of major genes with high heritability, implying that this trait is suitable for rapid selection in early generations. [ABSTRACT FROM AUTHOR]

Petioles of non-heading Chinese cabbage are not only an important edible part but also a conduit for nutrient transport, holding significant agricultural and research value. In this study, we conducted a comprehensive genetic analysis of petiole-related traits using a segregating population. Modern quantitative genetic approaches were applied to investigate the genetic regulation of petiole thickness. The results indicated that petiole thickness is a quantitative trait, and the identified genetic model was consistent with two pairs of additive-dominant main genes and additive-dominant polygenes (2MG-AD). BSA-seq analysis identified a major effect of QTL controlling petiole thickness on chromosome A09: 42.08–45.09 Mb, spanning 3.01 Mb, designated as QTL-BrLH9. Utilizing InDel markers, the interval was narrowed down to 51 kb, encompassing 14 genes with annotations for 10 of them. Within the interval, four mutated genes were detected. Combined with gene annotation, protein sequence analysis, and homology alignment, it was found that BraA09g063520.3C's homologous gene SMXL6 in Arabidopsis (Arabidopsis thaliana (L.) Heynh) is an inhibitor of the coding and synthesis of the strigolactone pathway. Strigolactone (SLs) plays an important role in plant growth and development. The cloning results showed that multiple frameshift mutations and non-synonymous mutations occurred on the exon. The qPCR results showed that the expression of the gene was significantly different between the two parents at the adult stage, so it was speculated that it would lead to changes in petiole thickness. BraA09g063520.3C was predicted as the final candidate gene. [ABSTRACT FROM AUTHOR]

Non-heading Chinese cabbage prefers cool temperatures, and heat stress has become a major factor for reduced yield. The proline transporter protein (ProT) is highly selective for proline transport, contributing to the heat tolerance of non-heading Chinese cabbage. However, there has been no systematic study on the identification and potential functions of the ProT gene family in response to heat stress in non-heading Chinese cabbage. We identified six BchProT genes containing 11–12 transmembrane helices characteristic of membrane proteins through whole-genome sequencing. These genes diverged into three evolutionary branches and exhibited similarity in motifs and intron/exon numbers. Segmental duplication is the primary driving force for the amplification of BchProT. Notably, many stress-related elements have been identified in the promoters of BchProT using cis-acting element analysis. The expression level of BchProT6 was the highest in petioles, and the expression level of BchProT1 was the highest under high-temperature stress. Subcellular localization indicated their function at cell membranes. Heterologous expression of BchProT1 in Arabidopsis plants increased proline transport synthesis under heat-stress conditions. This study provides valuable information for exploring the molecular mechanisms underlying heat tolerance mediated by members of the BchProT family. [ABSTRACT FROM AUTHOR]

[Objectives] This paper aimed to study the expression pattern of BCGR2. 1 in non-heading Chinese cabbage under photooxidative stress, and explore the effects of BcGR2.1 overexpression or silencing on the plant resistance to photooxidative stress, so as to clarify the function of the gene. [Methods] The CDS sequence of BcGR2. 1 gene was cloned from non-heading Chinese cabbage, and the phylogenetic tree analysis and conserved domain alignment of the amino acid sequence encoded by BcGR2.1 were performed. The expression level of BcGR2. 1 under photooxidative stress was analyzed by RT-qPCR. BcGR2. 1-overexpressing Arabidopsis thaliana and BcGR2. 1-silencing plants were obtained by Agrobacterium-mediated floral dip transformation and virus- induced gene silencing (VIGS) respectively, the effects of overexpression or silencing of BcGR2. 1 on the plant resistance to photooxidative stress were also tested. [Results] BcGR2.1 gene contained a 1 626 bp open reading frame (ORF), and had the closest evolutionary relationship with Brassica rapa. The expression level of BeGR2. 1 was up-regulated and then down-regulated under photooxidative stress induced by methyl viologen (MV) treatment. The content of ascorbic acid (AsA) increased in overexpression transgenic A. thaliana and decreased in BcGR2. I-silencing plants. Under photooxidative stress, H2O2 content was significantly lower in overexpression transgenic A. thaliana than that in wild type, while H2O2 content in BcGR2. 1-silencing plants was significantly higher than that in PTY empty plants. The chlorophyll fluorescence kinetics parameters such as FY/Fm Y(II), ETR and qP were significantly higher in BcGR2.1-overexpressing A. thaliana than those in wild type. [Conclusions] BcGR2.1 had positive regulation on AsA content in A. thaliana and non-heading Chinese cabbage, inhibited the H2O2 explosion under photooxidative stress, maintained the normal state of chlorophyll fluorescence parameters, and improved the tolerance of plants to photooxidative stress. [ABSTRACT FROM AUTHOR]

WRKY transcription factors (TFs) participate in plant defense mechanisms against biological and abiotic stresses. However, their regulatory role in heat resistance is still unclear in non-heading Chinese cabbage. Here, we identified the WRKY-IIe gene BcWRKY22(BraC09g001080.1), which is activated under high temperatures and plays an active role in regulating thermal stability, through transcriptome analysis. We further discovered that the BcWRKY22 protein is located in the nucleus and demonstrates transactivation activity in both the yeast and plant. Additionally, our studies showed that the transient overexpression of BcWRKY22 in non-heading Chinese cabbage activates the expression of catalase 2 (BcCAT2), enhances CAT enzyme activity, and reduces Hydrogen Peroxide (H2O2) accumulation under heat stress conditions. In addition, compared to its wild-type (WT) counterparts, Arabidopsis thaliana heterologously overexpresses BcWRKY22, improving thermotolerance. When the BcWRKY22 transgenic root was obtained, under heat stress, the accumulation of H2O2 was reduced, while the expression of catalase 2 (BcCAT2) was upregulated, thereby enhancing CAT enzyme activity. Further analysis revealed that BcWRKY22 directly activates the expression of BcCAT2 (BraC08g016240.1) by binding to the W-box element distributed within the promoter region of BcCAT2. Collectively, our findings suggest that BcWRKY22 may serve as a novel regulator of the heat stress response in non-heading Chinese cabbage, actively contributing to the establishment of thermal tolerance by upregulating catalase (CAT) activity and downregulating H2O2 accumulation via BcCAT2 expression. [ABSTRACT FROM AUTHOR]

Leaves are the predominant photosynthetic and edible organs in non-heading Chinese cabbage (Brassica campestris ssp. chinensis, NHCC), contributing significantly to yield, appearance, and desirability to consumers. However, the genetic basis of leaf shape and size in non-heading Chinese cabbage remains unclear. In this study, we developed a RIL population using ‘Maertou’, with slender leaves and narrow petioles, and ‘Suzhouqing’, with oval leaves and wide petioles, to construct a genetic linkage map and detect QTLs. To obtain stable and reliable QTLs, the 11 leaf-related traits, including the leaf length, leaf width, and fresh weight of the lamina and petiole and the thickness of petiole was observed on two locations—while the leaf shape, petiole shape, index of lamina/petiole length, and index of petiole fresh weight were calculated based on 7 leaf-related traits. QTL mapping illustrated that a total of 27 QTLs for leaf-related traits were preliminarily detected. The candidate genes were annotated and several genes involved in leaf development and leaf shape appeared in the overlapping regions of multiple loci, such as KRP2, GRF4, ARGOS, and SAUR9. This study lays the foundation for further exploration of the genetic mechanisms and development of effective molecular markers for leaf shape and size in NHCC.

Brassinosteroids (BRs) play critical roles in plant growth by promoting cell elongation and division, leading to increased leaf inclination angles. BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMS-SUPPRESSOR 1 (BES1) act as transcription factors in the brassinosteroid signaling pathway and are involved in several physiological activities regulated by BRs. In this study, we identified and cloned BcBZR1 from the heitacai non-heading Chinese cabbage (NHCC) cultivar. The sequence analysis showed that the coding sequence length of BcBZR1 is 996 bp, encoding 331 amino acid residues. Subcellular localization assays showed that BcBZR1 is localized in the nucleus and cytoplasm and that BcBZR1 protein is transported to the nucleus after receiving BR signals. Compared with Col-0, the leaf inclination angle was smaller in BcBZR1-OX. The EBR treatment experiment indicated that BRs regulate the differential expression of paclobutrazol resistance1 (PRE1) and ILI1 binding bHLH1 (IBH1) in the adaxial and abaxial cells of the petiole through BZR1, thus regulating the leaf inclination angle. The bimolecular fluorescence complementation (BiFC) assay indicated that BcBZR1 interacts with C-repeat Binding Factor2 (BcCBF2) and CBF3. Taken together, our findings not only validate the function of BcBZR1 in leaf inclination angle distribution in non-heading Chinese cabbage, but also contribute to the mechanism of leaf inclination angle regulation in this species under cold stress.

Clathrin is an evolutionarily highly conserved evolutionary protein consisting of clathrin light chains (CLC) and clathrin heavy chains (CHC), and these form its basic structure. Clathrin is an important host factor in the process of viral infection. In this study, we cloned the BcCLC1 gene and the BcCLC2 gene from the '49CX' variety of non-heading Chinese cabbage (NHCC, Brassica campestris L. ssp. chinensis Makino) and verified their functions. The results showed that BcCLC1 was mainly localized in the cytomembrane and cytoplasm, and only a small amount entered the nucleus. BcCLC2 encoded a protein comprising 265 amino acids that were distributed in the cytomembrane, nucleus, and cytoplasm. A BiFC assay and yeast two-hybrid (Y2H) analysis showed that BcCLCs (BcCLC1 and BcCLC2) could interact with several TuMV proteins. We further investigated the mechanism of BcCLCs in regulating TuMV virus infections in NHCC, and observed that BcCLCs gene silencing inhibited TuMV infections and overexpression of BcCLCs in Arabidopsis promoted TuMV infections in NHCC. Finally, mutants of Arabidopsis homologs of BcCLCs were also screened and subjected to TuMV inoculation tests. In conclusion, we speculate that BcCLCs confer Turnip mosaic virus (TuMV) resistance in NHCC by interacting with TuMV proteins to promote the intracellular transport of the virus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

As a widely cultivated vegetable in China and Southeast Asia, the breeding of non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) is widespread; more than 400 varieties have been granted new plant variety rights (PVRs) in China. Distinctness is one of the key requirements for the granting of PVRs, and molecular markers are widely used as a robust supplementary method for similar variety selection in the distinctness test. Although many genome-wide molecular markers have been developed, they have not all been well used in variety identification and tests of distinctness of non-heading Chinese cabbage. In this study, by using 423 non-heading Chinese cabbage varieties collected from different regions of China, 287 simple sequence repeat (SSR) markers were screened for polymorphisms, and 23 core markers were finally selected. The polymorphic information content (PIC) values of the 23 SSR markers ranged from 0.555 to 0.911, with an average of 0.693, and the average number of alleles per marker was 13.65. Using these 23 SSR markers, 418 out of 423 varieties could be distinguished, with a discrimination rate of 99.994%. Field tests indicated that those undistinguished varieties were very similar and could be further distinguished by a few morphological characteristics. According to the clustering results, the 423 varieties could be divided into three groups: pak-choi, caitai, and tacai. The similarity coefficient between the SSR markers and morphological characteristics was moderate (0.53), and the efficiency of variety identification was significantly improved by using a combination of SSR markers and morphological characteristics. [ABSTRACT FROM AUTHOR]

Clubroot disease is a kind of soil-borne disease that seriously infects Brassica species. In this study, we collected 121 varieties of non-heading Chinese cabbages. In order to better understand the genetic variation and to screen suitable clubroot disease-resistant parental material, we re-sequenced them to examine the population genetic structure, population genetic diversity, population differentiation index, and selective sweep based on SNPs. The mapping rate with the reference genome was high, and data quality analysis revealed that the sequencing quality was good. The annotated data indicated that intronic and intergenic areas held the majority of SNPs and indels. Four subgroups of 121 non-heading Chinese cabbages were identified using principal component analysis, phylogenetic tree, and genetic structure analysis. An examination of genetic diversity revealed that while selfing may happen in subgroups C and D, heterozygosity may exist in subgroups A and B. In subgroup B, self-fertilization is not possible. There was a moderate degree of genetic differentiation between subgroups B and C (Fst = 0.0744347). For genes in certain sweep regions, we also ran GO enrichment and KEGG enrichment analysis. Two disease resistance-related genes, BraA01g042910.3.5C and BraA06g019360.3.5C, were examined. These findings will serve as a theoretical foundation for developing novel, clubroot disease-resistant types of non-heading Chinese cabbages. [ABSTRACT FROM AUTHOR]

WRKY transcription factors (TFs) participate in plant defense mechanisms against biological and abiotic stresses. However, their regulatory role in heat resistance is still unclear in non-heading Chinese cabbage. Here, we identified the WRKY-IIe gene BcWRKY22(BraC09g001080.1), which is activated under high temperatures and plays an active role in regulating thermal stability, through transcriptome analysis. We further discovered that the BcWRKY22 protein is located in the nucleus and demonstrates transactivation activity in both the yeast and plant. Additionally, our studies showed that the transient overexpression of BcWRKY22 in non-heading Chinese cabbage activates the expression of catalase 2 (BcCAT2), enhances CAT enzyme activity, and reduces Hydrogen Peroxide (H2O2) accumulation under heat stress conditions. In addition, compared to its wild-type (WT) counterparts, Arabidopsis thaliana heterologously overexpresses BcWRKY22, improving thermotolerance. When the BcWRKY22 transgenic root was obtained, under heat stress, the accumulation of H2O2 was reduced, while the expression of catalase 2 (BcCAT2) was upregulated, thereby enhancing CAT enzyme activity. Further analysis revealed that BcWRKY22 directly activates the expression of BcCAT2 (BraC08g016240.1) by binding to the W-box element distributed within the promoter region of BcCAT2. Collectively, our findings suggest that BcWRKY22 may serve as a novel regulator of the heat stress response in non-heading Chinese cabbage, actively contributing to the establishment of thermal tolerance by upregulating catalase (CAT) activity and downregulating H2O2 accumulation via BcCAT2 expression.

Sesquiterpenes are important defensive secondary metabolites and aroma components. However, limited information is available on the mechanism of sesquiterpene formation and composition in the non-heading Chinese cabbage (NHCC) leaf. Therefore, headspace solid-phase microextraction/gas chromatography–mass spectrometry (HS-SPME/GC–MS) combined with transcriptome analysis was used to study the mechanism of volatile organic compound formation. A total of 26 volatile organic compounds were identified in two NHCC cultivars ‘SZQ’ and ‘XQC’ and their F1 hybrids. Among these, sesquiterpene β-caryophyllene was identified only in ‘XQC’ and F1. Five genes encoding caryophyllene synthase were identified. The candidate β-caryophyllene synthase genes BcTPSa11 and BcTPSa21 had high expression levels only in ‘XQC’ and F1. In addition, several transcription factors of MYB-related, MYB, bHLH, and AP2/ERF families were identified by co-expression, suggesting that they regulate β-caryophyllene biosynthesis. Our results provide a molecular basis for sesquiterpene biosynthesis as well as insights into the regulatory network of β-caryophyllene in NHCC.

Non-heading Chinese cabbage (Brassica campestris ssp. chinensis) is an important vegetable crop. In recent years, due to the unreasonable irrigation and cultivation methods, secondary salinization of soil in the facility has become more and more serious, which has caused adverse effects on vegetable production. Saline-alkali stress will also inhibit the normal growth of Chinese cabbage. In the past years, more attention has been paid to the effects of neutral salt on the growth and physiology of non-heading Chinese cabbage, while less attention has been paid to the effects of alkaline salt on the growth and physiology of non-heading Chinese cabbage. In this study, different salt and alkali stress treatments were set up through hydroponic experiment to study the different response mechanism of non-heading cabbage to medium and alkaline sodium salt stress. The results shown that the fresh weight, biomass, chlorophyll a, chlorophyll B, carotenoid and total chlorophyll contents of non-heading Chinese cabbage seedlings under alkaline carbonate stress decreased more than those under neutral salt stress at the same stress concentration. Compared with neutral salt, alkaline salt had greater influence on the antioxidant system of non-heading Chinese cabbage, with higher activity of peroxidase (POD) and superoxide dismutase (SOD), and higher MDA content and plasma membrane permeability (MI), while the activity of peroxidase (CAT) was lower under alkali stress. Compared with neutral salt stress, the accumulation of proline and soluble sugar in non-heading Chinese cabbage was higher under alkaline carbonate stress, which had a greater influence on the osmotic regulation system of non-heading Chinese cabbage. According to the experimental results, compared with neutral salt stress, alkaline carbonate stress has a stronger inhibitory effect on the growth of non-nodular cabbage, and has a greater effect on its physiology. [ABSTRACT FROM AUTHOR]

The primary domain/leucine zipper (bZIP) transcription factor, Elongated Hypocotyl (HY5), is crucial for the photomorphogenesis of seedlings. Here, we identified BcHY5 as a regulator of hypocotyl length from the non-heading Chinese cabbage (NHCC) variety 'Suzhouqing', which encoded a protein comprised 164 amino acid residues. Ectopic expression of BcHY5 in Arabidopsis shortens the length of the hypocotyl. Additionally, we discovered a protein called BcBBX24 containing the B-BOX (BBX) domain, which is the interacting partner of BcHY5. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and GST pull-down assays revealed that BcHY5 interacted with BcBBX24. Additionally, by physically binding to the promoter of BcHY5, BcBBX24 inhibited the transcriptional activity of BcHY5. Therefore, our findings reveal a transcriptional mechanism through which light response occurs in NHCC seedlings, where BcHY5 can interact with BcBBX24 and BcBBX24 can prevent BcHY5 transcription. [ABSTRACT FROM AUTHOR]

Non-heading Chinese cabbage originated in China. It is a popular vegetable widely planted in the middle and lower reaches of the Yangtze River with extremely rich germplasm resources, short growth cycle, high yield and rich nutrients. In the paper, based on the six varieties of non-heading Chinese cabbage, excellent germplasm resources, the development and application of breeding technology, the research progress and existing problems of genetic breeding technology and molecular biology of non-heading Chinese cabbage were discussed. To solve these problems, we suggest continuing to collect germplasm resources at the edge of the original habitat of non-heading Chinese cabbage and create more breeding materials in terms of germplasm resources, strengthening the research on genome-based molecular design breeding technology in terms of breeding technology, and establishing an efficient system of genetic transformation and gene editing in non-heading Chinese cabbage in terms of molecular biology. It is expected to provide a theoretical basis for the creation of new varieties of non-heading Chinese cabbage and the research of molecular biology. [ABSTRACT FROM AUTHOR]

Clathrin is an evolutionarily highly conserved evolutionary protein consisting of clathrin light chains (CLC) and clathrin heavy chains (CHC), and these form its basic structure. Clathrin is an important host factor in the process of viral infection. In this study, we cloned the BcCLC1 gene and the BcCLC2 gene from the ‘49CX’ variety of non-heading Chinese cabbage (NHCC, Brassica campestris L. ssp. chinensis Makino) and verified their functions. The results showed that BcCLC1 was mainly localized in the cytomembrane and cytoplasm, and only a small amount entered the nucleus. BcCLC2 encoded a protein comprising 265 amino acids that were distributed in the cytomembrane, nucleus, and cytoplasm. A BiFC assay and yeast two-hybrid (Y2H) analysis showed that BcCLCs (BcCLC1 and BcCLC2) could interact with several TuMV proteins. We further investigated the mechanism of BcCLCs in regulating TuMV virus infections in NHCC, and observed that BcCLCs gene silencing inhibited TuMV infections and overexpression of BcCLCs in Arabidopsis promoted TuMV infections in NHCC. Finally, mutants of Arabidopsis homologs of BcCLCs were also screened and subjected to TuMV inoculation tests. In conclusion, we speculate that BcCLCs confer Turnip mosaic virus (TuMV) resistance in NHCC by interacting with TuMV proteins to promote the intracellular transport of the virus.

Purple non-heading Chinese cabbage is one of the most popular vegetables, and is rich in various health-beneficial anthocyanins. Research related to genes associated with anthocyanin biosynthesis in non-heading Chinese cabbage is important. This study performed integrative transcriptome and metabolome analysis in the purple non-heading Chinese cabbage wild type (WT) and its green mutant to elucidate the formation of purple leaves. The anthocyanin level was higher in purple than in green plants, while the contents of chlorophyll and carotenoid were higher in the green mutant than in the purple WT. Twenty-five anthocyanins were identified in purple and green cultivars; eleven anthocyanin metabolites were identified specifically in the purple plants. RNA-seq analysis indicated that 27 anthocyanin biosynthetic genes and 83 transcription factors were significantly differentially expressed between the WT and its mutant, most of them with higher expression in the purple than green non-heading Chinese cabbage. Transcriptome and metabolome analyses showed that UGT75C1 catalyzing the formation of pelargonidin-3,5-O-diglucoside and cyanidin-3,5-O-diglucoside may play a critical role in purple leaf formation in non-heading Chinese cabbage. Therefore, these results provide crucial information for elucidating the formation of purple leaves in non-heading Chinese cabbage. [ABSTRACT FROM AUTHOR]

Excessively using fertilizers poses serious problems such as environmental pollution, soil degeneration, and quality and yield reduction of vegetables. This study aimed to illustrate the effect of different organic manure and inorganic fertilizers on the characteristics of soil, and the growth, yield, and quality of non-heading Chinese cabbage. There were 28 treatments in the first experiment: no fertilization (CK), conventional fertilization (100% nitrogen T1), 20% reduction of total nitrogen (T2), 30% reduction of total nitrogen (T3), and 20% or 30% reduction of total nitrogen with four kinds of fertilizers and three kinds of dosages (24 treatments). Six treatments, being selected from the first experiment based on growth of plants, were further applied to the second experiment. The results of the second experiment showed that the pH, nitrate nitrogen, and organic matter content of soil treated by N2 (20% reduction of total nitrogen with 1500 kg·ha−1 No.1: Bacillus-enriched bio-organic fertilizer) were significantly enhanced compared with T1 (100% nitrogen). The N2-treated plants showed an 11.66% increase in root activity, 9.24% enhancement in yield, 5.79% increase in vitamin C (VC), and 47.87% decrease in nitrate content compared with T1. Nitrogen reduction with bio-organic fertilizer significantly increased the dominant phyla of Gemmatimonadetes and Chytridiomycota and significantly decreased Ascomycota, and increased the dominant genera of Gemmatimonas and Bacillus and decreased Fusarium, indicating that this treatment altered the microbial community composition of soil. Redundancy analysis (RDA) showed that AP (available phosphorus), OM (organic matter), and UREA (urease activity) of the soil were significantly correlated with microbial community structure. Yield was significantly, positively correlated with Rhodanobacter and Olpidium. In conclusion, nitrogen reduction with bio-organic fertilizer benefited growth, yield, and quality of non-heading Chinese cabbage by improving the soil quality. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[ Objectives J The paper aimed to create a new material of autotetraploid non-heading Chinese cabbage ' Wuyueman' with high yield and disease resistance. [ Methods J The 2 g • L -i colchicine solution was used to treat the cotyledon growth points of nonheading Chinese cabbage 'Wuyueman' diploid seedlings. The autotetraploid plants were identified by methods of morphological, cytological, anatomical and flow cytometry analysis. Then, the morphological indexes, agronomic traits, nutritional quality, photosynthetic characteristics and disease resistance between diploid and tetraploid plants were compared and analyzed. [ Results] Compared with diploid plants,autotetraploid plants had lower plant expansion,lower length-width ratio of leaves. Meanwhile,autotetraploid plants had oblate leaf shape, markedly thickened petiole, enlarged seeds, flowers organs, and pods. Also, the leaf stoma became larger and the stomata density decreased in autotetraploids. Pollen grains of autotetraploids presented irregular shapes such as rectangle and diamond. The number of cell chromosomes and the fluorescence intensity of DNA of autotetraploids were two times that of diploids. The contents of soluble sugar and cellulose in autotetraploids significantly increased, while the contents of nitrate nitrogen and chlorophyll significantly decreased. The contents of soluble protein and organic acid between diploids and autotetraploids were not significantly changed. In autotetraploids, the light saturation point, light compensation point, and dark breathing rate had increased by 13.47%, 24.02% and 12.12% respectively, and the photosynthetic capacity was stronger than that in diploids. Under the same conditions, the expression level of Botrytis cinerea was lower, and the disease resistance was stronger in autotetraploids. [ Conclusions] A new material of autotetraploid non-heading Chinese cabbage 'Wuyueman' with high yield and disease resistance potential was created. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

The availability of a high-quality genome sequence of Brassica campestris ssp. chinensis NHCC001 has paved the way for deep mining of genome data. We used the B. campestris NHCC001 draft genome to develop a comprehensive database, known as the non-heading Chinese cabbage database, which provides access to the B. campestris NHCC001 genome data. The database provides 127,347 SSR, from which 382,041 pairs of primers were designed. NHCCDB contains information on 105,360 genes, which were further classified into 63 transcription factor families. Furthermore, NHCCDB provides eight kinds of tools for biological or sequencing data analyses, including sequence alignment tools, functional genomics tools, comparative genomics tools, motif analysis tools, genome browser, primer design, and SSR analysis tools. In addition, eight kinds of graphs, including a box plot, Venn diagram, corrplot, Q-Q plot, Manhattan plot, seqLogo, volcano plot, and a heatmap, can be generated rapidly using NHCCDB. We have incorporated a search system for efficient mining of transcription factors and genes, along with an embedded data submit function in NHCCDB. We believe that the NHCCDB database will be a useful platform for non-heading Chinese cabbage research and breeding. [ABSTRACT FROM AUTHOR]

Iron is a trace metal that is found in animals, plants, and the human body. Human iron absorption is hampered by plant iron shortage, which leads to anemia. Leafy vegetables are one of the most direct and efficient sources of iron for humans. Despite the fact that ferrotrophic disorder is common in calcareous soil, however, non-heading Chinese cabbage performs a series of reactions in response to iron deficiency stress that help to preserve iron homeostasis in vivo. In this study, we discovered that iron deficiency stress caused leaf yellowing and impeded plant development in both iron-deficient and control treatments by viewing or measuring phenotypic, chlorophyll content, and Fe2+ content in both iron-deficient and control treatments. We found a total of 9213 differentially expressed genes (DEGs) in non-heading Chinese cabbage by comparing root and leaf transcriptome data with iron deficiency and control treatments. For instance, 1927 DEGs co-expressed in root and leaf, including 897 up-regulated and 1030 down-regulated genes, respectively. We selected some key antioxidant genes, hormone signal transduction, iron absorption and transport, chlorophyll metabolism, and transcription factors involved in the regulation of iron deficiency stress utilizing GO enrichment, KEGG enrichment, multiple types of functional annotation, and Weighted Gene Co-expression Network Analysis (WGCNA). This study identifies prospective genes for maintaining iron homeostasis under iron-deficient stress, offering a theoretical foundation for further research into the molecular mechanisms of greater adaptation to iron-deficient stress, and perhaps guiding the development of iron-tolerant varieties. [ABSTRACT FROM AUTHOR]

Male sterility has been widely used in hybrid seed production in Brassica, but not in B. rapa ssp. chinensis, and genetic models of male sterility for this subspecies are unclear. We discovered a spontaneous mutant in B. rapa ssp. chinensis. A series of progeny tests indicated that male sterility in B. rapa ssp. chinensis follows a three-allele model with BrMsa, BrMsb, and BrMsc. The male sterility locus has been mapped to chromosome A07 in BC1 and F2 populations through genotyping by sequencing. Fine mapping in a total of 1,590 F2 plants narrowed the male sterility gene BrMs to a 400 kb region, with two SNP markers only 0.3 cM from the gene. Comparative gene mapping shows that the Ms gene in B. rapa ssp. pekinensis is different from the BrMs gene of B. rapa ssp. chinensis, despite that both genes are located on chromosome A07. Interestingly, the DNA sequence orthologous to a male sterile gene in Brassica napus, BnRf, is within 400 kb of the BrMs locus. The BnRf orthologs of B. rapa ssp. chinensis were sequenced, and one KASP marker (BrMs_indel) was developed for genotyping based on a 14 bp indel at intron 4. Cosegregation of male sterility and BrMs_indel genotypes in the F2 population indicated that BnRf from B. napus and BrMs from B. rapa are likely to be orthologs. The BrMs_indel marker developed in this study will be useful in marker-assisted selection for the male sterility trait.

Clubroot disease is a kind of soil-borne disease that seriously infects Brassica species. In this study, we collected 121 varieties of non-heading Chinese cabbages. In order to better understand the genetic variation and to screen suitable clubroot disease-resistant parental material, we re-sequenced them to examine the population genetic structure, population genetic diversity, population differentiation index, and selective sweep based on SNPs. The mapping rate with the reference genome was high, and data quality analysis revealed that the sequencing quality was good. The annotated data indicated that intronic and intergenic areas held the majority of SNPs and indels. Four subgroups of 121 non-heading Chinese cabbages were identified using principal component analysis, phylogenetic tree, and genetic structure analysis. An examination of genetic diversity revealed that while selfing may happen in subgroups C and D, heterozygosity may exist in subgroups A and B. In subgroup B, self-fertilization is not possible. There was a moderate degree of genetic differentiation between subgroups B and C (Fst = 0.0744347). For genes in certain sweep regions, we also ran GO enrichment and KEGG enrichment analysis. Two disease resistance-related genes, BraA01g042910.3.5C and BraA06g019360.3.5C, were examined. These findings will serve as a theoretical foundation for developing novel, clubroot disease-resistant types of non-heading Chinese cabbages.

Salt stress has long been a prominent obstacle that restricts crop growth, and increasing the L-ascorbic acid (ASA) content of crops is an effective means of alleviating this stress. 2-Keto-L-gulonic acid (2KGA) is a precursor used in industrial ASA production as well as an ASA degradation product in plants. However, to date, no study has investigated the effects of 2KGA on ASA metabolism and salt stress. Here, we evaluated the potential of using 2KGA to improve crop resistance to salt stress (100mM NaCl) through a cultivation experiment of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). The results showed that the leaf and root biomass were significantly improved by 2KGA application. The levels of metabolites and enzymes related to stress resistance were increased, whereas the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were decreased. Lipid peroxidation and cell membrane damage were alleviated following 2KGA treatment. Positive correlations were found between photosynthetic pigments and organic solutes, ASA and photosynthetic pigments, and ASA and antioxidant enzymes. In contrast, negative correlations were observed between antioxidant enzymes and H2O2/MDA. Moreover, the expression levels of L-gulono-1,4-lactone oxidase , GDP-mannose pyrophosphorylase , dehydroascorbate reductase-3 , and ascorbate peroxidase were increased by 2KGA treatment. These results suggested that exogenous 2KGA application can relieve the inhibitory effect of salt stress on plant growth, and the promotion of ASA synthesis may represent a critical underlying mechanism. Our findings have significant implications for the future application of 2KGA or its fermentation residue in agriculture. [ABSTRACT FROM AUTHOR]

Salt stress has long been a prominent obstacle that restricts crop growth, and increasing the L-ascorbic acid (ASA) content of crops is an effective means of alleviating this stress. 2-Keto-L-gulonic acid (2KGA) is a precursor used in industrial ASA production as well as an ASA degradation product in plants. However, to date, no study has investigated the effects of 2KGA on ASA metabolism and salt stress. Here, we evaluated the potential of using 2KGA to improve crop resistance to salt stress (100mM NaCl) through a cultivation experiment of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). The results showed that the leaf and root biomass were significantly improved by 2KGA application. The levels of metabolites and enzymes related to stress resistance were increased, whereas the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were decreased. Lipid peroxidation and cell membrane damage were alleviated following 2KGA treatment. Positive correlations were found between photosynthetic pigments and organic solutes, ASA and photosynthetic pigments, and ASA and antioxidant enzymes. In contrast, negative correlations were observed between antioxidant enzymes and H2O2/MDA. Moreover, the expression levels of L-gulono-1,4-lactone oxidase, GDP-mannose pyrophosphorylase, dehydroascorbate reductase-3, and ascorbate peroxidase were increased by 2KGA treatment. These results suggested that exogenous 2KGA application can relieve the inhibitory effect of salt stress on plant growth, and the promotion of ASA synthesis may represent a critical underlying mechanism. Our findings have significant implications for the future application of 2KGA or its fermentation residue in agriculture.

The primary domain/leucine zipper (bZIP) transcription factor, Elongated Hypocotyl (HY5), is crucial for the photomorphogenesis of seedlings. Here, we identified BcHY5 as a regulator of hypocotyl length from the non-heading Chinese cabbage (NHCC) variety ‘Suzhouqing’, which encoded a protein comprised 164 amino acid residues. Ectopic expression of BcHY5 in Arabidopsis shortens the length of the hypocotyl. Additionally, we discovered a protein called BcBBX24 containing the B-BOX (BBX) domain, which is the interacting partner of BcHY5. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and GST pull-down assays revealed that BcHY5 interacted with BcBBX24. Additionally, by physically binding to the promoter of BcHY5, BcBBX24 inhibited the transcriptional activity of BcHY5. Therefore, our findings reveal a transcriptional mechanism through which light response occurs in NHCC seedlings, where BcHY5 can interact with BcBBX24 and BcBBX24 can prevent BcHY5 transcription.

BackgroundNon-heading Chinese cabbage (Brassica rapa ssp. chinensis) is an important leaf vegetable grown worldwide. However, there has currently been not enough transcriptome and small RNA combined sequencing analysis of cold tolerance, which hinders further functional genomics research.ResultsIn this study, 63.43 Gb of clean data was obtained from the transcriptome analysis. The clean data of each sample reached 6.99 Gb, and the basic percentage of Q30 was 93.68% and above. The clean reads of each sample were sequence aligned with the designated reference genome (Brassica rapa, IVFCAASv1), and the efficiency of the alignment varied from 81.54 to 87.24%. According to the comparison results, 1,860 new genes were discovered in Pak-choi, of which 1,613 were functionally annotated. Among them, 13 common differentially expressed genes were detected in all materials, including seven upregulated and six downregulated. At the same time, we used quantitative real-time PCR to confirm the changes of these gene expression levels. In addition, we sequenced miRNA of the same material. Our findings revealed a total of 34,182,333 small RNA reads, 88,604,604 kinds of small RNAs, among which the most common size was 24 nt. In all materials, the number of common differential miRNAs is eight. According to the corresponding relationship between miRNA and its target genes, we carried out Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the set of target genes on each group of differentially expressed miRNAs. Through the analysis, it is found that the distributions of candidate target genes in different materials are different. We not only used transcriptome sequencing and small RNA sequencing but also used experiments to prove the expression levels of differentially expressed genes that were obtained by sequencing. Sequencing combined with experiments proved the mechanism of some differential gene expression levels after low-temperature treatment.ConclusionIn all, this study provides a resource for genetic and genomic research under abiotic stress in Pak-choi.

Background: Non-heading Chinese cabbage (Brassica rapa ssp. chinensis) is an important leaf vegetable grown worldwide. However, there has currently been not enough transcriptome and small RNA combined sequencing analysis of cold tolerance, which hinders further functional genomics research. Results: In this study, 63.43 Gb of clean data was obtained from the transcriptome analysis. The clean data of each sample reached 6.99 Gb, and the basic percentage of Q30 was 93.68% and above. The clean reads of each sample were sequence aligned with the designated reference genome (Brassica rapa, IVFCAASv1), and the efficiency of the alignment varied from 81.54 to 87.24%. According to the comparison results, 1,860 new genes were discovered in Pak-choi, of which 1,613 were functionally annotated. Among them, 13 common differentially expressed genes were detected in all materials, including seven upregulated and six downregulated. At the same time, we used quantitative real-time PCR to confirm the changes of these gene expression levels. In addition, we sequenced miRNA of the same material. Our findings revealed a total of 34,182,333 small RNA reads, 88,604,604 kinds of small RNAs, among which the most common size was 24 nt. In all materials, the number of common differential miRNAs is eight. According to the corresponding relationship between miRNA and its target genes, we carried out Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the set of target genes on each group of differentially expressed miRNAs. Through the analysis, it is found that the distributions of candidate target genes in different materials are different. We not only used transcriptome sequencing and small RNA sequencing but also used experiments to prove the expression levels of differentially expressed genes that were obtained by sequencing. Sequencing combined with experiments proved the mechanism of some differential gene expression levels after low-temperature treatment. Conclusion: In all, this study provides a resource for genetic and genomic research under abiotic stress in Pak-choi. [ABSTRACT FROM AUTHOR]

[Objectives] The purpose of this study was to verify the function of MDHAR2 in multigene family of monodehydroascorbate reductase (MDHAR) and to explore its effect on ascorbic acid (AA) content in non-heading Chinese cabbage. [Methods] The full length of BeMDHAR2 was amplified from cDNA of non-heading Chinese cabbage Suzhouqing. Bioinformatics was used to analyze the amino acid sequence. Sub cellular localization was analyzed by transient expression in tobacco, and real-time fluorescence quantitative PCR (RT-PCR) was used to detect the expression of the gene under different abiotic stresses and hormones treatments. The effect of the gene expression on the ascorbic acid content of Suzhouqing was verified by vinus induced gene silencing (VIGS). [Results] The gene contained an open reading frame(ORF) of 1 308 bp. encoding 435 amino acids. The phylogenetic tree analysis showed that BeMDHAR2 had the closest evolutionary relationship with Brassica rapa. BcMDHAR2 was located on the nucleus, cytoplasm and membrane. RT-qPCR showed that the expression of BeMDHARZ was up-regulated and then down-regulated under low temperature salt, heavy metal ions and exogenous hormones. The gene had a certain response to photoperiod, and the expression level increased with the light time. The expression level decreased after dark. After BMDHAR2 gene silencing, the expression level of this gene was significantly reduced, and AsA content also decreased. The expression levels of related genes in AsA-GSH cycle were also changed. The SOD activity increased in the plant. [Conclusions] The expression level of BeMDHAR2 changed with the changes of external conditions. BeMDHAR2 could be induced by different hormones and respond to different stresses, which had positive regulation on AsA content in non-heading Chinese cabbage. [ABSTRACT FROM AUTHOR]

In non-heading Chinese cabbage, the yield relies mostly on the health of leaves, which can be heavily impacted by turnip mosaic virus (TuMV). The virions or viral ribonucleoprotein complexes are transported through the phloem and xylem. Plasmodesmata are indispensable because they traverse cell walls and connect companion cells, allowing virus particles long distance movement. However, which complexes and genes participate in this process is still unknown. Plants can activate defense mechanisms and apply disease resistance genes to respond to pathogen attacks. In this study, we collected the stems and petioles infected by TuMV for 7 d (TuMV-7), 14 d (TuMV-14), and 21 d (TuMV-21). Using isobaric tags for relative and absolute quantification-based proteomic technology, 6 043 distinct proteins were identified and 323, 240, 285, 203, 253, and 363 differentially expressed proteins were found in the comparable pairs of TuMV-7/control, TuMV-14/TuMV-7, TuMV-14/control, TuMV-21/TuMV-7, TuMV-21/TuMV-14, and TuMV-21/control, respectively. We performed a functional annotation analysis of all identified proteins and a functional enrichment analysis of all differentially expressed proteins. The results indicated that the long distance movement of TuMV involved many complex regulatory pathways. The respective proteins were related to those occurring in plasmodesmata and to Ca2+ transporters. Further, we also found proteins related to heat shock proteins, pathogenesis-related proteins, and proteins scavenging reactive oxygen species.

Heptahelical protein (HHP) signalling pathway is involved in cold acclimation responses to low temperature and other stresses. The HHP transcription factor family is the key component regulating this signalling pathway. In this study, five HHP-like genes, BcHHP1, BcHHP2, BcHHP3, BcHHP4, and BcHHP5, were isolated from non-heading Chinese cabbage (Brassica rapa ssp. chinensis cv. Suzhouqing). Multiple sequence alignment and phylogenetic analysis showed that BcHHP proteins are highly homologous to HHP proteins from Arabidopsis thaliana, Glycine max, Oryza sativa, and Zea mays. Some of these HHP proteins might share similar functions in some aspects, which might be further proved by interaction network of BcHHP genes. Furthermore, real-time quantitative PCR showed that BcHHP1 was induced under cold and salt treatments. Besides, BcHHP1 was also accumulated in response to abscisic acid and salicylic acid, indicating that BcHHP1 gene might participate in response to hormone treatments. In addition, a BcHHP1-YFP fusion protein was localized to the nucleus and cytoplasm. These results indicated that five BcHHP genes might play important roles in a functional HHP signalling pathway responding to cold treatment. This work might be useful for future functional analysis of other HHP-like genes.

Here, we analyzed the transcriptomes of Brassica campestris L. ssp. chinensis Makino under the treatment of a combination of red (R) and blue (B) light. In total, 5,643 differentially expressed genes (DEGs) were found under the R and B combination compared with the white light. An analysis of the DEGs enriched in photosynthesis and antenna proteins showed that there were many more upregulated subunits in PSII than those in PSI. The DEGs encoding the cytochrome b6f complex, photosynthetic electron transport, ATP synthases, and key genes involved in chlorophyll (Chl) and carotenoids (Car) biosynthesis were also upregulated. In addition, real-time PCR indicated that 13 of 15 genes showed the similar expression patterns with the RNA-seq data. The R and B combination might promote photosynthesis by specifically regulating Chl biosynthesis, thus accelerating the growth and development of plants and providing a valuable reference for vegetable production in factories and variety breeding.

Purple non-heading Chinese cabbage is one of the most popular vegetables, and is rich in various health-beneficial anthocyanins. Research related to genes associated with anthocyanin biosynthesis in non-heading Chinese cabbage is important. This study performed integrative transcriptome and metabolome analysis in the purple non-heading Chinese cabbage wild type (WT) and its green mutant to elucidate the formation of purple leaves. The anthocyanin level was higher in purple than in green plants, while the contents of chlorophyll and carotenoid were higher in the green mutant than in the purple WT. Twenty-five anthocyanins were identified in purple and green cultivars; eleven anthocyanin metabolites were identified specifically in the purple plants. RNA-seq analysis indicated that 27 anthocyanin biosynthetic genes and 83 transcription factors were significantly differentially expressed between the WT and its mutant, most of them with higher expression in the purple than green non-heading Chinese cabbage. Transcriptome and metabolome analyses showed that UGT75C1 catalyzing the formation of pelargonidin-3,5-O-diglucoside and cyanidin-3,5-O-diglucoside may play a critical role in purple leaf formation in non-heading Chinese cabbage. Therefore, these results provide crucial information for elucidating the formation of purple leaves in non-heading Chinese cabbage.

Excessively using fertilizers poses serious problems such as environmental pollution, soil degeneration, and quality and yield reduction of vegetables. This study aimed to illustrate the effect of different organic manure and inorganic fertilizers on the characteristics of soil, and the growth, yield, and quality of non-heading Chinese cabbage. There were 28 treatments in the first experiment: no fertilization (CK), conventional fertilization (100% nitrogen T1), 20% reduction of total nitrogen (T2), 30% reduction of total nitrogen (T3), and 20% or 30% reduction of total nitrogen with four kinds of fertilizers and three kinds of dosages (24 treatments). Six treatments, being selected from the first experiment based on growth of plants, were further applied to the second experiment. The results of the second experiment showed that the pH, nitrate nitrogen, and organic matter content of soil treated by N2 (20% reduction of total nitrogen with 1500 kg·ha−1 No.1: Bacillus-enriched bio-organic fertilizer) were significantly enhanced compared with T1 (100% nitrogen). The N2-treated plants showed an 11.66% increase in root activity, 9.24% enhancement in yield, 5.79% increase in vitamin C (VC), and 47.87% decrease in nitrate content compared with T1. Nitrogen reduction with bio-organic fertilizer significantly increased the dominant phyla of Gemmatimonadetes and Chytridiomycota and significantly decreased Ascomycota, and increased the dominant genera of Gemmatimonas and Bacillus and decreased Fusarium, indicating that this treatment altered the microbial community composition of soil. Redundancy analysis (RDA) showed that AP (available phosphorus), OM (organic matter), and UREA (urease activity) of the soil were significantly correlated with microbial community structure. Yield was significantly, positively correlated with Rhodanobacter and Olpidium. In conclusion, nitrogen reduction with bio-organic fertilizer benefited growth, yield, and quality of non-heading Chinese cabbage by improving the soil quality.

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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).