Search Results (20)

Choy Sum (Brassica rapa subsp. chinensis var. parachinensis), also known as flowering Chinese cabbage, is an important stalk vegetable in Asia. However, the unique regulatory mechanism governing its “easy-bolting yet susceptible to premature bolting” trait remains poorly understood. The phosphatidyl ethanolamine-binding protein (PEBP) family serves as a central regulator of bolting, flowering, and growth development in plants. But this gene family has not been systematically identified and studied in Choy Sum yet. Therefore, this study systematically identified and analyzed the members of the PEBP gene family in Choy Sum using bioinformatics, transcriptomics, real-time fluorescence quantification, subcellular localization, and transgenic techniques. A total of 12 BcPEBP genes were identified and categorized into three subfamilies: FT-like, TFL1-like, and MFT-like. Phylogenomic analyses revealed family expansion through whole-genome duplication with strong purifying selection. Most members have highly conserved core motifs and gene structures. Protein sequence alignment showed that BcFT-2 and BcTFL-2 underwent non-synonymous mutations at key residues. The analysis of cis-acting elements suggests that the BcPEBP gene may be influenced by complex hormone and light regulatory networks. Expression profiling demonstrated leaf-specific upregulation of BcFT-1/2 during development and shoot apices-predominant expression of BcTFL1 genes, and the expression between homologous genes of BcTFL1-1/3 is more refined. Subcellular localization confirmed dual nuclear and plasma membrane targeting of BcFT-1/2 proteins. Overexpression of BcFT-1/2 in transgenic Arabidopsis promotes flowering. These findings establish BcPEBP genes as key bolting regulators and provide molecular targets for breeding-improved varieties. Full article

As a primary macronutrient, nitrogen is integral to plant growth and regulates their development; ammonium transporters (AMTs) mediate nitrogen absorption and its involvement in metabolism. In this study, nine BcAMT genes were identified in flowering Chinese cabbage (Brassica campestris) and were systematically categorized into two subfamilies. Their evolutionary relationships, conserved motifs, chromosomal distribution, cis-regulatory elements, and expression profiling were systematically characterized. RNA sequencing and quantitative real-time PCR (qRT-PCR) analyses demonstrated that BcAMT1.1 was abundantly expressed in roots, leaves, and stems of flowering Chinese cabbage and was markedly upregulated under nitrogen deficiency. Assessing subcellular location using GFP fusion demonstrated that BcAMT1.1 localized to the plasma membrane. Functional assays identified heterologous expression in the yeast mutant strain 31019b, and transgenic Arabidopsis validated that BcAMT1.1 acted as a functional ammonium transporter. Compared with the wildtype, overexpressing BcAMT1.1 promoted seedling growth, enhanced NH₄⁺ influxes and NO₃⁻ effluxes under low-nitrogen conditions, and significantly increased the transcription levels of key nitrogen assimilation genes (i.e., AtGLN1.1, AtGLN2, AtGDH2). Collectively, our findings enhance the fundamental understanding of BcAMT gene functions and highlight BcAMT1.1 as a crucial component in nitrogen uptake and assimilation under low-nitrogen conditions, providing valuable genetic resources for improving nitrogen efficiency in vegetable crops. Full article

Ogura cytoplasmic male sterility (CMS) in Chinese cabbage (Brassica rapa) is characterized by complete pollen abortion, wherein stamens fail to produce viable pollen while pistils retain normal fertility. This maternally inherited trait is valuable for hybrid breeding. This study employed integrated analysis of miRNA, transcriptome, and degradome sequencing data aligned to the Chinese cabbage reference genome to elucidate the molecular function of bra-miR9569 in Ogura CMS pollen fertility and explore its associated pathways. Subsequently, a bra-miR9569 overexpression vector was constructed and transformed into Arabidopsis thaliana. Phenotypic characterization of transgenic Arabidopsis lines, combined with anther viability assessment and quantification of ATP content and reactive oxygen species (ROS) levels in Chinese cabbage, was performed to analyze the effects of bra-miR9569. Our findings demonstrate that mutation of the mitochondrial gene orf138 in Ogura CMS lines leads to upregulation of bra-miR9569. This microRNA negatively regulates the expression of the ATP-related gene AHA6, resulting in reduced H⁺-ATPase activity. The consequent energy deficiency triggers cellular content degradation, ultimately causing failure of pollen wall formation and pollen abortion. Full article

Orphan genes (OGs), which are unique to a specific taxon and have no detectable sequence homology to any known genes across other species, play a pivotal role in governing species-specific phenotypic traits and adaptive evolution. In this study, 20 OGs of Chinese cabbage (Brassica rapa OGs, BrOGs) were transferred into Arabidopsis thaliana by genetic transformation to construct an overexpression library in which 50% of the transgenic lines had a delayed flowering phenotype, 15% had an early flowering phenotype, and 35% showed no difference in flowering time compared to control plants. There were many other phenotypes attached to these transgenic lines, such as leaf color, number of rosette leaves, and silique length. To understand the impact of BrOGs on delayed flowering, BrOG142OE, which showed the most significantly delayed flowering phenotype, was chosen for further analysis, and BrOG142 was renamed BOLTING RESISTANCE 4 (BR4). In BR4OE, the expression of key flowering genes, including AtFT and AtSOC1, significantly decreased, and AtFLC and AtFRI expression increased. GUS staining revealed BR4 promoter activity mainly in the roots, flower buds and leaves. qRT-PCR showed that BR4 primarily functions in the flowers, flower buds, and leaves of Chinese cabbage. BR4 is a protein localized in the nucleus, cytoplasm, and cell membrane. The accelerated flowering time phenotype of BR4OE was observed under gibberellin and vernalization treatments, indicating that BR4 regulates flowering time in response to these treatments. These results provide a foundation for elucidating the mechanism by which OGs regulate delayed flowering and have significance for the further screening of bolting-resistant Chinese cabbage varieties. Full article

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. Full article

Clubroot is a soil-borne disease caused by Plasmodiophora brassicae, which can seriously affect the growth and production of cruciferous crops, especially Chinese cabbage crops, worldwide. At present, few studies have been conducted on the molecular mechanism of this disease’s resistance response. In this experiment, we analyzed the bioinformation of bra-miR167a, constructed a silencing vector (STTM167a) and an overexpression vector (OE-miR167a), and transformed them to Arabidopsis to confirm the role of miR167a in the clubroot resistance mechanism of Arabidopsis. Afterwards, phenotype analysis and expression level analysis of key genes were conducted on transgenic plants. From the result, we found that the length and number of lateral roots of silence transgenic Arabidopsis STTM167a was higher than that of WT and OE-miR167a. In addition, the STTM167a transgenic Arabidopsis induced up-regulation of disease resistance-related genes (PR1, PR5, MPK3, and MPK6) at 3 days after inoculation. On the other hand, the auxin pathway genes (TIR1, AFB2, and AFB3), which are involved in maintaining the balance of auxin/IAA and auxin response factor (ARF), were down-regulated. These results indicate that bra-miR167a is negative to the development of lateral roots and auxins, but positive to the expression of resistance-related genes. This also means that the STTM167a can improve the resistance of clubroot by promoting lateral root development and the level of auxin, and can induce resistance-related genes by regulating its target genes. We found a positive correlation between miR167a and clubroot disease, which is a new clue for the prevention and treatment of clubroot disease. Full article

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. Full article

Leaf flattening plays a vital role in the establishment of plant architecture, which is closely related to plant photosynthesis and, thus, influences the product yield and quality of Chinese cabbage. In this study, we used the doubled haploid line ‘FT’ of Chinese cabbage as the wild type for ethyl methanesulfonate (EMS) mutagenesis and obtained a mutant cwm with stably inherited compact and wrinkled leaves. Genetic analysis revealed that the mutated trait was controlled by a single recessive nuclear gene, Brcwm. Brcwm was preliminarily mapped to chromosome A07 based on bulked segregant RNA sequencing (BSR-seq) and fine-mapped to a 205.66 kb region containing 39 genes between Indel12 and Indel21 using SSR and Indel analysis. According to the whole-genome re-sequencing results, we found that there was only one nonsynonymous single nucleotide polymorphism (SNP) (C to T) within the target interval on exon 4 of BraA07g021970.3C, which resulted in a proline to serine amino acid substitution. The mutated trait co-segregated with the SNP. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that BraA07g021970.3C expression was dramatically higher in ‘FT’ leaves than that in cwm leaves. BraA07g021970.3C is homologous to AT3G55000 encoding a protein related to cortical microtubule organization. A similar phenotype of dwarfism and wrinkled leaves was observed in the recessive homozygous mutant cwm-f1 of AT3G55000, and its T3 transgenic lines were restored to the Arabidopsis wild-type phenotype through ectopic overexpression of BraA07g021970.3C. These results verified that BraA07g021970.3C was the target gene essential for leaf flattening in Chinese cabbage. Full article

Late embryogenesis abundant (LEA) proteins are important developmental proteins in the response of plants to abiotic stress. In our previous study, BcLEA73 was differentially expressed under low-temperature stress. Herein, we combined bioinformatics analysis, subcellular localization, expression assays, and stress experiments (including salt, drought, and osmotic stress) to identify and analyze the BcLEA gene family. Gene cloning and functional analysis of BcLEA73 were performed in tobacco and Arabidopsis. Based on the sequence homology and the available conservative motif, 82 BrLEA gene family members were identified and were divided into eight subfamilies in the genome-wide database of Chinese cabbage. The analysis showed that the BrLEA73 gene was located on chromosome A09 and belonged to the LEA_6 subfamily. Quantitative real-time PCR analysis indicated that the BcLEA genes were differentially expressed to varying degrees in the roots, stems, leaves, and petioles of Wucai. The overexpressed BcLEA73 transgenic plants exhibited no significant differences in root length and seed germination rates compared to the wild-type (WT) plants under control conditions. Under salt and osmotic stress treatment, the root length and seed germination rates of the BcLEA73-OE strain were significantly greater than those of WT plants. Under salt stress, the total antioxidant capacity (T-AOC) of the BcLEA73-OE lines increased significantly, and the relative conductivity, (REL), hydrogen peroxide (H₂O₂) content, and superoxide anion (O₂⁻) production rate decreased significantly. Under drought treatment, the survival rate of the BcLEA73-OE lines was significantly higher than that of WT plants. These results showed that the BcLEA73 gene of Wucai functions in enhancing the tolerance of plants to salt, drought, and osmotic stress. This study provides a theoretical basis to explore the relevant functions of the BcLEA gene family members of Wucai. Full article

Nitrogen (N) is a major limiting factor for plant growth and vegetable production. Understanding the regulatory mechanisms of N uptake, transport, and assimilation is key to improving N use efficiency in plants. Ammonium transporters (AMTs) play an important role in plant N metabolism. In this study, we isolated an important AMT1 subfamily member (BcAMT1;5) with a highly conserved signatural AMT1 subfamily motif from flowering Chinese cabbage. Based on functional complementation in yeast mutant 31019b and overexpression of BcAMT1;5 in Arabidopsis, BcAMT1;5 is a functional AMT. Tissue expression analysis showed that BcAMT1;5 was mainly expressed in roots and showed multiple N regime transcript patterns to respond to varying nutritional conditions. This was up-regulated by N-deficiency and down-regulated by supplying NH₄⁺. The glucuronidase (GUS) activities of BcAMT1;5_pro::GUS showed a similar change in response to different N conditions. Overexpression of BcAMT1;5 accelerated the growth of transgenic seedlings, increased NH₄⁺ net influxes, and enhanced the content and accumulation of NH₄⁺ and NO₃⁻ at low N concentrations. Additionally, it increased the transcript levels of N assimilation-related genes in shoots. These results indicate that BcAMT1;5 may participate in N uptake and assimilation under various N conditions in flowering Chinese cabbage, but it was differed obviously from other AMT1s. Full article

Transposable elements (TEs) are DNA fragments that can be replicated or transposed within a genome. TEs make up a high proportion of the plant genome and contribute to genetic diversity and evolution, affecting genome structure or gene activity. Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous class II DNA transposable elements. MITEs have specific sequences, target site duplications (TSDs), and terminal inverted repeats(TIRs), which are characteristics of the classification of MITE families. In this study, a Stowaway-like MITE, PTE-2, was activated in transgenic Chinese cabbage lines. PTE-2 was revealed by in silico analysis as the putative activated element in transgenic Chinese cabbage lines. To verify the in silico analysis data, MITE insertion polymorphism (MIP) PCR was conducted and PTE-2 was confirmed to be activated in transgenic Chinese cabbage lines. The activation tendency of the copy elements of PTE-2 at different loci was also analyzed and only one more element was activated in the transgenic Chinese cabbage lines. Analyzing the sequence of MIP PCR products, the TSD sequence and TIR motif of PTE-2 were identified and matched to the characteristics of the Stowaway-like MITE family. In addition, the flanking region of PTE-2 was modified when PTE-2 was activated. Full article

Petal color is an important agronomic trait in cabbage (Brassica oleracea L. var. capitata). Although the key gene BoCCD4 has been functionally characterized, the underlying molecular regulatory mechanism of petal color formation in cabbage is still unclear. In this study, we applied the transcriptome analysis of yellow petals from the cabbage inbred line YL-1 and white petals from the Chinese kale inbred line A192-1 and the BoCCD4-overexpressing transgenic line YF-2 (YL-1 background), which revealed 1928 DEGs common to both the A192-1 vs. YL-1 and the YL-1 vs. YF-2 comparison groups. One key enzyme-encoding gene, BoAAO3, and two key TF-encoding genes, Bo2g151880 (WRKY) and Bo3g024180 (SBP), related to carotenoid biosynthesis were significantly up-regulated in both the A192-1 and YF-2 petals, which was consistent with the expression pattern of BoCCD4. We speculate that these key genes may interact with BoCCD4 to jointly regulate carotenoid biosynthesis in cabbage petals. This study provides new insights into the molecular regulatory mechanism underlying petal color formation in cabbage. Full article

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. Full article

Clubroot disease caused by Plasmodiophora brassicae is one of the major threats to Brassica crops. New clubroot resistant varieties of Chinese cabbage (B. rapa ssp. pekinensis) have been developed through breeding, but the underlying genetic mechanism of clubroot resistance is still unclear. In this study, two Chinese cabbage DH lines, clubroot-resistant Y635-10 and susceptible Y177-47 were crossed to develop F₂ population for fine mapping and cloning resistance gene CRq. After sequence analysis, the expression vector was constructed by gateway technology and transferred into Arabidopsis thaliana for functional characterization. Bulked segregant analysis sequencing (BSA-seq) confirmed that CRq is located in the 80 kb genomic region on chromosome A03 between markers GC30-FW/RV and BGA. In silico tools confirmed that the gene length was 3959 bp with 3675 bp coding sequences (CDs), and it has three exons and two introns. In addition, we found 72bp insertion in the third exon of CRq in the susceptible line. We developed and verified functional marker Br-insert1, by which genotyping results showed that 72bp insertion might lead to the destruction of the LRR region of Y177-47, resulting in a loss of resistance relative to clubroot. The results of genetic transformation showed that the roots for wild-type Arabidopsis thaliana were significantly enlarged compared with T₂ generation transgenic Arabidopsis after treatment by P. brassicae spores, and transgenic Arabidopsis had certain resistance. Therefore, CRq is a candidate gene of clubroot disease resistance in Chinese cabbage, which could be used as a reference for elucidating disease resistance mechanisms and the marker-assisted breeding of clubroot resistant varieties. Full article

Previous research has shown that miR398 contributed to plant thermotolerance by silencing its target gene COPPER/ZINC SUPEROXIDE DISMUTASE1 (CSD1) in Arabidopsis thaliana. However, the phylogenesis of miR398 and CSD1 in Brassica crop and their role in regulating leaf cell death under heat stress remains unexplored. Here, we characterized the homologous genes of miR398a and CSD1 in Brassica rapa ssp. pekinensis (Chinese cabbage) and found miR398a abundance was accumulated under heat stress (38 °C and 46 °C for 1 h) in Chinese cabbage, while the expression level of its targets BraCSD1-1 and BraCSD2-1 were downregulated. To further explore their role in heat response, we constructed the transgenic plants overexpressing artificial miR398a (aBra-miR398a), Bra-miR398a target mimic (Bra-MIM398a), and BraCSD1-1 in Chinese cabbage for genetic study. Under high temperatures, p35S::aBra-miR398a lines reduced the areas of leaf cell death and delayed the leaf cell death. By contrast, p35S::Bra-MIM398a and p35S::BraCSD1-1 plants enlarged the areas of leaf cell death and displayed the earliness of leaf cell death. Finally, we found that the expression level of stress-responsive genes BraLEA76, BraCaM1, BraPLC, BraDREB2A, and BraP5CS increased in transgenic plants overexpressing aBra-miR398a, which may contribute to their resistance to heat-induced leaf cell death. Taken together, these results revealed the function of Bra-miR398a in attenuating leaf cell death to ensure plant thermotolerance, indicating that the miR398-CSD1 module could be potential candidates for heat-resistant crop breeding. Full article