Transcriptome analysis reveals the potential lncRNA-mRNA modules involved in genetic male sterility and fertility of Chinese cabbage (brassica rapa L. ssp. pekinensis).

BACKGROUND: Long non-coding RNAs (lncRNAs) play a crucial role in regulating gene expression vital for the growth and development of plants. Despite this, the role of lncRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) pollen development and male fertility remains poorly understood. RESULTS: In this study, we characterized a recessive genic male sterile mutant (366-2 S), where the delayed degradation of tapetum and the failure of tetrad separation primarily led to the inability to form single microspores, resulting in male sterility. To analyze the role of lncRNAs in pollen development, we conducted a comparative lncRNA sequencing using anthers from the male sterile mutant line (366-2 S) and the wild-type male fertile line (366-2 F). We identified 385 differentially expressed lncRNAs between the 366-2 F and 366-2 S lines, with 172 of them potentially associated with target genes. To further understand the alterations in mRNA expression and explore potential lncRNA-target genes (mRNAs), we performed comparative mRNA transcriptome analysis in the anthers of 366-2 S and 366-2 F at two stages. We identified 1,176 differentially expressed mRNAs. Remarkably, GO analysis revealed significant enrichment in five GO terms, most notably involving mRNAs annotated as pectinesterase and polygalacturonase, which play roles in cell wall degradation. The considerable downregulation of these genes might contribute to the delayed degradation of tapetum in 366-2 S. Furthermore, we identified 15 lncRNA-mRNA modules through Venn diagram analysis. Among them, MSTRG.9997-BraA04g004630.3 C (ß-1,3-glucanase) is associated with callose degradation and tetrad separation. Additionally, MSTRG.5212-BraA02g040020.3 C (pectinesterase) and MSTRG.13,532-BraA05g030320.3 C (pectinesterase) are associated with cell wall degradation of the tapetum, indicating that these three candidate lncRNA-mRNA modules potentially regulate pollen development. CONCLUSION: This study lays the foundation for understanding the roles of lncRNAs in pollen development and for elucidating their molecular mechanisms in regulating male sterility in Chinese cabbage.

Genome-Wide Identification, Expression, and Protein Analysis of CKX and IPT Gene Families in Radish (Raphanus sativus L.) Reveal Their Involvement in Clubroot Resistance.

Cytokinins (CKs) are a group of phytohormones that are involved in plant growth, development, and disease resistance. The isopentenyl transferase (IPT) and cytokinin oxidase/dehydrogenase (CKX) families comprise key enzymes controlling CK biosynthesis and degradation. However, an integrated analysis of these two gene families in radish has not yet been explored. In this study, 13 RsIPT and 12 RsCKX genes were identified and characterized, most of which had four copies in Brassica napus and two copies in radish and other diploid Brassica species. Promoter analysis indicated that the genes contained at least one phytohormone or defense and stress responsiveness cis-acting element. RsIPTs and RsCKXs were expanded through segmental duplication. Moreover, strong purifying selection drove the evolution of the two gene families. The expression of the RsIPT and RsCKX genes distinctly showed diversity in different tissues and developmental stages of the root. Expression profiling showed that RsCKX1-1/1-2/1-3 was significantly upregulated in club-resistant materials during primary infection, suggesting their vital function in clubroot resistance. The interaction network of CKX proteins with similar 3D structures also reflected the important role of RsCKX genes in disease resistance. This study provides a foundation for further functional study on the IPT and CKX genes for clubroot resistance improvement in Raphanus.

Comparative Metabolome and Transcriptome Analysis Reveals the Defense Mechanism of Chinese Cabbage (Brassica rapa L. ssp. pekinensis) against Plasmodiophora brassicae Infection.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) ranks among the most cultivated and consumed vegetables in China. A major threat to its production is Plasmodiophora brassicae, which causes large root tumors, obstructing nutrient and water absorption and resulting in plant withering. This study used a widely targeted metabolome technique to identify resistance-related metabolites in resistant (DH40R) and susceptible (DH199S) Chinese cabbage varieties after inoculation with P. brassicae. This study analyzed disease-related metabolites during different periods, identifying 257 metabolites linked to resistance, enriched in the phenylpropanoid biosynthesis pathway, and 248 metabolites linked to susceptibility, enriched in the arachidonic acid metabolism pathway. Key metabolites and genes in the phenylpropanoid pathway were upregulated at 5 days post-inoculation (DPI), suggesting their role in disease resistance. In the arachidonic acid pathway, linoleic acid and gamma-linolenic acid were upregulated at 5 and 22 DPI in resistant plants, while arachidonic acid was upregulated at 22 DPI in susceptible plants, leading to the conclusion that arachidonic acid may be a response substance in susceptible plants after inoculation. Many genes enriched in these pathways were differentially expressed in DH40R and DH199S. The research provided insights into the defense mechanisms of Chinese cabbage against P. brassicae through combined metabolome and transcriptome analysis.

Integrative Transcriptome, miRNAs, Degradome, and Phytohormone Analysis of Brassica rapa L. in Response to Plasmodiophora brassicae.

Clubroot is an infectious root disease caused by Plasmodiophora brassicae in Brassica crops, which can cause immeasurable losses. We analyzed integrative transcriptome, small RNAs, degradome, and phytohormone comprehensively to explore the infection mechanism of P. brassicae. In this study, root samples of Brassica rapa resistant line material BrT24 (R-line) and susceptible line material Y510-9 (S-line) were collected at four different time points for cytological, transcriptome, miRNA, and degradome analyses. We found the critical period of disease resistance and infection were at 0-3 DAI (days after inoculation) and 9-20 DAI, respectively. Based on our finding, we further analyzed the data of 9 DAI vs. 20 DAI of S-line and predicted the key genes ARF8, NAC1, NAC4, TCP10, SPL14, REV, and AtHB, which were related to clubroot disease development and regulating disease resistance mechanisms. These genes are mainly related to auxin, cytokinin, jasmonic acid, and ethylene cycles. We proposed a regulatory model of plant hormones under the mRNA-miRNA regulation in the critical period of P. brassicae infection by using the present data of the integrative transcriptome, small RNAs, degradome, and phytohormone with our previously published results. Our integrative analysis provided new insights into the regulation relationship of miRNAs and plant hormones during the process of disease infection with P. brassicae.

bra-miR167a Targets ARF8 and Negatively Regulates Arabidopsis thaliana Immunity against Plasmodiophora brassicae.

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.

BrWAX2 plays an essential role in cuticular wax biosynthesis in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: Map-based cloning was used to identify the BrWAX2 gene, which was involved in the cuticular wax biosynthesis. The malfunction of BrWAX2 together with other reduced expression of genes in alkane-forming pathway caused the glossy phenotype. Cuticular wax covering the outer plant surface plays various roles in protecting against biotic and abiotic stresses. Wax-less mutant shows glossy in stem and leaf surface and plays important roles in enriching Chinese cabbage germplasm resources for breeding brilliant green varieties. However, genes responsible for the glossy trait in Chinese cabbage are rarely reported. In this study, we identified a glossy Chinese cabbage line Y1211-1. Genetic analysis indicated that the glossy trait in Y1211-1 was controlled by a single recessive locus, BrWAX2 (Brassica rapa WAX 2). Using bulked segregant sequencing (BSA-Seq) and kompetitive allele-specific PCR (KASP) assays, BrWAX2 was fine-mapped to an interval of 100.78 kb. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra032670, homologous to CER1 in Arabidopsis, was the most likely candidate gene for BrWAX2. The gene Bra032670 was absent in glossy mutant. Cuticular wax composition analysis and RNA-Seq analysis suggested that the absence of BrWAX2 together with the decreased expression of other genes in alkane-forming pathway reduced the wax amount and caused the glossy phenotype. Furthermore, we developed and validated the functional marker BrWAX2-sp for BrWAX2. Overall, these results provide insight into the molecular mechanism underlying cuticular wax biosynthesis and reveal valuable information for marker-assisted selection (MAS) breeding in Chinese cabbage.

BrWAX3, Encoding a ß-ketoacyl-CoA Synthase, Plays an Essential Role in Cuticular Wax Biosynthesis in Chinese Cabbage.

In this study, we identified a novel glossy mutant from Chinese cabbage, named SD369, and all wax monomers longer than 26 carbons were significantly decreased. Inheritance analysis revealed that the glossy trait of SD369 was controlled by a single recessive locus, BrWAX3. We fine-mapped the BrWAX3 locus to an interval of 161.82 kb on chromosome A09. According to the annotated genome of Brassica rapa, Bra024749 (BrCER60.A09), encoding a ß-ketoacyl-CoA synthase, was identified as the candidate gene. Expression analysis showed that BrCER60.A09 was significantly downregulated in all aerial organs of glossy plants. Subcellular localization indicated that the BrCER60.A09 protein functions in the endoplasmic reticulum. A 5567-bp insertion was identified in exon 1 of BrCER60.A09 in SD369, which lead to a premature stop codon, thus causing a loss of function of the BrCER60.A09 enzyme. Moreover, comparative transcriptome analysis revealed that the 'cutin, suberine, and wax biosynthesis' pathway was significantly enriched, and genes involved in this pathway were almost upregulated in glossy plants. Further, two functional markers, BrWAX3-InDel and BrWAX3-KASP1, were developed and validated. Overall, these results provide a new information for the cuticular wax biosynthesis and provide applicable markers for marker-assisted selection (MAS)-based breeding of Brassica rapa.

Global DNA Methylation and mRNA-miRNA Variations Activated by Heat Shock Boost Early Microspore Embryogenesis in Cabbage (Brassica oleracea).

Microspore culture, a type of haploid breeding, is extensively used in the cultivation of cruciferous crops such as cabbage. Heat shock (HS) treatment is essential to improve the embryo rate during the culture process; however, its molecular role in boosting early microspore embryogenesis (ME) remains unknown. Here we combined DNA methylation levels, miRNAs, and transcriptome profiles in isolated microspores of cabbage '01-88' under HS (32 °C for 24 h) and normal temperature (25 °C for 24 h) to investigate the regulatory roles of DNA methylation and miRNA in early ME. Global methylation levels were significantly different in the two pre-treatments, and 508 differentially methylated regions (DMRs) were identified; 59.92% of DMRs were correlated with transcripts, and 39.43% of miRNA locus were associated with methylation levels. Significantly, the association analysis revealed that 31 differentially expressed genes (DEGs) were targeted by methylation and miRNA and were mainly involved in the reactive oxygen species (ROS) response and abscisic acid (ABA) signaling, indicating that HS induced DNA methylation, and miRNA might affect ME by influencing ROS and ABA. This study revealed that DNA methylation and miRNA interfered with ME by modulating key genes and pathways, which could broaden our understanding of the molecular regulation of ME induced by HS pre-treatment.

Quantitative proteomic analyses reveal that energy metabolism and protein biosynthesis reinitiation are responsible for the initiation of bolting induced by high temperature in lettuce (Lactuca sativa L.).

BACKGROUND: Lettuce (Lactuca sativa L.), one of the most economically important leaf vegetables, exhibits early bolting under high-temperature conditions. Early bolting leads to loss of commodity value and edibility, leading to considerable loss and waste of resources. However, the initiation and molecular mechanism underlying early bolting induced by high temperature remain largely elusive. RESULTS: In order to better understand this phenomenon, we defined the lettuce bolting starting period, and the high temperature (33 °C) and controlled temperature (20 °C) induced bolting starting phase of proteomics is analyzed, based on the iTRAQ-based proteomics, phenotypic measurement, and biological validation by RT-qPCR. Morphological and microscopic observation showed that the initiation of bolting occurred 8 days after high-temperature treatment. Fructose accumulated rapidly after high-temperature treatment. During initiation of bolting, of the 3305 identified proteins, a total of 93 proteins exhibited differential abundances, 38 of which were upregulated and 55 downregulated. Approximately 38% of the proteins were involved in metabolic pathways and were clustered mainly in energy metabolism and protein synthesis. Furthermore, some proteins involved in sugar synthesis were differentially expressed and were also associated with energy production. CONCLUSIONS: This report is the first to report on the metabolic changes involved in the initiation of bolting in lettuce. Our study suggested that energy metabolism and ribosomal proteins are pivotal components during initiation of bolting. This study could provide a potential regulatory mechanism for the initiation of early bolting by high temperature, which could have applications in the manipulation of lettuce for breeding.

Genome-wide analysis of HSP70 family genes in cabbage (Brassica oleracea var. capitata) reveals their involvement in floral development.

BACKGROUND: Heat shock proteins have important functions in regulating plant growth and response to abiotic stress. HSP70 family genes have been described in several plant species, but a comprehensive analysis of the HSP70 family genes in cabbage has not been reported to date, especially their roles in floral development. RESULTS: In this study, we identified 52 BoHSP70 genes in cabbage. The gene structures, motifs, and chromosome locations of the BoHSP70 genes were analyzed. The genes were divided into seven classes using a phylogenetic analysis. An expression analysis showed that the BoHSP70 genes were highly expressed in actively growing tissues, including buds and calluses. In addition, six BoHSP70 genes were highly expressed in the binuclear-pollen-stage buds of a male fertile line compared with its near isogenic sterile line. These results were further verified using qRT-PCR. Subcellular localization analysis of the bud-specific gene BoHSP70-5 showed that it was localized in the cytoplasm. CONCLUSIONS: Our results help to elucidate the involvement of the BoHSP70 family genes in cabbage floral development and establish the groundwork for future research on the functions of these genes.

Fine mapping and candidate gene analysis of CRA8.1.6, which confers clubroot resistance in turnip (Brassica rapa ssp. rapa).

Clubroot disease poses a significant threat to Brassica crops, necessitating ongoing updates on resistance gene sources. In F2 segregants of the clubroot-resistant inbred line BrT18-6-4-3 and susceptible DH line Y510, the genetic analysis identified a single dominant gene responsible for clubroot resistance. Through bulk segregant sequencing analysis and kompetitive allele-specific polymerase chain reaction assays, CRA8.1.6 was mapped within 110 kb (12,255-12,365 Mb) between markers L-CR11 and L-CR12 on chromosome A08. We identified B raA08g015220.3.5C as the candidate gene of CRA8.1.6. Upon comparison with the sequence of disease-resistant material BrT18-6-4-3, we found 249 single-nucleotide polymorphisms, seven insertions, six deletions, and a long terminal repeat (LTR) retrotransposon (5,310 bp) at 909 bp of the first intron. However, the LTR retrotransposon was absent in the coding sequence of the susceptible DH line Y510. Given the presence of a non-functional LTR insertion in other materials, it showed that the LTR insertion might not be associated with susceptibility. Sequence alignment analysis revealed that the fourth exon of the susceptible line harbored two deletions and an insertion, resulting in a frameshift mutation at 8,551 bp, leading to translation termination at the leucine-rich repeat domain's C-terminal in susceptible material. Sequence alignment of the CDS revealed a 99.4% similarity to Crr1a, which indicate that CRA8.1.6 is likely an allele of the Crr1a gene. Two functional markers, CRA08-InDel and CRA08-KASP1, have been developed for marker-assisted selection in CR turnip cultivars. Our findings could facilitate the development of clubroot-resistance turnip cultivars through marker-assisted selection.

Map-Based Cloning and Characterization of Br-dyp1, a Gene Conferring Dark Yellow Petal Color Trait in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Flower color is an important trait in Brassica species. However, genes responsible for the dark yellow flower trait in Chinese cabbage have not been reported. In this study, we identified a dark-yellow-flowered Chinese cabbage line SD369. Genetic analysis indicated that the dark yellow flower trait in SD369 was controlled by a single recessive locus, Br-dyp1 (dark yellow petal color 1 in Brassica rapa). Using bulked segregant RNA sequencing and kompetitive allele-specific PCR assays, Br-dyp1 was fine-mapped to an interval of 53.6 kb on chromosome A09. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra037130 (BraA09.ZEP), which encodes a zeaxanthin epoxidase, was the most likely candidate gene for Br-dyp1. Carotenoid profile analysis suggested that Bra037130 (BraA09.ZEP) might participate in the epoxidation from zeaxanthin to violaxanthin. The 679 bp insertion in dark yellow petal caused premature stop codon, thus caused the loss-of-function of the enzyme zeaxanthin epoxidase (ZEP), which disturbed the carotenoid metabolism, and caused the increased accumulation of total carotenoid, and finally converted the flower color from yellow to dark yellow. Comparative transcriptome analysis also showed that the "carotenoid biosynthesis" pathway was significantly enriched, and genes involved in carotenoid degradation and abscisic acid biosynthesis and metabolism were significantly downregulated. Furthermore, we developed and validated the functional marker Br-dyp1-InDel for Br-dyp1. Overall, these results provide insight into the molecular basis of carotenoid-based flower coloration in B. rapa and reveal valuable information for marker-assisted selection breeding in Chinese cabbage.

Fine Mapping and Candidate Gene Identification of a White Flower Gene BrWF3 in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Flower color is an important trait in plants. However, genes responsible for the white flower trait in Chinese cabbage are rarely reported. In this study, we constructed an F2 population derived from the Y640-288 (white flower) and Y641-87 (yellow flower) lines for the fine mapping of the white flower gene BrWF3 in Chinese cabbage. Genetic analysis indicated that BrWF3 was controlled by a single recessive gene. Using BSA-seq and KASP assays, BrWF3 was fine-mapped to an interval of 105.6 kb. Functional annotation, expression profiling, and sequence variation analyses confirmed that the AtPES2 homolog, Bra032957, was the most likely candidate gene for BrWF3. Carotenoid profiles and transmission electron microscopy analysis suggested that BrWF3 might participate in the production of xanthophyll esters (particularly violaxanthin esters), which in turn disrupt chromoplast development and the formation of plastoglobules (PGs). A SNP deletion in the third exon of BrWF3 caused the loss of protein function, and interfered with the normal assembly of PGs, which was associated with reduced expression levels of genes involved in carotenoid metabolism. Furthermore, we developed and validated the functional marker TXBH83 for BrWF3. Our results provide insight into the molecular mechanism underlying flower color pigmentation and reveal valuable information for marker-assisted selection (MAS) breeding in Chinese cabbage.

Transcriptome and Coexpression Network Analyses Reveal Hub Genes in Chinese Cabbage (Brassica rapa L. ssp. pekinensis) During Different Stages of Plasmodiophora brassicae Infection.

Clubroot, caused by the soil-borne protist Plasmodiophora brassicae, is one of the most destructive diseases of Chinese cabbage worldwide. However, the clubroot resistance mechanisms remain unclear. In this study, in both clubroot-resistant (DH40R) and clubroot-susceptible (DH199S) Chinese cabbage lines, the primary (root hair infection) and secondary (cortical infection) infection stages started 2 and 5 days after inoculation (dai), respectively. With the extension of the infection time, cortical infection was blocked and complete P. brassica resistance was observed in DH40R, while disease scales of 1, 2, and 3 were observed at 8, 13, and 22 dai in DH199S. Transcriptome analysis at 0, 2, 5, 8, 13, and 22 dai identified 5,750 relative DEGs (rDEGs) between DH40R and DH199S. The results indicated that genes associated with auxin, PR, disease resistance proteins, oxidative stress, and WRKY and MYB transcription factors were involved in clubroot resistance regulation. In addition, weighted gene coexpression network analysis (WGCNA) identified three of the modules whose functions were highly associated with clubroot-resistant, including ten hub genes related to clubroot resistance (ARF2, EDR1, LOX4, NHL3, NHL13, NAC29, two AOP1, EARLI 1, and POD56). These results provide valuable information for better understanding the molecular regulatory mechanism of Chinese cabbage clubroot resistance.

An SNP Mutation of Gene RsPP Converts Petal Color From Purple to White in Radish (Raphanus sativus L.).

Along with being important pigments that determining the flower color in many plants, anthocyanins also perform crucial functions that attract pollinators and reduce abiotic stresses. Purple and white are two different colors of radish petals. In this study, two cDNA libraries constructed with purple and white petal plants were sequenced for transcriptome profiling. Transcriptome results implied that the expression level of the genes participating in the anthocyanin biosynthetic pathway was commonly higher in the purple petals than that in the white petals. In particular, two genes, F3'H and DFR, had a significantly higher expression pattern in the purple petals, suggesting the important roles these genes playing in radish petal coloration. BSA-seq aided-Next Generation Sequencing of two DNA pools revealed that the radish purple petal gene (RsPP) was located on chromosome 7. With additional genotyping of 617 F2 population plants, the RsPP was further confined within a region of 93.23 kb. Transcriptome and Sanger sequencing analysis further helped identify the target gene, Rs392880. Rs392880 is a homologous gene to F3'H, a key gene in the anthocyanin biosynthetic pathway. These results will aid in elucidating the molecular mechanism of plant petal coloration and developing strategies to modify flower color through genetic transformation.

Root Transcriptome and Metabolome Profiling Reveal Key Phytohormone-Related Genes and Pathways Involved Clubroot Resistance in Brassica rapa L.

Plasmodiophora brassicae, an obligate biotrophic pathogen-causing clubroot disease, can seriously affect Brassica crops worldwide, especially Chinese cabbage. Understanding the transcriptome and metabolome profiling changes during the infection of P. brassicae will provide key insights in understanding the defense mechanism in Brassica crops. In this study, we estimated the phytohormones using targeted metabolome assays and transcriptomic changes using RNA sequencing (RNA-seq) in the roots of resistant (BrT24) and susceptible (Y510-9) plants at 0, 3, 9, and 20 days after inoculation (DAI) with P. brassicae. Differentially expressed genes (DEGs) in resistant vs. susceptible lines across different time points were identified. The weighted gene co-expression network analysis of the DEGs revealed six pathways including "Plant-pathogen interaction" and "Plant hormone signal transduction" and 15 hub genes including pathogenic type III effector avirulence factor gene (RIN4) and auxin-responsive protein (IAA16) to be involved in plants immune response. Inhibition of Indoleacetic acid, cytokinin, jasmonate acid, and salicylic acid contents and changes in related gene expression in R-line may play important roles in regulation of clubroot resistance (CR). Based on the combined metabolome profiling and hormone-related transcriptomic responses, we propose a general model of hormone-mediated defense mechanism. This study definitely enhances our current understanding and paves the way for improving CR in Brassica rapa.

Comparative Transcriptome Analysis of Early- and Late-Bolting Traits in Chinese Cabbage (Brassica rapa).

Chinese cabbage is one of the most important and widely consumed vegetables in China. The developmental transition from the vegetative to reproductive phase is a crucial process in the life cycle of flowering plants. In spring-sown Chinese cabbage, late bolting is desirable over early bolting. In this study, we analyzed double haploid (DH) lines of late bolting ("Y410-1" and "SY2004") heading Chinese cabbage (Brassica rapa var. pekinensis) and early-bolting Chinese cabbage ("CX14-1") (B. rapa ssp. chinensis var. parachinensis) by comparative transcriptome profiling using the Illumina RNA-seq platform. We assembled 721.49 million clean high-quality paired-end reads into 47,363 transcripts and 47,363 genes, including 3,144 novel unigenes. There were 12,932, 4,732, and 4,732 differentially expressed genes (DEGs) in pairwise comparisons of Y410-1 vs. CX14-1, SY2004 vs. CX14-1, and Y410-1 vs. SY2004, respectively. The RNA-seq results were confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs revealed significant enrichment for plant hormone and signal transduction as well as starch and sucrose metabolism pathways. Among DEGs related to plant hormone and signal transduction, six unigenes encoding the indole-3-acetic acid-induced protein ARG7 (BraA02g009130), auxin-responsive protein SAUR41 (BraA09g058230), serine/threonine-protein kinase BSK11 (BraA07g032960), auxin-induced protein 15A (BraA10g019860), and abscisic acid receptor PYR1 (BraA08g012630 and BraA01g009450), were upregulated in both late bolting Chinese cabbage lines (Y410-1 and SY2004) and were identified as putative candidates for the trait. These results improve our understanding of the molecular mechanisms underlying flowering in Chinese cabbage and provide a foundation for studies of this key trait in related species.

Proteomic variations after short-term heat shock treatment reveal differentially expressed proteins involved in early microspore embryogenesis in cabbage (Brassica oleracea).

Microspore embryogenesis (ME), a widely used haploid breeding method that can considerably shorten the breeding cycle, provides an efficient mean of cultivating many important Brassica crops, such as cabbage, Chinese cabbage, and oilseed rape. For cabbage, in many cases, short-term heat shock treatment can strongly increase the embryogenesis rate, however, the underlying mechanism of this effect has not been elucidated. In this study, we compared the proteomics of isolated microspores with samples pretreated at 32 °C for 24 h and 25 °C for 24 h using two cabbage accessions (Zhonggan 628 and 87-534) showing highly different embryogenic rates. The embryo yield was 19.7 embryos/bud in Zhonggan 628 after 32 °C treatment, while no embryoid was observed in Zhonggan 628 after 25 °C treatment as well as in 87-534 at both temperatures. We identified a total of 363 and 282 differentially expressed proteins (DEPs) for Zhonggan 628 and 87-534 via a label-free proteomics technology. There were 97 DEPs specifically identified only in Zhonggan 628 but not in 87-534 after 32 °C heat-shock treatment that may be related to heat shock-induced embryogenesis in vitro culture. These DEPs were primarily enriched in carbon metabolic process, protein synthesis and degradation process, and signal transduction. Based on protein-protein interaction and pathway enrichment analyses, we proposed that SGT1 homolog A and B(SGT1), heat shock 70 kDa protein 5 (HSP70), cell division control protein 48 homolog A (CDC48) and fatty acyl-CoA reductase (FAR) might play important roles in microspore embryogenesis. This proteomic study may contribute to our molecular understanding of cabbage microspore embryogenesis and help to build a high-efficiency haploid breeding system.

Morphological, transcriptomics and phytohormone analysis shed light on the development of a novel dwarf mutant of cabbage (Brassica oleracea).

Plant dwarf mutants generally exhibit delayed growth, delayed development, short internodes, and abnormal leaves and flowers and are ideal materials to explore the molecular mechanism of plant growth and development. In the current study, we first discovered a spontaneous cabbage (Brassica oleracea) dwarf mutant 99-198dw, which exhibits a dwarf stature, wrinkled leaves, non-heading, and substantially reduced self-fertility compared with the wild-type 99-198; however, the underlying molecular mechanism of its dwarfism is unknown. Here, we performed comparative phenotype, transcriptome and phytohormone analyses between 99-198 and 99-198dw. Cytological analysis showed that an increase in cell size, a reduction in cell layers, chloroplast degradation and a reduction in mitochondria were observed in 99-198dw. RNA-Seq showed that a total of 3801 differentially expressed genes (DEGs) were identified, including 2203 upregulated and 1598 downregulated genes in the dwarf mutant. Key genes in stress-resistant pathways were mostly upregulated, including salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), ethylene (ET), etc., while the DEGs reported to be related to plant height, such as those involved in the gibberellin (GA), brassinolide (BR), indole-3-acetic acid (IAA), and strigolactone (SL) pathways were mostly downregulated. In addition, the DEGs in the cell division pathway were all downregulated, which is consistent with the cytokinesis defects detected by cytological analysis. The changes in the GA4, JA, ET, SA and ABA contents measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) absolute quantification were consistent with the transcriptome analysis. Further hormone treatment tests showed that the exogenous application of GA, BR, 6BA, paclobutrazol (PC), etc. did not rescue the phenotype, implying that the change in phytohormones is due to but not the cause of the dwarf trait. It was speculated that mutation of certain DEG related to cell division or participating in signalling pathway of phytohormones like GA, BR, IAA, and SL were the cause of dwarf. These results are informative for the elucidation of the underlying regulatory network in 99-198dw and enrich our understanding of plant dwarf traits at the molecular level.

Comparative Analysis of Genome Wide DNA Methylation Profiles for the Genic Male Sterile Cabbage Line 01-20S and Its Maintainer Line.

Methylation modifications play an important role in multiple biological processes. Several studies have reported altered methylation patterns in male sterile plants such as rice and wheat, but little is known about the global methylation profiles and their possible roles in the cabbage (Brassicaoleracea) male sterile line. In this study, single-base-resolution bisulfite sequencing (BS-Seq) was adopted to identify the pattern and degree of cytosine methylation in the male sterile line 01-20S and its near-isogenic fertile line 01-20F. Similar methylation patterns were profiled, with some changes observed in local positions. In total, 505 differentially methylated genomic regions (DMRs) and 106 DMR-associated genes were detected. Nine genes related to pollen development were discovered and further validated by a quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Among these, four were downregulated in 01-20S. In particular, Bol039180 (an invertase/pectin methylesterase inhibitor family protein) is likely involved in pectin degradation, and might play an important role in the pollen separation defects of 01-20S. This study facilitates a better understanding of DNA methylation alterations and their possible roles in genic male sterility in cabbages.