Project description:Female-sterile mutants are ideal materials for studying pistil development in plants. Here, we identified a female-sterile mutant fsm in Chinese cabbage. This mutant, which exhibited stable inheritance, was derived from Chinese cabbage DH line 'FT' using a combination of isolated microspore culture and ethyl methanesulfonate mutagenesis. Compared with the wild-type line 'FT,' the fsm plants exhibited pistil abortion, and floral organs were also relatively smaller. Genetic analysis indicated that the phenotype of fsm is controlled by a single recessive nuclear gene. Morphological observations revealed that the presence of abnormal ovules in fsm likely influenced normal fertilization process, ultimately leading to female sterility. Comparative transcriptome analysis on the flower buds of 'FT' and fsm using RNA-Seq revealed a total of 1,872 differentially expressed genes (DEGs). Of these, a number of genes involved in pistil development were identified, such as PRETTY FEW SEEDS 2 (PFS2), temperature-induced lipocalin (TIL), AGAMOUS-LIKE (AGL), and HECATE (HEC). Furthermore, GO and KEGG pathway enrichment analyses of the DEGs suggested that a variety of biological processes and metabolic pathways are significantly enriched during pistil development. In addition, the expression patterns of 16 DEGs, including four pistil development-related genes and 12 floral organ development-related genes, were analyzed using qRT-PCR. A total of 31,272 single nucleotide polymorphisms were specifically detected in fsm. These results contribute to shed light on the regulatory mechanisms underlying pistil development in Chinese cabbage.
Project description:Cytoplasmic male sterility (CMS) is universally utilized in cruciferous vegetables. However, the Chinese cabbage hau CMS lines, obtained by interspecific hybridization and multiple backcrosses of the Brassica juncea (B. juncea) CMS line and Chinese cabbage, show obvious leaf etiolation, and the molecular mechanism of etiolation remains elusive. Here, the ultrastructural and phenotypic features of leaves from the Chinese cabbage CMS line 1409A and maintainer line 1409B are analyzed. The results show that chloroplasts of 1409A exhibit abnormal morphology and distribution. Next, RNA-sequencing (RNA-Seq) is used to identify 485 differentially expressed genes (DEGs) between 1409A and 1409B, and 189 up-regulated genes and 296 down-regulated genes are found. Genes that affect chloroplasts development, such as GLK1 and GLK2, and chlorophyll biosynthesis, such as PORB, are included in the down-regulated DEGs. Quantitative real-time PCR (qRT-PCR) analysis validate that the expression levels of these genes are significantly lower in 1409A than in 1409B. Taken together, these results demonstrate that leaf etiolation is markedly affected by chloroplast development and pigment biosynthesis. This study provides an effective foundation for research on the molecular mechanisms of leaf etiolation of the hau CMS line in Chinese cabbage (Brassica rapa L. ssp. pekinensis).
Project description:Pistil development is a complicated process in plants, and female sterile mutant is an ideal material for screening and cloning the pistil development-related genes. In our previous study, a female sterile mutant fsm (namely fsm1 here) was obtained from a Chinese cabbage DH line ‘FT’ using a combination of isolated microspore culture and ethyl methanesulfonate (EMS) mutagenesis. BraA04g009730.3C was predicted as the candidate gene for mutant fsm1. BraA04g009730.3C encoded STERILE APETALA (SAP), a transcriptional regulator, which played a role in regulating floral organ development. In this study, another female sterile mutant (namely fsm2) was derived from a population combining EMS mutagenesis and germinating seeds of ‘FT’. The phenotype of mutant fsm2 was consistent with that of fsm1, exhibiting pistil abortion, and smaller floral organs. Genetic analysis indicated that the phenotype of mutant fsm2 was controlled by a single recessive nuclear gene. Allelism testing showed that the mutant genes of fsm1 and fsm2 were allelic, named as Brfsm. A single-nucleotide mutation (G-to-A) in the first exon of BraA04g009730.3C caused a missense mutation from GAA (glutamic acid) to GGA (glycine) in mutant fsm2. Comparative transcriptome analysis on the pistils of wild-type ‘FT’ and mutant fsm1 revealed that a total of 3,855 differentially expressed genes (DEGs) were obtained, among which 29 genes related to ovule development and 16 genes related to organ size were identified. Based on the validation of qRT-PCR, we proposed the possible regulatory pathways whereby SAP may mediate pistil development in the fsm mutant. The mutation of BraA04g009730.3C in fsm plants was involved in the pistil abortion and smaller floral organ in Chinese cabbage. These results lay a solid foundation for elucidating the molecular mechanism of pistil development in Chinese cabbage. Overall design: The RNA from the pistils of a female sterile mutant fsm1 and its wild type‘FT’at the full-bloom stage in Chinese cabbage were sequenced by RNA-Seq, in triplicate.
Project description:The female sterility mutants are ideal materials for studying the pistil development in plants. We identified a female sterility mutant (fsm) exhibiting stable inheritance, which was derived from a Chinese cabbage DH line ‘FT’ using the combination of isolated microspore culture and ethyl methanesulfonate (EMS) mutagenesis. Genetic analysis indicated that the phenotype of fsm was controlled by a single recessive nuclear gene. The morphological observations revealed that the differences in the floral organs were significant between fsm and its wild type ‘FT’, the pistil of fsm was smaller and shorter, especially the ovary; and the degenerated ovule in the ovary may directly result in the female sterility. Comparative transcriptome analysis of ‘FT’ and fsm were performed using RNA-Seq. A total of 1,872 differentially expressed genes were identified between ‘FT’ and fsm. Of these, a number of genes involved in the ovule development were found, such as PRETTY FEW SEEDS 2 (PFS2) and temperature-induced lipocalin (TIL), and these genes were all up-regulated in the fsm vs. ‘FT’ comparison. Furthermore, GO and KEGG pathway enrichment analyses of DEGs suggested a variety of biological processes and metabolic pathways were significantly enriched during the pistil development. In addition, the expression patterns of eighteen DEGs were analyzed using qRT-PCR to confirm the accuracy of the RNA-seq data. A total of 31,272 single nucleotide polymorphisms (SNPs) were specifically detected in fsm, which could be directly related to the female sterility phenotype. These results contribute to increase our understanding of the molecular mechanisms of pistil development in Chinese cabbage. The RNA from the developing flower buds of a female sterility mutant (fsm) and its wild type‘FT’at the full-bloom stage in Chinese cabbage were sequenced by RNA-Seq, in triplicate.
Project description:BACKGROUND: Chinese cabbage (Brassica rapa ssp. pekinensis) is a member of one of the most important leaf vegetables grown worldwide, which has experienced thousands of years in cultivation and artificial selection. The entire Chinese cabbage genome sequence, and more than forty thousand proteins have been obtained to date. The genome has undergone triplication events since its divergence from Arabidopsis thaliana (13 to 17 Mya), however a high degree of sequence similarity and conserved genome structure remain between the two species. Arabidopsis is therefore a viable reference species for comparative genomics studies. Variation in the number of members in gene families due to genome triplication may contribute to the broad range of phenotypic plasticity, and increased tolerance to environmental extremes observed in Brassica species. Transcription factors are important regulators involved in plant developmental and physiological processes. The AP2/ERF proteins, one of the most important families of transcriptional regulators, play a crucial role in plant growth, and in response to biotic and abiotic stressors. Our analysis will provide resources for understanding the tolerance mechanisms in Brassica rapa ssp. pekinensis. RESULTS: In the present study, 291 putative AP2/ERF transcription factor proteins were identified from the Chinese cabbage genome database, and compared with proteins from 15 additional species. The Chinese cabbage AP2/ERF superfamily was classified into four families, including AP2, ERF, RAV, and Soloist. The ERF family was further divided into DREB and ERF subfamilies. The AP2/ERF superfamily was subsequently divided into 15 groups. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns, and interaction networks of the AP2/ERF transcription factor superfamily were predicted and analyzed. Distribution mapping results showed AP2/ERF superfamily genes were localized on the 10 Chinese cabbage chromosomes. AP2/ERF transcription factor expression levels exhibited differences among six tissue types based on expressed sequence tags (ESTs). In the AP2/ERF superfamily, 214 orthologous genes were identified between Chinese cabbage and Arabidopsis. Orthologous gene interaction networks were constructed, and included seven CBF and four AP2 genes, primarily involved in cold regulatory pathways and ovule development, respectively. CONCLUSIONS: The evolution of the AP2/ERF transcription factor superfamily in Chinese cabbage resulted from genome triplication and tandem duplications. A comprehensive analysis of the physiological functions and biological roles of AP2/ERF superfamily genes in Chinese cabbage is required to fully elucidate AP2/ERF, which provides us with rich resources and opportunities to understand crop stress tolerance mechanisms.
Project description:This study aimed to comprehensively examine the interface between primary and secondary metabolites in oval- and rectangular-shaped Chinese cabbage (Brassica rapa ssp. pekinensis) using gas chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS) and high-performance liquid chromatography (HPLC). In addition to differences in shape, there was significant morphological variation between the two cultivars. The rectangular variety had greater height and deeper green color, whereas the oval variety had more leaves and greater width. A total of 42 primary metabolites identified by GC-TOFMS were subjected to partial least-squares discriminant, which indicated significant differences in the primary and secondary metabolisms of the two cultivars. Furthermore, total glucosinolate and phenolic contents were higher in the oval cultivar, whereas the rectangular cultivar contained a higher level of total carotenoids. This metabolome study comprehensively describes the relationship between primary and secondary metabolites in the oval and rectangular cultivars of Chinese cabbage and provides information useful for developing strategies to enhance the biosynthesis of glucosinolates, phenolics, and carotenoids in Chinese cabbage. Additionally, this work highlights that HPLC and GC-TOFMS-based metabolite profiling is suitable techniques to determine metabolic differences in Chinese cabbage.
Project description:Although drought stress is one of the most limiting factors in growth and production of Chinese cabbage (Brassica rapa L. ssp. pekinensis), the underlying biochemical and molecular causes are poorly understood. In the present study, to address the mechanisms underlying the drought responses, we analyzed the transcriptome profile of Chinese cabbage grown under drought conditions. Drought stress transcriptionally activated several transcription factor genes, including AP2/ERFs, bHLHs, NACs and bZIPs, and was found to possibly result in transcriptional variation in genes involved in organic substance metabolic processes. In addition, comparative expression analysis of selected BrbZIPs under different stress conditions suggested that drought-induced BrbZIPs are important for improving drought tolerance. Further, drought stress in Chinese cabbage caused differential acclimation responses in glucosinolate metabolism in leaves and roots. Analysis of stomatal aperture indicated that drought-induced accumulation of glucosinolates in leaves directly or indirectly controlled stomatal closure to prevent water loss, suggesting that organ-specific responses are essential for plant survival under drought stress condition. Taken together, our results provide information important for further studies on molecular mechanisms of drought tolerance in Chinese cabbage.
Project description:Chinese cabbage (Brassica rapa ssp. pekinensis) is a major crop that is widely cultivated, especially in Korea, Japan, and China. With the advent of next generation sequencing technology, the cost and time required for sequencing have decreased and the development of genome research accelerated. Genome sequencing of Chinese cabbage was completed in 2011 using the variety Chiifu-401-42, and since then the genome has been continuously updated. In the present study, we conducted whole-genome sequencing of Chinese cabbage inbred line CT001, a line widely used in traditional or molecular breeding, to improve the accuracy of genetic polymorphism analysis. The constructed CT001 pseudomolecule represented 85.4% (219.8 Mb) of the Chiifu reference genome, and a total of 38,567 gene models were annotated using RNA-Seq analysis. In addition, the spontaneous mutation rate of CT001 was estimated by resequencing DNA obtained from individual plants after sexual propagation for six generations to estimate the naturally occurring variations. The CT001 pseudomolecule constructed in this study will provide valuable resources for genomic studies on Chinese cabbage.
Project description:Calmodulin-like (CML) proteins are a primary family of plant-specific Ca2+ sensors that specifically bind to Ca2+ and deliver a Ca2+ signal. CML proteins have been identified and characterized in many plant species, such as the model plant Arabidopsis and rice. Based on considerable evidence, the roles of CML proteins are crucial in plant growth and development and in the response to various external stimuli. Nevertheless, the characterization and expression profiling of CML genes in Chinese cabbage (Brassica rapa L. ssp. pekinensis) remain limited.In this study, a genome-wide search and comprehensive analysis were performed, and a total of 79 BrCML genes were identified in Chinese cabbage. Gene structure analysis revealed that these BrCML genes contained two to four conserved EF-hand motifs. Phylogenetic analysis showed that CML homologs between Chinese cabbage and Arabidopsis shared close relationships. The identified BrCML genes were located across ten chromosomes and three different subgenomes of Chinese cabbage. Moreover, 126 pairs of orthologous CML genes were found among Chinese cabbage, Arabidopsis and Brassica oleracea. Expression analysis revealed that the expression of some BrCML genes was tissue-specific and that of some was susceptible to temperature stress. A putative interaction network of BrCML proteins was proposed, which suggested that BrCML2, BrCML6, BrCML15 and BrCML25 were co-expressed and might play roles in flower development and other relevant biological processes of Chinese cabbage.The results of this study increased the understanding and characterization of BrCML genes in Chinese cabbage, and will be a rich resource for further studies to investigate BrCML protein function in various developmental processes of Chinese cabbage.
Project description:BACKGROUND: Growth regulating factors (GRFs) have been shown to play important roles in plant growth and development. GRF genes represent a large multigene family in plants. Recently, genome-wide structural and evolutionary analyses of the GRF gene families in Arabidopsis, rice, and maize have been reported. Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the most important vegetables for agricultural production, and a full genome assembly for this plant has recently been released. However, to our knowledge, the GRF gene family from Chinese cabbage has not been characterized in detail. RESULTS: In this study, genome-wide analysis was carried out to identify all the GRF genes in Chinese cabbage. Based on the complete Chinese cabbage genome sequence, 17 nonredundant GRF genes, named BrGRFs, were identified and classified into six groups. Phylogenetic analysis of the translated GRF protein sequences from Chinese cabbage, Arabidopsis, and rice indicated that the Chinese cabbage GRF proteins were more closely related to the GRF proteins of Arabidopsis than to those of rice. Expression profile analysis showed that the BrGRF genes had uneven transcript levels in different organs or tissues, and the transcription of most BrGRF genes was induced by gibberellic acid (GA3) treatment. Additionally, over-expression of BrGRF8 in transgenic Arabidopsis plants increased the sizes of the leaves and other organs by regulation of cell proliferation. CONCLUSIONS: The data obtained from this investigation will contribute to a better understanding of the characteristics of the GRF gene family in Chinese cabbage, and provide a basis for further studies to investigate GRF protein function during development as well as for Chinese cabbage-breeding programs to improve yield and/or head size.