RNA-seq of Brassica napus seeds at different developmental stages
Ontology highlight
ABSTRACT: Understanding the regulation of lipid metabolism is vital for genetic engineering of Brassica napus (B. napus) to increase oil yield or modify oil composition. We report the application of Illumina Hiseq 2000 for transcriptome profiling of seeds of B. napus at different developmental stages, which may uncover the dynamic changes in lipid metabolism and reveal key genes involved in lipid biosynthesis and degradation. Total RNA from developing seeds at 2, 4, 6, and 8 weeks after pollination (WAP) were isolated and sequenced separately. The gene expression levels of all samples were quantified and normalized by the DESeq normalization. We found that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. Two genes, encoding for acetyl-CoA carboxylase and acyl-ACP desaturase, might be critical for fatty acid biosynthesis in oil rape seeds. This study provides insight into the mechanism underlying lipid metabolism and reveals candidate genes that are worthy of further investigation for their values in genetic engineering of B. napus. Whole Transcriptome profiling of developing Brassica napus seeds at 2, 4, 6, 8 WAP by RNA sequencing using Illumina HiSeq 2000.
Project description:Understanding the regulation of lipid metabolism is vital for genetic engineering of Brassica napus (B. napus) to increase oil yield or modify oil composition. We report the application of Illumina Hiseq 2000 for transcriptome profiling of seeds of B. napus at different developmental stages, which may uncover the dynamic changes in lipid metabolism and reveal key genes involved in lipid biosynthesis and degradation. Total RNA from developing seeds at 2, 4, 6, and 8 weeks after pollination (WAP) were isolated and sequenced separately. The gene expression levels of all samples were quantified and normalized by the DESeq normalization. We found that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. Two genes, encoding for acetyl-CoA carboxylase and acyl-ACP desaturase, might be critical for fatty acid biosynthesis in oil rape seeds. This study provides insight into the mechanism underlying lipid metabolism and reveals candidate genes that are worthy of further investigation for their values in genetic engineering of B. napus.
Project description:MicroRNAs (miRNAs) are important post-transcriptional regulators of plant development and seed formation. In Brassica napus, an important edible oil crop, valuable lipids are synthesized and stored in specific seed tissues during embryogenesis. The miRNA transcriptome of B. napus is currently poorly characterized, especially at different seed developmental stages. This work aims to describe the miRNAome of developing seeds of B. napus by identifying plant-conserved and novel miRNAs and comparing miRNA abundance in mature versus developing seeds. A total of 62 miRNA families were detected through a computational analysis of a large number of reads obtained from deep sequencing two small RNA and two RNA-seq libraries of (i) pooled immature developing stages and (ii) mature B. napus seeds. Among these miRNA families, 17 families are currently known to exist in B. napus; additionally, 32 families not reported in B. napus but conserved in other plant species were identified by alignment with known plant mature miRNAs. The contigs from the assembled mRNA-seq data allowed for a search for putative new precursors and led to the identification of 13 novel miRNA families. Differential expression between the libraries was determined through a statistical analysis of normalized miRNA reads and revealed several miRNAs and isomiRNAs that were more abundant during the developing stages. The predicted miRNA target genes encode a broad range of proteins related to seed development and energy storage. This work presents a comprehensive study of the miRNA transcriptome of B. napus seeds and will provide a basis for future research on more targeted studies of individual miRNAs and their functions in embryogenesis, seed maturation and lipid accumulation in B. napus. RNA profiles in 2 different seed libraries (mature seeds and a pool of developing seed stages) of Brassica napus by deep sequencing (Illumina HiSeq2000).
Project description:MicroRNAs (miRNAs) are important post-transcriptional regulators of plant development and seed formation. In Brassica napus, an important edible oil crop, valuable lipids are synthesized and stored in specific seed tissues during embryogenesis. The miRNA transcriptome of B. napus is currently poorly characterized, especially at different seed developmental stages. This work aims to describe the miRNAome of developing seeds of B. napus by identifying plant-conserved and novel miRNAs and comparing miRNA abundance in mature versus developing seeds. A total of 62 miRNA families were detected through a computational analysis of a large number of reads obtained from deep sequencing two small RNA and two RNA-seq libraries of (i) pooled immature developing stages and (ii) mature B. napus seeds. Among these miRNA families, 17 families are currently known to exist in B. napus; additionally, 32 families not reported in B. napus but conserved in other plant species were identified by alignment with known plant mature miRNAs. The contigs from the assembled mRNA-seq data allowed for a search for putative new precursors and led to the identification of 13 novel miRNA families. Differential expression between the libraries was determined through a statistical analysis of normalized miRNA reads and revealed several miRNAs and isomiRNAs that were more abundant during the developing stages. The predicted miRNA target genes encode a broad range of proteins related to seed development and energy storage. This work presents a comprehensive study of the miRNA transcriptome of B. napus seeds and will provide a basis for future research on more targeted studies of individual miRNAs and their functions in embryogenesis, seed maturation and lipid accumulation in B. napus. microRNA profiles in 2 different seed libraries (mature seeds and a pool of developing seed stages) of Brassica napus by deep sequencing (Illumina HiSeq2000).
Project description:In Brassicaceae and other species, the developing embryo develops in a spatially constrained environment, modulating embryo´s shape and size. It is unknown how embryonic cells adapt their growth and metabolism to the apparent mechanical cues, and in particular if embryo maturation is affected by physical constraints. To address this question, we performed topographical analysis (magnetic resonance imaging, infrared microspectroscopy, immunolabelling), metabolite, transcript and proteome profiling in developing seeds of Brassica napus. Here we describe how the imposition of mechanical stress during development causes a stimulation of oil and protein storage activity, characteristic of embryo maturation.
Project description:MicroRNAs and siRNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still less understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in a high oil content and a low oil content Brassica napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 11 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large amount of 23-nt small RNAs with specific nucleotide composition preference were also identified, which may present new classes of functional small RNAs.
Project description:High temperature stress results in yield loss and alterations to seed composition during seed filling in oilseed rape (Brassica napus). However, the mechanism underlying this heat response is poorly understood. In this study, we employed a microarray analysis with silique walls and seeds from the developing siliques (20 days after flowering) of Brassica napus that had undergone heat stress. Two-condition experiment, control vs heat stress, 2 time points
Project description:The rapeseed crop (Brassica napus) is valued mainly for animal feed, food oil and biofuel production. This plant is particularly susceptible to many pathogens like parasitic plants, fungi or animals attacking aerial or root parts. Conventional plant protection products, used intensively in agriculture, have a negative impact on the environment (air and water quality, biodiversity, etc.) as well as on human health. There is therefore a growing demand for the development of new, more planet-friendly alternative protection methods such as biocontrol compounds. Natural rhamnolipids (RLs) can be used as elicitors of plant defense mechanisms. Indeed, these glycolipids, from bacteria secretome, are biodegradable, non-toxic and they are known for their stimulating and protective effects, in particular on rapeseed against filamentous fungi. Characterizing the organ responsiveness to defense stimulating compounds like RLs is missing. This analysis is crucial in the frame of optimizing the effectiveness of RLs against various diseases. A TMT labeling of the proteins extracted from the shoots and roots of B. napus has been performed and showed a differential pattern of protein abundance between them. These results have allowed identifying several protein families possibly involved in the differential responses observed in shoots and roots of rapeseed upon RLs treatment. In particular, quantitative proteomic analysis highlighted differential accumulation of parietal and cytoplasmic defense or stress proteins in response to treatments with RLs with a clear effect of the type of application (foliar spraying or root absorption). These results must be considered for further use of RLs to fight specific rapeseed pathogens.
Project description:To broadly identify genes regulated by Transparent Testa16 in Brassica napus In order to broadly identify genes regulated by BnTT16s, microarray technology was employed to compare gene expression levels in developing seeds (2-DAP) of Bntt16 RNAi and wild-type plants.
Project description:Background: The biological control agent Pseudomonas chlororaphis PA23 is effective at protecting Brassica napus (canola) from the necrotrophic fungus Sclerotinia sclerotiorum via direct antagonism. Despite the growing importance of biocontrol bacteria in plant protection from fungal pathogens, little is known about how the host plant responds to bacterial priming on the leaf surface or about changes in gene activity genome-wide in the presence and absence of S. sclerotiorum. Results: PA23 priming of mature canola plants reduced the number of lesion forming petals by 90%. Global RNA sequencing of the host pathogen interface showed a reduction in the number of genes uniquely upregulated in response to S. sclerotiorum by 16-fold when pretreated with PA23. Upstream defense-related gene patterns suggest MAMP-triggered immunity via surface receptors detecting PA23 flagellin and peptidoglycans. Although systemic acquired resistance was induced in all treatment groups, a response centered around a glycerol-3-phosphate (G3P)-mediated pathway was exclusively observed in plants treated with PA23 alone. Activation of these defense mechanisms by PA23 involved mild reactive oxygen species production as well as pronounced thylakoid membrane structures and plastoglobule formation in leaf chloroplasts. Conclusion: Further to the direct antibiosis that it exhibits towards the pathogen S. sclerotiorum, PA23 primes defense responses in the plant through the induction of unique local and systemic defense regulatory networks. This study has shed light on the potential effects of biocontrol agents applied to the plant phyllosphere. Understanding these interactions will aid in the development of biocontrol systems as a viable alternative to chemical pesticides in the protection of important crop systems. Mature canola leaf tissue treated with combinations of PA23 or S. sclerotiorum ascospores (3 treatment groups) was compared to a water treated control (all treatments done in triplicate).
Project description:MicroRNAs and siRNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still less understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in a high oil content and a low oil content Brassica napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 11 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large amount of 23-nt small RNAs with specific nucleotide composition preference were also identified, which may present new classes of functional small RNAs. Examination of small RNA profiles in 2 different seed oil content rapeseed culvitars at 2 locations.