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Kinetic complexities of triacylglycerol accumulation in developing embryos from Camelina sativa provide evidence for multiple biosynthetic systems.

ABSTRACT: Quantitative flux maps describing glycerolipid synthesis can be important tools for rational engineering of lipid content and composition in oilseeds. Lipid accumulation in cultured embryos of Camelina sativa is known to mimic that of seeds in terms of rate of lipid synthesis and composition. To assess the kinetic complexity of the glycerolipid flux network, cultured embryos were incubated with [¹⁴C/¹³C]glycerol, and initial and steady state rates of [¹⁴C/¹³Cglyceryl] lipid accumulation were measured. At steady state, the linear accumulations of labeled lipid classes matched those expected from mass compositions. The system showed an apparently simple kinetic precursor-product relationship between the intermediate pool, dominated by diacylglycerol (DAG) and phosphatidylcholine (PC), and the triacylglycerol (TAG) product. We also conducted isotopomer analyses on hydrogenated lipid class species. [¹³C₃glyceryl] labeling of DAG and PC, together with estimates of endogenous [¹²C₃glyceryl] dilution, showed that each biosynthetically active lipid pool is ∼30% of the total by moles. This validates the concept that lipid sub-pools can describe lipid biosynthetic networks. By tracking the kinetics of [¹³C₃glyceryl] and [¹³C₂acyl] labeling, we observed two distinct TAG synthesis components. The major TAG synthesis flux (∼75%) was associated with >95% of the DAG/PC intermediate pool, with little glycerol being metabolized to fatty acids, and with little dilution from endogenous glycerol; a smaller flux exhibited converse characteristics. This kinetic heterogeneity was further explored using postlabeling embryo dissection and differential lipid extractions. The minor flux was tentatively localized to surface cells across the whole embryo. Such heterogeneity must be recognized in order to construct accurate gene expression patterns and metabolic networks describing lipid biosynthesis in developing embryos.

SUBMITTER: Pollard M

PROVIDER: S-EPMC8715117 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCamelina sativa is an emerging dedicated oilseed crop designed for biofuel and biodiesel applications as well as a source for edible and general-purpose oils. Such valuable oilseed crop is subjected to plant breeding programs and is suggested for large-scale production of better seed and oil quality. To accomplish this objective and to further enhance its oil content, a better understanding of lipid metabolism at the molecular level in this plant is critical. Here, we applied tissue transcriptomics and lipid composition analysis to identify and profile the genes and gene networks associated with triacylglycerol (TAG) biosynthesis, and to investigate how those genes are interacting to determine the quantity and quality of Camelina oil during seed development.ResultsOur Camelina transcriptome data analysis revealed an approximate of 57,854 and 57,973 genes actively expressing in developing seeds (RPKM ≥ 0.1) at 10-15 (Cs-14) and 16-21 (Cs-21) days after flowering (DAF), respectively. Of these, 7932 genes showed temporal and differential gene expression during the seed development (log2 fold change ≥1.5 or ≤-1.5; P ≤ 0.05). The differentially expressed genes (DEGs) were annotated and were found to be involved in distinct functional categories and metabolic pathways. Furthermore, performing quantitative real-time PCR for selected candidate genes associated with TAG biosynthesis validated RNA-seq data. Our results showed strong positive correlations between the expression abundance measured using both qPCR and RNA-Seq technologies. Furthermore, the analysis of fatty-acid content and composition revealed major changes throughout seed development, with the amount of oil accumulate rapidly at early mid seed development stages (from 16-28 DAF onwards), while no important changes were observed in the fatty-acid profile between seeds at 28 DAF and mature seeds.ConclusionsThis study is highly useful for understanding the regulation of TAG biosynthesis and identifying the rate-limiting steps in TAG pathways at seed development stages, providing a precise selection of candidate genes for developing Camelina varieties with improved seed and oil yields.

Project description:Chia (Salvia hispanica L.), a member of the mint family (Lamiaceae), is a rediscovered crop with great importance in health and nutrition and is also the highest known terrestrial plant source of heart-healthy omega-3 fatty acid, alpha linolenic acid (ALA). At present, there is no public genomic information or database available for this crop, hindering research on its genetic improvement through genomics-assisted breeding programs. The first comprehensive analysis of the global transcriptome profile of developing Salvia hispanica L. seeds, with special reference to lipid biosynthesis is presented in this study. RNA from five different stages of seed development was extracted and sequenced separately using the Illumina GAIIx platform. De novo assembly of processed reads in the pooled transcriptome using Trinity yielded 76,014 transcripts. The total transcript length was 66,944,462 bases (66.9 Mb), with an average length of approximately 880 bases. In the molecular functions category of Gene Ontology (GO) terms, ATP binding and nucleotide binding were found to be the most abundant and in the biological processes category, the metabolic process and the regulation of transcription-DNA-dependent and oxidation-reduction process were abundant. From the EuKaryotic Orthologous Groups of proteins (KOG) classification, the major category was "Metabolism" (31.97%), of which the most prominent class was 'carbohydrate metabolism and transport' (5.81% of total KOG classifications) followed by 'secondary metabolite biosynthesis transport and catabolism' (5.34%) and 'lipid metabolism' (4.57%). A majority of the candidate genes involved in lipid biosynthesis and oil accumulation were identified. Furthermore, 5596 simple sequence repeats (SSRs) were identified. The transcriptome data was further validated through confirmative PCR and qRT-PCR for select lipid genes. Our study provides insight into the complex transcriptome and will contribute to further genome-wide research and understanding of chia. The identified novel UniGenes will facilitate gene discovery and creation of genomic resource for this crop.

Project description:Rice (Oryza sativa) is an excellent model monocot with a known genome sequence for studying embryogenesis. Here we report the transcriptome profiling analysis of rice developing embryos using RNA-Seq as an attempt to gain insight into the molecular and cellular events associated with rice embryogenesis. RNA-Seq analysis generated 17,755,890 sequence reads aligned with 27,190 genes, which provided abundant data for the analysis of rice embryogenesis. A total of 23,971, 23,732, and 23,592 genes were identified from embryos at three developmental stages (3-5, 7, and 14 DAP), while an analysis between stages allowed the identification of a subset of stage-specific genes. The number of genes expressed stage-specifically was 1,131, 1,443, and 1,223, respectively. In addition, we investigated transcriptomic changes during rice embryogenesis based on our RNA-Seq data. A total of 1,011 differentially expressed genes (DEGs) (log(2)Ratio ? 1, FDR ? 0.001) were identified; thus, the transcriptome of the developing rice embryos changed considerably. A total of 672 genes with significant changes in expression were detected between 3-5 and 7 DAP; 504 DEGs were identified between 7 and 14 DAP. A large number of genes related to metabolism, transcriptional regulation, nucleic acid replication/processing, and signal transduction were expressed predominantly in the early and middle stages of embryogenesis. Protein biosynthesis-related genes accumulated predominantly in embryos at the middle stage. Genes for starch/sucrose metabolism and protein modification were highly expressed in the middle and late stages of embryogenesis. In addition, we found that many transcription factor families may play important roles at different developmental stages, not only in embryo initiation but also in other developmental processes. These results will expand our understanding of the complex molecular and cellular events in rice embryogenesis and provide a foundation for future studies on embryo development in rice and other cereal crops.

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Kinetic complexities of triacylglycerol accumulation in developing embryos from Camelina sativa provide evidence for multiple biosynthetic systems.

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