Metabolomic and transcriptomic analysis of the controls on carbon partitioning into TAG reserves in oilseeds

Plant oils supply about 25% of the calories in our diet. Increased consumption of oils and fats in the human diet is regarded as unhealthy, as it leads to obesity. However, the composition of plant oils influences whether they are beneficial or detrimental to human health. For example, there is evidence that consumption of long chain polyunsaturated fatty acids (LC-PUFAs), which are mainly supplied in our diet from fish oils, can improve our metabolism of fats in a beneficial way. In addition to their use as foodstuffs, plant oils are becoming increasingly important as replacements for petrochemicals in a wide range of industrial applications such as in the production of alternative fuels and lubricants. Despite the obvious uses and benefits of plant oils, we do not fully understand what controls oil yield and composition in agricultural crops. In these crops, the oils are produced in seeds. Therefore, in order to understand and optimize oil production in plants, we need to understand how oils are produced and stored in seeds. Oils in seeds are stored as triacylglycerols (TAGs), which are made up of three fatty acid molecules linked to glycerol. For many years, plant scientists have studied the biochemistry of TAG synthesis in oilseeds, with the aim to understand what metabolic pathway is responsible for TAG accumulation, and how it is regulated. However, it has become apparent that the process of TAG synthesis is only one component of overall lipid metabolism in plants, such that there are several competing pathways for the biochemical intermediates that are required to make oils. In addition, the picture is further complicated by recent work that has shown that there are multiple biochemical routes for TAG synthesis, and that TAG breakdown (catabolism) probably occurs at the same time as synthesis. Using molecular genetics approaches, researchers have identified many of the genes and metabolic intermediates that are important in lipid synthesis in plants. However, in order to understand how TAG synthesis is specifically regulated in oilseeds, we need to evaluate which genes and metabolites are primarily or specifically involved in TAG metabolism and which are involved in other areas of lipid metabolism. To answer this question, we plan to grow and harvest seeds from the model oilseed plant Arabidopsis at different developmental stages where TAG synthesis is known to be up- or down-regulated. There are hundreds of existing datasets that show how global gene expression (the transcriptome) varies over these developmental stages, and we plan to mine these data to find genes that show correlations with TAG synthesis. We also plan to generate some of our own transcriptomic data using Arabidopsis mutants where TAG synthesis is altered. Analysis of this data will uncover additional genes that may be important in regulating TAG synthesis. In order to correlate changes in gene expression with actual TAG synthesis during seed development, it is important to know how much TAG is present at any one stage, what the fatty acid composition of this TAG is, and how other lipid-related biochemical intermediates change in concentration. In addition, it is necessary to monitor how apparently unrelated biochemical pathways are changing, as some of the metabolites in these pathways may be indirectly regulating TAG biosynthesis. All these measurements can be accomplished using metabolomics, where the biochemical composition of small molecules is measured using a range of analytical techniques. Arabidopsis mutants that are deficient is specific lipid metabolism pathways will be selected for metabolome analysis, in order to understand how these pathways are linked to TAG synthesis. The results of this research will improve our understanding of lipid metabolism in plants, which will ultimately enable us to improve oil yields and the fatty acid composition of plants for dietary and industrial uses.

This project aims to identify genes and metabolites that are key in regulating triacylglycerol (TAG) content in oilseeds. We will achieve this by analysing metabolome and transcriptome data from wild type and mutant Arabidopsis seed tissues sampled over six developmental stages that are representative of differential TAG synthesis and breakdown. Both global metabolomic analysis (NMR, GC-MS) and specialised targeted profiling methods for key lipid related metabolites such as acyl-CoAs, TAGs and sphingolipids will be performed. This will give rise to the first comprehensive metabolomic dataset for developing and germinating Arabidopsis seeds. Ten pre-selected mutants disrupted in biochemical pathways directly related to the acyl-CoA pool and/or in the regulation of TAG synthesis during seed maturation will also be profiled in the first phase of the work. Comprehensive transcriptomic datasets already exist for four of the selected seed stages and we will generate additional datsets for the two remaining stages and for two seed development stages of the abscisic acid insensitive abi3 regulatory mutant which is altered in seed maturation and storage reserve synthesis. This data will be combined with all public domain Affymetrix based transcriptomic data (currently amounting to 1,700 non-redundant datasets) in an in-house fully relational my SQL database. This database will be used to identify putative TAG related transcriptional modules and it will also be used to house the metabolomic datasets. Metabolite-specific temporal patterns across the developmental series of maturing and germinating seeds will be generated and dedicated pattern matching procedures employed to identify similarities between metabolite and transcriptomic data. Based on this approach a further set of mutants will be selected for the second phase metabolomic and targeted gene expression analysis. This research will provide a platform of knowledge that will facilitate the rational design of metabolic engineering of economically valuable oils in crop plants.