Fatty acid metabolism and intracellular lipid trafficking in plants

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IntroductionIn plants, fatty acids serve multiple functions as structural components, as storage compounds, and as signaling substances. Given the multiple roles of fatty acids a complex regulatory network is obviously essential to match supply and demand of the diverse pathways of fatty acid metabolism. The regulation involves control over fatty acid biosynthesis, the activation, and the transport of fatty acids within the cell.ObjectivesIntracellular transport and enzymatic activation of fatty acids are cornerstones of the regulatory network of plant fatty acid metabolism and both will be addressed in the proposed project. The ER and the plastid are major sites of plant lipid metabolism and substrate trafficking between both subcellular domains was shown to be essential. In one part of the proposed project we will focus on identifying new components of the lipid transport machinery which is still poorly understood. In another part, the role of free fatty acids on transcriptional control will be analyzed.The metabolic availability of fatty acids in the cytoplasm is strongly dependent on their enzymatic activation by acyl-CoA synthetases (LACS). In the third part of the project we will employ LACS mutant lines with modified fatty acid availability to gain insight into the intricate network of plant fatty acid metabolism.MethodsWe identified recently an Arabidopsis mutant line characterized by impaired lipid trafficking between ER and plastids and by elevated levels of free fatty acids. We will analyze this mutant by whole transcriptome shotgun sequencing (RNA-seq) in order to identify new components of the lipid trafficking machinery. By the same experiment we will investigate the effects of elevated levels of free fatty acids on the transcriptome. As alternative approach to further characterize the lipid transport machinery we will use immunoaffinity purification of tagged versions of proteins involved in the transport in order to co-purify associated proteins.In Arabidopsis we observed functional redundancy between activities responsible for the activation of free fatty acids. To enable a reverse-genetic approach for the gene family we generated a mutant collection comprising more than 130 multiple mutant lines. Mutant lines with concise phenotypes will be analyzed by a set of biochemical, genetic, and cell biological methods in order to understand the biological roles of specific cellular fatty acid pools.OutlookThe data obtained will reveal insight into the flux of fatty acids within the cellular metabolism and will support future efforts aiming at the modification of plant lipid metabolism. Improving the understanding on lipid trafficking between different subcellular compartments will also allow us to test for similar mechanisms in other model organisms.