¹⁴C-Tracing of Lipid Metabolism

Lipids are produced through a dynamic metabolic network involving branch points, cycles, reversible reactions, parallel reactions in different subcellular compartments, and distinct pools of the same lipid class involved in different parts of the network. For example, diacylglycerol (DAG) is a biosynthetic and catabolic intermediate of many different lipid classes. Triacylglycerol can be synthesized from DAG assembled de novo, or from DAG produced by catabolism of membrane lipids, most commonly phosphatidylcholine. Quantification of lipids provides a snapshot of the lipid abundance at the time they were extracted from the given tissue. However, quantification alone does not provide information on the path of carbon flux through the metabolic network to synthesize each lipid. Understanding lipid metabolic flux requires tracing lipid metabolism with isotopically labeled substrates over time in living tissue. [¹⁴C]acetate and [¹⁴C]glycerol are commonly utilized substrates to measure the flux of nascent fatty acids and glycerol backbones through the lipid metabolic network in vivo. When combined with mutant or transgenic plants, tracing of lipid metabolism can provide information on the molecular control of lipid metabolic flux. This chapter provides a method for tracing in vivo lipid metabolism in developing Arabidopsis thaliana seeds, including analysis of ¹⁴C labeled lipid classes and fatty acid regiochemistry through both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) approaches.