Testing models of fatty acid transfer and lipid synthesis in spinach leaf using in vivo oxygen-18 labeling

Oxygen-18 labeling has been applied to the study of plant lipid biosynthesis for the first time. [(13)C(2)(18)O(2)]Acetate was incubated with spinach (Spinacia oleracea) leaves and the (18)O content in fatty acid methyl esters isolated from different lipid classes measured by gas chromatography-mass spectometry. Fatty acids isolated from lipids synthesized within the plastid, such as monogalactosyldiacylglycerol, show an (18)O content consistent with the exogenous acetate undergoing a single activation step and with the direct utilization of acyl-acyl carrier protein by the acyl transferases of the chloroplast. In contrast, fatty acids isolated from lipids assembled in the cytosol, such as phosphatidylcholine, show a 50% reduction in the (18)O content. This is indicative of export of the fatty acyl groups from the plastid via a free carboxylate anion, and is consistent with the acyl-acyl carrier protein thioesterase:acyl-coenzyme A (CoA) synthetase mediated export mechanism. If this were not the case and the acyl group was transferred directly from acyl-acyl carrier protein to an acyl acceptor on the cytosolic side, there would be either complete retention of (18)O or, less likely, complete loss of (18)O, but not a 50% loss of (18)O. Thus, existing models for fatty acid transfer from the plastid and for spatially separate synthesis of "prokaryotic" and "eukaryotic" lipids have both been confirmed.