Fatty acid metabolism in higher plants: Probing the mechanism of fatty acid transport across the chloroplast envelope

Fatty acids (FA's) play a variety of crucial roles in living organisms. Despite the important roles in various aspects of life, FA transport remains one of the most poorly understood areas of FA metabolism. The main objective of this thesis is to begin to determine the mechanism(s) of FA transport in plants. Lipid metabolism within the cell requires massive movements of acyl chains between intracellular organelles. One such example is the export of FA from the plastid. De novo fatty acid synthesis (FAS) in plants occurs almost exclusively in the plastids. The free fatty acid (FFA) released at the final step of FAS by hydrolysis of acyl-acyl carrier protein (ACP) crosses the double envelope membrane to be re-esterified to acyl-coenzyme A in the cytosol. The maximum half life for FFA in this export pool in pea and spinach leaves was determined to be ≤1 s. Kinetic labeling experiments with isolated pea chloroplasts indicated that the measured long-chain acyl CoA synthesis (LACS) reaction using freely diffusing bulk FFA substrate cannot account for the efficient LACS reaction that must occur at the very low in vivo concentrations of FFA. The "nascent" FFA was also shown to be protected from binding to extraplastidial bovine serum albumin before being converted to the long-chain acyl CoA. These data discount a free diffusion model of FFA export and indicate a channeled delivery of FFA to the LACS.; Plant cells are capable of incorporating, elongating and desaturating exogenously provided short- and medium-chain FA's. The existence of a pathway in the plastid for the esterification of exogenous FFA to ACP has only been inferred from past studies. In this thesis the plastid was demonstrated to be the major site for the elongation of the exogenous FFA into C16 and C18 FA's. An Arabidopsis T-DNA insertion mutant, aae15, was isolated through a reverse genetics approach, and this mutant displayed as much as 80% reduction in elongation of exogenous [1-14C]laurate to C16 and C18 FA's.; The gene (AAE15 (acyl activating enzyme15)) disrupted in the mutant is a homolog of long-chain acyl-CoA synthetases, is represented by 11 expressed sequence tags (EST's) and contains a plastid targeting sequence. The findings indicate the existence of a novel pathway of FFA metabolism in the plastid. It is likely that specific membrane proteins are involved in lipid traffic across the envelope. Despite the importance and complexity of the plastid envelope, only a small fraction of integral envelope proteins have been purified or characterized. Through a bioinformatics strategy 541 proteins were selected from the Arabidopsis thaliana nuclear genome as potential candidates of the plastid inner envelope membrane proteins (AtPEM candidates). A web based database was developed with detailed information about the predicted proteins, including their expression profiles based on EST databases. Twenty ATP Binding Cassette transporter proteins were selected as candidates for plastid envelope lipid transporters and 11 T-DNA insertion mutants were analyzed for FA compositions and contents. The knock-outs of At1g70610 and At5g64940 consistently showed reduced oil contents (about 50--70% of wild type) in seeds and therefore are candidates for further analysis as potential lipid transporters.