Compartment-specific metabolism associated with acetyl-CoA and acyl carrier protein
A characteristic feature of plant cells is the subcellular compartmentation of metabolism. Duplicated enzymes or cofactors occur in the same or distinct compartments, posing a challenge to define the complex metabolic networks that are central to biological functions. Our knowledge regarding the compartment-specific metabolism in plants has been hampered by the limitations of current analytical methods to determine the subcellular location of metabolites. In this dissertation, we integrate reverse genetic and metabolomic analyses to characterize the physiological roles of several compartment-specific enzymes and cofactors.
Two distinctly localized Arabidopsis acetate-activating enzymes, the plastidic acetyl-CoA synthetase (ACS) and the peroxisomal acetate non-utilizing 1 (ACN1), are functionally redundant, but their roles in metabolism are not clear. Mutations in both ACS and ACN1 lead to abnormal phenotypes of delayed growth and infertility, which are associated with hyperaccumulation of acetate levels and decreased accumulation of acetyl-CoA-derived metabolites. Cellular acetate is generated from either the oxidation of ethanol or the non-oxidative decarboxylation of pyruvate via the common intermediate acetaldehyde. These processes are induced by hypoxia, suggesting the role of ACS and ACN1 in reducing the carbon loss in the form of ethanol after hypoxia. Using 13C-acetate as a tracer, we demonstrate that the acetate metabolized by the plastidic ACS is used for the de novo synthesis of fatty acids and leucine, whereas the acetate activated by the peroxisomal ACN1 enters the glyoxylate cycle that generates the organic acid intermediates for amino acid biosynthesis. Collectively, these studies establish the significant role of these two enzymes in protecting plant cells from the toxic accumulation of excess acetate.
Typical of plants, Arabidopsis expresses two distinct Type II fatty acid synthases (FASs), one mitochondrial and the other in plastids. These two systems are supported by a small, phosphopantetheinylated protein cofactor, acyl carrier protein (ACP). The Arabidopsis genome contains eight ACP-coding genes. We demonstrate that three of these genes encode mitochondrial ACP (mtACP) isozymes, supporting the mitochondrial fatty acid synthase (mtFAS) system. Functional redundancy among the three mtACPs was dissected by a genetic strategy, which demonstrate that the simultaneous loss of all three mtACP genes is associated with an embryo-lethal phenotype. Characterization of double mutant combinations revealed unequal functional redundancy among the three mtACP isoforms, with mtACP3 being the least effective of the three in supporting the mtFAS system.