Modelling the peroxisomal carbon leak during lipid mobilization in Arabidopsis

Abstract

Abstract Mutation of the ACN1 (acetate non-utilizing 1) locus of Arabidopsis results in altered acetate assimilation into gluconeogenic sugars and anapleurotic amino acids and leads to an overall depression in primary metabolite levels by approx. 50% during seedling development. Levels of acetyl-CoA were higher in acn1 compared with wild-type, which is counterintuitive to the activity of ACN1 as a peroxisomal acetyl-CoA synthetase. We hypothesize that ACN1 recycles free acetate to acetyl-CoA within peroxisomes in order that carbon remains fed into the glyoxylate cycle. When ACN1 is not present, carbon in the form of acetate can leak out of peroxisomes and is reactivated to acetyl-CoA within the cytosol. Kinetic models incorporating estimates of carbon input and pathway dynamics from a variety of literature sources have proven useful in explaining how ACN1 may prevent the carbon leak and even contribute to the control of peroxisomal carbon metabolism. Lipid mobilization in oilseeds The processes by which a seed starts to germinate and quickly establishes itself as a fully self-nourishing organism, as well as the mechanisms that prevent these processes when growth conditions prove unfavourable, are truly remarkable. For seed to become seedling, the seed must imbibe, experience favourable environmental signals to germinate and then produce a set of machinery for the breakdown of reserves. In a few days after germination this machinery is degraded and the photosynthetic machinery takes over to make a self-sufficient plant. The processes that govern dormancy and germination have been studied extensively both physiologically and molecularly The control of lipid mobilization The efficient degradation of lipid stores requires the coordinate use of fatty acids by β-oxidation and the subsequent assimilation of acetyl-CoA. The induction of enzyme activities in both pathways matches the profile of TAG (triacylglycerol) degradation Overall, very few studies have been directed toward standard metabolic control processes regulating lipid mobilization in plants. One study attempted to investigate the regulation of the glyoxylate cycle using castor bean endosperm Creating models and fitting parameters We chose a relatively simple kinetic model in which metabolite concentrations are governed by mass flow and enzymatic rate constants E-MEXP-2493). A number of assumptions had to be made to form the model and assign rate constants. A major assumption of the model was a potential efflux of acetate from peroxisomes (k T ). Tobacco plants expressing a yeast acetyl-CoA hydrolase in mitochondria had elevated acetyl-CoA levels, which probably was due to acetate export and reactivation in the cytosol Output from the model The goal of the modelling was to produce realistic values for rate constants that would explain the observations that acetylCoA levels and metabolites were lower in acn1 than wild-type at day 3. By HPLC In conclusion, the model has allowed us to hypothesize that ACN1 prevents a leak of carbon as acetate from peroxisomes. We can extend this hypothesis to ACN1 serving a role to help regulate the flow of acetyl-CoA between β-oxidation and the glyoxylate cycle as part of a synthetase/thioesterase substrate cycle. Further refinement of the model will be possible by studying the fate of isotopically labelled fatty acids, acetate and other substrates in acn1 and other mutants, and obtaining metabolite levels in experiments conducted over the developmental time course