Poster abstracts
Poster number 112 submitted by Enkhtuul Tsogtbaatar
13C-labeling of pennycress (Thlaspi arvense L.) embryos to reveal a bottleneck in oil synthesis
Enkhtuul Tsogtbaatar (The Ohio State Biochemistry Program ), Jean-Christophe Cocuron (Department of Molecular Genetics, Center for Applied Plant Sciences, The Ohio State University ), Ana P. Alonso (Department of Molecular Genetics)
Abstract:
Pennycress (Thlaspi arvense L.) produces oil up to 35% of the total seed biomass, and its overall fatty acid composition has shown to be suitable for industrial products and biodiesel. However, for this plant to become an economically viable, its oil production needs to be improved. In pennycress embryos, fatty acid synthesis (FAS) requires carbon skeletons, energy, and reducing power; all of these are provided by central metabolism. We hypothesize that one or more steps is/are limiting FAS. To test this hypothesis, we have previously conducted a metabolomics study that identified the main carbon and nitrogen sources, and the active biochemical pathways during FAS in pennycress embryos. We are now performing 13C-Metabolic Flux Analysis to quantify in vivo carbon fluxes through each metabolic pathway, which will pinpoint potential bottleneck(s). First, this approach requires establishing in vivo culture conditions that mimic the development of pennycress embryos in planta. Given that the endosperm provides embryos for necessary substrates, the endosperm composition was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to design the culture growth medium. Second, the efficiency with which pennycress embryos convert carbon into biomass was measured to be 93%. Third, pennycress embryos were incubated with 13C-labeled substrates until metabolic and isotopic steady states are attained. The labeling abundances in intracellular metabolites were then quantified by LC-MS/MS. The main findings from the 13C-glucose labeling experiment show the higher activity of oxidative pentose phosphate pathway in cytosol than in plastid, and reversibility of glycolysis due to aldolase activity. In addition, the 13C-glutamine labeling experiment results indicate no occurrence of gluconeogenesis, but reversibility of isocitrate dehydrogenase in Krebs cycle and active NADP-dependent malic enzyme.
Future plans include the incorporation of all the labeling information into a mathematical model to generate a flux map, which will identify the bottleneck(s) in FAS. Understanding the biochemical basis of oil synthesis in pennycress embryos is fundamental to advance future breeding and/or metabolic engineering efforts aiming at increasing FAS.
Keywords: Pennycress, Metabolic Flux Analysis, oil metabolism