Poster: Metabolic Engineering

Abs # 1014: A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus seeds

Presenter:	Schwender, Joerg , schwend2@msu.edu
Authors	Schwender, Joerg  (A)   Shachar-Hill, Yair  (A)   Ohlrogge, John B (A)
Affiliations:	(A): Michigan State University, Department of Plant Biology

Metabolic flux analysis by steady-state stable isotope labeling provides a tool to understand and quantitate central carbon metabolism in plant tissues which are unperturbed by transgenic or other manipulations. We are developing these methods to identify and quantify fluxes through the central reaction networks of developing oilseed embryos. To quantify relative carbon flux through the oxidative pentose phosphate pathway (OPPP) and glycolysis, fluxes through transaldolase and transketolase as well as triose cycling must be quantified. Embryos were cultured on liquid media containing [U-¹³C₆]glucose, [1-¹³C]glucose, [6-¹³C]glucose or [1,2-¹³C₂]glucose and the labeling patterns in amino acids, fatty acids, sucrose and starch were measured by GC/MS and NMR. The data were used to establish and verify a flux model of central carbon metabolism distributed between the cytosol and plastid. Computer-aided simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit flux parameters to experimental data. The distribution of label in cytosolic vs plastidic metabolites indicated that key intermediates of glycolysis/OPPP have similar labeling in the two compartments. Thus exchange of metabolites between compartments is rapid compared to net flux into lipids. Cycling rates between hexose-phosphate and triose-phosphate as well as transketolase were similar to net glycolytic flux. Flux through OPPP accounts for about 10 % of hexose influx into developing embryos. Therefore reductant produced by OPPP accounts for at most a third of NADPH needed for B. napus embryo fatty acid synthesis.