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J. Biol. Chem., Vol. 278, Issue 32, 29442-29453, August 8, 2003
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A Flux Model of Glycolysis and the Oxidative Pentosephosphate Pathway in Developing Brassica napus Embryos*
From the Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824
Developing oilseeds synthesize large quantities of triacylglycerol from sucrose and hexose. To understand the fluxes involved in this conversion, a quantitative metabolic flux model was developed and tested for the reaction network of glycolysis and the oxidative pentose phosphate pathway (OPPP). Developing Brassica napus embryos were cultured with [U-13C6]glucose, [1-13C]glucose, [6-13C]glucose, [U-13C12]sucrose, and/or [1,2-13C2]glucose and the labeling patterns in amino acids, lipids, sucrose, and starch were measured by gas chromatography/mass spectrometry and NMR. Data were used to verify a reaction network of central carbon metabolism distributed between the cytosol and plastid. Computer simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit metabolic flux parameters to the experimental data. The observed distribution of label in cytosolic and plastidic metabolites indicated that key intermediates of glycolysis and OPPP have similar labeling in these two compartments, suggesting rapid exchange of metabolites between these compartments compared with net fluxes into end products. Cycling between hexose phosphate and triose phosphate and reversible transketolase velocity were similar to net glycolytic flux, whereas reversible transaldolase velocity was minimal. Flux parameters were overdetermined by analyzing labeling in different metabolites and by using data from different labeling experiments, which increased the reliability of the findings. Net flux of glucose through the OPPP accounts for close to 10% of the total hexose influx into the embryo. Therefore, the reductant produced by the OPPP accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis.
Received for publication, April 2, 2003
* This work was supported by Department of Energy Grant DE-FG02-87ER13729, National Science Foundation Grant MCB 0224655, and United States Department of Agriculture Grant 83786. This work was also supported by the Michigan Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Plant Biology, Michigan
State University, Wilson Dr., East Lansing, MI 48824-1312. Tel.: 517-355-5237;
Fax: 517-353-1926; E-mail:
Schwend2{at}msu.edu.
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