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First published online October 1, 2004; 10.1104/pp.104.050625 Plant Physiology 136:3043-3057 (2004) © 2004 American Society of Plant Biologists Quantification of Compartmented Metabolic Fluxes in Developing Soybean Embryos by Employing Biosynthetically Directed Fractional 13C Labeling, Two-Dimensional [13C, 1H] Nuclear Magnetic Resonance, and Comprehensive Isotopomer Balancing1,[w]Departments of Chemical Engineering (G.S., V.V.I., J.V.S.), Biochemistry, Biophysics and Molecular Biology (D.B.F.), Agronomy (J.M.P., R.Z., M.E.W.), and Genetics, Development and Cell Biology (M.H.S.), Iowa State University, Ames, Iowa 50011
Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis tool that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-13C Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [13C, 1H] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 13C isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain SDs. A high flux was found through the oxidative pentose phosphate pathway (19.99 ± 4.39 µmol d–1 cotyledon–1, or 104.2 carbon mol ± 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 ± 5.00 µmol d–1 cotyledon–1, or 113.2 carbon mol ±26.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.
1 This work was supported by the Division of Bioengineering and Environmental Systems (BES) of the National Science Foundation (grant no. BES–0224600), by the Plant Sciences Institute of Iowa State University, and by the Iowa Soybean Promotion Board. [w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.050625. * Corresponding author; e-mail jshanks{at}iastate.edu; fax 515–294–2689. Received July 26, 2004; returned for revision August 6, 2004; accepted August 6, 2004. This article has been cited by other articles:
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