(485p) Design of Isotope Labeling Experiments to Measure Metabolic Flux through the Gamma Aminobutyric Acid (GABA) Shunt in Plants

The gamma-aminobutyric acid (GABA) shunt is a bypass of the tricarboxylic acid (TCA) cycle that is present in several organisms including plants and animals. In plants, this pathway is involved in key metabolic and regulatory processes and carries substantial carbon flux, especially under conditions of stress (Fait et al., Trends Plant Sci., 13, 14-19, 2008).

We report the design of isotope-assisted metabolic flux analysis (isotope MFA) to determine carbon fluxes through the GABA shunt and related pathways in the model plant Arabidopsis thaliana. Isotope MFA is a powerful investigative tool for the estimation of intracellular carbon fluxes and construction of flux maps toward the elucidation of metabolic control and identification of metabolic engineering targets. In isotope MFA, the organism or tissue of interest is fed a designed mixture of isotopically labeled carbon sources and fluxes are evaluated from the ensuing isotopic labeling patterns by advanced computational techniques. Because the evaluation of fluxes through this technique is critically dependent on the selection of appropriate mixtures of isotopically labeled carbon sources, the selection of such a mixture becomes an important task in experimental design.

In this presentation, we will report the use of detailed analysis of the GABA shunt and related metabolic networks as well as isotopomer balancing and statistical analyses toward selection of isotopic carbon sources to accurately measure flux through the GABA shunt. Additionally, we will also report data indispensable to flux analysis such as determination of the biomolecular composition of the tissue of interest. Because of the importance of the GABA shunt in plants and the current lack of quantitative understanding of this pathway, we anticipate that this study will contribute significantly toward an improved understanding of this pathway.