(130a) Following Carbon Traffic through the Pentose Phosphate Pathways in Plant Systems with Statistically Designed Isotope Labeling Experiments

The pentose phosphate pathway (PPP) is an integral part of primary carbon metabolism in plants and most microorganisms. It has a twofold function: its oxidative branch produces nicotine adenine dinucleotide phosphate (NADPH) which is crucial to the biosynthesis of amino acids and fatty acids, whereas its reductive branch synthesizes five-carbon intermediates that form the backbones of nucleotides, amino acids, phenylpropanoids and lignin (Kruger and von Schaewen, Curr. Opin. Plant Biol. 6: 236-246, 2003). Two features of the PPP enable plants to regulate carbon flow through this pathway and thereby respond efficiently to changing environmental conditions. First, the topology of the PPPs is malleable because of substrate non-specificity of the enzymes and reversibility of reactions involved in the reductive branch. Furthermore, the PPP in plants is duplicated in two intracellular compartments ? the cytosol and the plastid, with transfer of common metabolites across intracellular membranes. Extensive investigation of the PPPs such as assessing the impact of environmental conditions or genetic variations as well as comparative studies between plant species is required to shed light on the physiological function of the this complex pathway. Isotope assisted metabolic flux analysis (isotope MFA) is capable of providing valuable information needed to visualize carbon traffic through the PPP as fluxes offer a dynamic view of metabolic networks. We propose to perform isotope MFA to comparatively and quantitatively study the PPP in two lines of plant suspension cells under varying environmental conditions. The sophistication of the PPPs and their incompletely known topology pose additional challenges in conducting isotope MFA. Bearing this in mind we used published statistical methods (Mollney et al., Biotech. Bioeng. 66: 86-103, 1999) to design isotope labeling experiments (ILEs) that would maximize the identifiabilities of the fluxes in the PPP. A statistical design criterion known as the A-optimality criterion was used to compare the efficacies of commercially available glucose isotopomers as well as isotopomer measurements made by NMR and mass spectrometry (MS) toward elucidating PPP fluxes. The study identified optimal glucose isotopomers and showed that isotopomer abundances measured by MS were superior to those measured by NMR owing mainly to the higher accuracy of MS. In this presentation, we will report the metabolic flux maps for the PPP arising from the designed ILEs described above. We expect that these studies will complement and augment information reported by previous pioneering studies of the pathway (Schwender et al., J. Biol. Chem. 278: 29442-29453, 2003; Sriram et al., Plant Physiol. 136: 3043-3057, 2004; Masakapalli et al., Plant Physiol. 152: 602-619, 2009).