(197d) Metabolic Flux Analysis Using Designed Isotope Labeling Experiments Reveals Effects of Light On Metabolic Fluxes In Heterotrophic Plant Suspension Cells

It is well known that light regulates plant metabolism substantially and in several different ways. For example, energy from light is crucial to the generation of ATP and the electron carrier nicotine adenine dinucleotide phosphate (reduced) (NADPH) during photosynthesis. The important role of light in plant primary metabolism is exemplified by its induction of glutamine synthetase (which plays an essential role in nitrogen assimilation; Peterman and Goodman, Mol. Gen. Genet. 230: 145-154, 1991) and repression of glucose-6-phosphate dehydrogenase (which catalyzes the first, NADPH-providing step of the oxidative pentose phosphate pathway; Scheibe et al., Arch. Biochem. Biophys. 274: 290-297, 1989; Hutchings et al., J. Exp. Bot. 56: 577-585, 2005). Furthermore, the absence of light causes accumulation of vegetative storage proteins (Berger et al., Plant Mol. Biol. 27: 933-942, 1995) whereas its presence causes accumulation of starch (Hendriks et al., Plant Physiol. 133: 838-849, 2003). Hence light affects various facets of plant metabolism including carbon and nitrogen fixation, the accumulation of various metabolites as well as growth and development, at the systems level (Bläsing et al., The Plant Cell 17: 3257 -3281, 2005). To gain a systemwide perspective on the metabolic changes occurring in plant cells under differential light treatments we performed isotope-assisted metabolic flux analysis (isotope MFA) on heterotrophically grown plant suspension cells that were subjected to contrasting light conditions.

Isotope MFA uses isotopic tracers to map carbon traffic (fluxes) through metabolic pathways which provide valuable information in understanding cell physiology. We used statistical design methods to judiciously choose the isotopic tracers (in this study, differently ¹³C-labeled isomers of glucose), and determined the isotopically labeled metabolite measurements that can provide the most information on primary metabolic fluxes. On the basis of these results, three independent tracer experiments with 100% 1-¹³C, 100% 1,2-¹³C or 30% U-¹³C glucose were conducted on the plant suspension cells grown under continuous light or dark. On attainment of isotopic and metabolic steady state, several intracellular metabolites were analyzed for ¹³C labeling patterns using both gas chromatography-mass spectrometry and 2-D [¹³C, ¹H] NMR. The experimental data thus obtained from the three independent tracer experiments were collectively fitted to a multicompartmental plant metabolic network model consisting of primary carbon metabolic pathways. In this presentation we will compare the metabolic flux maps obtained under the two contrasting light conditions and discuss the implications of the differences. We expect that these flux maps will complement and augment the information obtained by previous MFA studies on plant suspension cells (Williams et al., Plant Physiol. 148: 704-718, 2008; Masakapalli et al., Plant Physiol. 152: 602-619, 2009).