(746f) Isotopically Nonstationary 13C Flux Analysis of Arabidopsis Thaliana Rosettes At Varying Light Intensities

Photoautotrophic metabolism represents the primary source of all food on earth as well as raw materials for bio-based production of fuels and chemicals.  Efforts to improve photosynthetic efficiency would be aided by more quantitative descriptions of primary metabolism in plants.  We have conducted isotopically nonstationary ¹³C metabolic flux analysis (INST-MFA) studies in Arabidopsis thaliana rosettes to quantify metabolism under photoautotrophic conditions using two different light intensities of 200 (normal light) and 500 µmol/m²/s (high light).  Using LC-MS/MS and GC-MS profiling techniques on isotopically labeled intracellular metabolites, along with measurements of net photosynthetic CO­­₂ uptake and starch production rate, we have created comprehensive flux maps of the central carbon metabolism in Arabidopsis rosettes under both normal and high light conditions.  The resulting flux maps provide a subcellular compartmentalized description of the carbon fixation pathways, which is the first application of INST-MFA to a terrestrial plant system in planta.  We show that modeling subcellular compartmentation is necessary to account for differences in glucose-6-phosphate labeling patterns in the chloroplast and cytosol, which give rise to starch and sucrose, respectively.  The INST-MFA results reveal that Calvin cycle and sucrose synthesis fluxes were increased two-fold in high light relative to normal light conditions, while flux towards starch synthesis remained the same.  However, photorespiration increased from 13% of photosynthesis in normal light to 28% in high light. We hypothesize that this increase in photorespiration under high light serves to dissipate surplus ATP and NADPH.   This study shows that INST-MFA is a feasible approach for quantifying photosynthetic metabolism in Arabidopsis rosettes and provides a quantitative framework to examine genetic and environmental perturbations that impact leaf metabolism.