Isotopically Nonstationary 13C Metabolic Flux Analysis of Arabidopsis thaliana Rosettes at Altered Light Conditions

Photoautotrophic metabolism represents the primary source of all food on earth as well as raw materials for bio-based production of fuels and chemicals. In particular, the ability to quantify the flow and fate of carbon in biochemical networks using ¹³C flux analysis is important for engineering desired metabolic phenotypes. Therefore, efforts to improve photosynthetic efficiency would be aided by more quantitative descriptions of primary metabolism in plants. There are few comprehensive methods that quantitatively describe leaf metabolism, though such information would be valuable for increasing photosynthetic capacity, enhancing biomass production, and rerouting carbon flux toward desirable end products.

Isotopically nonstationary ¹³C metabolic flux analysis (INST-MFA) has been previously applied to map carbon fluxes in photoautotrophic bacteria, which involves model-based regression of transient ¹³C-labeling patterns of intracellular metabolites. We have recently conducted in vivo isotopic labeling of Arabidopsis thaliana rosettes with ¹³CO₂ at two differing light intensities of 200 (normal light) and 500 (high light) µmol/m²/s, with and without prior acclimation, in order to quantify fluxes through photosynthetic leaf metabolism. 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 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. The results suggest that subcellular compartmentation was necessary to include products from glucose-6-phosphate (i.e. starch and sucrose) that are spatially resolved in the chloroplast and cytosol.  Photorespiration flux increased from 15% to 34% of net CO₂ assimilation with increasing light, despite concomitant increases in carboxylation flux that led to more sucrose production. Interestingly, we observed an inverse relationship between intermediate pool sizes and Calvin cycle fluxes as light intensity increased. Additionally, we identified enhanced hexose exchange between the chloroplast and cytosol as a potential short-term adaptation to high light that was suppressed by acclimation. This study shows that INST-MFA is a feasible approach to quantifying photosynthetic metabolism in Arabidopsis rosettes and gives the framework for comparison in future studies involving perturbed environmental conditions, as well as transgenics with improved photosynthetic efficiency.