(640g) Isotopically Nonstationary 13C Flux Analysis of Changes in Arabidopsis thaliana Leaf Metabolism Due to Environmental and Genetic Perturbations

Photoautotrophic metabolism represents the primary source of all food on earth as well as raw materials for bio-based production of fuels and chemicals. Therefore, improving plant productivity is an important aim for metabolic engineering. 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. Our group is developing novel approaches that use ¹³C metabolic flux analysis (MFA) to quantitatively assess in vivo metabolic phenotypes of photoautotrophs. Previously, we applied isotopically nonstationary MFA (INST-MFA) to map carbon fluxes in photoautotrophic bacteria, which involves model-based regression of transient ¹³C-labeling patterns of intracellular metabolites. The flux analysis revealed unanticipated photosynthetic inefficiencies tied to oxidative metabolic pathways, despite minimal photorespiration.

We have now applied a similar modeling approach to map autotrophic metabolism of Arabidopsis thaliana leaves, which is the first application of INST-MFA to a terrestrial plant system in planta. Metabolism was quantified under two different acclimated photoautotrophic conditions: 200 µmol/m²/s (low light, LL) and 500 µmol/m²/s (high light, HL). LC-MS/MS and GC-MS profiling of isotopically labeled intracellular metabolites, net flux measurements of CO₂ uptake and starch production, as well as output flux ratios of amino acids and sucrose tied by vascular exudate measurements were use to create comprehensive flux maps of central carbon metabolism in Arabidopsis leaves under both LL and HL conditions. The results quantitatively describe alteration in carbon partitioning by acclimation and light conditions. Despite a doubling in the RuBisCO carboxylation rate, the photorespiratory flux increased from 17% to 28% of net CO₂ assimilation with high light acclimation and was independently validated by ¹⁴C-labeling. The concentrations of multiple Calvin cycle intermediates were reduced during high light acclimation, indicating an inverse relationship between intermediate pool sizes and fluxes.

Our ongoing work involves extending the ¹³C INST-MFA approach to examine and describe differences in transgenic lines of Arabidopsis thaliana that have been engineered with an artificial carbon concentration mechanism to enhance photosynthetic carbon fixation.  Quantification of the global impact of these genetic perturbations on photosynthetic carbon fluxes will be required to guide further rounds of engineering. The resulting improvements in photosynthetic capacity in Arabidopsis leaves will provide a scientific framework for similarly transformative steps in crops that are important for biofuel and chemical feedstock needs.