Validation of carbon isotopologue distribution measurements by GC-MS and application to¹³C-metabolic flux analysis of the tricarboxylic acid cycle inBrassica napusleaves

Abstract

The estimation of metabolic fluxes in photosynthetic organisms represents an important challenge that has gained interest over the last decade with the development of 13C-Metabolic Flux Analysis at isotopically non-stationary steady-state. This approach requires a high level of accuracy for the measurement of Carbon Isotopologue Distribution in plant metabolites, which has still not been accurately evaluated at the isotopoloque level for GC-MS. Thus, uncertainties remain about the accuracy of the CID measured and subsequently, the metabolic fluxes associated. Moreover, a significant part of our knowledge about the functioning and the regulation of plant TCA cycle is solely based on 13C-labeling experiments analyzed with Nuclear Magnetic Resonnance. But this method is not very sensitive at low 13C abundance and can hardly fully resolve isotopologue dynamics inherent to the TCA cycle compared to MS. Here, we developed a workflow to validate the measurements of CIDs from plant metabolites with GC-MS by producing tailor-made E. coli standard extracts harboring a predictable binomial CID for all metabolites. Most of our TMS-derivatives mass fragments were validated with these standards and at natural isotope abundance in plant matrices. Then, we applied this validated MS method to investigate the light/dark regulation of plant TCA cycle metabolic fluxes by incorporating U-13C-pyruvate to Brassica napus leaf discs. We took advantage of pathway-specific isotopologues/isotopomers to showed that the TCA cycle has a nearly four-fold higher contribution to Citrate and Malate production than phosphoenolpyruvate carboxylase (PEPc). Overall, the dynamic of 13C-enrichment in Citrate, Glutamate, Succinate and Malate ruled out the hypothesis of the stored citrate contribution to TCA cycle and highlithed the major contribution of the forward plant TCA cycle to malate biosynthesis in both dark and light conditions. Interestingly, the light-dependent 13C-incorporation into Glycine and Serine showed that nearly 5% of TCA cycle-derived CO2 was actively reassimilated by the photosynthesis. We also observed an increase of PEPc contribution to malate production in the light while there was a higher involvement of PEPc-derived carbons to citrate production in dark condition. The results suggested an important role for Malate import into mitochondria in sustaining TCA cycle in dark conditions.