13c isotope labelling to follow the flux of photorespiratory intermediates

Measuring the carbon flux through metabolic pathways in intact illuminated leaves re-mains challenging because of, e.g., isotopic dilution by endogenous metabolites, the impossibility to reach isotopic steady state, and the occurrence of multiple pools. In the case of photorespiratory intermediates, our knowledge of the partitioning between photorespiratory recycling, storage, and utilization by other pathways is thus rather limited. There has been some controversy as to whether photorespiratory glycine and serine may not be recycled, thus changing the apparent stoichiometric coefficient between photorespiratory O2 fixation and CO2 release. We describe here an isotopic method to trace the fates of glycine, serine and glycerate, taking advantage of positional13C content with NMR and isotopic analyses by LC–MS. This technique is well-adapted to show that the pro-portion of glycerate, serine and glycine molecules escaping photorespiratory recycling is very small.