¹³CO₂ Labeling and Mass Spectral Analysis of Photorespiration

Photorespiratory metabolism is compartmented over the chloroplast, peroxisome, cytosol, and mitochondria, and due to its complex structure it is often the case that metabolite levels alone are not able to fully describe photorespiration. Metabolic fluxes represent a more meaningful biological description of metabolism, adding to metabolite levels and often revealing different aspects of the system such as the presence of inactive metabolic pools of photorespiratory intermediates. We describe here a protocol for the ¹³CO₂ feeding of Arabidopsis and tracing of ¹³C enriched metabolites for metabolic fluxes estimation, which allows high throughput analysis of labeling pattern on different metabolites involved in photorespiration and downstream processes.