Combining Gas Exchange and Rapid Quenching of Leaf Tissue for Mass Spectrometry and NMR Analysis Using an External...

We describe here a method to study and manipulate photorespiration in intact illuminated leaves. When the CO₂/O₂ mole fraction ratio changes, instant sampling is critical, to quench leaf metabolism and thus trace rapid metabolic modification due to gaseous conditions. To do so, we combine ¹³CO₂ labeling and gas exchange, using a large custom leaf chamber to facilitate fast sampling by direct liquid nitrogen spraying. Moreover, the use of a high chamber surface area (about 130 cm²) allows one to sample a large amount of leaf material to carry out ¹³C-nuclear magnetic resonance (NMR) analysis and complementary analyses, such as isotopic analyses by high-resolution mass spectrometry (by both GC and LC-MS). ¹³C-NMR gives access to absolute ¹³C amounts at the specific carbon atom position in the labeled molecules and thereby provides an estimate of ¹³C-flux of photorespiratory intermediates. Since NMR analysis is not very sensitive and can miss minor metabolites, GC or LC-MS analyses are useful to monitor metabolites at low concentrations. Furthermore, ¹³C-NMR and high-resolution LC-MS allow to estimate isotopologue distribution in response to ¹³CO₂ labeling while modifying photorespiration activity.