Combining Gas Exchange and Rapid Quenching of Leaf Tissue for Mass Spectrometry Analysis Directly in Gas Exchange Cuvette.

Isotopically nonstationary metabolic flux analysis (INST-MFA) is a powerful technique for studying plant central metabolism, which involves introducing a ¹³ CO ₂ tracer to plant leaves and sampling the labeled metabolic intermediates during the transient period before reaching an isotopic steady state. The metabolic intermediates involved in the C ₃ cycle have exceptionally fast turnover rates, with some intermediates turning over many times a second. As a result, it is necessary to rapidly introduce the label and then rapidly quench the plant tissue to determine concentrations in the light or capture the labeling kinetics of these intermediates at early labeling time points. Here, we describe a rapid quenching (0.1-0.5 s) system for ¹³ CO ₂ labeling experiments in plant leaves to minimize metabolic changes during labeling and quenching experiments. This system is integrated into a commercially available gas exchange analyzer to measure initial rates of gas exchange, precisely control ambient conditions, and monitor the conversion from ¹² CO ₂ to ¹³ CO ₂ .
(© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)