Xinyu Fu (Walker lab)
Date & Location: September 22, 2020, at 12p; Virtual talk
Subject: Isotopically nonstationary metabolic flux analysis of photorespiration in tobacco leaves
Abstract: Quantifying fluxes through the plant metabolic network is fundamental for engineering plants with improved photosynthetic efficiency and productivity. Recent advances in metabolomics and computational modeling have enabled us to map carbon fluxes through photosynthetic metabolism using 13CO2 as a tracer. However, experimental and computational challenges have limited its application to an integrated understanding of carbon partitioning within the complex plant metabolic network. Here, we presented an improved framework for isotopically nonstationary metabolic flux analysis (INST-MFA), interfaced with gas exchange measurements, to quantify the roles of photorespiration on the carbon economy of the photosynthetic cells.
To perform in vivo 13CO2 labeling of tobacco (Nicotiana tabacum) leaves, we outfitted a gas exchange cuvette with a custom gas mixing system and a cryospray injection port to quench metabolism within 0.3 second. 13CO2 labeling experiments were performed on leaves acclimated at high (40% O2), normal (21% O2), and low photorespiratory (2% O2) conditions. Using various GC-MS and LC-MS/MS platforms, we analyzed the mass isotopomer distributions of 50 fragment ions from 35 metabolites, representing intermediates involved in the Calvin Benson cycle, photorespiration, tricarboxylicacid cycle, and synthesis of starch and sucrose. Metabolic models were solved to estimate ~100 network-wide fluxes based on the isotopomer measurements, net CO2 assimilation, synthesis rates of starch and sucrose, and levels of vascular sucrose and amino acids. Rates of rubisco carboxylation and oxygenation were estimated from gas exchange data to provide accurate constraints to the final flux solutions. This study expands the potential for INST-MFA to provide a mechanistic understanding of photorespiration and how it interacts with central metabolism, possibly elucidating routes for improving the efficiency of carbon fixation.
Speaker Lab: Dr. Berkley Walker