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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #330558

Research Project: Soybean Seed Improvement Through Quantitative Analysis of Phenotypic Diversity in Response to Environmental Fluctuations

Location: Plant Genetics Research

Title: Isotopically nonstationary metabolic flux analysis (INST-MFA) of photosynthesis and photorespiration in plants

Author
item Fangfang, Ma - Danforth Plant Science Center
item Jazmin, Lara - Vanderbilt University
item Young, Jamey - Vanderbilt University
item Allen, Douglas - Doug

Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 11/1/2017
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Photorespiration is a central component of photosynthesis; however to better understand its role it should be viewed in the context of an integrated metabolic network rather than a series of individual reactions that operate independently. Isotopically nonstationary 13C metabolic flux analysis (INST-MFA), which is based on transient labeling studies at metabolic steady state, offers a comprehensive platform to quantify plant central metabolism. In this chapter, we describe the application of INST-MFA to investigate metabolism in leaves. Leaves are an autotrophic tissue, assimilating CO2 over a diurnal period implying that the metabolic steady state is limited to less than 12 hours and thus requiring an INST-MFA approach. This strategy results in a comprehensive unified description of photorespiration, Calvin cycle, sucrose and starch synthesis, tricarboxylic acid (TCA) cycle, and amino acid biosynthetic fluxes. We present protocols of the experimental aspects for labeling studies: transient 13CO2 labeling of leaf tissue, sample quenching and extraction, mass spectrometry (MS) analysis of isotopic labeling data, measurement of sucrose and amino acids in vascular exudates, and provide details on the computational flux estimation using INST-MFA.