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Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants

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Photorespiration

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1653))

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 h 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.

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Acknowledgements

Any product or trademark mentioned here does not imply a warranty, guarantee, or endorsement by the authors or their affiliations over other suitable alternatives. Mass spectrometry methods were developed using instrumentation in part supported by National Science Foundation grants (DBI-1427621 and DBI-0521250 for acquisition of QTRAP LC-MS/MS instruments). We acknowledge support from the Department of Energy (DE-AR0000202) to F.M. and D.K.A.

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Authors and Affiliations

  1. Donald Danforth Plant Science Center, St. Louis, MO, USA

    Fangfang Ma & Doug K. Allen

  2. Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA

    Lara J. Jazmin & Jamey D. Young

  3. United States Department of Agriculture, Agricultural Research Service, St. Louis, MO, USA

    Doug K. Allen

Authors
  1. Fangfang Ma
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  2. Lara J. Jazmin
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  3. Jamey D. Young
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  4. Doug K. Allen
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Corresponding author

Correspondence to Doug K. Allen .

Editor information

Editors and Affiliations

  1. Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany

    Alisdair R. Fernie

  2. Plant Physiology Department, University of Rostock, Rostock, Germany

    Hermann Bauwe

  3. Institute of Plant Biochemistry, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Düsseldorf, Germany

    Andreas P.M. Weber

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Ma, F., Jazmin, L.J., Young, J.D., Allen, D.K. (2017). Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants. In: Fernie, A., Bauwe, H., Weber, A. (eds) Photorespiration. Methods in Molecular Biology, vol 1653. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7225-8_12

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  • DOI: https://doi.org/10.1007/978-1-4939-7225-8_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7224-1

  • Online ISBN: 978-1-4939-7225-8

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