Photosynthesis pp 141-154 | Cite as

Measurement of O2 Uptake and Evolution in Leaves In Vivo Using Stable Isotopes and Membrane Inlet Mass Spectrometry

  • Steven M. Driever
  • Neil R. Baker
Part of the Methods in Molecular Biology book series (MIMB, volume 1770)


Oxygen is both product and substrate of photosynthesis and metabolism in plants, by oxygen evolution through water splitting and uptake by photorespiration and respiration. It is important to investigate these processes simultaneously in leaves, especially in response to environmental variables, such as light and temperature. To distinguish between processes that evolve or take up O2 in leaves in the light, in vivo gas exchange of stable isotopes of oxygen and membrane inlet mass spectrometry is used. A closed-cuvette system for gas exchange of leaf disks is described, using the stable isotopes 16O2 and 18O2, with a semipermeable membrane gas inlet and isotope mass separation and detection by mass spectrometry. Measurement of evolution and uptake, as well as CO2 uptake, at a range of light levels allows composition of a light–response curve, here described for French bean and maize leaf disks.

Key words

Oxygen evolution Oxygen uptake Stable isotopes Membrane inlet mass spectrometry MIMS 



This work was initiated and done under the supervision of Prof. Neil R. Baker (University of Essex, UK) and supported by a research studentship from the Department of Biological Sciences at the University of Essex to Dr. Steven M. Driever. We thank Prof. Suzanne von Caemmerer (Australian National University) for providing details on the leaf cuvette as used by Maxwell et al. [15] and Ruuska et al. [12].


  1. 1.
    Hunt S (2003) Measurements of photosynthesis and respiration in plants. Physiol Plantarum 117(3):314–325. CrossRefGoogle Scholar
  2. 2.
    Delieu T, Walker DA (1981) Polarographic measurement of photosynthetic oxygen evolution by leaf-disks. New Phytol 89(2):165–178. CrossRefGoogle Scholar
  3. 3.
    Delieu TJ, Walker DA (1983) Simultaneous measurement of oxygen evolution and chlorophyll fluorescence from leaf pieces. Plant Physiol 73(3):534–541. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    van Gorkom HJ, Gast P (1996) Measurement of photosynthetic oxygen evolution. In: Biophysical techniques in photosynthesis. Springer, Dordrecht, pp 391–405CrossRefGoogle Scholar
  5. 5.
    Davey PA, Hunt S, Hymus GJ, DeLucia EH, Drake BG, Karnosky DF, Long SP (2004) Respiratory oxygen uptake is not decreased by an instantaneous elevation of [CO2], but is increased with long-term growth in the field at elevated [CO2]. Plant Physiol 134(1):520–527. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Willms JR, Dowling AN, Dong ZM, Hunt S, Shelp BJ, Layzell DB (1997) The simultaneous measurement of low rates of CO2 and O2 exchange in biological systems. Anal Biochem 254(2):272–282. CrossRefPubMedGoogle Scholar
  7. 7.
    Cousins AB, Pracharoenwattana I, Zhou WX, Smith SM, Badger MR (2008) Peroxisomal malate dehydrogenase is not essential for photorespiration in arabidopsis but its absence causes an increase in the stoichiometry of photorespiratory CO2 release. Plant Physiol 148(2):786–795. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Cousins AB, Walker BJ, Pracharoenwattana I, Smith SM, Badger MR (2011) Peroxisomal hydroxypyruvate reductase is not essential for photorespiration in arabidopsis but its absence causes an increase in the stoichiometry of photorespiratory CO2 release. Photosynth Res 108(2–3):91–100. CrossRefPubMedGoogle Scholar
  9. 9.
    Walker BJ, Cousins AB (2013) Influence of temperature on measurements of the CO2 compensation point: differences between the Laisk and O2-exchange methods. J Exp Bot 64(7):1893–1905. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Biehler K, Haupt S, Beckmann J, Fock H, Becker TW (1997) Simultaneous CO2- and 16O2/18O2-gas exchange and fluorescence measurements indicate differences in light energy dissipation between the wild type and the phytochrome-deficient aurea mutant of tomato during water stress. J Exp Bot 48(312):1439–1449. CrossRefGoogle Scholar
  11. 11.
    Driever SM, Baker NR (2011) The water-water cycle in leaves is not a major alternative electron sink for dissipation of excess excitation energy when CO2 assimilation is restricted. Plant Cell Environ 34(5):837–846. CrossRefPubMedGoogle Scholar
  12. 12.
    Ruuska SA, Badger MR, Andrews TJ, von Caemmerer S (2000) Photosynthetic electron sinks in transgenic tobacco with reduced amounts of Rubisco: little evidence for significant Mehler reaction. J Exp Bot 51:357–368. CrossRefPubMedGoogle Scholar
  13. 13.
    Haupt-Herting S, Fock HP (2000) Exchange of oxygen and its role in energy dissipation during drought stress in tomato plants. Physiol Plant 110(4):489–495. CrossRefGoogle Scholar
  14. 14.
    Haupt-Herting S, Fock HP (2002) Oxygen exchange in relation to carbon assimilation in water-stressed leaves during photosynthesis. Ann Bot 89:851–859. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Maxwell K, Badger MR, Osmond CB (1998) A comparison of CO2 and O2 exchange patterns and the relationship with chlorophyll fluorescence during photosynthesis in C3 and CAM plants. Aust J Plant Physiol 25(1):45–52CrossRefGoogle Scholar
  16. 16.
    Shirao M, Kuroki S, Kaneko K, Kinjo Y, Tsuyama M, Forster B, Takahashi S, Badger MR (2013) Gymnosperms have increased capacity for electron leakage to oxygen (Mehler and PTOX reactions) in photosynthesis compared with angiosperms. Plant Cell Physiol 54(7):1152–1163. CrossRefPubMedGoogle Scholar
  17. 17.
    Siebke K, Ghannoum O, Conroy JP, Badger MR, von Caemmerer S (2003) Photosynthetic oxygen exchange in C4 grasses: The role of oxygen as electron acceptor. Plant Cell Environ 26(12):1963–1972. CrossRefGoogle Scholar
  18. 18.
    Arnon D, Hoagland D (1940) Crop production in artificial culture solutions and in soils with special reference to factors influencing yields and absorption of inorganic nutrients. Soil Sci 50:463–485Google Scholar
  19. 19.
    Coe RA, Lin H (2018) Light-response curves in land plants. In: Covshoff S (ed) Photosynthesis: methods and protocols, Methods in molecular biology, vol 1770. Springer, New YorkCrossRefGoogle Scholar
  20. 20.
    Radmer RJ, Kok B (1976) Photoreduction of O2 primes and replaces CO2 assimilation. Plant Physiol 58(3):336–340. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Steven M. Driever
    • 1
  • Neil R. Baker
    • 2
  1. 1.Centre for Crop System AnalysisWageningen University and ResearchWageningenThe Netherlands
  2. 2.School of Biological SciencesUniversity of EssexColchesterUK

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