Measurements of Electron Partitioning Between Cytochrome and Alternative Oxidase Pathways in Plant Tissues
Plant respiration is characterized by the existence of the alternative oxidase pathway (AOP) that competes with cytochrome oxidase pathway (COP) for the electrons of the ubiquinone pool of the mitochondrial electron transport chain, thus reducing ATP synthesis. The oxygen (O2) isotope fractionation technique is the only available to determine the electron partitioning between the two pathways and their in vivo activities in plant tissues. In this chapter, the basis of the O2 isotope fractionation technique and its derived calculations are carefully explained together with a detailed description of the dual-inlet isotope ratio mass spectrometry (DI-IRMS) system and the protocol developed at the University of Balearic Islands. The key advantages of the DI-IRMS over other systems are highlighted as well as the potential problems of this technique. Among these problems, those associated with leakage, diffusion, and inhibitor treatments are noted and solutions to prevent, detect, and repair these problems are detailed.
Key wordsOxygen isotope fractionation Respiration In vivo mitochondrial electron partitioning Cytochrome oxidase pathway Alternative oxidase pathway
We would like to thank all the staff at the Serveis Cientifico-Tecnics of the Universitat de les Illes Balears for their help while running the experiments on the IRMS.
- 10.Dawson TE, Brooks PD (2001) Fundamentals of stable isotope chemistry and measurement. In: Unkovich M et al (eds) Stable isotope techniques in the study of biological processes and functioning of ecosystems. Springer, Netherlands, pp 1–18Google Scholar
- 11.Ribas-Carbo M, Robinson SA, Giles L (2005) The application of the oxygen-isotope technique to assess respiratory pathway partitioning. In: Lambers H, Ribas-Carbo M (eds) Plant respiration: from cell to ecosystem, vol 18. Advances in photosynthesis and respiration series. Springer, Dordrecht, The Netherlands, pp 31–42CrossRefGoogle Scholar
- 13.Cheah MH, Millar AH, Myers RC et al (2014) Online oxygen kinetic isotope effects using membrane inlet mass spectrometry can differentiate between oxidases for mechanistic studies and calculation of their contributions to oxygen consumption in whole tissues. Anal Chem 86:5171–5178CrossRefPubMedGoogle Scholar
- 14.Del-Saz NF, Florez-Sarasa I, Clemente-Moreno MJ et al (2016) Salinity tolerance is related to cyanide-resistant alternative respiration in Medicago truncatula under sudden severe stress. Plant Cell Environ. doi: 10.1111/pce.12776
- 15.Florez-Sarasa I, Ribas-Carbo M, Del-Saz NF et al (2016) Unravelling the in vivo regulation and metabolic role of the alternative oxidase pathway in C3 species under photoinhibitory conditions. New Phytol. doi: 10.1111/nph.14030