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.
Keywords: Alternative oxidase pathway; Cytochrome oxidase pathway; In vivo mitochondrial electron partitioning; Oxygen isotope fractionation; Respiration.