389 From: Methods in Molecular Biology, vol. 372: Mitochondria: Practical Protocols Edited by: D. Leister and J. M. Herrmann © Humana Press Inc., Totowa, NJ 28 Oxidative Stress and Plant Mitochondria Nicolas L. Taylor and A. Harvey Millar Summary Mitochondria not only are a source of reactive oxygen species (ROS) but also are sites of oxidative damage. In plants, mitochondria must normally operate when there are high levels of ROS produced during photosynthesis and photorespiration. These levels are further enhanced during biotic and abiotic stress of plants. Excessive stress can lead to mitochondrial damage, which may then lead to induction of programmed cell death in plants. We outline methods for imposing oxidative stress in plants, provide methods for measurements of its severity, and then explain assays for assessing plant mitochondrial oxidative damage and measuring the capacity of key stress defense and response pathways. Key Words: Aconitase; alternative oxidase; glycine decarboxylase; lipid peroxidation; lipoic acid; reactive oxygen species; superoxide dismutase. 1. Introduction Mitochondria form a focus for much oxidative stress research as not only they are the sites of oxygen consumption and a significant source of cellular reactive oxygen species (ROS), but also oxidative damage of the organelle perturbs the cell’s energy supply required for repair mechanisms. Consequently, the nature of oxidative damage to mitochondria is under investigation in a variety of organisms. These studies are providing information on the general susceptibilities of these organelles to damage, as well as uncovering a range of defense mechanisms specific to experimental conditions and the mitochondrial protein profile found in different organisms. Concomitant with imposed oxidative damage, specific proteins are either synthesized or lost from mitochondria. This includes loss of, or replacement of, tricarboxylic acid (TCA) cycle enzymes and selected subunits of the respiratory chain and induction of peroxiredoxins and defense machinery. Significant manipulation of mitochondrial functions can also influence oxidative damage elsewhere in the cell, and this can have wide-reaching