Department of Plant Bioenergetics, Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland.
Methods Mol Biol. 2022;2363:199-213. doi: 10.1007/978-1-0716-1653-6_15.
Mitochondria actively participate in oxygenic metabolism and are one of the major sources of reactive oxygen species (ROS) production in plant cells. However, instead of measuring ROS concentrations in organelles it is more worthwhile to observe active ROS generation or downstream oxidation products, because the steady state level of ROS is easily buffered. Here, we describe how to measure the in vitro production of superoxide anion radicals (O·) by mitochondria and the release of O· into the cytosol. A method to determine glutathione, which is the most abundant mitochondrial low-mass antioxidant, is presented since changes in the redox state of glutathione can be indicative of the oxidative action of ROS. The identification of oxidative damage to mitochondrial components is the ultimate symptom that ROS homeostasis is not under control. We present how to determine the extent of oxidation of membrane lipids and the carbonylation of mitochondrial proteins. In summary, oxidative stress symptoms have to be analyzed at different levels, including ROS production, scavenging capacity, and signs of destruction, which only together can be considered markers of mitochondrial ROS status.
线粒体积极参与需氧代谢，是植物细胞中活性氧（ROS）产生的主要来源之一。然而，与其测量细胞器中的 ROS 浓度，不如观察活性 ROS 的产生或下游氧化产物，因为 ROS 的稳态水平很容易被缓冲。在这里，我们描述了如何测量线粒体中超氧阴离子自由基（O·）的体外产生以及 O·向细胞质的释放。由于谷胱甘肽（glutathione）的氧化还原状态的变化可以指示 ROS 的氧化作用，因此介绍了一种确定谷胱甘肽的方法，谷胱甘肽是最丰富的线粒体低分子量抗氧化剂。线粒体成分氧化损伤的鉴定是 ROS 动态平衡失控的最终症状。我们介绍了如何确定膜脂的氧化程度和线粒体蛋白的羰基化程度。总之，氧化应激症状必须在不同的水平上进行分析，包括 ROS 的产生、清除能力和破坏的迹象，只有这些综合起来才能被认为是线粒体 ROS 状态的标志物。
