Histochemical Detection of Superoxide and H2O2 Accumulation in Brassica juncea Seedlings

Plant cells continually produce reactive oxygen species (ROS) as a by-product of aerobic metabolism. Increased production of ROS occurs under unfavorable conditions imposed by various abiotic and biotic factors. Accumulation of ROS is damaging to various cellular components and macromolecules including plasma membrane, nucleic acids, and proteins and eventually leads to cell death. In this protocol, we describe the histochemical detection of hydrogen peroxide (H2O2) and superoxide (O2-) anion, two of the most important ROS, in Brassica juncea seedlings by using 3,3ʹ-Diaminobenzidine (DAB) and Nitrotetrazolium blue chloride (NBT) as the chromogenic substrate. DAB is oxidized by H2O2 in the presence of peroxidases and produces reddish brown precipitate. NBT reacts with O2- to form a dark blue insoluble formazan compound. The protocol can be used in other plant species and for different plant tissues., 植物细胞不断产生活性氧（ROS）作为有氧代谢的副产物。增加的ROS的产生发生在由各种非生物和生物因素施加的不利条件下。 ROS的积累损害各种细胞组分和大分子，包括质膜，核酸和蛋白质，并最终导致细胞死亡。在该方案中，我们描述了过氧化氢（H 2 O 2 O 2）和超氧化物（O 2 2 O 2）的组织化学检测，通过使用3,3'-二氨基联苯胺（DAB）和硝基四唑氯化蓝（NBT）作为显色底物，在芥菜籽苗中培养两种最重要的ROS。 DAB在过氧化物酶存在下被H 2 O 2 O 2氧化并产生红棕色沉淀。 NBT与O 2反应以形成深蓝色不溶性甲an化合物。该方案可用于其他植物物种和不同的植物组织。