Subcellular Localization Of Hydrogen Peroxide Generation Induced By Abscisic Acid In Guard Cell Of Vicia Faba L

It is well documented that H₂O₂, as the second messenger, is involved in the signal transduction in the ABA-induced stomatal closure. However, little is known about the subcellular location of H₂O₂ production in the guard cells. In this article, we investigate the subcellular localization of H₂O₂ generation induced by ABA in guard cell of Vicia faba L by Laser Scanning Confocol Microscope (LSCM) and Transmision Electron Microscope (TEM). To analysis the kinetics of H₂O₂ generation and detect the subcellular localization of H₂O₂ production in guard cells, the fully-grown leaves of Vicia faba were treated with 10 mmol/L ABA for 10, 15, 20, 30, 60 and 120 min, respectively. After section the leaves, we examined the accumulation sites of electron-dense deposits with Ce-H₂O₂ reaction in the subcellular parts of guard cells. The observations indicated that startling accumulation of H₂O₂ was mainly on the dorsal wall for 10 min after ABA treatment, and some on the vendral wall at15,20 and 30 min, respectively. After 90 and 120min treatment with ABA, the electron-dense deposits were found in chloroplast stroma, not in the mitochondrion and plasma membrane. These results are similar to that observed in the H₂O₂ treatment for10min. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, can significantly inhibit the accumulation of electron-dense deposits on the cell wall of guard cell, while the accumulation can be partly abolished by the application of catalase. Based on the enzyme source of H₂O₂ generation catylased by superoxide-generating NADPH oxidase, this suggests that H₂O₂ is rapidly synthesized in plasma membrane and released into the apoplast, such as dorsal wall. The deposits both on the cell wall and chloroplasts were reduced by addition of ascorbic acid, which rich in the chloroplasts to effectively eliminate the active oxygen specieses. Thus, we conclude that the sites of H₂O₂ generation induced by ABA in guard cells are plasma membrane and chloroplasts.In addition, we also observed electron-dense deposits in the epidermal cells and mesophyll cells in the experiment of subcellular analyses. It is found that there are deposits in the epidermal cells, while no deposits in the mesophyll cells within 20 min by ABAtreatment. In contrast, time course experiments based on the electron-dense deposits showed that such deposits in the epidermal cells were decreased gradually and tiny amount of deposits were found in the mesophyll cells in the time-dependent manner. . To confirm the results obtained from Ce-H₂O₂ reaction, the fluorescence probe dichlorofluoresein was loaded into guard cells with epidermal strips of Vicia faba and detect the generation of H₂O₂ during ABA-induced stomatal closure by LSCM. Exogenous ABA-induced the increases in fluorescence intensity in guard cells occurred in choroplasts and plasma membrane, which is significant earlier than other regions of guard cells. At the same time, the ABA-induced change of fluorescence intentsity in guard cells was abolished by application of catalase, DPI and ascorbic acid, which is the same as the earlier observation in our laboratory.