Real-time monitoring of superoxide anion radical generation in response to wounding: electrochemical study
Ankush Prasad,
Aditya Kumar,
Ryo Matsuoka,
Akemi Takahashi,
Ryo Fujii,
Yamato Sugiura,
Hiroyuki Kikuchi,
Shigeo Aoyagi,
Tatsuo Aikawa,
Takeshi Kondo,
Makoto Yuasa,
Pavel Pospíšil,
Shigenobu Kasai
Affiliations
Ankush Prasad
Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic
Aditya Kumar
Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic
Ryo Matsuoka
Hokuto Denko Corporation, Tokyo, Japan
Akemi Takahashi
Graduate Department of Environmental Information Engineering, Tohoku Institute of Technology, Sendai, Japan
Ryo Fujii
Graduate Department of Environmental Information Engineering, Tohoku Institute of Technology, Sendai, Japan
Yamato Sugiura
Graduate Department of Environmental Information Engineering, Tohoku Institute of Technology, Sendai, Japan
Hiroyuki Kikuchi
Graduate Department of Environmental Information Engineering, Tohoku Institute of Technology, Sendai, Japan
Shigeo Aoyagi
Hokuto Denko Corporation, Tokyo, Japan
Tatsuo Aikawa
Department of Pure and Applied Chemistry, Tokyo University of Science, Noda, Chiba, Japan
Takeshi Kondo
Department of Pure and Applied Chemistry, Tokyo University of Science, Noda, Chiba, Japan
Makoto Yuasa
Department of Pure and Applied Chemistry, Tokyo University of Science, Noda, Chiba, Japan
Pavel Pospíšil
Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic
Shigenobu Kasai
Biomedical Engineering Research Center, Tohoku Institute of Technology, Sendai, Japan
Background The growth and development of plants is deleteriously affected by various biotic and abiotic stress factors. Wounding in plants is caused by exposure to environmental stress, mechanical stress, and via herbivory. Typically, oxidative burst in response to wounding is associated with the formation of reactive oxygen species, such as the superoxide anion radical (O2•−), hydrogen peroxide (H2O2) and singlet oxygen; however, few experimental studies have provided direct evidence of their detection in plants. Detection of O2•− formation in plant tissues have been performed using various techniques including electron paramagnetic resonance spin-trap spectroscopy, epinephrine-adrenochrome acceptor methods, staining with dyes such as tetrazolium dye and nitro blue tetrazolium (NBT); however, kinetic measurements have not been performed. In the current study, we provide evidence of O2•− generation and its kinetics in the leaves of spinach (Spinacia oleracea) subjected to wounding. Methods Real-time monitoring of O2•− generation was performed using catalytic amperometry. Changes in oxidation current for O2•− was monitored using polymeric iron-porphyrin-based modified carbon electrodes (φ = 1 mm) as working electrode with Ag/AgCl as the reference electrode. Result The results obtained show continuous generation of O2•− for minutes after wounding, followed by a decline. The exogenous addition of superoxide dismutase, which is known to dismutate O2•− to H2O2, significantly suppressed the oxidation current. Conclusion Catalytic amperometric measurements were performed using polymeric iron-porphyrin based modified carbon electrode. We claim it to be a useful tool and a direct method for real-time monitoring and precise detection of O2•− in biological samples, with the potential for wide application in plant research for specific and sensitive detection of O2•−.
