Real-time monitoring of superoxide anion radical generation in response to wounding: electrochemical study.

Abstract

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 (O 2 •- ), hydrogen peroxide (H 2 O 2 ) and singlet oxygen; however, few experimental studies have provided direct evidence of their detection in plants. Detection of O 2 •- 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 O 2 •- generation and its kinetics in the leaves of spinach ( Spinacia oleracea ) subjected to wounding. Methods Real-time monitoring of O 2 •- generation was performed using catalytic amperometry. Changes in oxidation current for O 2 •- 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 O 2 •- for minutes after wounding, followed by a decline. The exogenous addition of superoxide dismutase, which is known to dismutate O 2 •- to H 2 O 2 , 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 O 2 •- in biological samples, with the potential for wide application in plant research for specific and sensitive detection of O 2 •- .

Plant phenotyping relevance

植物の傷害応答におけるスーパーオキシド生成を、電気化学センサーでリアルタイム測定する手法の開発・検証が中心であり、植物生理状態の表現型測定に該当する。

Code and data availability

The paper's raw electrochemical phenotyping data (Data S5–S7), leaf photographs (Data S1), and DPV/EPR supplements are described as public supplementary files, but their PeerJ supplement URLs are not among the allowed_urls, so no actionable paper-specific asset can be listed. The only allowed URL (an ECS conference web

No evidence-backed public reproduction asset is currently recorded.