Real-Time Monitoring of Superoxide Anion Radical and Hydrogen Peroxide Generation in Higher Plants in Response to Wounding: In-Vivo Electrochemical Study

Abstract

The growth and development in plants are deleteriously affected by various biotic and abiotic stress factors. Wounding in plants occurs with exposure to environmental mechanical stresses or via herbivory. The response of plants towards wounding have been categorized based on the response time; the fast response includes oxidative burst accompanied by generation of reactive oxygen species (ROS) or delayed response which includes transcriptional activation of wound-induced genes, deposition of callose, accumulation of proteinase inhibitors and hydrolytic enzymes etc. The oxidative burst in response to wounding is known to be associated with the formation of various ROS such superoxide anion radical (O2 •−), singlet oxygen and hydrogen peroxide (H2O2). In the current study, we provide experimental evidences and real time monitoring of O2 •− and H2O2 generation following wounding in leaves of spinach (Spinacia oleracea). Real-time monitoring of ROS generation was achieved using amperometric method utilizing different electrodes. Non-specific real-time measurements of ROS were done by monitoring the changes in reduction current using carbon electrode while qualitative and quantitative measurement of O2 •− and H2O2 generationwere done using newly developed iron-porphyrin based modified carbon electrodes and osmium-horseradish peroxidase (Os-HRP) based modified carbon electrodes, respectively. The results obtained showed continuous generation of O2 •− and H2O2 for minutes after injury followed by a decline. The activation of lipoxygenase after wounding; its involvements in the production of H2O2 and the contribution of H2O2 in generation of local response in injured plant tissue besides the initiation of systemic response caused by hormones such as jasmonic acid, ethylene, salicylic acid, and abscisic acid will be disscussed.