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 (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^•-.

Keywords: Electrochemical detection; Polymeric iron-porphyrin-based modified carbon electrode; Superoxide anion radical; Wounding.

Figure 1. Reaction mechanism and experimental setup.

(A) Schematic illustration of the reaction mechanism for the amperometric detection of O₂^•− using the polymeric iron-porphyrin-based modified carbon electrode depicting the reduction-oxidation cycle leading to generation of the oxidation current. (B and C) Schematic illustration of the experimental setup for the electrochemical measurements. The stimulation was performed using a glass capillary, and the polymeric iron-porphyrin-based modified carbon electron was positioned at a distance of 1 mm using a motor-driven XYZ microscopic stage (B). The in vivo generation of O₂^•− was measured using a polymeric iron-porphyrin-based modified carbon electron (working electrode, WE), platinum wire (counter electrode, CE) and Ag/AgCl (reference electrode, RE) (C).

Figure 2. Calibration curve.

Changes in oxidation current measured using iron-porphyrin-based modified carbon electrode by exogenous addition of a standard known concentration of O₂^•− generated in situ using X/XO system in the concentration range of 0.4–1.3 µM.

Figure 3. Real-time monitoring of the oxidation current of O₂^•− from spinach leaves.

The kinetics of the production of O₂^•− were measured using a polymeric iron-porphyrin-based modified carbon electrode on non-wounded spinach leaves.

Figure 4. Real-time monitoring of oxidation current for O₂^•− during wounding.

The kinetics of the production of O₂^•− were measured using a polymeric iron porphyrin based modified carbon electrode during wounding in spinach leaves. The wounding in spinach leaves was done one time (A) and multiple times (B) close to the site of electrode during the measurement and oxidation current for O₂^•− was measured.

Figure 5. Real-time monitoring of the oxidation current of O₂^•− during wounding.

The kinetics of the production of O₂^•− were measured using a polymeric iron-porphyrin-based modified carbon electrode during wounding in spinach leaves. The wounding of spinach leaves was performed during the measurement, and the oxidation current for O₂^•− was measured for approximately 30 min. The effect of SOD on the oxidation current was measured in the presence of SOD (400 U ml⁻¹) added exogenously during the measurement.

Figure 6. Real-time monitoring of oxidation current for O₂^•− during wounding.

The kinetics of the production of O₂^•− was measured using a polymeric iron porphyrin based modified carbon electrode during wounding in spinach leaves. The wounding in spinach leaves was done during the measurement and oxidation current for O₂^•− was measured for a duration of about 30 min. Effect of SOD was measured in the presence of SOD (400 U ml⁻¹) added exogenously during the measurement at the point of maximum oxidation current.