Quantifying Oxidative Stress Markers in Plants

Herein, 1 mg/mL Di-amino benzidine (DAB) and 1 mg/mL nitroblue tetrazolium (NBT) were used to stain the 5th to 8th rosette leaves from 7-week-old seedlings for H₂O₂ and O₂⁻, respectively [49 (link)], according to the previous method [16 (link)]. The measurements of H₂O₂, O₂⁻, and MDA were performed according to the previous studies [50 (link),51 (link)]. Briefly, leaf samples used for determination of H₂O₂ and O₂⁻ were extracted by 50 mmol/L phosphate buffer. Then, the supernatant was mixed with titanium sulphate and determined at 415 nm to calculate the concentration H₂O₂, while the supernatant was mixed with sulphanilic acid and α-naphthylamine and detected at the absorbance of 530 nm to determine the concentration of O₂⁻. MDA was determined by trichloroacetic acid and thiobarbituric acid, and the absorbance was determined at 532, 600, and 450 nm.
In addition, the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were detected using the Total Superoxide Dismutase Assay Kit (Nanjing Jiancheng, A001-1, Nanjing, China), the Catalase Assay Kit (Nanjing Jiancheng, A007-1, Nanjing, China), and the Peroxidase Assay Kit (Nanjing Jiancheng, A084-3, Nanjing, China), respectively.