SOD, POD, CAT, and MDA Content in Flag Leaves

One half gram of fresh flag leaves was ground in a mortar filled with 5 mL extraction buffer comprising 100 mM potassium phosphate buffer (pH 7.0), 1% (w/v) polyvinylpyrrolidone (PVPP), and 1 mM ethylenediaminetetraacetic acid (EDTA). After centrifugation at 20,000 rpm and 4°C for 20 min, the supernatant was collected for antioxidant enzyme analysis. The activity of superoxide dismutase (SOD; EC 1.15.1.1) was assayed by the inhibition of nitro blue tetrazolium (NBT) photoreduction as previously described (Wang et al., 2021). The optical density of the product was measured at 560 nm. One unit of SOD corresponded to the amount of enzyme inhibiting 50% of the NBT photoreduction. Catalase (CAT; EC 1.11.1.6) activity was determined from H₂O₂ decomposition over a 3-min interval. Absorbance of the product was measured at 240 nm as previously reported (Wang et al., 2021). Guaiacol peroxidase (POD; EC 1.11.1.7) activity was evaluated by guaiacol oxidation. Absorbance of the product was read at 470 nm according to the method described by Wang and Huang (2000).

Based on a previous study (Lv X. K. et al., 2017), 0.5 g of leaf tissue was homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 20,000 × g and 4°C for 20 min. The MDA content was determined by the method of Wang and Huang (2000) using the following equation: