ROS staining
Histochemical staining for the qualitative detection of superoxide and peroxide radicals was performed with nitro blue tetrazolium (NBT, Sangon Biotech Co., Ltd, Shanghai, China) and 3,3-diaminobenzidine (DAB, Sigma-aidrich Inc., Darmstadt, Germany), respectively, as described previously (Zhang et al., 2013). Seedlings were excised at the base with a razor blade and supplied through the cut ends with NBT (0.5 mg mL-1) or DAB (2 mg mL-1) solutions for eight hours. Leaves were then decolorized in boiling ethanol (95 %) for 20 min. At least three leaves were used for each treatment.
Antioxidant enzyme activity assay
Enzymes activity was measured by grinding 0.5 g of maize leaves in 2 mL ice-cold 25 mM HERPES buffer (pH7.8) containing 0.2 mM EDTA, 2 Mm ascorbate, and 2% PVP. The homogenates were centrifuged at 4 ℃ for 15 min at 13,000 g, and the supernatant was used for enzyme activities assay. All steps in the preparation of the enzyme extract were carried out at 4℃. POD and SOD activities were measured as previously described (Zhang et al., 2016). 1 U of CAT activity was considered a decrease in absorbance of 0.01 at OD240 nm minute-1(Wang et al., 2017b).
H2O2, superoxide,and MDA quantification
H2O2 activity was evaluated according to previous studies (Bawa et al., 2019). Plant tissues (0.4 g) were homogenized in 1 mL of 0.1% (w/v) trichloroacetic acid (TCA) on ice. The homogenate was centrifuged at 12000 g for 15 min. 1 cm3 of potassium phosphate buffer, and 1 cm3 of potassium iodide (KI) were added to 0.5 cm3 aliquot of the supernatant. The absorbance of the supernatant was measured at 390 nm, and H2O2 content was calculated using a standard curve. Superoxide was determined by the hydroxylamine method (Elstner & Heupel, 1976). The MDA content was measured with thiobarbituric acid reactive substances (Cao et al., 2009). 0.5 g leaves sample was homogenized in 5 cm3 of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 10 000 × g for 20 min. To 1 cm3 aliquot of the supernatant, 4 cm3 of 0.5% thiobarbituric acid (TBA), dissolved in 20% TCA, was added. The resulting mixture was heated at 95 ºС for 30 min and then quickly cooled in an ice bath. Then, the supernatant was centrifugated at 10,000 g for 15 min, and the absorbance was measured at 532 and 600 nm. The value of non-specific absorption at 600 nm was subtracted from that of 532 nm. The concentration of MDA was calculated using an absorption coefficient of 155 nm.