Measurement of Oxidative Damage and Antioxidant Enzyme Activity

Leaf sampling of pearl millet seedlings for the measurement of MDA and H₂O₂ was carried out at 1, 3, and 7 h of salt treatment. The MDA contents were assessed according to the protocol of Heath and Packer (1968) as lipid peroxidation. In total, 500 mg of fresh leaf tissue samples in triplicates per treatment were taken and homogenized by adding TCA (10%) and 2-thiobarbituric acid (0.65%), followed by heating at 95°C for 60 min. After that, the mixture was kept at room temperature and allowed to cool and then centrifuged at 10,000x g for 10 min at room temperature. The supernatant was collected, and the absorbance was recorded at 532 nm. The difference in blank and sample absorbance was used to calculate MDA contents. On the other hand, the H₂O₂ was determined by using methods described by Khan et al. (2021a). In total, 500 mg fresh leaf tissue samples in triplicates per treatment were taken and homogenized in an ice bath with 5 ml of0.1% (w/v) trichloracetic acid (TCA). The homogenized mixture was centrifuged for 15 min at 12,000x g at room temperature. After that, 0.5 ml of supernatant was taken and mixed with 1 ml of potassium iodide (1M) and0.5 ml phosphate buffer (10 mM) (pH 7). The absorbance of the mixture was measured at 390 nm. The difference in blank and sample absorbance was used to calculate H₂O₂ concentration. In addition, the proline content was measured according to the protocol given by (Bates et al., 1973).

Moreover, to estimate the antioxidant enzyme activities, pearl millet leaf samples were crushed with the help of a pestle and mortar. The crushed material was homogenized with0.5 M phosphate buffer (pH 7.8) and was filtered. The filtrate was subjected to centrifugation for 10 min at 12,000 × g at 4°C. The supernatant was separated, collected, and used to measure the activities of antioxidant enzymes. The SOD and POD activities were measured by following the method of Zhang (1992). The CAT activity was estimated according to the protocol described by Aebi (1984). The antioxidant activities are expressed in the unit of U mg^{– 1}.