*4.4. Analysis of MDA, Proline and ROS Contents*

The MDA content of the leaf was measured according to the method of Zhou and Leul [91]. Proline concentration was determined by a spectrophotometer according to the method of Li [92]. Briefly, 100 mg of leaf was homogenized with 5 mL of 3% sulfosalicylic acid and centrifuged at 5000× *g* for 10 min. The supernatant was treated with acid-ninhydrin and acetic acid, after which the supernatant was boiled for 1 h at 100 ◦C. Absorbance was determined at 520 nm and the proline content was expressed as μg gFW−1. For determination of the H2O2 content, 0.5 g of leaf was homogenized with 5.0 mL of 0.1% trichloroacetic acid (TCA) using an ice bath, and then the homogenate was centrifuged for 15 min at 12,000× *g* [93]. The H2O2 content in the supernatant was read using a spectrophotometer at 390 nm. The content of O2 − was measured according to Jiang and Zhang's method [94]. The content of OH− in the leaf was determined according to our previous study [89].
