*2.3. Relative Leakage Ratio, MDA, and H2O2 Contents*

Relative leakage ratio (RLR) was measured indirectly as leakage of UV-absorbing substances according to the method of Redmann et al. [38]. Five leaf segments (R = 0.5 cm) with three replicates were kept in 10 mL distilled water for 24 h and measured at 280 nm (A1). Samples were treated in liquid nitrogen and shaken for another 24 h in incubation water. The samples were measured at 280 nm (A2) and the RLR was calculated as A1/A2 [38]. Malondialdehyde (MDA) content (nmol g−<sup>1</sup> FW) was determined (0.1 g leaf samples with 3 replicates) as described by Hodges et al. [39] and MDA was calculated using the extinction coefficient (157 mM−<sup>1</sup> cm<sup>−</sup>1). To determine the amount of H2O2 (nmol g−<sup>1</sup> FW), leaf samples (0.1 g and 3 replicates) were extracted in 0.1% TCA with 0.1 M Tris-HCl (pH 7.6). The extracts were treated with potassium iodide reagent and kept in the dark for 90 min. Samples were measured at 390 nm and calculated using the standard curve [40].

#### *2.4. Antioxidant Enzyme Activities*

Soluble protein was extracted from leaves (0.5 g with 3 replicates) to determine the enzyme activities. The Bradford method [41] was used to determine the protein concentration and the leaf samples were extracted in the corresponding extraction buffer. 1 mL of buffer solution (9 mM Tris-HCl and 13.6% glycerol) was added to the powdered samples with liquid nitrogen and the total SOD activity (EC 1.15.1.1) (U mg protein−1) was determined [42]. The buffer solution of the leaves homogenized for POD (EC 1.11.1.7) and CAT (EC 1.11.1.6) included 100 mM potassium phosphate buffer (pH 7.0), 2% PVP, and 1 mM Na2EDTA. The POD activity was determined by measuring the oxidation of guaiacol (ε = 26.6 mM cm−1) by H2O2 (nmol H2O2 min−<sup>1</sup> mg protein−1) at 470 nm [43]. The CAT activity was calculated as nmol H2O2 min−<sup>1</sup> mg protein−1, with the absorbance values at 240 nm decreasing according to the dissociation of H2O2 [44].
