The malondialdehyde (MDA) and H2O2 contents, and GR activity were measured with the assay kits of Jiangsu Keming Biotechnology Institute (Suzhou, China). For the determination of MDA content and GR activity, 0.1 g of leaf tissues were ground to powder with 1 ml buffer I [50 mM phosphate buffer (pH 7.8), containing 0.5% (w/v) Triton-100, 0.1 mM EDTA, and 2% PVP], centrifuged at 10,000 rpm for 20 min at 4 °C and then the supernatant was used for determination according to the manufacturer’s protocol. For the determination of H2O2 contents, 1 ml acetone replaced the extraction buffer I.

The production rate of O2·— was measured according to the method described by Elstner and Heupel [52]. In brief, about 0.1 g leaf was homogenized with 3 mL 65 mM potassium phosphate buffer (PBS) (pH 7.8), and centrifuged at 10,000 rpm at 4 °C for 20 min. Then, 0.5 mL supernatant was mixed with 0.1 mL 10 mM hydroxylamine hydrochloride and 0.5 mL PBS and incubated at 25 °C for 20 min. Subsequently, 1 mL 7 mM α-naphthylamine and 1 mL 58 mM sulfonamide were added to the incubation mixture and incubated at 25 °C for another 20 min. Then, 3 mL chloroform was added and centrifuged 10,000 rpm for 5 min. The absorbance was measured at 530 nm.

For the measurement of POD, CAT and SOD activities, about 0.2 g of leaf tissues were homogenized in 50 mM phosphate buffer solution (pH 7.8). The extracts were centrifuged at 10,000 rpm at 4 °C for 20 min. Supernatants were collected for enzymes activities analysis as described by Hou et al. [53].

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