Protein extraction and SOD activity analysis
Yeast lysates were extracted by the glass bead lysis method (Culotta et al., 1997). Arabidopsis leaf extracts were prepared with a grinding buffer of 150 mM Tris (pH 7.2). The homogenate was centrifuged at 12,000 rpm at 4°C for 10 min. Total protein was separated on 10% non-denaturing polyacrylamide gel, and the photochemical method of in-gel activity assay was used to analyze SOD activity (Beauchamp & Fridovich, 1971; Kliebenstein et al., 1998; Kuo, Huang, Shih, & Jinn, 2013c). The SOD activity was quantified by analyzing activity gel with the UVP ChemStudio PLUS imaging system (Analytik Jena US), and verified by KCN and H2O2 treatments (Kuo et al., 2013c). KCN is an inhibitor of CuZnSOD, and H2O2 inhibits both CuZnSOD and FeSOD. MnSOD activity is not affected by either chemical (Balzan, Bannister, Hunter, & Bannister, 1995; Chen, Weng, Ho, Cheng, & Lai, 2000; Kuo et al., 2013c).