In-gel superoxide dismutase (SOD) activity assay and nitroblue tetrazolium (NBT) staining

Native soluble proteins were extracted from leaves harvested from poplar plants cultivated in hydroponic culture in control medium and supplemented with 500 µM ZnSO₄ grinding fresh tissues in 0.15 M Tris, pH 7.5. Three independent poplar transgenic lines carrying 35S::ScZRC1 or pRbcS::ScZRC1 were analysed. After centrifugation, the supernatant was recovered and the total protein content was estimated using the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated at 4 °C by native 10% poly-acrylamide gel electrophoresis (PAGE) in Tris-glycine native buffer (25 mM Tris, 0.192 M glycine). SOD activity was then determined in-gel as described by Beauchamp and Fridovich (1971). The band intensity was determined by scanning the gels and processing images with Quantity OneR software v4.4.1 (Bio-Rad); intensities were normalized on actual protein loading, as estimated by Coomassie staining of a replica gel. O₂⁻ in plants was detected by treating leaves with NBT and visualizing the blue spots according to Rao and Davis (1999).