Stress tolerance analyses of plants overexpressing upstream regulatory factors

The pROKII-35S::gene and pROKII-35S empty vector were separately transformed into one-month-old birch seedlings using the transient transformation method. The transient transgenic plants were treated with 150 mM NaCl or 200 mM mannitol for 12 h. Control plants were treated with water. The detached leaves of birch plants were incubated with 0.5 mg/mL nitroblue tetrazolium (NBT, dissolved in phosphate buffer, pH 7.8) and 0.5 mg/mL 3’-diaminobenzidine (DAB, dissolved in phosphate buffer, pH 3.8) as described in a previous study (Zhang et al., 2011). Evans blue (1.0 mg/mL, dissolved in sterile deionized water) staining was conducted to detect cell death following previously published procedures (Kim et al., 2003). The activity of superoxide dismutase (SOD) and peroxidase (POD), the content of H₂O₂, and electrolyte leakage were measured following previously described methods (Liu et al., 2015; Wang et al., 2015). The concentration of protein in plants was detected using a kit produced by Nanjing Jiancheng Bioengineering Institute. Three independent biological replicates were conducted.