Quantitative GUS Activity Assay in Arabidopsis

The quantitative GUS activity was measured using the Lu’s methods with slight modification [53 (link)]. GUS activity was detected in 1-month-old Arabidopsis leaf tissues (10 mg) from three independent transgenic lines and six individuals in each line. Total proteins were extracted using 300 µL GUS extraction buffer (50 mM phosphate buffer, pH 7.0; 10 mM EDTA, pH 8.0; 0.1 % Sodium Dodecvl Sulfate; 10 mM β-mercaptoethanol). BCA Protein Assay (Beyotime Biotechnology, China) was used to measure the protein concentrations. Extraction (100 µL) was added to 900 µL GUS extraction buffer containing 1 mM 4-methylumbelliferyl glucuronide (MUG, Sigma) and incubated at 37 °C. The 900 µL stop solution (1 M Sodium Carbonate) immediately added into 100 µL the above reaction mixture and 60 min later, respectively. Fluorescence of 4-methylumbelliferone (MU) was monitored using Tecan Infnite™ at 455 nm emission and 365 nm excitation. GUS activity was expressed as µmoles 4-methylumbelliferone (MU) min^− 1 mg^− 1 protein.