GUS fluorometric assay

The protoplasts were collected by centrifugation at 100 × g for 2 min, and the extraction buffer (0.05 M NaH₂PO₄ with pH 7.0, 0.1% SDS, 0.01 M EDTA with pH 8.0, 20% methanol, 0.1% Triton X-100 and 0.1% 2-mercaptoethanol) was added to the protoplasts, which were vortexed to homogeneity. The homogenate was centrifuged at 1,000 × g for 10 min at 4°C, and the supernatant was used for the protein fluorometric quantification assay [30]. The total protein concentration in the extracts was determined by the Bradford method (Protein Assay Kit; Bio-Rad, Hercules, CA, USA). The fluorescence was then measured with excitation at 365 nm and emission at 455 nm on a HITACHI F-4500 spectrofluorimeter. The fluorimeter was calibrated with freshly prepared MU standards in the same buffer. Triplicate assays were conducted in the experiment, and all experiments were repeated three times.