Cell‐free protein degradation assay

The cell‐free protein degradation assay was performed as described previously (Wang et al., 2009). Plants were grown at 22℃ in long‐day conditions (12 h light/12 h dark cycles) and the 3‐week‐old leaves were ground to powder in liquid nitrogen. Total proteins were extracted with a cell‐free degradation buffer (25 mmol/L Tris‐HCl pH 7.5, 10 mmol/L NaCl, 10 mmol/L MgCl₂, 4 mmol/L PMSF, 5 mmol/L DTT, and 10 mmol/L adenosine triphosphate) and cell debris was removed by centrifugation at 15,000 × g for 10 min at 4℃. Total protein extracts from each of the plant materials were measured the concentration by Bicinchoninic Acid Kit for Protein Determination (Sigma Aldrich) and adjusted to equal concentrations with the degradation buffer. Then, 1,000 ng of recombinant MBP‐BES1 proteins were added in 1,000 μL plant extracts (containing 100 mg total proteins) for further reaction. The reaction mixtures were incubated at 30℃ for different periods, and 200 μL reaction mixtures were taken from the tube at each sample and analyzed by immunoblots with MBP antibody.