Sumoylation assays

To determine the global sumoylation status of MdMYB2-OE, antiMdMYB2, MdSIZ1, antiMdSIZ1, MdMYB2-OE/antiMdSIZ1, antiMdMYB2/MdSIZ1-OE, and 35S::GFP (empty vector) transgenic calli, calli grown on medium at 25°C were treated at either 25°C or 17°C for 1 h. Total proteins were then extracted for western blot assays with anti-SUMO1 antibodies (Abmart, Shanghai, China).

For in vivo sumoylation assays, 15-day-old MdMYB1-GFP, antiMdSIZ1/MdMYB1-GFP, antiMdMYB2/MdMYB1-GFP, antiMdMYB2/pIR-MdSIZ1/MdMYB1-GFP, and antiMdSIZ1/pIR-MdMYB2/MdMYB1-GFP transgenic calli that had been grown at 25°C were treated at either 25°C or 17°C for 1 h before immunoprecipitation of total proteins with anti-GFP antibodies using a Pierce classic IP kit (Thermo, Shanghai, China) following the manufacturer’s instructions. Western blot analyses were subsequently performed with anti-GFP (Abmart, Shanghai, China) or anti-SUMO1 (Abmart) antibodies.