Figure 9

OsOTUB1 promotes the degradation of OsSPL14. (A) Accumulation of OsSPL14 in ZH11 and ZH11-npt1 plants. The abundance of the HSP90 protein was used as a loading control. (B) Treatment with the proteasome inhibitor MG132 stabilizes OsSPL14. Total protein was extracted from young panicles (< 0.2 cm in length) of ZH11 plants exposed to either 0 or 50 μM MG132. The immunoblot was probed with either anti-OsSPL14 or anti-HSP90 antibodies. (C) OsOTUB1 destabilizes OsSPL14. The lysates from young panicles of ZH11 and ZH11-npt1 plants were co-incubated with GST-OsSPL14 in the presence or absence of His-OsOTUB1. The lysates were harvested at various times and immunoblotted to assess the accumulation of OsSPL14 and HSP90. (D) Ubiquitination of OsSPL14. The protein extracts from young panicles were immunoprecipitated using an anti-Myc antibody, then analysed using anti-ubiquitin, anti-K48-linked ubiquitin or anti-K63-linked ubiquitin chain conjugates. (E) Flag-OsSPL14 can be modified via K48-ubiquitin linkage. Rice protoplasts were co-transfected with Flag-OsSPL14 and HA-ubiquitin (either HA-tagged WT or K48R ubiquitins), and the ubiquitinated forms of Flag-OsSPL14 were immunoprecipitated using an anti-Flag antibody and then analysed using an anti-HA antibody. (F) The K63-linked ubiquitination of OsSPL14 is regulated by OsOTUB1. Rice protoplasts were co-transfected with Flag-OsSPL14 and HA-ubiquitin (either HA-tagged WT, K48R, K63R, K48O or K63O ubiquitins) in the presence or absence of Myc-OsOTUB1; lysates were then harvested and immunoblotted to assess the accumulation of OsSPL14 and analysed for ubiquitinated forms of Flag-OsSPL14, as described in E.