Figure 4.

SMXL2 Undergoes Ubiquitination after GR24^4DO, KAR₁, or GR24^ent-5DS Treatment.

(A) Ubiquitination of SMXL2-GFP proteins in 7-d-old 35S:SMXL2-GFP transgenic plants after GR24^4DO, GR24^ent-5DS, or KAR₁ treatment. Seedlings were treated with 50 µM MG132 for 1 h and then with 2 µM GR24^4DO, GR24^ent-5DS, or KAR₁ for 20 min in 0.5× MS liquid medium.

(B) Ubiquitination of SMXL2-GFP proteins in 7-d-old 35S:SMXL2-GFP transgenic plants with 50 µM MG132 for 1 h and then with 2 µM KAR₁ for 2 h or 2 µM GR24^4DO for 20 min in 0.5× MS liquid medium. Mock treatments were 2 h.

(C) Ubiquitination of SMXL2-GFP in wild-type, d14-1, and kai2-2 Arabidopsis seedlings containing 35S:SMXL2-GFP transgene. Seedlings were treated after 7 d of growth with 50 µM MG132 for 1 h and then with 2 µM GR24^4DO or GR24^ent-5DS for 20 min in 0.5× MS liquid medium.

(D) Ubiquitination of GFP-SMXL2 in protoplasts made from wild-type and d14-1 kai2-2 seedlings.

(E) Ubiquitination of GFP-SMXL6 in protoplasts made from wild-type, d14-1, and kai2-2 seedlings.

In (D) and (E), protoplasts were transformed with 35S:GFP-SMXL2 or 35S:GFP-SMXL6 plasmid, incubated overnight for protein synthesis, and then pretreated with 50 µM MG132 for 1 h and treated with 50 µM GR24^4DO and GR24^ent-5DS for 1.5 h.

Proteins were detected by immunoblotting with anti-ubiquitin (Ubi) polyclonal antibody or anti-GFP monoclonal antibody. All experiments were repeated at least three times with similar results. IP, coimmunoprecipitation assay.