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. 2020 Apr 20;30(8):1410–1423.e3. doi: 10.1016/j.cub.2020.01.089

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© 2020 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Brassinosteroids Promote BZR1 SUMOylation and ULP1a Degradation

(A) BL induces BZR1 SUMOylation. Immunoprecipitation (IP:αGFP) experiments were carried out with αGFP beads from total proteins derived from transgenic lines expressing _proBZR1::BZR1-GFP in Col-0 background after treatment with 1 μM BL at time points of 0, 30, and 60 min. Immunoblots were probed with αGFP (IB: αGFP) or αSUMO1/2 (IB: αSUMO1) antibodies.

(B) BRZ inhibits SUMOylation of BZR1. Immunoprecipitation (IP: αGFP) experiments were carried out with αGFP beads from total proteins derived from transgenic lines expressing _proBZR1::BZR1-GFP in Col-0 background after treatment with 2 μM BRZ at time points of 0, 30, and 60 min. Immunoblots were probed with αGFP (IB: αGFP) or αSUMO1/2 (IB: αSUMO1) antibodies.

(C) BZR1 is SUMOylated in planta. Transiently co-expressed BZR1-GFP or BZR1^2K/R-GFP transiently co-expressed with SUMO1-HA in leaves of N. benthamiana is shown. Immunoprecipitation (IP: αGFP) experiments were carried out with αGFP beads from total protein derived from these leaves. Immunoblots were probed with αGFP (IB: αGFP) or αSUMO1/2 (IB: αSUMO1) antibodies. GFP was used as a negative control.

(D) BZR1 is SUMOylated in stable Arabidopsis transgenic lines. Immunoprecipitation (IP: αGFP) experiments were carried out with αGFP beads from total protein derived from transgenic lines expressing _proBZR1::BZR1-GFP or _proBZR1::BZR1^2K/R-GFP in Col-0 background. Immunoblots were probed with αGFP (IB: αGFP) or αSUMO1/2 (IB: αSUMO1) antibodies. See also Figure S3.

(E and F) BL does not induce the accumulation of BZR1^2K/R. 10-day-old transgenic Arabidopsis seedlings carrying _proBZR1::BZR1-GFP or _proBZR1::BZR1^2K/R-GFP were treated with a combination of 200 μM cycloheximide + 1 μM BL (E) or 2 μM BRZ (F), and total proteins extracted at indicated time points were immunoblotted with αGFP (IB: αGFP) antibody. Ponceau staining for Rubisco levels was used as a loading control.

See also Figure S3.