Figure 3.

BZR1 stability is regulated by COP1 in a 26S proteasome-dependent manner. (a) Immunoblot analysis of BZR1 proteins. Seven-day-old light-grown seedlings expressing BZR1pro:BZR1-HA were treated or not with MG132 in the dark for 6 h. Actin was used as a loading control. (b) Immunoblot analysis results of the 10-day-old seedlings grown under continuous light and subsequently subjected to LD (light to dark for 12 h) or LDL (light to dark for 12 h and back to light for 12 h) growth conditions. The total protein extracted from 10-day-old seedlings was immunoblotted with anti-BZR1. Actin was used as a loading control. (c) Coimmunoprecipitation assay to test for the interaction between BZR1 and COP1 in 35Spro:BZR1-HA 35Spro:COP1-GFP Arabidopsis seedlings. Seedlings were grown under long-day conditions (with a photon fluence rate of 103 μmol m⁻² s⁻¹), and then transferred to the dark for 2 days. Total proteins were first immunoprecipitated with anti-GFP, and were subsequently immunoblotted with anti-HA and anti-GFP. (d) Yeast two hybrid analysis. –LW indicates growth media lacking leucine (L) and tryptophan (W). DBD and AD are the vectors harboring the DNA binding domain and the DNA activation domain, respectively. Vector indicates the empty vector. (e) qRT-PCR analyses of ACS5, GER1, PRE5 and PRE6 in Col-0 and cop1-4 treated with the same growth and light conditions as described in (b).