Fig. 4: DAY regulates BRI1 stability.

a DAY deficiency triggers BRI1 instability. DAY silencing causes aggregation of BRI1-GFP in Arabidopsis roots. The pBRI1::BRI1-GFP × DAYi seedlings were grown in medium containing ethanol or 1 µM DEX in the light for 9 days, and their roots were observed by CLSM. Scale bar, 20 µm. b DAY silencing decreases BRI1 protein levels. 9-day-old seedlings of WT (Ws-2 and Col-0), bri1 mutants (bri1–5 and bri1-116), and DAYi plants grown in the presence of ethanol or 1 μM DEX were subjected to immunoblotting with anti-BRI1 and anti-cpHSP70 antibodies. c DAY silencing increases phosphorylated BZR1. 9-day-old DAYi seedlings grown in the presence of ethanol or 1 μM DEX were subjected to immunoblotting using anti-BZR1 and anti-cpHSP70 antibodies. d, The dominant mutation bzr1-1D rescues the short petiole phenotype, but not chloroplast defect, of DAY-silenced seedlings. Seedlings from DAYi and DAYi x bzr1-1D lines were grown on 1/2 MS medium for 5 days at 22 °C and then transferred to medium containing ethanol or 1 µM DEX at 28 °C for 7 days. Scale bar, 1 cm. e, f, Overexpression of DAY partially rescues a BR signaling mutant. Plants were grown in soil for 4 weeks at 22 °C (e, left panel) or 2 weeks at 22 °C and then transferred to 24 °C for 2 weeks (e, middle panel). Expression of DAY-GFP protein in bri1-301 mutant was shown by immunoblotting with anti-GFP antibody (e, right panel). Morphology of 5-day-old dark-grown seedlings of wild-type, bri1-301, 35 S::DAY-GFP / bri1-301 #1 and #2 transgenic lines on medium in the absence (f, left panel) or presence of 250 nM brassinazole (Brz) (f, middle panel). Hypocotyl lengths are quantified relative to those of mock-treated samples (f, right panel). Genotypes are indicated. Dots represent individual ratio values (%) between mock-treated and Brz-treated independent seedlings. n denotes numbers of biologically independent hypocotyls, Brz- (left number), and mock-treated (right number). Statistical significance was determined by one-way ANOVA, Dunnett’s multiple comparisons test ****P < 0.0001. Scale bars: 3 cm (e) or 1 cm (f).