Figure 6.

Stabilization of HFR1 by LAF1. (A) HFR1 protein levels are increased by induced LAF1 expression. (Panel a) Two independent transgenic lines containing 35S-HFR1-3HA and XVE-LAF1-6Myc were treated with MG132 (10 μM) with and without induction of LAF1-6Myc expression by inducer (10 μM β-estradiol) for 12 h. HFR1 and LAF1 expression levels were detected by anti-HA and anti-Myc, respectively. These protein extracts were analyzed with anti-COP1 and anti-phyA antibodies. Tubulin expression was used to normalize loading. (Panel b) Northern blot analysis of transgenic plants containing 35S-HFR1-3HA and XVE-LAF1-6Myc-2. Total RNAs were isolated from transgenic plants treated as in panel a. Transgenic HFR1 transcript levels were detected using a full-length HFR1 cDNA probe. Ethidium bromide staining (EtBr) of total RNAs (3 μg per lane) was used to monitor RNA loading. (B) HFR1 protein levels are not affected by LAF1(R97A) expression. (Panel a) Two independent transgenic lines containing 35S-HFR1-3HA and XVE-LAF1(R97A)-6Myc were treated as in A. Antibodies for detection of HFR1-3HA, LAF1(R97A)-6Myc, and loading control were identical to those of A, except anti-COP1 and anti-phyA antibodies. (Panel b) Three independent transgenic lines containing 35S-HFR1(C)-3HA and XVE-LAF1-6Myc were treated with and without induction of LAF1-6Myc expression by inducer (10 μM β-estradiol) for 12 h. Antibodies for detection of HFR1(C)-3HA, LAF1-6Myc, and loading control were identical to those of A, except anti-COP1 and anti-phyA antibodies. (C) Phenotypes of HFR1/LAF1 double overexpressing transgenic seedlings under FR light. (Panel a) Hypersensitivity of HFR1/LAF1 double overexpressing transgenic seedlings under FR light. Seedlings were grown for 4 d under FR light (1.5 μmol m⁻² sec⁻¹) on media without sucrose. Data are presented as average hypocotyl length ± standard deviations (SD; n > 100). Seedlings are shown above the histograms. (*) HFR1/LAF1 double overexpressing transgenic seedlings are significantly shorter than HFR1 or LAF1 single overexpression seedlings (P < 0.01, Student’s t-test; n > 100). Western blot analysis of transgenic Arabidopsis seedlings overexpressing HFR1-6Myc and LAF1-3HA (panel b) or HFR1-6Myc and LAF1(R97A)-3HA (panel c) under FR irradiation with or without MG132. After treatment with 25 μM MG132 or mock treatment, seedlings were incubated for a further 12 h under continuous FR light (1.5 μmol m⁻² sec⁻¹). Tubulin levels were used as loading controls.