Figure 1.
Characterization of hfr1-23 mutants.
(A) T-DNA insertion identified in HFR1 gene. A schematic diagram representing HFR1 gene with its intron/exon structrure, the encoded protein, and the location of T-DNA insertion in hfr1-23 mutant. N, bHLH and C represent the N-terminus, basic helix-loop-helix domain and C-terminus of HFR1 protein, respectively. (B) Seven-day-old Arabidopsis seedlings of phyA, hfr1, cry1 and WT in different light conditions. Seedlings from left to right are phyA, hfr1, cry1 and WT. a–d: Seedlings grown in the dark; e–h: Seedlings grown in red light (30 µmol m−2 s−1); i–l: Seedlings grown in far-red light (10.3 µmol m−2 s−1); m–q: Seedlings grown in blue light (30 µmol m−2 s−1). (C) Hypocotyl elongation of WT, phyA, cry1 and hfr1 seedlings in blue (15 µmol m−2 s−1) and far-red light (10.3 µ mol m−2 s−1). All seedlings were grown in continuous light for 4 days before measurement. 20 seedlings were used in each sample. Error bars represent standard deviations. (D) HFR1 protein level in hfr1-23 mutants and col-0. β-Tubulin was used as control. The seedlings were grown in blue light (50 µmol m−2 s−1) for 3 days.
Figure 2.
Gene expression profiling in WT, cry1 and hfr1 mutants in the dark and 1 h blue light (50 µmol m−2 s−1).
(A) Evaluation of the profile by Principle Component Analysis (PCA). All genes on ATH1 chips were used to generate the PCA plot. WT, cry1 and hfr1 represented the global gene expression in WT, cry1B104 and hfr1-23 mutants. Three biological replicates in the same genetic background for each treatment were colored differently (red: WT, yellow: cry1 and blue: hfr1). All samples in dark treatment were grouped in a green circle. All samples in blue light treatment were shown in a blue circle. (B) Genes regulated by blue light in a cry1- and HFR1-dependent manner. INDUCTION: Genes induced by blue light and dependent on cry1 and HFR1; REPRESSION: genes repressed by blue light and dependant on cry1 and HFR1. Genes regulated by blue light in cry1-dependent manner (blue circle) were determined by 2-way ANOVA with p-value lower than the false discovery rate (3.87E-05) in the comparison of WT/cry1; genes regulated by blue light in HFR1-dependent manner (red circle) were determined by 2-way ANOVA with p-values lower than the false discovery rate (4.62E-05) in WT/hfr1 comparison. All genes were applied as universe. Numbers in each portion showed the gene distribution. (C) Blue light induction of genes in WT, cry1 and hfr1 mutants. 293 blue light-induced genes, dependant on both cry1 and HFR1 from (B), were divided into four groups based on their mean fold induction (MFI) of cry1 in blue light (MFI≥100, 10–100, 5–10 and 2–5). Genes in each group were plotted in rank order of their relative response to 1 h blue light compared to the dark treatment. Curves represented MFI values for WT (red), cry1 (yellow) and hfr1 (blue) mutants. (D) Distribution of genes induced more than 2-fold by cry1 in 1 h blue light among functional categories, shown as a percentage of the total annotated genes within each group. ET: electron transport; ME: metabolism; P/C: photosysthesis/chloroplast; TP: transport; S/D: stress/defense; TR: transcription; G/D: growth/development; KI: protein kinases; HR: hormones; UN: unknown.
Figure 3.
Dynamic change of gene expression from dark, 30 min and 1 h blue light.
RT-PCR of selected genes from 4-day-old dark-grown WT, cry1 and hfr1 mutant seedlings treated by dark, 30 min and 1 h blue light (50 µmol m−2 s−1). Relative expression level of each gene was normalized to GAPDH. Expression level was from the average of three biological replicates.
Table 1.
Motifs overrepresented in genes induced by blue light and dependent on both cry1 and HFR1.
Figure 4.
Activity of the CYP82C2 promoter depends on both cry1 and HFR1 and requires blue light.
(A) GFP protein level in independent PCYP82C2::GFP transgenic lines grown in blue light (50 µmol m−2 s−1) for 7 days. WT was used as a negative control for GFP detection; w1 and w10: independent transgenic lines in col-0 background; c2, c3 and c4: independent transgenic lines in cry1 background; h3 and h4: independent transgenic lines in hfr1 background. Tubulin was used as loading control. (B) GFP expression on RNA and protein level in w1, c2 and h3 transgenic plants after 1 h blue light (50 µmol m−2 s−1) treatment. GAPDH was used as the control for RT-PCR; tubulin was used as control in western blot. (C) GFP fluorescence in 7-day-old transgenic seedlings grown in darkness and continuous blue light (50 µmol m−2 s−1). 1 and 4: PCYP82C2::GFP/col-0; 2 and 5: P CYP82C2::GFP/cry1; 3 and 6: P CYP82C2::GFP/hfr1.
Figure 5.
cry1 influences HFR1 on both RNA and protein level.
(A) RT-PCR showing the relative expression level of HFR1 normalized to GAPDH. Expression levels were the average of three biological replicates. (B) Western blot showing cry1 and HFR1 protein level in WT, cry1 and hfr1 mutants in the dark, 30 min and 1 h blue light (50 µmol m−2 s−1). Tubulin was used as the control.