Article Figures & Data
Figures
- Figure 1.
The GST–HY5 Protein Specifically Binds to the Consensus G-Box Tetramer in Vitro.
(A) Sequences of the three consensus LREs examined in this study.
(B) Gel shift analysis with a 53-bp DNA fragment of the consensus G-box tetramer (4G) that was used as a probe. The amount of protein added in each reaction was none (lane 1), 4 μg of glutathione S-transferase (GST; lane 2), and 0.8 μg of GST–HY5 (lanes 3 to 8). The amounts of unlabeled competitors (COMP) were 100, 200, and 300 ng of 4G in lanes 4, 5, and 6, respectively, and 300 ng of GATA and GT1 tetramers (4GATA and 4GT1) in lanes 7 and 8, respectively. Increasing concentrations of the competitor are indicated by the triangle. Plus and minus signs indicate presence and absence of competitors, respectively.
- Figure 2.
GST–HY5 Binds to the RBCS-1A Minimal Light-Responsive Promoter.
A 196-bp DNA fragment of the RBCS-1A minimal promoter was used as a probe. The amount of protein in each reaction was none (lane 1), 4 μg of GST (lane 2), and 0.8 μg of GST–HY5 (lanes 3 to 11). The amounts of unlabeled competitors (COMP) were G, 26-bp double-stranded DNA (5′-AATTATCTTCCACGTGGCATTATTCC-3′; underlining indicates the hexameric G-box core motif) oligonucleotide containing the G-box from the RBCS-1A promoter (80, 160, and 320 ng in lanes 4, 5, and 6, respectively), and 4G, consensus G-box tetramer (80, 160, and 320 ng in lanes 7, 8, and 9, respectively), and 320 ng of 4GATA and 4GT1 in lanes 10 and 11, respectively. Increasing concentrations of the competitor are indicated by the triangles.
- Figure 3.
DNase I Footprinting Analysis of the 196-bp Minimal Light-Responsive Promoter Region of the RBCS-1A Gene with GST–HY5 Protein.
Lane 1 shows the A+G Maxam and Gilbert sequencing ladder of the same labeled fragment (bottom strand). Lanes 2 to 5 show the DNase I cleavage pattern with 0, 5, 10, and 15 μg of the GST–HY5 protein (increasing concentrations of the protein are indicated by the triangle). The sequence of the G-box–containing region in the promoter fragment is shown at right. The hypersensitive nucleotides are indicated with stars, and the protected nucleotides are indicated with open circles.
- Figure 4.
Effect of the hy5 Mutation on Light-Activated Expression of the G-Box–Containing Promoters.
Promoter activity in the wild type (WT) and hy5 mutant (hy5) backgrounds was estimated by quantitative GUS activity assays. The chimeric promoter–GUS reporter transgenes are diagrammed above each graph. The activities are the average of four independent repeats in one representative experiment (out of four), and the error bars indicate standard deviations.
(A) GUS activity of the G/NOS101–GUS transgene in 6-day-old seedlings grown under complete darkness (CD) or constant white light (cWL).
(B) GUS activity of the GATA/NOS101–GUS transgene in 6-day-old light- and dark-grown seedlings.
(C) and (D) Light induction kinetics of dark-grown seedlings containing G-GATA/NOS101–GUS and RBCS-1A promoter–GUS transgenes, respectively. Four-day-old dark-grown seedlings exposed to white light for 0, 12, and 48 hr and 6-day-old light-grown seedlings (cWL) were used for the GUS activity assay.
- Figure 5.
Red Light Pulse Induction and Its Far-Red Light Pulse Reversibility of Dark-Grown Seedlings Containing G-GATA/NOS101–GUS or the RBCS-1A Promoter–GUS Transgenes.
Four-day-old dark-grown seedlings were exposed to 2 min of red light (RL) alone or immediately followed by exposure to 10 min of far-red light (RL+FR). After the light treatment, the seedlings were transferred to complete darkness (CD) for an optimal time period for each line (18 hr for RBCS-1A–GUS and 48 hr for G-GATA/NOS101–GUS) before being harvested for the GUS assay. The activities are the average of four independent repeats in one representative experiment (out of four), and the error bars indicate standard deviations.
(A) GUS activity of the G-GATA/NOS101–GUS transgene.
(B) GUS activity of the RBCS-1A promoter–GUS transgene.
- Figure 6.
Effects of the hy5 Mutation on Tissue-Specific Expression of G-Box–Containing Promoters in Light-Grown Plants.
In (A) to (J), the wild type is shown to the left or at top and the hy5 mutant is shown to the right or at bottom.
(A) to (C) Six-day-old seedlings carrying the G/NOS101–GUS, G-GATA/NOS101–GUS, and RBCS-1A promoter–GUS transgenes, respectively. Arrows indicate the dramatic GUS staining changes at the junctions of cotyledons and hypocotyls.
(D) to (F) Leaves of 16-day-old plants carrying the G/NOS101, G-GATA–NOS101, and RBCS-1A promoter–GUS transgenes, respectively.
(G) and (H) Stems of 16-day-old plants carrying the G/NOS101 and RBCS-1A promoter–GUS transgenes, respectively.
(I) and (J) Roots of 16-day-old plants carrying the G/NOS101 and RBCS-1A promoter–GUS transgenes, respectively.
- Figure 7.
Comparison of GUS Activities in the Leaves, Stems, and Roots of 16-Day-Old Light-Grown Plants.
(A) Plants containing the G-GATA/NOS101–GUS transgene.
(B) Plants containing the RBCS-1A promoter–GUS transgene. For further details, see the legend to Figure 4 and Methods.
- Figure 8.
Nuclear Localization of GFP–HY5 in Arabidopsis.
(A) and (B) Dark- and far-red light–grown hypocotyl cells.
(C) and (D) Dark- and far-red light–grown root cells.
Fluorescence in the nuclei of 4-day-old dark- and far-red light–grown hypocotyl and root cells of transgenic lines expressing the S65TGFP full-length HY5 fusion protein is shown. In all cases, green fluorescence is located exclusively in the nuclei, as determined by a direct comparison of the confocal and Nomarski microscopic images of the same cells. For example, see the insert and the insert within the insert in (A). The arrows indicate the nuclei. The bar in the insert in (A) = 50 μm. The bar in (B) = 0.2 mm for (A) to (D).
