Fig 1.
Expression patterns of HY5 and HYH and phenotypic analysis in response to light.
(A) Expression patterns of HY5::GUS and HYH::GUS reporter lines in the shoots of plants grown under light, dark, and dark-to-light conditions for 7 d. Scale bar: 1 cm. (B) The increase in hypocotyl length of four-day-old etiolated wild-type (WT), hy5-2, hyh, and hy5-2 hyh seedlings exposed to light for an additional day (n>12). Error bars represent SD (standard deviation). (C) Expression patterns of HY5::GUS and HYH::GUS reporter lines in the roots of plants grown in the light for 7 d. The middle and right panels are cross sections of the roots shown in the left panels, at positions denoted by the upper and lower dashed lines, respectively. Scale bar: 50 μm. (D and E) Primary root length (D) and root apical meristem (RAM) size (E) of four-day-old etiolated WT, hy5-2, hyh, and hy5-2 hyh seedlings transferred to constant light (n = 20). Error bars represent SD. In C, D, and E, error bars with different letters indicate a significant difference at p<0.05 (t test).
Fig 2.
HY5 and HYH transcripts and proteins were induced in the root in response to light.
(A–H) Expression of HY5 promoter fusion (A–D) and protein fusion (E–H) lines in roots under light, dark, light-to-dark and dark-to-light transition conditions. (I–P) Expression of the HYH promoter fusion (I–L) and protein fusion (M–P) lines in roots under light, dark, light-to-dark, and dark-to-light transition conditions. Scale bar represents 50 μm.
Fig 3.
Root-autonomous regulation of HY5 and HYH expression in response to light.
(A) Diagram of the decapitation procedure. Dashed lines mark the cutting positions. Letters in brackets represent the corresponding panels. (B,C,F,G) Expression of HY5 protein fusion lines in roots of decapitated or intact plants under dark-to-light and light-to-dark transitions. (D,E,H,I) Expression of HYH protein fusion lines in roots of decapitated or intact plants under dark-to-light and light-to-dark transitions. (J,K) Expression of HY5 protein fusion lines in the roots of intact seedlings under dark and light growth conditions. (L,M) Expression of HYH protein fusion lines in the roots of intact seedlings under dark and light growth conditions. Images were acquired using a fluorescence stereomicroscope. The dotted line indicates the root outline. Scale bar in (B): 50 μm.
Fig 4.
Expression of HYH transcripts and proteins in the shoots and roots of WT and hy5-2 seedlings.
(A,B) Expression of the HYH::GUS fusion in shoots of 7-d-old light-grown WT (A) and hy5-2 (B) seedlings. Scale bar in (A,B): 1 mm. (C,D) Expression of the HYH::HYH-GFP fusion in the cotyledons of WT (C) and hy5-2 (D) seedlings. (E,F) Expression of the HYH::HYH-GFP fusion in the hypocotyls of WT (E) and hy5-2 (F) seedlings. (G,H) Expression of the HYH::ERGFP fusion in the roots of WT (G) and hy5-2 (H) seedlings. (I,J) Expression of the HYH::HYH-GFP fusion in the roots of WT (I) and hy5-2 (J) seedlings. Scale bars in (C–J): 50 μm.
Fig 5.
HY5 binds to the HYH promoter.
(A) Schematic model of HYH genomic sequences. Four fragments of the HYH promoter region (frag 1–4; indicated by lines) were selected for use in a yeast one-hybrid assay (Y1H). Asterisks represent the putative HY5 binding sites. The “ACGT” sequences targeted by HY5 are highlighted for frag 3. (B) Y1H demonstrating that HY5 binds to the HYH promoter at frag 3. COP1 serves as a positive control [27] and the empty vector expressing the AD domain alone is the negative control. (C) An EMSA confirmed the binding of HY5 to the promoter of HYH. Scheme showing the structure of the frag 3 region. The squares indicate ACGT-containing elements (ACE) in frag 3. The corresponding electrophoretic mobility shift assay (EMSA) probes are illustrated as frags 3–1, 3–2, and 3–3. HY5 was produced in an in vitro expression system. The labeled probes were 3' FAM oligonucleotides, while non-labeled probes served as competitors. Arrows indicate the shifted bands.