Fig. 3. HY5 directly binds and regulates the expression of STOMAGEN.

a, b Gene expression analyses of STOMAGEN in WT, hy5-215, hy5-51 (a only), and HY5-OX by RT-qPCR. In (a), RNA was extracted from 3-day-old seedlings grown under the light. In (b), seedlings were grown in darkness for 4 days and were exposed to light for 0, 2, and 4 h before harvest. Values are mean + /− SEM, n = 3 biological replicates. One-way (a) or two-way (b) ANOVA with Tukey’s multiple comparisons test, P < 0.01. c Co-expression of HY5 and STOMAGEN in the mesophyll layer. Confocal analysis of 3-day-old abaxial cotyledons of a transgenic seedling harboring both HY5pro:HY5-YFP and STOMAGENpro:H2B-mScarlet-I. From left: YFP signals (yellow), mScarlet-I signals (magenta), autofluorescence (cyan) and merged image of all three channels. Scale bar, 50 μm. Three independent cotyledons were examined with similar results (d) Gene structure of STOMAGEN. Arrow indicates the translational start site. Vertical bars mark the position of a Z-box (upstream of TSS only). P1 to 3 represent region(s) tested by EMSA (e), DNA pull down (f) and ChIP-qPCR (g). e EMSA analysis showing the binding of HY5 to a promoter fragment of STOMAGEN (P2). Recombinant MBP (control) and MBP-HY5 were assayed for binding with the biotin-labeled P2 probe. An unlabeled probe (competitor) was used to determine binding specificity (lanes 4–6). f DNA pull-down analysis showing the binding of HY5 to P2 and its dependence on the Z-box. Biotin-labeled probes, including a P2 probe with a mutated Z-box (mP2), were used to pull-down recombinant MBP (control) and MBP-HY5. Results were analyzed by western blotting using an anti-MBP antibody. g ChIP-qPCR assays were performed on WT and HY5pro:HY5-YFP using an anti-GFP antibody. Seedlings were grown for 4 days in darkness before exposed to light for 4 h or kept in the dark. Promoter regions of STOMAGEN (see d) were tested. A genomic region downstream of STOMAGEN and IR1 (see “Methods”) were used as negative controls. Values are mean + /− SEM, n = 3 technical replicates. Assay was repeated with similar results. h GUS reporter assay of two independent lines of STOMAGENpro:GUS and mSTOMAGENpro:GUS, which carries a mutated Z-box in the P2 region, in WT and hy5-215. Seedlings were grown for 3 days under the light. Scale bar, 2 mm. Light intensity used: 200 (a) or 100 µmol m⁻² s⁻¹ (b, c, g, h). The experiments in (e) and (f) were carried out two times with similar results.