Fig. 1.
Enhanced UV-B photomorphogenesis in plants expressing constitutively monomeric UVR8G101S. (A) UVR8 amino acid sequence of residues 94 to 108 in wild type (WT) and uvr8-17D. The G101S mutation in uvr8-17D is indicated in bold. Asp-96 and -107 involved in dimer interaction are indicated in red. (B) Representative images of wild-type (Col-0), uvr8-6 null mutant, and uvr8-17D seedlings grown in white light or white light supplemented with UV-B. (Scale bar, 5 mm.) (C) Quantification of hypocotyl length (n > 60); shared letters indicate no statistically significant difference in the means (P > 0.05). (D) Anthocyanin concentration; values of independent measurements (red bars), means, and SEM are shown (n = 3). (E) Immunoblot analysis of CHS and actin (loading control) protein levels in Col-0, uvr8-6, and uvr8-17D grown in white light for 4 d and then white light supplemented with UV-B for 0 to 12 h. (F) Survival of Col-0, uvr8-6, and uvr8-17D seedlings after UV-B stress. Seedlings were grown for 7 d in white light (nonacclimated) or white light supplemented with UV-B (acclimated), then exposed to varying durations (0 to 1.5 h) of broadband UV-B stress. Pictures were taken after a 7-d recovery period. (G) Rosette phenotype of Col-0 and uvr8-17D grown for 56 d under short-day conditions in white light or white light supplemented with UV-B. (H) Immunoblot analysis of UVR8 and actin (loading control) protein levels in 7-d-old Col-0, uvr8-6, and uvr8-17D seedlings. (I) UVR8 dimer/monomer status in Col-0 and uvr8-17D. Protein samples were extracted from dark-grown seedlings either exposed or not exposed to 15 min of saturating UV-B and analyzed using immunoblot analysis of samples separated through SDS-PAGE without prior heat denaturation. Actin is shown as loading control. (J) Immunoblot analysis of UVR8 in DSP cross-linked extracts (Top, –β-ME) of dark-grown Col-0, uvr8-6, and uvr8-17D seedlings either exposed or not exposed to 15 min of saturating UV-B. Cross-linking was reversed by treating with 5% β-ME (Bottom, +β-ME). UGPase is shown as loading control. β-ME, β-mercaptoethanol. (K) Size-exclusion chromatography of recombinant UVR8, UVR8G101S, and UVR8D96N,D107N proteins purified from Sf9 insect cells, with and without UV-B treatment.