Figure legends
Fig.1 COL13 RNA accumulates to high levels in hypocotyl. (a) Quantitative real time-PCR analysis of AtCOL13 transcript abundance in different tissues. R=Root, S=Stem, L=Leaf, SAM=Shoot apical meristem, H=Hypocotyl, F=Flower. (b) Activity of COL13 promoter revealed by β-glucuronidase (GUS) staining in Arabidopsis seedlings. Bar=100 mm.
Fig.2 COL13 regulates hypocotyl elongation under red-light conditions. (a) Relative expression of COL13 in Col-0 and overexpression (OX) lines. (b) Relative expression of COL13 in Col-0, T-DNA mutant (col13) and RNAi lines (R1-1 etc.). (c)-(e) Phenotypic analysis seedlings of the indicated genotypes were grown in the presence of red light. Images of representative seedlings are shown in (c). The hypocotyl lengths of the indicated genotypes were measured and are shown in (d) and (e). Error bars indicate SD (n >15). Asterisks indicate that hypocotyl lengths in OX9 and col13, COL13 RNAi are significantly different with WT under red light (P < 0.05).
Fig. 3 Genetic interaction and physiological characterization of hypocotyl elongation. (a) Semi-quantitative RT-PCR analyses of COL13 expression in phyB , col3 , hy5 and cop1 mutants. (b) qRT-PCR analyses of COL13 expression in phyB , col3 ,hy5 and cop1 mutants. (c) Activity of the COL13 promoter revealed by β-glucuronidase (GUS) staining in WT and col3 mutant backgrounds. (d) Hypocotyl length in WT, single- and double-mutant plants. (e) Hypocotyl length in WT and col3 plants compared to transgenic plants with COL13 RNAi or COL13 overexpression (OX) in the col3 background. Error bars indicate SD (n >15). Lower-case letters indicate significantly different data groups (hypocotyl length) of the indicated seedlings grown in red light.
Fig.4 Analysis of the binding of HY5 to COL3 promoter, and COL3 to COL13 promoter truncations. (a) Diagram of constructs used. The AD-HY5 or AD-COL3 fusion gene driven by the 35S promoter produces a potential effector protein, while the AD protein alone represents a negative control for basal activity of COL3 promoter or each COL13 promoter truncation. The LUC gene driven by the series of COL3 promoter or COL13 promoter truncations tests the ability of the AD-HY5 or AD-COL3 fusion protein to bind to each promoter truncation. (b) The fusion protein AD-HY5, but not AD alone, can effect LUC expression from the COL3 promoter truncations, and the fusion protein AD-COL3, but not AD alone, can effect LUC expression from some of the COL13 promoter truncations. (c) Electrophoretic mobility shift assay (EMSA) analysis showing the binding of COL3 to COL13 the -1421 to -1184 bp promoter (probe 2) in vitro. The black arrow indicates binding of COL3 to the biotin-labeled COL13 promoter. The + and – represent the presence and absence of corresponding components, respectively.
Fig.5 Subcellular localization of COL13. (a) COL13-CFP localizes to the nucleus in protoplasts. (d) COL13-GFP localizes to the nucleus in root tip cells.
Fig.6 COL13 interacts with COL3. (a) Yeast Two-Hybrid assay between COL13 and COL3. DDO, Double Dropout; QDO, Quadruple Dropout; pGADT7, prey plasmid; pGBKT7, bait plasmid. (b) Co-immunoprecipitation (Co-IP) in Arabidopsis. Immunoprecipitations (IPs) were performed on protein extracted from 10-d-old Arabidopsis seedlings grown under long-day illumination (16L: 8D) at 22˚C. Leaf tissues were harvested 1 h after the light cycle commenced. IP was performed using anti-HA antibody and COL13 was co-immunoprecipitated with anti-GFP antibody. A 5% input was used. Western blots were performed on 10% (wt/vol) precast gels (Bio-Rad). (c) COL3-CFP and COL13-YFP colocalize to the nucleus in protoplasts in light and dark. (d-f) FRET between CFP-COL3 and YFP-COL13 analyzed by acceptor bleaching in the nucleus. The top panels in (d) show a representative pre-bleach nucleus coexpressing YFP-COL13 and CFP-COL3 excited with either a 514- or a 405-nm laser in light and dark, resulting in emission from YFP (yellow) or CFP (blue), respectively. The bottom panels in (d) show the same nucleus post-bleaching after excitation with a 514- or a 405-nm laser. The relative intensities of both YFP and CFP were measured before and after bleaching, as indicated in (e) and (f).
Fig.7 COL13 promotes the interaction between COL3 and COP1. (a) Yeast three-hybrid analysis of the COP1-COL3 interaction in the presence of COL13. Normalized Miller Units were calculated as a ratio of α-galactosidase activity in yeast. Additionally, normalized Miller Units here are reported separately for yeast grown on media without or with 1 mM methionine (Met), corresponding to induction (-Met) or repression (+Met) of Met25 promoter-driven COL13 expression, respectively. Means and SEM for three biological repetitions are shown. Lower-case letters indicate significant difference of α-galactosidase. (b) A model representing the HY5-COL3-COL13 regulatory chain and COP1-dependent COL3-COL13 feedback pathway in regulation of hypocotyl elongation.