Figure legends
Fig. 1 COL13 RNA accumulates at high levels in the hypocotyl. (a) Quantitative real-time PCR analysis of COL13transcript abundance in different tissues. R=Root; S=Stem; L=Leaf; SAM=Shoot apical meristem; H=Hypocotyl; F=Flower. (b) Activity of the COL13 promoter revealed by β-glucuronidase (GUS) staining inArabidopsis 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.). Asterisks indicate that the expression of COL13 in OX9, OX11, OX20,col13 , and COL13 RNAi lines are significantly different with Col-0 under red light (P < 0.05). (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), white bar=0.5cm. The hypocotyl lengths of the indicated genotypes were measured at the 5th or 3rd day, and are shown in (d) and (e), respectively. Error bars indicate SD (n >15). Asterisks indicate that hypocotyl lengths in OX9, OX11, OX20, col13 , and COL13 RNAi lines are significantly different with Col-0 under red light (P < 0.05).
Fig. 3 Genetic interaction and physiological characterization of hypocotyl elongation. (a) Semi-quantitative reverse transcriptase (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 theCOL13 promoter revealed by β-glucuronidase (GUS) staining in WT and col3 mutant backgrounds. (d ) Hypocotyl length in WT, single- and double-mutant plants. Here we use the F1hybrid of Col-0×WS as WT. (e ) Hypocotyl length in WT andcol3 plants compared to transgenic plants with COL13 RNAi or COL13 overexpression (OX) in the col3 background. Here we use WS as WT. 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 the COL3 promoter and COL3 to COL13 promoter truncations. (a) Diagram of constructs used in this study. For luciferase system, the AD-HY5 or AD-COL3 fusion proteins driven by the 35S promoter produces a potential effector protein, while the AD protein alone represents a negative control for basal activity ofCOL3 promoter or each COL13 promoter truncation. TheLUC gene driven by the series of COL3 promoter orCOL13 promoter truncations tests the ability of the AD-HY5 or AD-COL3 fusion protein to bind to each promoter truncation. For yeast-one hybrid system, the GAD-HY5 or GAD-COL3 fusion proteins driven by the GAL1 (PGAL1) promoter serves as effectors. The GAD protein served as negative control to see if there exits the self activity of COL3 or COL13 promoters. The LacZgene driven by COL3 or COL13 promoter truncations served as the reporter to test the binding activity of the GAD-HY5 or GAD-COL3 fusion protein to individual promoter truncations. (b) For luciferase assay, the fusion protein AD-HY5 can up-regulate LUC expression from the COL3 promoter, but not from COL13 promoter; and the fusion protein AD-COL3, but not AD alone, can up-regulate LUCexpression from some of the COL13 promoter truncations. For Yeast-one hybrid assay, the fusion protein GAD-HY5 can strongly bind to the promoter of COL3 , but not COL13 to direct LacZexpression in yeast cells that turns 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside to blue compound; and the fusion protein AD-COL3, but not AD alone, can bind to some of theCOL13 promoter truncations. (c) The 1059-bp promoter (-1675 bp to -616 bp ) of COL13 was devided into five overlapping regions: -1675 to -1391 bp (probe 1), -1421 to -1184 bp (probe 2), -1201 to -1040 bp (probe 3), -1060 to -868 bp (probe 4), and -898 to -616 bp (probe 5). (d) Electrophoretic mobility shift assay (EMSA) analysis showing the binding of COL3 on the -1421 to -1184 bp promoter of COL13 (probe 2) in vitro. The + and represent the positive and negative control, respectively. (e ) Electrophoretic mobility shift assay (EMSA) analysis showing the binding of COL3 on the -1421 to -1184 bp promoter of COL13 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. Competition experiments were carried out by adding 5-, 10- and 25-fold excessive competitor. (f) In probe 2, there were three light responsive elements (ATCT-motif, G-Box and TCT-motif) and one core promoter element for transcription start (TATA-box). (g) The fusion protein GAD-COL3, but not GAD alone, can strongly bind to a promoter truncation of COL13 to direct LacZ expression in yeast cells. The 238-bp promoter region (-1421 bp to -1184 bp ) ofCOL13 was devided into four fragments: -1421 to -1356 bp, -1355 to -1307 bp, -1306 to -1242 bp, and -1241 to -1184 bp. The colour rectangles represented indicated promoter elements in (f).
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 proteins 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 an anti-HA antibody and COL13 was co-immunoprecipitated with an 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 the light and dark. Bar=5um. (d-f) FRET between CFP-COL3 and YFP-COL13 analyzed by acceptor bleaching in the nucleus. Bar=5um. The top panels in (d) show a representative pre-bleach nucleus co-expressing 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), respectively.