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.