The HY5-COL3-COL13 regulatory chain
Based on the genetic data, col3 hy5 double mutant behaved like
the hy5 mutation (Datta et al.,
2006), and COL13 might be the downstream of COL3 in
regulating hypocotyl elongation, we hypothesized that there is a
HY5-COL3-COL13 regulatory chain for controlling hypocotyl growth.To test this hypothesis, HY5 and COL3 coding sequence, as well as
a deletion series of the COL13 promoter were cloned into the
dual-luciferase system, respectively (Fig. 4a). As shown in Figure 4b,
these dual-luciferase experiments confirmed the ability of HY5 to bind to
the COL3 promoter and COL3 to bind to the COL13 promoter.
In addition, these experiments also map the COL3 target regions to
between -1675 bp and -616 bp of the COL13 promoter (Fig. 4b). The
in vivo interaction of COL3 with this
1059-bp
sequence of the COL13 promoter was further confirmed by
electrophoresis mobility shift assay (EMSA; Fig. 4c). To investigate
core-binding motif of the 1059-bp region, a series of EMSAs involving
deletions of this region were done. We divided the 1059-bp promoter
sequence 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), and showed that probe
2 (-1421 to -1184 bp) was essential for binding of COL3 to theCOL13 promoter (Fig. S2).
COL13
is located in the nucleus
Transformation of Arabidopsis protoplasts with a construct
expressing COL13-CFP indicated that COL13 is located in the nucleus
(Fig. 5a), and a similar result was obtained when the root apical cells
of stable COL13-GFP transgenic plants were examined (Fig. 5b).
COL13 interacts with
COL3 ,
but not COP1
According to previous reports, both COL3 and COL13 are CONSTANS
(CO)-like proteins, they are related to CO
(Robson et al., 2001), and as shown for
COL13 above, COL3 also positively regulates red light-mediated
inhibition of hypocotyl elongation in Arabidopsis(Datta et al., 2006). We also demonstrated
above that COL13 shares the same subcellular localization as COL3 (Fig.
5a, b). Given that COL3 can interact with BBX32 and that COL13 also
belongs to the BBX zinc finger TF family, we hypothesized that COL3
might interact with COL13. This idea was supported by a two-hybrid assay
revealing that COL3 was able to interact with COL13 protein in yeast
(Fig. 6a). Next, we examined the interaction in transgenic plants
expressing both COL3 and COL13, and showed that COL13 was
co-immunoprecipitated with COL3 from seedling tissues (Fig. 6b). The
interaction between COL13 and COL3 was also demonstrated in plant cells
in a fluorescence resonance energy transfer (FRET) assay (Fig. 6c-f). As
shown in Fig. 6c, both cyan fluorescent protein (CFP)-fused COL3 and
yellow fluorescent protein (YFP)-fused COL13 were observed in the nucleus
after excitation with a 405-nm or a 514-nm laser, respectively. After
bleaching an area of interest with the 514-nm laser, YFP-COL13
fluorescence was reduced dramatically, whereas there was a clear
increase in CFP-COL3 emission in the same area (Fig. 6d), indicating
that FRET had occurred. The relative intensities of emissions from
CFP-COL3 and YFP-COL13 in the area of interest, before and after
bleaching, are shown in Fig. 6e,f.
COL13
promotes the interaction between COL3 and COP1
Interestingly, although COL13 and COL3 have similar structures,
containing two N-terminal tandemly repeated B-box domains and a CCT
domain in the C-terminal, only COL3 can interact with COP1, COL13 does
not bind to COP1
(Fig.6a).
The results were also demonstrated by FRET assay (Fig. S3a-h). To
investigate if COL13 influences the interaction between COP1 and COL3,
we performed a yeast three-hybrid assay. In this yeast system,
COL3-COL13-pBridge construct allows expression of theCOL3-BD /bait and COL13 in yeast, and COL13 only
expressed in the absence of methionine (Met). As shown in Fig.7a, the
growth of yeast carrying indicated constructs on selective medium (+Met
or -Met) along with an α-galactosidase assay showed that COP1 and COL3
had stronger binding activity with the expression of COL13. Based on
previous report, COP1 interacted with COL3 and inhibited the production
of COL3(Datta et al., 2006). By combining
our results above, we proposed a possible COP1-dependent COL3-COL13
feedback pathway (Fig.7b), which involved in regulation of hypocotyl
elongation.