HY5-COL3-COL13 regulatory chain
Based on the genetic data, the col3 hy5 double mutant behaved
like the hy5 mutation (Datta et al., 2006), and COL13might be downstream of COL3 in regulating hypocotyl elongation.
In addition, it has been well established that HY5 binds to “CACG” and
“GACGTG” in the promoters of light-responsive genes (Lee et al., 2007;
Nawkar et al., 2017). COL3 is a light-responsive gene, and we examined
the promoter sequence of the COL3 gene and found that it contains two
“CACG” and one “GACGTG”. Thus, we hypothesized that there would be
an HY5-COL3-COL13 regulatory chain for controlling hypocotyl
growth. To test this hypothesis, the HY5 and COL3 coding
sequences, as well as a deletion series of the COL13 promoter,
were cloned into the dual-luciferase system (Fig. 4a). As shown in
Figure 4b, these dual-luciferase experiments and yeast-one hybrid assays
confirmed the ability of HY5 to bind to the COL3 promoter and COL3
to bind to the COL13 promoter. Additionally, these experiments
also mapped the COL3 target regions (1059 bp) to between -1675 bp and
-616 bp of the COL13 promoter (Fig. 4b). To investigate the
core-binding motif of the 1059 bp region, a series of EMSAs involving
deletions of this region were performed. We divided the 1059 bp promoter
sequence into five overlapping regions (Fig. 4c): -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
the COL13 promoter (Fig. 4d). The in vivo interaction of
COL3 with probe 2 was further confirmed by EMSA competition experiments
that were conducted by adding excess amounts of the competitor (5-, 10-,
and 25-fold higher amounts) (Fig. 4e). Next, we analyzed the sequence of
probe 2, and found that in the 238 bp fragment, there were three light
responsive elements (ATCT-motif, G-Box and TCT-motif) and one core
promoter element for transcription start (TATA-box) (Fig. 4f). Then we
wondered if these promoter elements were required for binding with COL3.
To address this question, yeast one-hybrid assays were performed and the
results showed that a 49-bp region of COL13 promoter containing
G-Box and TCT-motif were the binding requirement (Fig, 4g).
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, which 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 inArabidopsis (Datta et al., 2006). We also demonstrated 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
interacts 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 phenotypes of 35S:COL3-HA and 35S:COL13-GFP
transgenic plants were the same as 35S:COL3 and 35S:COL13 transgenic
plants, respectively, which produced shorter hypocotyl than WT grown in
the presence of red light (Fig. S2). The interaction between COL13 and
COL3 was also demonstrated in plant cells in a 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 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 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, and COL13
does not bind to COP1
(Fig.
6a). These results were also demonstrated by the FRET assay (Fig. S3).
To investigate whether COL13 influences the interaction between COP1 and
COL3, we performed a yeast three-hybrid assay. In this yeast system, the
COL3-COL13-pBridge construct allowed expression of COL3-BD /bait
and COL13 in yeast, and COL13 was expressed only 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 that showed that COP1 and COL3 had a
stronger binding activity with the expression of COL13. Based on a
previous report, COP1 interacted with COL3 and inhibited the production
of COL3 (Datta et al., 2006). By combining our results above, we propose
a possible COP1-dependent COL3-COL13 feedback pathway (Fig. 7b), which
is involved in the regulation of hypocotyl elongation.