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.