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.