Identification And Function Preliminary Analysis Of BIC2, A Blue Light Photoreceptor CRY2’s Interacting Protein

From the moment of germination, the Arabidopsis start to receive the signal of light via photoreceptors and regulate its own growth and development according to the cue of light signal. Thus, it could better take advantage of light energy to synthesis the organic matter that meet the requirement of its regular metabolism. Such biological process described in the former passage, we defined it as photo morphogenesis. In the process of photo morphogenesis, the Arabidopsis mainly sense two ranges of light wavelength, UV to visible blue light and red to far red. The protein that could achieve these two wavelength of light, we called them photoreceptor. Photoreceptors are mainly divided into four classes. Among them, the cryptochrome is main kind of blue light photoreceptor. Cryptochrome, this small gene family has three candidates, CRY1, CRY2 and CRY3. CRY1 and CRY2 are mainly responsible for sensing blue light and regulating the photo morphogenesis under blue light, as well as long day dependent flowering. CRY1 takes most part of function in regulating inhibition of hypocotyl length and expansion of cotyledon. While CRY2 shares redundant function with CRY1 under some specific condition, CRY2 mainly involves in the regulation of long day dependent flowering. Some researches indicate that when CRY2 activated by blue light, they could form dimers or oligomers and further become photobodies. In the meantime, CRY2 undergo phosphorylation and ubiquitination, two kinds of post-translational modification and then are degraded through 26 S proteasome pathway. In these processes, dimerization has great important relation with CRY2’s function.Yoshito and his colleagues screened the full-length c DNA overexpression Arabidopsis library and discovered BIC1, which has the phenotype of CRY1-loss of-function. In our research, we apply improved GFP binding protein to achieve immune-precipitate in GFP-CRY2 and BIC2-GFP overexpression transgenic plants and then tandem with LC-MS to verify the protein interaction between these two proteins, and perform parallel reaction monitor, one kind of MS method, to acquire protein abundance information by targeted acquisition strategy. We discovered that if we use GFP-CRY2 as bait protein to perform immune-precipitation assay, the BIC2 abundance would be in a blue light dependent manner. However, if we use BIC2-GFP as bait protein, the abundance of CRY2 would not have any blue light dependent pattern. Then we investigate the phenotype of BIC2-GFP overexpression lines and bic1bic2 double mutant. We observed that BIC2 overexpression lines performed obvious late flowering in long day and long hypocotyl length under blue light,and bic1bic2 double mutant appeared reversed phenotype with BIC2-GFP lines. This phenomenon suggests that BIC2 may inhibit the physiological function of CRY2. Then we expressed CRY2 and BIC2 in HEK293 T cell line, and through western verification we discovered that BIC2 could inhibit CRY2’s dimerization under denatured and reducing reagent containing condition. Therefore, we deduced that BIC2 might influence CRY2’s physiological function via inhibition of CRY’2 dimerization. Finally, we used size exclusion chromatography to identify CRY2’s complex under nature condition. We found that CRY2 stays at dimer formation under both blue and dark condition, and BIC2 couldn’t diminish CRY2 dimer in this system. Our discovery suggested that there might be other mechanisms to regulate CRY2’s dimerization and BIC2’s function. Our experiment provided clues for further research on CRY2’s photochemistry characteristics under blue light.