The Study Of Arabidopsis Cryptochrome2Dimerization Mechanism And Function Of CRY2Inhibitors (BICs) Using Arabidopsis Mesophyll Protoplasts

Plants possess seven blue light receptors, including two cryptochromes (CRY1and CRY2), two phototropins (phot1and phot2), and three LOV/F-box/Kelchdomainproteins (ZTL, FKF, and LKP2). Cryptochromes are photolyase-like flavoproteinsand composed of two major domains, the N-terminal PHR (forPhotolyase-Homologous Region) domain and the C-terminal extension CCE (forCryptochrome C-terminal Extension) domain. The PHR domain with highlyconserved sequence, shares strong sequence similarity to DNA photolyases. The PHRdomain binds noncovalently to the chromophore FAD (flavin adenine dinucleotide) toperceive blue light. The CCE domain, a unique domain of cryptochromes, is theeffector domain of cryptochromes and it has no specific structure. By now, we havegot some understanding about Arabidopsis CRY1and CRY2mediated blue lightsignal transduction pathway, however, the molecular mechanism of photoreaction anddesensitization of CRYs is still unclear. Both of these two processes are important inblue light signal transduction pathway. Evidence has suggested that thephosphorylation and dimerization of Arabidopsis CRYs is essential for their initialblue light signal transduction, however, the relationship between various primaryreactions is still not clear. For example, the dimerization of CRYs is required by itsfunction, however, it has been reported that the dimerization of CRYs is not blue lightdependent. Someone speculated that it is due to desensitization mediated by relevantproteins. Therefore, it is important to discover cryptochrome dimerization andsensitization related proteins and identify their function for revealing initial blue lightsignal transduction mechanism in plants. Based on the possible CRY2interactingproteins BICs(blue-light-dependent inhibitor of cryptochromes) screened by our laboratory and collaborators via FOX hunting system, we proved that BICs caninteract with CRY2both in vitro and in vivo. And through optimized Arabidopsismesophyll proplasts isolation and transfection system, we found CRY2blue lightdependent dimerization and proved BICs can inhibit CRY2blue light dependentdimerization, CRY2photobody formation and CRY2interaction with its interactionprotein CIB1, and then affect CRY2mediated blue light signal transduction. Mainresults of this essay are as follows:(1) Through yeast two hybrid and co-immunoprecipitation experiments, wedemonstrated that BIC1, BIC2can interact with CRY2both in vitro and in vivo. BIC1interacts with CRY2in a constitutively way, however, BIC2interacts with CRY2in ablue light dependent way.(2) We optimized Arabidopsis mesophyll protoplasts isolation and transfectionsysterm. Using bimolecular fluorescence complementary (BiFC) in Arabidopsismesophyll protoplasts, we proved CRY2can form homodimers in plants and theformation of homodimers is blue light dependent. We also proved that both BIC1andBIC2proteins can inhibit CRY2homodimer formation.(3) We explored the CRY2photobodies using Arabidopsis mesophyll protoplastsand found that both BIC1and BIC2proteins can inhibit CRY2blue light dependentphotobody formation.(4) We proved that both BIC1and BIC2can inhibit the interaction betweenCRY2and its interacting protein CIB1, and then prevent the CRY2-mediated bluelight signal transduction using bimolecular fluorescence complementarytechnology(BiFC).