Biochemistry Analysis Of Blue Light Receptor Interaction With Spa1and The Mechanism In Arabidopsis

Blue light receptors cryptochromes are that mediate light regulation of geneexpression in all major evolution lineages, but the molecular mechanism underlyingcryptochrome signal transduction remains not fully understood. It has been reportedthat cryptochromes suppress activity of the multifunctional E3ubiquitin ligaseCONSTITUTIVE PHOTOMORPHOGENIC1(COP1) to regulate gene expressionin response to blue light. But how plant cryptochromes mediate light suppression ofCOP1activity remains unclear. We report here that Arabidopsis cryptochrome1(CRY1) or cryptochrome2(CRY2) undergo blue light-dependent interaction withthe COP1-interacting protein SUPPRESSOR OF PHYTOCHROME A1(SPA1).For CRY2-SPA1interaction, we analyzed the domain structures of CRY2andSPA1required for their interaction. CRY2has two domains, the N-terminalphotolyase homologous region (PHR) domain and the C-terminal cryptochromeC-terminal region (CCR) domain. PHR is the evolutionarily conservedchromophore-binding domain; CCR is an effector domain that interacts withCOP1.SPA1is composed of three domains: the N-terminal kinaselike domain, thecentral coiled-coil domain, and the C-terminal WD-repeat domain. It has beendemonstrated that the kinase-like domain is a regulatory domain for SPA1whereasthe coiled-coil domain and the WD-repeat domain that interact with COP1and theCOP1substrates, respectively. We analyzed the PHR domain of CRY2and kinasedomain of SPA1is required for the CRY2-SPA1interaction. Those domains alsoundergo blue light-dependent interaction with CRY2or SPA1. We next investigatedhow CRY2-SPA1interaction in response to blue light may affect the CRY2function.Given that CRY2undergoes blue light-dependent interaction with SPA1. SPA1mediates CRY2regulation of flowering. time, and SPA1and CRY2physicallyinteract with COP1to activate or suppress the COP1activity, respectively, wereasoned that blue light may affect interactions of these three mutually interactiveproteins(CRY2, SPA1, and COP1). We further demonstrate that SPA1actsgenetically downstream from CRY2to mediate blue light suppression of theCOP1-dependent proteolysis of the flowering-time regulator CONSTANS (CO). Weshow that blue light-dependent CRY2-SPA1interaction stimulates CRY2-COP1interaction. For CRY1-SPA1interaction, we also analyzed the domain structures of CRY1and SPA1required for their interaction. CRY1has two domains: the N-terminal PHR(photolyase homologous region) domain and the C-terminal CCR1(cryptochrome F3C-terminal extension) domain.The PHR is the evolutionarily conservedchromophore-binding domain; the CCR domain interacts with COP1and is criticalfor the function of CRY1in deetiolation responses. We analyzed the CCR1domain ofCRY1and WD-40domain of SPA1is required for the CRY1-SPA1interaction. Wethen asked the question of how blue-light-dependent CRY1–SPA1interactionconveys CRY1-mediated blue light response in plants. We reasoned that, in the dark,SPA1interacts with COP1to positively regulate COP1activity demonstrating that inthe blue light, CRY1mediates blue-light suppression of the SPA1–COP1interaction.Because blue light stimulates the CRY1–SPA1interaction, it is conceivable thatphotoexcited CRY1acts as a competitive inhibitor of the SPA1–COP1interaction tosuppress COP1activity in response to blue light. Our study shows that theCRY1–SPA1interaction suppresses the SPA1–COP1interaction andCOP1-dependent degradation of the transcription factor HY5.Our results reveal for the first time a wavelength-specific mechanism by whichblue light photoreceptor cryptochrome1or cryptochrome2mediates light regulation ofHY5or CO protein degradation to modulate developmental timing in Arabidopsis.These results demonstrated that a cryptochrome could undergo blue light-dependentinteraction with COP1or inhibit SPA1-COP1interaction. The fact that the blue lightdependence of cryptochrome-COP1interaction was not observed in previous reportsfor CRYs but was observed in our study for CRY1or CRY2suggests a differentmode of action of the two cryptochromes.