Molecular Mechanism Of Uv-b Signal Transduction Mediated By The Plant UV-B Photoreceptor UVR8

Sunlight,as the primary energy source of life activities on the earth,is used for photosynthesis of plants.At the same time,it is also an important environmental signal factor to regulate the growth and development of plants.Plants respond to UV-B radiation in the 280-315 nm band of sunlight through the unique UV-B receptor protein UVR8.UVR8 exists as a stable homodimer in the ground state.The three tryptophans W285,W233,and W337 as chromophores located at the dimer interface sense UV-B,resulting in the dissociation of UVR8 dimers into active monomers.The monomeric UVR8 interacts with the central repressor of photomorphogenesis COP1-SPA complex to form UVR8-COP1-SPA complex.Further inhibiting the activities of the E3 ubiquitin ligase COP1-SPA complex to degrade transcription factors such as HY5 in downstream photomorphogenesis,thereby stabilizing transcription factors such as HY5 to promote plant photomorphogenesis.On the other hand,continuous high-energy UV radiation is an environmental stress on plants.Therefore,plants have also evolved negative regulatory factors that inhibit UV-B signal transduction to tolerate this stress.In Arabidopsis thaliana,the homologous proteins RUP1 and RUP2 with WD40 domain negatively regulate UVR8-mediated UV-B signal transduction.Although the molecular mechanism by which UVR8 senses UV-B signal has been elucidated,the molecular mechanism of UV-B signaling mediated by UV-B-activated UVR8 interaction with COP1-SPA complex and RUP is not clear.In this thesis,biochemistry and structural biology were used to study these questions.The following progress has been made:1.In this study,the recombinant proteins HY5,RUP,and COP1-SPA4^1-464 were obtained in heterologous expression system.UVR8-mediated UV-B signal transduction pathway biochemical system was reconstructed in vitro.The results of gel filtration chromatography assay showed that UV-B radiation caused the dissociation of the dimeric UVR8 into a monomer,and UV-B-activated UVR8 competes to bind to COP1-SPA4^1-464 in the COP1-SPA4^1-464-HY5 complex to reform the UVR8-COP1-SPA4^1-464 complex.However,in the UV-B signaling pathway,the negative feedback regulator RUP2competitively binds to UVR8 in the UVR8-COP1-SPA4^1-464 complex,which leads to promoting the dimerization of UVR8 and inhibiting the UV-B signal transduction.In this study,the biochemical assays of RUP-mediated UVR8 redimerization were also reconstructed in vitro.The results exhibited that when RUP1 and RUP2 were added to the UV-B-activated UVR8 protein,respectively,most UVR8 monomers revert to ground state dimers at about 4 h.Therefore,RUP facilitates the regeneration of UVR8 dimers,which is consistent with the in vivo results reported.The pull-down assay analyzed that the interaction between RUP and wildtype UVR8 was UV-B-dependent,while the interaction with the constitutive mutant UVR8^W285A was UV-B-independent.Moreover,the co-transfected expression of UVR8^W285A and RUP2 is effective in mammalian cell expression system.2.In this study,the cryo-EM structure of UV-B-activated UVR8-COP1^WD40 complex with a resolution of 3.1（?）was resolved for the first time.The structure displays that COP1^WD40 interacts with the UV-B-activated UVR8 core domain and C27 subregion to form two different interfaces,and the R420 of COP1^WD40 plays an important role in the interaction.Combined with pull-down and competitive binding assays,it is found that these two different interfaces are essential for involving UVR8 interaction with COP1 and competing for the substrate HY5 of COP1.In the structure of UV-B-activated UVR8-COP1^WD40 complex,key residues in the UVR8 core domain exhibit UV-B-activated open conformation.Compared with the reported structure of wildtype or mutant UVR8 core domain,it was found that the conformation of key residues of combined mutants UVR8^{W285A,D96N,D107N},UVR8^W285A,G101S was similar to that of UV-B activated UVR8.3.In this study,the crystal structure of the RUP2-UVR8^W285A complex with a resolution of 2.0（?）displays was determined for the first time.Its structure shows that RUP2^WD40 forms two interfaces with the core domain and C27 subregion of UVR8^W285A,respectively.Structural alignment and recovery experiments showed that D129 of UVR8played a key role in redimerization.COP1^WD40-UVR8 and RUP2-UVR8^W285A all adopt similar interaction modes.The RUP2-UVR8^W285A complex has a larger buried surface areas and more hydrogen/salt bonds,which partly explains the reason why RUP2 can dissociate UVR8 from COP1-SPA4^1-464-UVR8 complex.Combined with the phenotypic investigation of mutants in vivo and the recovery and interaction biochemical assays in vitro,this study proves that the two interfaces of RUP2 and UVR8^W285A are essential for the formation of the RUP2-UVR8^W285A complex and RUP2 promoting the redimerization of UVR8.In summary,results of this study reveal the molecular mechanism of activation and negative feedback regulation in the UVR8-mediated UV-B signal transduction pathway,and provide a theoretical basis for the functional and applied research of UVR8.