Molecular Mechanism Of Antagonistic Regulation Of UV-B Response By Two E3 Ligases In Arabidopsis

During emergence from the soil,plants develop a strategy termed photomorphogenesis to organize the development of tissues and organs in response to environmental light stimuli.Plants have evolved sophisticated light signaling networks to adapt to various light signals and control photomorphogenesis.Nondamaging UV-B light signal facilitates photomorphogenesis of Arabidopsis thaliana seedlings,and prevents them from stress caused by strong UV-B irradiation.However,how the regulatory factors deliver UV-B signaling cascades in seedling photomorphogenesis remains unclear.To investigate the regulation of protein stability of crucial players involved in UV-B-induced photomorphogenesis,we performed biochemical experiments to show two sets of E3 ligases co-regulate the stability of HY5,the hub transcription factor in the light signaling pathway.We also uncovered their molecular mechanisms which positively or negatively regulate the prolonged signaling circuits of UV-B-induced photomorphogenesis.Firstly,we analyzed the interactions between RUP1/RUP2,the negative regulators of UV-B signaling,and transcription factor HY5 or scaffold protein of E3 ligase DDB1 using yeast-two hybrid,co-immunoprecipitation assays and so on.Together with the phenotypic analyses of cul4cs rup1-1 rup2-1 mutant and FLAGmRUP2/rup2-1 transgenic seedlings under UV-B light,we demonstrate that RUP1/RUP2 acts as the substrate receptor of CUL4-DDB1 E3 ligase to repress photomorphogenesis by mediating the degradation of HY5.Secondly,we reveal that COP1,the positive regulator of UV-B signaling,directly interactes with RUP1/RUP2 using in vitro pull-down,co-immunoprecipitation assays and so on.We also examined RUP1/RUP2 protein stability to confirm that COP1 targets RUP1/RUP2 for ubiquitination and degradation.Therefore,COP1 promotes UV-B-induced photomorphogenesis viadestabilizing RUP1/RUP2 and in turn stabilizing HY5.Thirdly,the UV-B inducible accumulation of RUP1/RUP2 interrupts the interaction between COP1 and HY5,preventing HY5 from COP1 mediated degradation.Furthermore,we detect that COP1 interferes the DNA binding efficiency of HY5 by EMSA assays,and changes the transcription of some UV-B responsive genes.In summary,this study reveals that two E3-substrate modules,CUL4-DDB1RUP1/RUP2-HY5 and COP1-RUP1/RUP2,function as antagonistic regulators in UV-B signaling,by balancing the protein levels of RUP1,RUP2 and HY5.COP1 is also involved in the transcriptional regulation by modulating the transcription factor activity of HY5,so as to precisely control plant UV-B responses at multiple layers.