The Functional Analysis Of Arabidopsis UV-B Photoreceptor UVR8

In Arabidopsis, UV-B induced photomorphogenesis is initiated by a unique photoreceptor UVR8(UV RESISTANCE LOCUS 8). Different from any other know photoreceptors, UVR8 utilizes its tryptophan residues as internal chromophore to sense UV-B. After UV-B light perception, UVR8 undegoes conformational change from homodimer which is maintained by its arginine to monomer. Then UVR8 sequesters COP1-SPA(CONSTITUTIVELY PHOTOMORPHOGENIC 1-SUPPRESSOR OF PHYA) core complex from CUL4-DDB1(CULLIN 4-DAMAGED DNA BINDING PROTEIN 1) E3 apparatus. The newly formed UV-B dependent UVR8-COP1-SPA complex facilitates the role switch of COP1 from repressor in visible light to promoter in UV-B light. However, the biological significance of key residues,light-absorbing residus W233, W285 and dimer-stabilizing residues R286, R338,in UVR8 has not been fully understood. Previous studies about the function of UVR8 are either carried out in yeast or from structural analysis. Our study focus on the biological significance of key residues for UVR8 activity in UV-B induced photomorphogenesis. We have got several results:Firstly, we take advantage of site-directed mutagenesis to generate UVR8 variant proteins in Arabidopsis. These UVR8 variant proteins can be divided into two groups: light-absorbing residues mutants W233 A, W233 F, W285 A, W285 F and dimer-stabilizing mutants R286 A and R338 A. We generate the transgenic plants harboring different UVR8 variants through Agrobacterium infiltration method. We select the lines which are single insertion and show similar protein expression level for all the later experiments.Secondly, we detecte the conformational change and interaction with COP1 of different UVR variants in response to photomorphogenic UV-B in yeast. In consistent with previous reports, UVR8 WT show UV-B dependent monomerization and interaction with COP1. The constitutive dimer UVR8W285 F exhibits no interaction with COP1. And other UVR8 variants appear as monomers and associate with COP1 irrespective of UV-B treatment.Thirdly, UVR8 variants alter physiological responses to photomorphogenic UV-B in Arabidopsis. Through comparison of two physiological response,UV-B induced hypocotyl growth and anthocyanin accumulation, we have found that in response to UV-B, the hypocotyl shortening is most obvious in UVRWT, similar to wild type Col. And the UVR8R338 A also show a significant shortening than all the other UVR8 variants, though the level is less than UVR8 WT.Fourthly, UVR8 mutations affect UV-B-responsive gene expression. We examined the expression pattern of several UV-B-responsive marker genes, ELIP2(EARLY LIGHT-INDUCIBLE PROTEIN 2), UGT84A1 UGT84A1(UDP-GLYCOSYLTRANSFERASE 84A1) and CHS(CHALCONE SYNTHASE). UV-B is able to induce these genes expression in UVRWT, and also very obvious in UVR8R338 A but largely impaired in other UVR8 variants. We have found that similar UV-B induction of COP1 and HY5(LONG HYPOCOTYL 5) mRNA, except that HY5 mRNA also induced in UVR8W233 F. However, the accumulation of COP1 and HY5 protein mediated by photomorphogenic UV-B was scarcely detected in YFP-UVR8W285 F and YFP-UVR8R286 A. We have also found that the accumulation of REPPRESSOR OF RUP1(UV-BPHOTOMORPHOGENESIS 1) and RUP2 mRNA was apparently reduced in all UVR8 variants except UVR8R338 A.Fifthly, UVR8 variants impair UV-B light perception, UVR8 monomerization and UVR8-COP1 association. By measuring UV-B absorbance at 310 nm, we have found that mutations in either residue of the internal chromophore, W233 or W285, led to a complete loss of UV-B absorption and mutations in either residue of the dimer-stabilizing residues, R286 or R338, retained the ability to sense UV-B, but at significantly diminished levels. Through dimer/monomer assay, we have found that the conformational profiles of wild-type and variant UVR8 observed in Arabidopsis were consistent with those found in yeast. However, the wild-type and variant UVR8 interaction with COP1 have some discrepancy in plant and yeast. Independent of UV-B, UVR8W285 F and UVR8R286 A scarcely interact with COP1, while UVR8W233 A and UVR8W233 F co-immunoprecipitated very low levels of COP1. UVR8W285 A and UVR8R338 A co-immunoprecipitated medium levels of COP1 under-UV-B, while UVR8R338 A being able to co-immunoprecipitate more COP1 under +UV-B.Furthermore, constitutive activity of UVR8 is dependent on constant UVR8-COP1 interaction. Two UVR8 variants UVR8W285 A and UVR8R338 A displayed shorter hypocotyl, higher accumulation of anthocyanin and UV-B responsive genes than UVR8 WT under-UV-B. After UV-B treatment, UVR8W285 A shows no response to UV-B, while UVR8R338 A is still able to response to UV-B in these aspects, which have tightly connections to the UVR8W285A/UVR8R338 A and COP1 interaction. Morever, in dark condition, UVR8W285 A and UVR8R338 A displayed open cotyledons, suggesting that these two UVR8 variants were capable of inducing constitutive photomorphogenesis. Photobiological activities of UVR8W285 A and UVR8R338 A are enhanced by the repression of CUL4, a negative regulator in this pathway.Based on all the evidence mentioned above, we propose that key residues in UVR8 are coordinated to fulfill UVR8 function and the UVR8-COP1 affinity in plants critically determines the photomorphogenic UV-B signal transduction coupling with UVR8-mediated UV-B light perception.