Molecular Mechanism Of The Subcellular Localization And Activity Of Arabidopsis UV-B Photoreceptor

Light is the most important environmental cue for plant growth and development The ultraviolet-B(UV-B)wavelength(280-315 nm)is an indispensable component of the sunlight.Long-wavelength and low-fluence UV-B specifically induces plant photomorphogenesis and UV-B acclimation,and thus promotes plant growth and development.In Arabidopsis,UV-B light is perceived by UV RESISTANCE LOCUS 8(UVR8),a plant-specific chromoprotein.Upon UV-B irradiation,homodimeric UVR8 performs rapid monomerization to initiate UV-B light signaling.UVR8 is present in both the cytoplasm and the nucleus.However,it is not clear regarding the molecular mechanism by which UV-B regulates the subcellular localization and activity of UVR8.Therefore,we carried out the project in the following two aspects.(1)UV-B light regulates the subcellular localization of UVR8.Firstly,we used transgenic Arabidopsis plants expressing yellow fluorescence protein(YFP)-fused UVR8,and analyzed the subcellular localization of UVR8 by confocal microscopy.We have found that UV-B light promotes the nuclear accumulation of UVR8 in both cotyledons and hypocotyls.We next performed nuclear fractionation assays and have found that UV-B light rapidly induces the nuclear accumulation of UVR8 monomers instead of its dimers.Subsequently,to investigate the effect of UVR8 conformation on its subcellular localization,we examined engineered UVR8 proteins with constitutively dimeric or monomeric conformation.By confocal microscopy and nuclear fractionation assays,we have further proved that UV-B light promotes the accumulation of UVR8 monomers in the nucleus.(2)UV-B light regulates the activity of UVR8 by directing its subcellular localization.To study how UV-B light regulates UVR8 activity at the subcellular level,we conditionally localized UVR8 in distinct subcellular fractions by generating transgenic Arabidopsis plants expressing YFP-UVR8 fused with glucocorticoid receptor(GR).The application of dexamethasone(DEX)drives cytosolic UVR8 fusion proteins into the nucleus.We have found that the nuclear localization of UVR8 induces photomorphogenesis with UV-B treatment,with the seedlings showing the inhibition of hypocotyl elongation,the promotion of anthocyanin accumulation,and the expression of UV-B responsive genes.In addition,UVR8 conformation is determined by UV-B light rather than its subcellular localization.As the photoactivated UVR8-CONSTITUTIVELY PHOTOMORPHOGENIC 1(COP1)interaction is an initiative step in photomorphogenic UV-B signaling,we analyzed the endogenous association of YFP-GR-UVR8 and its variants with COP1 using in vivo co-immunoprecipitation(co-IP)assays.It is suggested that the formation of the UVR8-COP1 complex is dependent on UVR8 monomerization but is independent of UVR8 subcellular localization,and it is facilitated by nuclear UVR8 localization.These results illustrate that the molecular activity of UVR8 to mediate UV-B-induced photomorphogenesis relies on each of the three prerequisites,its monomeric states,nuclear localization,and activation by UV-B.Together,this study has revealed the molecular mechanism by which UV-B light regulates the subcellular localization and activity of UVR8.It is proposed that dual-source nuclear UVR8 monomers direct UV-B-induced photomorphogenesis in Arabidopsis.Upon UV-B illumination,nuclear UVR8 dimers rapidly switch to monomers to give rise to a basal stock of monomers so as to initiate UV-B signaling.As the exposure to UV-B prolongs,cytoplasmic UVR8 dimers are monomerized and then translocate into the nucleus to bulk up the nuclear pool of UVR8 monomers which mediate sustained UV-B signaling.Our findings have provided new insights into the subcellular regulation of photoreceptor activity in plants.