The Hinge Region Of Arabidopsis PhyA Regulates PhyA Phosphorylation And Functions

Plants convert the energy from the sunlight into chemical energy and organics by photosynthesis.Light is one of the key environmental signals that influences plant growth and development.In addition to being the primary energy source for plants,light also controls multiple developmental processes in the plant life cycle,including seed germination,seedling de-etiolation,leaf expansion,stem elongation,stomata and chloroplast movement,phototropism,circadian rhythms and shade avoidance.Thus,the unravel of the light signaling mechanisms can not only contribute to a better understanding of how plants respond to their dynamic light environment,but also provide new insights into the improvements of crop yield and quality through better utilization of the sunlight.Phytochrome A(phyA)is the only plant photoreceptor that perceives far-red(FR)light and then mediates various responses to this signal.Upon light exposure,photo-activated phyA disrupts the COP1/SPA complex,thus leading to the accumulation of photomorphogenesis-promoting factors(such as the transcription factor HY5)and the initiation of photomorphogenesis.Phosphorylation and dephosphorylation of oat phyA have been extensively studied,and a phosphorylation site in the hinge region of oat phyA has been identified to be important for regulating phyA function.Here,we show that three sites in the hinge region of Arabidopsis phyA,i.e.S590,T593 and S602,play important roles in regulating phyA function.Mutating all three of these sites to alanines(designated as phyAAAA)or aspartic acids(phyADDD)disrupted phyA function,and phyAAAA displayed worst impairement of phyA function.Yeast two-hybrid assay data showed that phyAAAA and phyADDD both showed stronger interaction with FHY1 and FHL,two chaperon proteins essential for phyA nuclear accumulation.However,both phyAAAA-GFP and phyADDD-GFP entered the nucleus and formed the nuclear bodies as phyAWT-GFP upon light exposure.Yeast three-hybrid assay data indicated that the Pfr forms of both phyAAAA and phyADDD displayed normal inhibition of the COP1-SPA1 interaction as phyAWT in yeast cells,suggesting that both phyAAAA and phyADDD could form the biologically active Pfr form to disrupt the COP1-SPA complexes.In addition,our immunoblot data indicated that both phyAAAA and phyADDD mutants were degraded more slowly,and more FHY1 proteins accumulated in both phyAAAAA and phyADDD seedlings than in phyAWT,consistent with the conclusion that phyA function is impaired in both phyAAAA and phyADDD lines.It was recently reported that a phosphorylated form of Arabidopsis phyA accumulates in vivo upon FR light irradiation.The immunoblot data indicated that the abundance of the phosphorylated phyA form was decreased in the nucleus in phyADDD and phyAAAA lines,indicating that the formation of the phosphorylated phyA was influenced by phyAAAA and phyADDD mutations.It was previously shown that the phosphorylated phyA form may serve as a preferred substrate of the COP1/SPA complex-mediated degradation.Our co-immunoprecipitation(co-IP)data indicated that the pool of phyA interacting with COP1 contained less ratios of phosphorylated form in phyAAAA and phyADDD seedlings in FR light.Nuclear-cytoplasmic fractionation assays indicated that the phosphorylated phyA form was produced in the nucleus.In addition,the phosphorylated phyA form was not detectable in the fhyl fhl and fhy3 far1 mutants,in both of which phyA is localized exclusively in the cytosol,indicating that nuclear localization is required for the formation of this phosphorylated phyA form.Moreover,in the phyA-NLS-GFP seedlings in which phyA is constitutively localized in the nucleus,the phosphorylated phyA form was only detectable in seedlings grown in FR light;by contrast,in phyA(Y242H)-NLS-YFP seedlings in which phyA is constitutively locked in the Pfr form,the phosphorylated phyA form was observed not only in darkness,but also in FR light.These data demonstrate that the phosphorylated phyA form is produced by the nuclear-localized Pfr form,and the abundance of the phosphorylated phyA form displayed a tight correlation with the extent of phyA activity.Collectively,our data suggest that the phosphorylated phyA form may represent a more active form of phyA in vivo essential for inducing the FR light response.Thus,less ratios of phosphorylated phyA form produced in both phyAAAA and phyADDD lines may accout for the reduced function of these phyA mutants.Together,our data demonstrate that three sites in the hinge region of Arabidopsis phyA,i.e.S590,T593 and S602,play important roles in regulating phyA phosphorylation and function,thus linking specific residues in the hinge region to the regulatory mechanisms of phyA phosphorylation.In addition,our study also indicates that the phosphorylation and function of the hinge region may differ between oat and Arabidopsis phyA photoreceptors.