The Molecular Mechanism Of TOPP4 Regulating Photomorphogenesis In Arabidopsis

Light is a key environmental cue that controls plant growth and development.Photomorphogenesis is started by photoreceptors receiving different light singaling.Activated phytochrome(phy)interacts with the downstream of phytochrome interacting factors(PIF),and induces rapid phosphorylation and degradation of PIF by the ubiquitin-proteasome system.These responses are the early molecular events in phy signaling pathway.However,the phosphatase which could dephosphorylate PIF has not yet been found,and how the dephosphorylated PIF affecting phytochrome signaling pathway is unclear.In this paper,we studied a topp4-1 mutant screened by our lab which displayed growth and developmental defects and verified that topp4-1 mutant is sensitive to red light.The mutant showed short hypocotyl and larger cotyledon hook under red light,while seedlings overexpression TOPP4-OX exhibited significantly longer hypocotyl elongation under low-intensity of red light.Overexpressing TOPP4 gene in topp4-1 mutants could recover the cotyledon angle and hypocotyl length of topp4-1 mutant,and overexpressing the mutant gene topp4-1 into wild-type plants could recapitulate the phenotype of topp4-1 mutant which is sensitive to red light,suggesting that the mutant protein topp4-1 reveals a dominant negative effect in the regulating of photomorgenesis in Arabidopsis.pTOPP4-GUS expression indicates that light induces TOPP4 expression in hypocotyl.And red light-induced expression of TOPP4 is largely mediated by phyB in the hypocotyls.Subsequently,we detected the phenotype of double mutant phyB-9 topp4-1 and found that the phenotype of topp4-1 mutant needs the normal function of phyB,while TOPP4 mutation weakens the closed cotyledon of phyB-9 in a certain extent.Further studies showed that overexpression of TOPP4 significantly repressed the shortened hypocotyls of phyB-GFP.These evidences indicated that TOPP4 interact with phyB genetically,and TOPP4 regulates photomorphogenesis.By yeast two-hybrid,BiFC,and pull-down analysis,we proved that TOPP4 directly interacts with phyB,which is essential for locating TOPP4 within photobodies.Genetic experiments verified that TOPP4 locates upstream of PIF.By yeast two-hybrid,pull-down,and Co-IP analysis,we confirmed that TOPP4 interacts with PIF5 abviously.In vivo and in vitro phosphorylation experiments showed that PIF5 is a substrate of TOPP4.Further more,we examined the protein levels of PIF5 in topp4-land TOPP4-OX seedlings,and reveal that TOPP4 inhibits the degradation of PIF5.This action ultimately affects photomorphogenesis in Arabidopsis.Taken together,this study demonstrated that PIF5 dephosphorylation is a key regulatory mechanism for PIF5 degradation during photomorphogenesis.This is a new signal mechanism in phytochrome signaling pathway,which provides valuable insights for further research in this field.