Positive Regulator Of Iron Homeostasis1,OsPRI1 Facilitates Iron Homeostasis

Iron(Fe)is one of the essential mineral elements for plant growth and development.As a cofactor for a wide variety of functional proteins,it affects many biochemical processes,including chlorophyll biosynthesis,photosynthesis,and respiration.In humans,Fe deficiency is one of the most prevalent nutritional disorders worldwide.Grains,fruits and vegetables are one of the major Fe sources for humans.Thus,characterizing the mechanism underlying Fe homeostasis in plants may have important consequences for human health.Higher plants have evolved two sophisticated strategies to take up Fe from soils,namely the reduction strategy(Strategy I)and chelation strategy(Strategy II).The reduction strategy is employed by nongraminaceous plants and the chelation strategy is utilized by graminaceous plants.As a specific graminaceous species,rice(Oryza sativa)employs Strategy ?,but it also uses a partial Strategy I.Rice plants not only secrete mugineic acid(MA)family phytosiderophores to chelate ferric Fe,but also directly absorb ferrous Fe with OsIRT1(IRON TRANSPORTERIS and OsIRT2.However,it is unclear how rice senses Fe status and regulates the expression of Fe homeostasis-associated genes.Recently,OsHRZ1 and OsHRZ2 were characterized as putative iron-binding sensors that negatively regulate Fe acquisition under Fe-sufficient conditions.However,how OsHRZ1 and OsHRZ2 affect the expression of Fe-deficiency-responsive genes has not been determined.Both OsHRZ1 and OsHRZ2 exhibit E3 ligase activity,implying that they function by ubiquitinating and degrading substrates.Therefore,identifying and characterizing the substrates of OsHRZ1 and OsHRZ2 may provide novel insights into rice responses to Fe deficiency.In this present study,a bHLH transcription factor,which was identified as an interacting partner of OsHRZ1 by yeast-two-hybrid assays,was named POSITIVE REGULATOR OF IRON HOMEOSTASIS1(OsPRI1).Transient expression experiments suggest that both OsHRZ1 and OsPRI1 are localized to the nucleus of plant cells.Pull-down assay in vitro also confirms the physical interaction between OsHRZ11 and OsPRI1.To investigate the functions of OsPRI1,we used CRISPR/Cas9 technology to edit the OsPRI1 gene,resulting in three different loss-of-function mutants,all of which displayed similar Fe-deficiency phenotypes.Measurement of Fe concentration found that the roots of pril mutants contain higher Fe concentration and the shoots contain lower Fe concentration,compared with the wild type.Further expression analysis suggests that some Fe-deficiency-responsive genes,OsIRT1,OsIRT2,OsNAS1,OsNAS2,OsYSL2,OsYSL15,OsIR02 and OsIR03,are strongly downregulated in the pril mutants.Yeast-one-hybrid and electrophoretic mobility shift assays suggest that OsPRI1 directly binds the promoters of OsIRO2 and OsIRO3.Considering OsHRZ1 exhibits E3 ligase activity and interacts with OsPRI1,we speculated that OsPRI1 is ubiquitinated by OsHRZ1.We induced the expression of GST-OsHRZ1 and His-OsPRI1 in E.coli cells and purified the fusion proteins.A subsequent in vitro ubiquitination assay indicated the ubiquitination of OsPRI1 by OsHRZ1.These results suggest that OsHRZ1 can regulate the ubiquitination of OsPRI1.Cell-free degradation assays revealed that the stability of OsPRI1 decreased in wild-type roots extracts but slightly decreased in the hrz1-2 mutant roots extracts.Additionaly,the degradation of OsPRI1 can be completely suppressed by MG 132.Collectively,these data suggest that OsHRZ1 mediates the ubiquitination and degradation of OsPRI1 in the 26S proteasome pathway.The hrz1-2 seedlings were insensitive to Fe-deficient conditions.When the pril-1 mutation was introduced into hrzl-2 mutants,the prilhrzl double mutant was more sensitive to Fe deficiency than the hrzl-2 mutant.Collectively,these data indicate that OsPRI1 functions downstream of OsHRZ1 to maintain Fe homeostasis in rice plants.In summary,these results indicate that OSPRI1,whose stability is negatively regulated by OsHRZ1,mediates rice responses to Fe deficiency by positively regulating OsIRO2 and sIR03 expression as part of the OsHRZ1-OsPRI1-OsIRO2/3 signal transduction cascade.