The Functional Study Of OsIIP3 And OsIIP4 In Response To Iron Deficiency In Rice

Iron（Fe）is one of the essential micronutrients for plant growth and development.As a cofactor for many functional plant proteins,it participates in chlorophyll synthesis,mitochondrial respiration,and many other biochemical processes.Iron deficiency causes chlorosis,slow growth,and decreased yield among new leaves of plants.Although Fe is the second largest metal element in the earth’s crust,it usually exists in the soil in the form of insoluble trivalent iron,which is not easily transported by plants.Besides,iron deficiency anemia seriously threatens the health of nearly one-third of the global population（especially women and children）.Plant foods are one of the main sources of human iron take.Therefore,studying the regulation of the iron deficiency response of plants can provide a theoretical basis for the cultivation of iron-deficiency-tolerant and iron-fortified crops.OsIRO3 is a key gene that regulates the response to iron deficiency in rice（Oryza sativa,L.）,and its mutants and over-expression transgenic plants are all hypersensitive to iron deficiency,indicating that OsIRO3 is strictly regulated in the response to iron deficiency in rice.In our previous studies,we had found multiple OsIRO3 interaction proteins through yeast two-hybrid assay.In this research work,we studied the functions of OsIIP3（OsIRO3 Interaction Protein 3）and OsIIP4 in response to iron deficiency in rice.OsIIP3 is an injury stress protein,and OsIIP4 is calcium-dependent protein kinase CPK1 adapter protein 2,However their function in response to iron deficiency is unknown.In this study,the functions of OsIIP3 and OsIIP4 in response to iron deficiency in rice were studied through biochemical reaction,cytology and genetics.Using luciferase complementation imaging assay（LCI）,biomolecular fluorescence complementation（Bi FC）and in vitro Pull-down experiments proved that both OsIIP3 and OsIIP4 can interact with OsIRO3.The tissue expression patterns of OsIIP3 and OsIIP4 and the gene expression patterns in response to iron deficiency were analyzed by qRT-PCR（Quantitative real-time PCR）and GUS staining.It was found that OsIIP3 and OsIIP4 were expressed in various tissues of rice at different growth stages,and OsIIP3 was the highest and expressed in leaf sheaths,while the expression level of OsIIP4 was high in leaves,leaf sheaths,and seeds.The expression of OsIIP3 in roots and shoots was induced by iron deficiency,but the expression of OsIIP4 was induced in shoots only by iron deficiency.Through the tobacco epidermal cellsrice、rice protoplasts transient expression analysis and complement transgene fluorescence,it was found that OsIIP3 was localized in the nucleus;OsIIP4 was localized in the prevacuolar compartment.Furthermore,We used CRISPR-Cas9 technology to obtain OsIIP3 and OsIIP4homozygous mutants（osiip3 and osiip4）.Under the condition of iron supply（2μM Fe SO₄）,the shoot length,root length and dry weight of osiip3 and osiip4 were significantly lower than those of wild type（WT）;after iron deficiency treatment,the shoot length,leaf SPAD value and root morphology indicators（total root length,root surface area,root volume,total root tip number,forks）the phenotypic differences of osiip3 and osiip4 as compared with wild type were further increased,indicating that osiip3 and osiip4 are more sensitive to the iron deficiency than the WT.The Fe content of WT,osiip3 and osiip4 were analyzed by ICP-MS,and it was found that the Fe content of roots and shoots of osiip3 and osiip4 were significantly lower than that of the wild type regardless of iron sufficient or deficiency condition.qRT-PCR results showed that under iron deficiency conditions,the expression of iron deficiency-response genes OsNAS1,OsNAS2,OsNAS3,OsIRO2,OsIRO3 and OsNNAT1 in osiip3 were significantly higher than that of WT,while under iron sufficient conditions,only OsNAS3 had a significantly higher expression as compared with WT,while osiip4 was only found to express higher levels of OsNAS3 relatively to WT under iron-deficiency conditions.Through the dual-luciferase reporter assay,it was found that OsIIP4 can inhibit OsNAS3 promoter activity through OsIRO3.Secondly,by constructing OsIIP3 and OsIIP4 over-expression lines（OsIIP3-OE and OsIIP4-OE）and observing under iron deficiency treatment,it was found that OsIIP3-OE and OsIIP4-OE were also sensitive to iron deficiency.In Comparison to the wild type,the shoot length,The root length,and leaf SPAD value were significantly reduced.The results of ICP-MS showed that compared with WT,the Fe content in OsIIP3-OE roots increased significantly under iron-deficient conditions,the iron content in OsIIP4-OE did not change significantly,while the Zn content in roots increased,and the Zn content in the shoots decreased,and the Fe and Zn content of OsIIP3-OE and OsIIP4-OE brown rice were significantly increased.This study clarified that OsIIP3 and OsIIP4 are the interaction proteins of OsIRO3,a negative regulator of rice iron deficiency response.A detailed analysis of their mutants and over-expression lines under iron deficiency-induced physiological changes and iron deficiency response-related gene expression revealed that both OsIIP3 and OsIIP4 are involved in the regulation of rice iron deficiency responses,dual-luciferase report experiment showed that OsIIP4 can inhibit the expression of OsNAS3 through OsIRO3.In summary,this study preliminarily proves that the OsIRO3 interaction proteins OsIIP3 and OsIIP4 play an important role in the growth and development of rice and the response to iron deficiency.