The Study Of Function And Mechanism Of Transcription Factors OsIRO3 And OsIRO4 In Regulation Of Iron Homeostasis In Rice

Iron(Fe)is one of essential micronutrient for plant growth and development.However,Fe overaccumlation is toxic for plants and inhibits plant growth.Consequently,plants must maintain Fe homeostasis.Fe nutritional deficiencies are widespread in the humans,especially in preganent women and children.The Fe nutrition absorbed by people are directly or indirectly derived from plants.Rice(Oryza sativa,L.)is a staple food source for nearly half of the world’s population and an important model plant.Therefore,elucidating the regulation mechanism of Fe homeostasis in rice will be beneficial for the cultivation of tolerance to low/high Fe crops and have a great significance to human nutrition and health.In recent years,great progress has been made in regulation of rice Fe homeostasis,and it has been found that OsPRIl(Positive regulator iron homeostasis 1)can be ubiquitinated and degraded by the Fe potential receptor OsHRZ1(Oryza sativa Haemerythrin motifcontaining Really Interesting New Gene(RING)-and Zinc-finger proteinl);OsPRI1 can directly regulate the expression of key downstream genes OsIRO2(Iron-related transcription factor 2)and OsIRO3(Iron-related transcription factor 3),initially forming the OsHRZ1OsPRI1-OsIRO2/3 Fe signaling pathway.However,the molecular mechanism of how rice responds to fluctuation of external Fe levels and regulates the expression of Fe homeostasisrelated genes is still unclear.Here,the study of the functions and molercular mechanisms of transcription factors OsIRO3 and OsIRO4 in regulation of rice Fe homeostasis was performed.This research will further improve understanding the regulatory network of Fe homeostasis in rice.The main findings are as follows:OsIRO3 is ubiquitously expressed,and its expression level in leaves is higher than that in other tissues.Through CRISPR/Cas9 technology,loss-of-function mutants of OsIRO3 were obtained.Knockout of OsIRO3 resulted in severe necrosis of the new leaves,abnormal root development,and increase of hydrogen peroxide and superoxide anion concentration in the new leaves under Fe-deficient conditions.Under Fe-sufficient conditions,there was no significant difference in the shoot or root Fe concentration between the iro3 mutants and the WT(wild type);Under Fe-deficient conditions,while there was no difference in the root Fe concentration,the shoot Fe concentration of the iro3 mutant was significantly higher than that of the WT.Gene expression analysis showed that knockout of OsIRO3 led to increase of some Fe deficiency-induced genes expression under Fe-sufficient conditions.The iro3 mutants had higher expression levels of OsNAS3 than the WT,while there was no significant difference in other Fe deficiency-induced genes expression between the WT and the iro3 mutants after Fe deficiency treatment for 4 d.Furthermore,yeast one-hybrid,ChIP-qPCR,and tobacco transient expression experiments confirmed that OsIRO3 can bind to the promoter of OsNAS3 directly and negatively regulate its expression.Analysis of NA(Nicotianamine)and DMA(2’-Deoxymugeneic acid)concentration in the WT and the iro3 mutants showed that loss-of-function mutation to OsIRO3 significantly increased the NA concentration in roots under Fe-sufficient and-deficient conditions and significantly increased the DMA concentration in rice under Fe-deficient conditions.Thus,we conclude that OsIRO3 plays a key role in responding to Fe deficiency and regulates NA and DMA levels by directly,negatively regulating the OsNAS3 expression.OsIRO4,a homologous gene of OsIRO3,expression is suppressed by Fe deficiency and induced by high Fe.OsIRO4is mainly expressed in the root cap and leaves under Fe-deficient conditions,however,it is ubiquitously expressed in various tissue structures under Fesufficient conditions.Furthermore,staining transgenic plants expressing the genome fusion with GUS gene in solution supplied with different Fe concentration showed that the protein level of OsIRO4 was also repressed by Fe deficiency and induced by high Fe.The subcellular localization results showed that OsIRO4 was localized in the nucleus and cytoplasm.Then,through analyzing the phenotype and Fe concentration of OsIRO4 knockout mutants and OsIRO4 overexpression lines under both Fe-sufficient and-deficient conditions,it was found that the iro4 mutants were more sensitive to high Fe,however,the overexpression of OsIRO4 lines were hypersensitive to Fe deficiency,compared with the WT.Loss-of-function mutation to OsIRO4 resulted in increase of the shoot Fe concentration under Fe-replete conditions.Contradictory,overexpression of OsIRO4 significantly decreased the shoot Fe concentration,but increased the root Fe concentration under both Fe-replete and Fe-deplete conditions.Meanwhile,the effects of OsIRO4 mutation or overexpression on the Fe deficiency-induced genes expression were analyzed.The RT-qPCR(Reverse transcription quantitative PCR)results showed that the expression of Fe deficiency-induced genes was induced in the iro4 mutants but repressed in the OsIRO4 overexpression lines,compared with the WT.Through analyzing the Fe concentration and gene expression in the priliro4 double mutants,it was found that OsIRO4 negatively regulated the expression of OsIRO2 and OsIROsIRO3 and Fe accumulation in the shoot mainly by OsPRI1.OsIRO4 inhibited the promoter activity of OsIRO2 and OsIRO3 through OsPRIl was further confirmed by tobacco transient expression experiments.Using the yeast two-hybrid,LUC fluorescence recovery and bimolecular fluorescence complementation experiments,then,the result of OsIRO4 can interact with OsPRIl in the nucleus was confirmed.Moreover,OsIRO4 can interact with transcriptional co-repressors OsTPL/TPRs(TOPLESS/TOPLESS-RELATED)through the EAR(Ethylene response factor associated amphiphilic repression)motif.These findlings indicate that the transcript and protein abundances of OsIRO4 are inhibited by Fe deficiency and induced by high Fe.OsIRO4 can interact with OsPRI1,and it also can recruit transcriptional co-repressor OsTPL/TPRs through the EAR motif to negatively regulate the expression of OsIRO2 and OsIRO3 and Fe homeostasis through OsPRI1.In short,OsIRO3 is a key regulator of Fe deficiency responses and regulates NA and DMA levels by directly,negatively regulating the expression of OsNAS3 in rice.OsIRO4,a high Fe-induced protein,interacts with OsPRI1 and recruits transcriptional co-repressors OsTPL/TPRs by the EAR motif to negatively regulate the expression of key downstream genes OsIRO2 and OsIRO3 of OsPRI1 and rice shoot Fe accumlution through OsPRI1,which is critical for preventing Fe excess accumulation in rice shoot.