Preliminary Functional Study Of Rtce Transcription Factors OsbHLH57,OsbHLH58,OsbHLH59 In Response To Iron Deficiency

As one of the trace elements necessary for normal growth and development of plants,iron is rich in soil but is difficult for plants to use.In order to cope with the external iron-deficient environment,plants have evolved a set of sophisticated signal regulation networks that strictly regulate iron absorption,transport and storage to maintain the dynamic balance of iron.Rice is one of the most important crops in the world and an important source of human to iron intake.Therefore,studying the regulation mechanism of rice iron deficiency response can not only let people understand the theoretical knowledge of plants responding to changes in the external environment,but also facilitate the cultivation of iron-tolerant crop.In rice,the bHLH transcription factor OsbHLH60(named as OsPRI1)can be recognized and bound by OsHRZ1(considered to be one of the iron receptors,a homologous protein of Arabidopsis BTS),and then degraded by OsHRZ1 ubiquitination.OsbHLH60 positively regulated the expression of two downstream bHLH transcription factor genes,OsIRO2 and OsIRO3,and regulated the iron deficiency response process of rice through OsHRZl-OsbHLH60-OsIRO2/3 signal transduction cascade.Phylogenetic tree analysis found that rice OsbHLH57,OsbHLH58,OsbHLH59 and OsbHLH60 are homologous proteins.Therefore,we believed that OsbHLH57,OsbHLH58,and OsbHLH59 may be involved in the iron deficiency response process of rice.This article mainly included the expression pattern and subcellular localization of OsbHLH57,OsbHLH58,OsbHLH59;the interaction between OsbHLH57,OsbHLH58,OsbHLH59 and OsHRZ1/2,OsbHLH60;the function of OsbHLH058 in iron deficiency response.In order to study the tissue expression pattern of OsbHLH57,OsbHLH58 and OsbHLH59,we analyzed the expression of these genes in different tissues at different stages of wild-type rice growth by fluorescent quantitative PCR.The results showed that OsbHLH57 and OsbHLH59 were commonly expressed,during tillering stage the expression level in leaves is higher;the expression level of OsbHLH58 in roots is significantly higher than in other tissues.We further analyzed the transgenic material in which the promoters of OsbHLH57,OsbHLH58 and OsbHLH59 were fused with the reporter gene GUS,and found that both OsbHLH57 and OsbHLH59 were expressed in leaves,while OsbHLH58 was mainly expressed in roots.To investigate whether the expressions of OsbHLH57,OsbHLH58,and OsbHLH59 were induced by iron deficiency,we analyzed the expression of these genes in the shoots and roots of wild-type rice under normal and iron deficiency conditions by fluorescent quantitative PCR.The results showed that OsbHLH57 and OsbHLH59 were not induced by iron deficiency signal,the expression of OsbHLH58 is inhibited by iron deficiency signal.In order to study the subcellular localization of OsbHLH57,OsbHLH58 and OsbHLH59,we performed subcellular localization analysis in rice heterologous and homologous systems,as tobacco epidermal cells and rice protoplasts.The results showed that OsbHLH57,OsbHLH58,OsbHLH59 are all nuclear localization protein.OsbHLH60 can interact with OsHRZ1 protein,and they are two important components in the process of rice iron deficiency signal transduction.In order to study whether OsbHLH57,OsbHLH58,OsbHLH59 also participate in the related iron deficiency signal transduction process,it is necessary to clarify the interaction between them and OsHRZ protein,OsbHLH60.Yeast two-hybrid experiment showed that OsbHLH57,OsbHLH58,OsbHLH59 can interact with OsHRZ1,OsHRZ2,OsbHLH60.We also verified the results through the luciferase complementation experiment,the results showed that OsbHLH57,OsbHLH58,OsbHLH59 interacted with OsHRZ1,OsHRZ2;OsbHLH58 interacted with OsbHLH60,OsbHLH59 interacted with OsbHLH60 weakly,OsbHLH57 didn’t interact with OsbHLH60.From these we speculated that OsbHLH57,OsbHLH58 and OsbHLH59 may be located downstream of OsHRZ protein like OsbHLH60,and are modified by OsHRZ ubiquitination;in addition,OsbHLH57,OsbHLH58,OsbHLH59 may form a dimer with OsbHLH60 and participate as a dimer in rice iron deficiency response process.Finally,we took OsbHLH58 as an example to preliminary study its role in the iron deficiency response of rice,and provided a reference for further functional research of OsbHLH57 and OsbHLH59.Because OsbHLH58 interacted with OsHRZ2,in order to study the function of OsbHLH58 in response to iron deficiency and the regulatory relationship with OsHRZ2,we constructed single and double mutant rice transgenic lines of OsbHLH58 and OsHRZ2.The mutant and wild-type rice lines were cultured under+Fe/-Fe conditions respectively,and their growth status and metal element content were analyzed.The results showed that under iron deficiency conditions,compared to the wild type,the iron content of the mutant bhlh58 was significantly reduced,while the iron content of the single mutant hrz2 and double mutant bhlh58hrz2 strains was significantly increased;under normal conditions,compared to the wild type,the bhlh58 and bhlh58hrz2 mutants iron content of shoot decreased significantly,and the iron content of bhlh58hrz2 roots increased significantly.These results indicated that OsbHLH58 is located downstream of OsHRZ2 and is regulating the iron deficiency response of rice,affecting iron absorption and iron transport from roots to shoot.This research layed a solid foundation for further elucidating the mechanism of OsbHLH57,OsbHLH58 and OsbHLH59 in the response of rice to iron deficiency,and for further improving the regulatory network of rice iron deficiency signal.