Preliminary Study On The Function Of Fe-Related Transcription Factors OsIRO3 And OsbHLH062 In Rice

Iron is one of the essential micronutrients for the survival and proliferation of plants. It is an important component in chlorophyll biosynthesis and various enzymes. To maintain iron homeostasis under different environments, plants have evolved a complicated signaling network in regulation Fe uptake and mobilization. Analyzing the genes that are involved in Fe uptake, metabolism and homeostasis at molecular level is pivotal for breeding crops with increased iron content.In a previous research, we have characterized an iron deficiency-induced bHLH (basic Helix-loop-Helix) gene in rice, OsIRO3, which negatively regulates Fe deficiency signaling in rice (Zheng et al.,2010). OsbHLH062, a highly homologue of OsIRO3, has been identified by phylogenetic analysis here. This study have analyzed the expression patterns and constructed the overexpression and mutant genetic materials for these two genes.Microarray data for Genevestigator showed that OsIRO3 is widely expressed in all rice tissues and OsbHLHO62 is mainly expressed in seeds, roots and flowers, and relatively low in leaves, stems and internodes. Promoter::GUS lines of OsIRO3 and OsbHLH62 demonstrated that the two genes were selectively expressed in the vascular tissues. As expected, OsIRO3 and OsbHLH62 fused GFP proteins localized in nuclear in the onion epidermal cells and rice protoplast. However, only the OsIRO3 showed transcription activation activity in yeast cells but not OsbHLH62. A further Y2H assay indicated that OsbHLH62 could interact with itself and OsIRO3. The protein-protein interaction between OsIRO3 and OsbHLH62 was further proved by BiFC in rice protoplasts. In order to analyze the function of OsIRO3 and OsbHLH062 in iron signaling network, overexpression and mutant lines were generated by transformation and CRISPR/Cas9, respectively.In summary, this study confirmed the tissue expression patterns, subcellular localization and protein interactions of OsIRO3 and OsbHLHO62. In the future research, the detailed molecular and physiological function of OsIRO3 and OsbHLHO62 will be analyzed by using the generated overexpression and mutant lines.