OsIRO3 negatively regulates Fe homeostasis by repressing the expression of OsIRO2

Iron (Fe) is crucial for crop productivity and quality. However, Fe deficiency is prevalent worldwide, particularly in alkaline soil. Plants have evolved sophisticated mechanisms to withstand Fe-deficient conditions. Oryza sativa IRON-RELATED BHLH TRANSCRIPTION FACTOR 3 (OsIRO3/OsbHLH63) has been identified as a negative regulator of Fe deficiency response signaling; however, the underlying mechanism remains unclear. In the present study, we constructed two iro3 mutants, which developed leaves with necrotic lesions under Fe-deficient conditions. Loss-of-function of OsIRO3 caused upregulation of Fe deficiency-associated genes in the root. Fe concentration measurements showed that the iro3 mutants had increased shoot Fe concentration only under Fe-deficient conditions. Further analysis revealed that OsIRO3 directly regulated the expression of IRON-RELATED BHLH TRANSCRIPTION FACTOR 2 (OsIRO2), which encodes a positive regulator of the Fe uptake system. Further investigation demonstrated that OsIRO3 interacted with POSITIVE REGULATOR OF IRON HOMEOSTASIS 1(OsPRI1) and OsPRI2, and. OsIRO3 repressed their transcription activation towards OsIRO2. OsIRO3 contains an EAR motif, which recruits the TOPLESS/TOPLESS-RELATED (OsTPL/OsTPRs) corepressors. Mutation of the EAR motif attenuated the repression ability of OsIRO3. This work sheds light on the molecular mechanism by which OsIRO3 modulates Fe homeostasis in rice.