<scp>OsbHLH062</scp> negatively regulates Fe homeostasis by enhancing <scp>OsHRZ1</scp> targeting <scp>OsPRIs</scp> for degradation

Iron (Fe) is crucial for plant growth and development, and many transcription factors are involved in the maintenance of Fe homeostasis. In rice Fe signaling, the basic helix-loop-helix (bHLH) IVc proteins (OsPRI1/OsbHLH060, OsPRI2/OsbHLH058, OsPRI3/OsbHLH059, and OsPRI4/OsbHLH057) act as positive regulators, while Oryza sativa IRON-RELATED BHLH TRANSCRIPTION FACTOR 3 (OsIRO3/OsbHLH063) functions as a negative regulator. Additionally, HEMERYTHRIN MOTIF-CONTAINING REALLY INTERESTING NEW GENE AND ZINC-FINGER PROTEIN1 (OsHRZ1) plays a role in targeting OsPRIs for degradation. Here, we investigated the role of OsbHLH062 in response to Fe deficiency. We revealed that OsbHLH062 directly binds to the promoter region of Oryza sativa IRON-RELATED bHLH TRANSCRIPTION FACTOR 2 (OsIRO2), thereby repressing its activity through the recruitment of corepressors TOPLESS/TOPLESSRELATED (OsTPL/OsTPRs). When both OsbHLH062 and OsIRO3 are mutated simultaneously, it results in stunted root growth, reduced plant height, elevated Fe concentration, and the upregulation of Fe deficiency inducible genes. OsbHLH062 is found to be localized in both the cytoplasm and the nucleus. The interaction of OsbHLH062 with OsPRIs promotes its accumulation in the nucleus. Our findings also reveal that both OsbHLH062 and OsIRO3 physically interact with OsHRZ1, which in turn enhances the interaction between OsHRZ1 and OsPRI3, leading to the degradation of OsPRI3 by OsHRZ1. These findings shed light on a new function of OsbHLH062 and OsIRO3 as potential cofactors of OsHRZ1, which contribute to the reduction in OsPRI proteins and, consequently, the repression of Fe signaling pathways.