Iron (Fe) homeostasis in plants is governed by a complex network of regulatory elements and transcription factors (TFs), as both Fe toxicity and deficiency negatively impact plant growth and physiology. The Fe homeostasis network is well characterized in Arabidopsis thaliana and remains poorly understood in monocotyledon species such as rice (Oryza sativa L.). Recent investigation of the rice Fe homeostasis network revealed OsIRO3, a basic Helix–Loop–Helix (bHLH) TF as a putative negative regulator of genes involved in Fe uptake, transport, and storage. We employed CRISPR-Cas9 gene editing to target the OsIRO3 coding sequence and generate two independent T-DNA-free, loss-of-function iro3 mutants in rice cv. Nipponbare. The iro3 mutant plants had similar phenotype under nutrient-sufficient conditions and had stunted growth under Fe-deficient conditions, relative to a T-DNA free, wild-type control (WT). Under Fe deficiency, iro3 mutant shoots had reduced expression of Fe chelator biosynthetic genes (OsNAS1, OsNAS2, and OsNAAT1) and upregulated expression of an Fe transporter gene (OsYSL15), relative to WT shoots. We place our results in the context of the existing literature and generate a model describing the role of OsIRO3 in rice Fe homeostasis and reinforce the essential function of OsIRO3 in the rice Fe deficiency response.

Effective maintenance of plant iron (Fe) homoeostasis relies on a network of transcription factors (TFs) that respond to environmental conditions and regulate Fe uptake, translocation, and storage. The iron-related transcription factor 3 (IRO3), as well as haemerythrin motif-containing really interesting new gene (RING) protein and zinc finger protein (HRZ), are major regulators of Fe homeostasis in diploid species like Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.), but remain uncharacterised in hexaploid bread wheat (Triticum aestivum L.). In this study, we have identified, annotated, and characterised three TaIRO3 homoeologs and six TaHRZ1 and TaHRZ2 homoeologs in the bread wheat genome. Protein analysis revealed that TaIRO3 and TaHRZ proteins contain functionally conserved domains for DNA-binding, dimerisation, Fe binding, or polyubiquitination, and phylogenetic analysis revealed clustering of TaIRO3 and TaHRZ proteins with other monocot IRO3 and HRZ proteins, respectively. Quantitative reverse-transcription PCR analysis revealed that all TaIRO3 and TaHRZ homoeologs have unique tissue expression profiles and are upregulated in shoot tissues in response to Fe deficiency. After 24 h of Fe deficiency, the expression of TaHRZ homoeologs was upregulated, while the expression of TaIRO3 homoeologs was unchanged, suggesting that TaHRZ functions upstream of TaIRO3 in the wheat Fe homeostasis TF network.