CRISPR/Cas9 editing of the wheat iron sensor TaHRZ1 confirms its conserved role in iron homeostasis and allocation in grains

Abstract

Plants rely on specialized sensing systems, including transcriptional regulators, to maintain iron (Fe) homeostasis. Among these, Hemerythrin RING Zinc finger (HRZ) proteins have emerged as key regulators of Fe homeostasis. In this study, six Triticum aestivum (wheat) HRZ homoeologs belonging to TaHRZ1 and TaHRZ2, were identified by BLAST search using rice (Oryza sativa) HRZ sequences and mapped to chromosomes 1 and 3. These encode proteins with conserved N-terminal Hemerythrin (HHE) domains and C-terminal CHY-RING and Zn-ribbon motifs. Phylogenetic analysis grouped these genes into distinct clades, while expression profiling revealed strong root-specific and Fe-responsive expression patterns, indicating roles in nutrient sensing. Functional conservation was demonstrated by complementation of the Arabidopsis thaliana bts-1 mutant, where both wheat genes restored normal Fe regulation. Full-length TaHRZ1 and TaHRZ2 interacted with members of wheat bHLH IVc transcription factors, while truncated versions lacking the RING domain did not, emphasizing their conserved role in protein interactions. CRISPR-Cas9 editing of the conserved HHE3 domain in all the TaHRZ1 homoeologs, coupled with GRF4-GIF1 chimeric protein, achieved 6.4-8.8% regeneration efficiency in wheat. Elemental analysis indicated enhanced Fe loading in the grains of the edited lines, particularly in the scutellum, suggesting improved iron partitioning compared to the wild type. Additionally, qRT-PCR revealed upregulation of TaFIT and TaIRO3, and downregulation of IDEF1 in edited lines, supporting an important regulatory role for TaHRZ1 in Fe homeostasis signalling. These findings position TaHRZ1 as a valuable target for biofortification strategies to enhance Fe content in wheat grains.