ofung@student.unimelb.edu.au

Iron (Fe) deficiency affects 30-50% of the world’s population. Genetic biofortification of staple crops is a promising strategy for improving human nutrition, but the number of effective precision breeding targets for Fe biofortification is small.

Recent studies have shown that 50% of genes in some eukaryotes possess short (<100 amino acid long) upstream Open Reading Frames (uORFs) located within the 5' leader sequence (LS). These uORFs can be translated and function as negative regulators by downregulating the translation of the primary ORF (pORF). We aligned publicly available rice (Oryza sativa L.) ribo-seq datasets and transcriptomes to identify non-canonical translation within the 5’ LS of important Fe homeostasis genes.

A dual luciferase assay (DLA) was used to determine whether 5’ LS translation indicated from ribo-seq datasets corresponded to uORFs that repressed the pORF. We identified two positive regulators of the Fe-deficiency response that contained a repressive uORF region: IDEF1 and IDEF2. The IDEF2-uORF peptide was highly conserved among monocots and a mutation series in the 5’ LS of the bread wheat (Triticum aestivum L.) TaIDEF2-A1 gene demonstrated variable pORF derepression. To fine-tune TaIDEF2 expression in bread wheat, we utilised CRISPR-Cas9 gene editing to target and mutate the TaIDEF2-uORF homoeologs in cv.

Fielder with the aim to increase expression of the TaIDEF2 pORF and activate the plant’s Fe deficiency response. Three T1 mutations in the TaIDEF2-A1-uORF and TaIDEF2-D1-uORF homoeologs have been identified and are predicted to inhibit the uORF’s repressive function.The effect of TaIDEF2-uORF mutations on plant growth under Fe deficiency and grain nutrition is now being assessed via hydroponic and glasshouse studies.

Together these findings highlight the potential of manipulating uORFs to achieve precise control over gene expression in plants using CRISPR-Cas9 gene editing.