A Model to Incorporate the bHLH Transcription Factor OsIRO3 within the Rice Iron Homeostasis Regulatory Network.

Oscar Carey-Fung,Martin O’Brien,Alexander A T Johnson,Jesse T Beasley

doi:10.3390/ijms23031635

Oscar Carey-Fung, Martin O’Brien + Show 2 more

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https://doi.org/10.3390/ijms23031635

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Abstract

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.

Highlights

Plants respond to abiotic stresses by modulating the expression of target genes through the activity of transcription factors (TFs)
A CRISPR-Cas9 construct containing two guide RNA sequences targeting exon 3 and exon 4 of OsIRO3 was transformed into rice to generate loss-of-function mutants of OsIRO3 (Figures 1b,c and S1)
We isolated a transgene-free wild-type (WT) line from a segregating population containing no mutation at either guide RNA (gRNA), which served as our control genotype in the experiments

Summary

Introduction

Plants respond to abiotic stresses by modulating the expression of target genes through the activity of transcription factors (TFs). All bHLH proteins contain a conserved DNA-binding basic region and two alpha helices separated by a variable loop region, and homo/heterodimerise with other TFs prior to binding DNA. IVb bHLH TF, iron-related transcription factor 3 (OsIRO3), is upregulated in response to environmental Fe deficiency, and overexpression of OsIRO3 reduces the expression of Fe chelator biosynthetic genes and decreases plant growth and shoot Fe concentrations under Fe deficiency [7,8]. For these reasons, OsIRO3 was initially characterised as a putative negative regulator of the Fe deficiency response in rice. Within the rice Fe homeostasis regulatory network, OsIRO3 sits downstream of the master regulators iron deficiencyresponsive element-binding factors 1 (OsIDEF1) and Hemerythrin motif-containing Really

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