The bHLH protein OsIRO3 is critical for plant survival and iron (Fe) homeostasis in rice (Oryza sativa L.) under Fe-deficient conditions

Abstract

ABSTRACT Iron (Fe) is an essential element for most organisms since it is involved in various biological reactions. Fe deficiency is a major constraint for plant growth and leads to low crop yields and poor nutritional quality. Fe uptake and translocation in plants require the coordinated expression of multiple genes, which is regulated by various transcription factors (TFs), especially members of the basic helix-loop-helix (bHLH) family. OsIRO3 was first reported as a repressor related to Fe homeostasis gene regulation in roots based on evidence in overexpressing rice. However, its functions in regulating rice growth and Fe homeostasis between roots and shoots during Fe deficiency is poorly understood. Here, we developed osiro3 knockout mutants by the CRISPR/Cas9 system. The leaves of osiro3 knockout mutants showed abnormal growth and development after Fe-deficiency treatment. Strong lesions occurred on the young leaves of osiro3 knockout mutants, which even led to plant death during severe Fe deficiency. Furthermore, accumulation of reactive oxygen species (ROS), Fe and manganese concentrations were all elevated in the shoots of osiro3 knockout mutants under the Fe deficiency stress. Quantitative RT-PCR showed that OsNAC4 for ROS induced plant hypersensitive cell death and OsFerritins for Fe storage and overload were all significantly activated in osiro3 knockout shoots after Fe deficiency treatment. In osiro3 knockout mutants, Fe-deficiency response genes were induced in roots, but suppressed in shoots, and furthermore Fe sufficiency/overload genes were induced in shoots under Fe-deficient conditions according to DNA microarray. Yeast two-hybrid analysis revealed a positive interaction between OsIRO3 and the homologous protein OsbHLH062, which was also transcriptionally increased in Fe-deficient roots. OsIRO3 is essential for maintaining plant survival in rice under Fe-deficient conditions and plays an important role in signal transmission from shoots to roots which is critical for plants to prevent Fe overload.