Iron (Fe) is an essential micronutrient that is frequently inaccessible to plants. Rice (Oryza sativa L.) plants employ the Combined Strategy for Fe uptake, which is composed by all features of Strategy II, common to all Poaceae species, and some features of Strategy I, common to non-Poaceae species. To understand the evolution of Fe uptake mechanisms, we analyzed the root transcriptomic response to Fe deficiency in O. sativa and its wild progenitor O. rufipogon. We identified 622 and 2,017 differentially expressed genes in O. sativa and O. rufipogon, respectively. Among the genes up-regulated in both species, we found Fe transporters associated with Strategy I, such as IRT1, IRT2 and NRAMP1; and genes associated with Strategy II, such as YSL15 and IRO2. In order to evaluate the conservation of these Strategies among other Poaceae, we identified the orthologs of these genes in nine species from the Oryza genus, maize and sorghum, and evaluated their expression profile in response to low Fe condition. Our results indicate that the Combined Strategy is not specific to O. sativa as previously proposed, but also present in species of the Oryza genus closely related to domesticated rice, and originated around the same time the AA genome lineage within Oryza diversified. Therefore, adaptation to Fe²⁺ acquisition via IRT1 in flooded soils precedes O. sativa domestication.

NAC transcription factors are plant-specific proteins involved in many processes during the plant life cycle and responses to biotic and abiotic stresses. Previous studies have shown that stress-induced OsNAC5 from rice (Oryza sativa L.) is up-regulated by senescence and might be involved in control of iron (Fe) and zinc (Zn) concentrations in rice seeds. Aiming a better understanding of the role of OsNAC5 in rice plants, we investigated a mutant line carrying a T-DNA insertion in the promoter of OsNAC5, which resulted in enhanced expression of the transcription factor. Plants with OsNAC5 enhanced expression were shorter at the seedling stage and had reduced yield at maturity. In addition, we evaluated the expression level of OsNAC6, which is co-expressed with OsNAC5, and found that enhanced expression of OsNAC5 leads to increased expression of OsNAC6, suggesting that OsNAC5 might regulate OsNAC6 expression. Ionomic analysis of leaves and seeds from the OsNAC5 enhanced expression line revealed lower Fe and Zn concentrations in leaves and higher Fe concentrations in seeds than in WT plants, further suggesting that OsNAC5 may be involved in regulating the ionome in rice plants. Our work shows that fine-tuning of transcription factors is key when aiming at crop improvement.