Iron (Fe) is an important micronutrient for plant growth and development but any excess of Fe is toxic because of the Fe-dependent generation of reactive oxygen species (ROS). Thus, Fe homeostasis must be tightly regulated. In Arabidopsis thaliana, a cascade of transcription factors has been identified as involved in the regulation of this process by modulating the expression of genes related to Fe uptake, transport, and storage. Recently, it was demonstrated that in response to Fe deficiency, bHLH121/URI (UPSTREAM REGULATOR OF IRT1) directly activates the expression of several genes involved in this regulatory network. It was also shown that bHLH121 interacts with ILR3 (bHLH105) and its homologs. Herein it is shown that bHLH121 is necessary for the expression of the main markers of the plant responses to Fe excess, the ferritin genes (i.e. FER1, FER3, and FER4). bHLH121 regulates ferritin genes expression by directly binding to their promoters, at the same locus than the ILR3-PYE repressive complex. Therefore, this study highlight that bHLH121, PYE, and ILR3 form a chain of antagonistic switches that regulate the expression of ferritin genes. The implication of this finding is discussed.

Iron (Fe) deficiency is prevalent in plants grown in neutral or alkaline soil. Plants have evolved sophisticated mechanisms that regulate Fe homeostasis, ensuring survival. In Arabidopsis, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) is a crucial regulator of Fe-deficiency response. FIT is activated indirectly by basic helix-loop-helix (bHLH) IVc transcription factors (TFs) under Fe deficiency; however, it remains unclear which protein(s) act as the linker to mediate the activation of FIT by bHLH IVc TFs. In this study, we characterize the functions of bHLH121 and demonstrate that it directly associates with the FIT promoter. We found that loss-of-function mutations of bHLH121 cause severe Fe-deficiency symptoms, reduced Fe accumulation, and disrupted expression of genes associated with Fe homeostasis. Genetic analysis showed that FIT is epistatic to bHLH121 and FIT overexpression partially rescues the bhlh121 mutant. Further investigations revealed that bHLH IVc TFs interact with and promote nuclear accumulation of bHLH121. We demonstrated that bHLH121 has DNA-binding activity and can bind the promoters of the FIT and bHLH Ib genes, but we did not find that it has either direct transcriptional activation or repression activity toward these genes. Meanwhile, we found that bHLH121 functions downstream of and is a direct target of bHLH IVc TFs, and its expression is induced by Fe deficiency in a bHLH IVc-dependent manner. Taken together, these results establish that bHLH121 functions together with bHLH IVc TFs to positively regulate the expression of FIT and thus plays a pivotal role in maintaining Fe homeostasis in Arabidopsis.