bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis

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 Fed eficiency;how ever,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 Fedeficiency symptoms,reduced Feaccum ulation,and disrupted expression of genes associated with Fehomeostasis.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 prom oters of the FIT and bHLHlb genes,but we did not find that it has either direct transcriptional activation or repression activity tow ard these genes.Meanw hile,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 IV c-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.

Oryza sativa FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR(OsFIT/OsbHLH156)interacts with OsIRO2 to regulate iron homeostasis

Iron(Fe)is indispensable for the growth and development of plants.It is well known that FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR(FIT)is a key regulator of Fe uptake in Arabidopsis.Here,we identify the Oryza sativa FIT(also known as Osb HLH156)as the interacting partner of IRON-RELATED BHLH TRANSCRIPTION FACTOR 2(OsIRO2)that is critical for regulating Fe uptake.The OsIRO2 protein is localized in the cytoplasm and nucleus,but OsFIT facilitates the accumulation of OsIRO2 in the nucleus.Loss-of-function mutations of OsFIT result in decreased Fe accumulation,severe Fe-deficiency symptoms,and disrupted expression of Feuptake genes.In contrast,OsFIT overexpression promotes Fe accumulation and the expression of Fe-uptake genes.Genetic analyses indicate that OsFIT and OsIRO2 function in the same genetic node.Further analyses suggest that OsFIT and OsIRO2 form a functional transcription activation complex to initiate the expression of Fe-uptake genes.Our findings provide a mechanism understanding of how rice maintains Fe homeostasis.