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.

铁（Fe）缺乏症普遍存在于中性或碱性土壤中。植物已经进化出调节铁稳态，确保生存的复杂机制。在拟南芥中，类似铁缺乏症的转录因子（FIT）是铁缺乏反应的重要调节剂。FIT在铁缺乏时被碱性螺旋-环-螺旋（bHLH）IVc转录因子（TFs）间接激活；但是，尚不清楚哪种蛋白作为介导bHLH IVc TFs激活FIT的接头。在这项研究中，我们表征bHLH121的功能，并证明它与FIT启动子直接相关。我们发现bHLH121的功能丧失突变会导致严重的铁缺乏症症状，减少铁的积累，并破坏与铁稳态相关的基因的表达。遗传分析表明，FIT对bHLH121具有上位性，而FIT的过表达可部分拯救bhlh121突变体。进一步的研究表明，bHLH IVc TF与bHLH121相互作用并促进其蓄积。我们证明bHLH121具有DNA结合活性，可以结合FIT和bHLH Ib基因的启动子，但我们没有发现它对这些基因具有直接的转录激活或抑制活性。同时，我们发现bHLH121bHLH IVc TFs的下游功能是bHLH IVc TF的下游，并且是bHLH IVc TFs的直接靶标，Fe缺乏以bHLH IVc依赖性方式诱导其表达。综上所述，这些结果证明bHLH121与bHLH IVc TF一起起作用以正向调节FIT的表达，因此在维持拟南芥中的Fe稳态中起着关键作用。