bHLH121 and clade IVc bHLH transcription factors synergistically function to regulate iron homeostasis in Arabidopsis thaliana

Iron is an essential micronutrient for plant growth and development. In Arabidopsis thaliana, an intricate regulatory network involving several basic helix-loop-helix (bHLH) transcription factors controls the homeostasis of iron. Among these transcription factors, bHLH121 plays a crucial role. bHLH121 interacts in vivo with clade IVc bHLH transcription factors and activates the expression of FIT and clade Ib bHLH transcription factors to stimulate the uptake of iron. How bHLH121 and clade IVc bHLH transcription factors function collectively and efficiently to maintain iron homeostasis is still unclear. Herein, we found that double loss-of-function mutants involving bhlh121 and one of the clade IVc bHLH transcription factors displayed more severe iron deficiency-associated growth defects than each of the single mutants. We also found that among the four clade IVc bHLH transcription factors, only bHLH34 and bHLH105 could partially complement the iron-associated growth defects of bhlh121 when overexpressed. These data, together with protein localization analysis, support that bHLH121 and clade IVc bHLH transcription factors act synergistically to regulate iron homeostasis and that different bHLH121/clade IVc and clade IVc/clade IVc protein complexes are involved in this process.