POPEYE directly regulates bHLH Ib genes and its own expression

Iron (Fe) is an essential trace element for plants. When suffering from Fe deficiency, plants modulate the expression of Fe deficiency responsive genes. POPEYE (PYE) is a key bHLH transcription factor involved in Fe homeostasis. However, the molecular mechanism of PYE regulating the Fe deficiency response remains elusive. We found that the over-expression of PYE attenuates the expression of Fe deficiency responsive genes. PYE directly represses the transcription of bHLH Ib genes ( bHLH38, bHLH39, bHLH100 , and bHLH101 ) by associating with their promoters. Although PYE contains an Ethylene response factor-associated Amphiphilic Repression (EAR) motif, it does not interact with the transcriptional corepressors TOPLESS/TOPLESS-RELATED (TPL/TPRs). Subcellular localization analysis indicated that PYE localizes in both the cytoplasm and nucleus. PYE contains a Nuclear Export Signal (NES) which is required for the cytoplasmic localization of PYE. The mutation of NES amplifies the repression function of PYE, resulting in downregulation of Fe deficiency responsive genes. Co-expression assays indicated that bHLH IVc members (bHLH104, bHLH105/ILR3, and bHLH115) facilitate the nuclear accumulation of PYE. Conversely, PYE indirectly represses transcription activation ability of bHLH IVc. Additionally, PYE directly negatively regulates its own transcription. This study provides insights into the complicated Fe deficiency response signaling pathway and enhances the understanding of PYE functions. Short summary PYE is a negative regulator of Fe homeostasis; however, it was still unclear how PYE integrates the Fe deficiency response signaling. Our study shows that conditional nuclear localization of PYE is crucial for Fe homeostasis. PYE not only negatively regulates FIT-dependent Fe uptake genes by directly targeting bHLH Ib genes, but also negatively regulates its own expression. ### Competing Interest Statement The authors have declared no competing interest.