IRON MAN interacts with BRUTUS to maintain iron homeostasis in Arabidopsis.

IRON MAN (IMA) peptides, a family of small peptides, control iron (Fe) transport in plants, but their roles in Fe signaling remain unclear. BRUTUS (BTS) is a potential Fe sensor that negatively regulates Fe homeostasis by promoting the ubiquitin-mediated degradation of bHLH105 and bHLH115, two positive regulators of the Fe deficiency response. Here, we show that IMA peptides interact with BTS. The C-terminal parts of IMA peptides contain a conserved BTS interaction domain (BID) that is responsible for their interaction with the C terminus of BTS. Arabidopsis thaliana plants constitutively expressing IMA genes phenocopy the bts-2 mutant. Moreover, IMA peptides are ubiquitinated and degraded by BTS. bHLH105 and bHLH115 also share a BID, which accounts for their interaction with BTS. IMA peptides compete with bHLH105/bHLH115 for interaction with BTS, thereby inhibiting the degradation of these transcription factors by BTS. Genetic analyses suggest that bHLH105/bHLH115 and IMA3 have additive roles and function downstream of BTS. Moreover, the transcription of both BTS and IMA3 is activated directly by bHLH105 and bHLH115 under Fe-deficient conditions. Our findings provide a conceptual framework for understanding the regulation of Fe homeostasis: IMA peptides protect bHLH105/bHLH115 from degradation by sequestering BTS, thereby activating the Fe deficiency response.

The transcription factor POPEYE negatively regulates the expression of bHLH Ib genes to maintain iron homeostasis.

Iron (Fe) is an essential trace element for plants. When suffering from Fe deficiency, plants modulate the expression of Fe deficiency-responsive genes to promote Fe uptake. POPEYE (PYE) is a key bHLH (basic helix-loop-helix) transcription factor involved in Fe homeostasis. However, the molecular mechanism of PYE regulating the Fe deficiency response remains elusive in Arabidopsis. We found that the overexpression 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 co-repressors TOPLESS/TOPLESS-RELATED (TPL/TPRs). Sub-cellular 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. Mutation of the NES amplifies the repression function of PYE, resulting in down-regulation of Fe deficiency-responsive genes. Co-expression assays indicated that three bHLH IVc members (bHLH104, bHLH105/ILR3, and bHLH115) facilitate the nuclear accumulation of PYE. Conversely, PYE indirectly represses the transcription activation ability of bHLH IVc. Additionally, PYE directly negatively regulates its own transcription. This study provides new insights into the Fe deficiency response signalling pathway and enhances the understanding of PYE functions in Arabidopsis.