IRON MAN interacts with Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR 1 to maintain copper homeostasis.

Copper (Cu) is essential for plant growth and development. IRON MAN (IMA) is a family of small peptides that can bind both iron (Fe) and Cu ions. It was reported that IMAs mediate Fe homeostasis in Arabidopsis thaliana. However, it remains unclear whether IMAs are involved in Cu homeostasis. The transcript abundance of IMA genes decreased in response to Cu deficiency. The combined disruption of all IMA genes caused enhanced tolerance to Cu deficiency and resulted in an increase in the transcript abundance of Cu uptake genes, whereas the overexpression of IMA1 or IMA3 led to the opposite results. Protein interaction assays indicated that IMAs interact with Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR1 (CITF1), which is a positive regulator of the Cu uptake genes. Further studies showed that IMAs not only interfere with the DNA binding of CITF1 but also repress the transcriptional activation activity of CITF1, hence resulting in downregulation of the Cu uptake genes. Genetic analyses indicated that IMAs modulate Cu homeostasis in a CITF1-dependent manner. Our findings indicate that IMAs inhibit the functions of CITF1 in regulating Cu deficiency responses, thereby providing a conceptual framework for comprehending the regulation of Cu homeostasis.