Posttranslational regulation of the iron deficiency basic helix-loop-helix transcription factor FIT is affected by iron and nitric oxide.

Understanding iron (Fe) sensing and regulation is important for targeting key genes for important nutritional traits like Fe content. The basic helix-loop-helix transcription factor FIT (for FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) controls Fe acquisition genes in dicot roots. Posttranscriptional regulation of transcription factors allows rapid adaptation to cellular changes and was also described for FIT. However, the mechanisms behind this regulation of FIT were for a long time not known. Here, we studied the posttranscriptional control mechanisms of FIT in Arabidopsis (Arabidopsis thaliana) and identified nitric oxide as a stabilizing stimulus for FIT protein abundance. Using cycloheximide, we confirmed that the level of FIT protein was regulated by way of protein turnover in wild-type and hemagglutinin-FIT plants. Upon cycloheximide treatment, FIT activity was hardly compromised, since Fe deficiency genes like IRON-REGULATED TRANSPORTER1 and FERRIC REDUCTASE OXIDASE2 were still inducible by Fe deficiency. A small pool of "active" FIT was sufficient for the induction of Fe deficiency downstream responses. Nitric oxide inhibitors caused a decrease of FIT protein abundance and, in the wild type, also a decrease in FIT activity. This decrease of FIT protein levels was reversed by the proteasomal inhibitor MG132, suggesting that in the presence of nitric oxide FIT protein was less likely to be a target of proteasomal degradation. Independent of FIT transcription, FIT protein stability and FIT protein activity, therefore, were targets of control mechanisms in response to Fe and nitric oxide. We summarize our results in a model that explains the different steps of FIT regulation integrating the plant signals that control FIT.

Interaction between the bHLH transcription factor FIT and ETHYLENE INSENSITIVE3/ETHYLENE INSENSITIVE3-LIKE1 reveals molecular linkage between the regulation of iron acquisition and ethylene signaling in Arabidopsis.

Understanding the regulation of key genes involved in plant iron acquisition is of crucial importance for breeding of micronutrient-enriched crops. The basic helix-loop-helix protein FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), a central regulator of Fe acquisition in roots, is regulated by environmental cues and internal requirements for iron at the transcriptional and posttranscriptional levels. The plant stress hormone ethylene promotes iron acquisition, but the molecular basis for this remained unknown. Here, we demonstrate a direct molecular link between ethylene signaling and FIT. We identified ETHYLENE INSENSITIVE3 (EIN3) and ETHYLENE INSENSITIVE3-LIKE1 (EIL1) in a screen for direct FIT interaction partners and validated their physical interaction in planta. We demonstrate that the ein3 eil1 transcriptome was affected to a greater extent upon iron deficiency than normal iron compared with the wild type. Ethylene signaling by way of EIN3/EIL1 was required for full-level FIT accumulation. FIT levels were reduced upon application of aminoethoxyvinylglycine and in the ein3 eil1 background. MG132 could restore FIT levels. We propose that upon ethylene signaling, FIT is less susceptible to proteasomal degradation, presumably due to a physical interaction between FIT and EIN3/EIL1. Increased FIT abundance then leads to the high level of expression of genes required for Fe acquisition. This way, ethylene is one of the signals that triggers Fe deficiency responses at the transcriptional and posttranscriptional levels.