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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.

Iron is an essential element for life on Earth and its shortage, or excess, in the living organism may lead to severe health disorders. Plants serve as the primary source of dietary iron and improving crop iron content is an important step towards a better public health. Our review focuses on the control of iron acquisition in dicotyledonous plants and monocots that apply a reduction-based strategy in order to mobilize and import iron from the rhizosphere. Achieving a balance between shortage and excess of iron requires a tight regulation of the activity of the iron uptake system. A number of studies, ranging from single gene characterization to systems biology analyses, have led to the rapid expansion of our knowledge on iron uptake in recent years. Here, we summarize the novel insights into the regulation of iron acquisition and internal mobilization from intracellular stores. We present a detailed view of the main known regulatory networks defined by the Arabidopsis regulators FIT and POPEYE (PYE). Additionally, we analyze the root and leaf iron-responsive regulatory networks, revealing novel potential gene interactions and reliable iron-deficiency marker genes. We discuss perspectives and open questions with regard to iron sensing and post-translational regulation.

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Highlights d Iron-regulated and calcium-dependent protein kinase CIPK11 interacts with FIT d CIPK11 and calcium sensors CBL1/9 promote FIT-dependent Fe deficiency responses d FIT is phosphorylated in plants, and CIPK11 phosphorylates FIT at Ser272 d Mutation at Ser272 modulates FIT activity, affecting seed iron content

Dicotyledonous plants growing under limited iron availability initiate a response resulting in the solubilization, reduction, and uptake of soil iron. The protein factors responsible for these steps are transmembrane proteins, suggesting that the intracellular trafficking machinery may be involved in iron acquisition. In search for components involved in the regulation of Arabidopsis thaliana iron deficiency responses, we identified the members of the SORTING NEXIN (SNX) protein family. SNX loss-of-function plants display enhanced susceptibility to iron deficiency in comparison to the wild type. The absence of SNX led to reduced iron import efficiency into the root. SNX1 showed partial colocalization with the principal root iron importer IRON-REGULATED TRANSPORTER1 (IRT1). In SNX loss-of-function plants, IRT1 protein levels were decreased compared with the wild type due to enhanced IRT1 degradation. This resulted in diminished amounts of the IRT1 protein at the plasma membrane. snx mutants exhibited enhanced iron deficiency responses compared with the wild type, presumably due to the lower iron uptake through IRT1. Our results reveal a role of SNX1 for the correct trafficking of IRT1 and, thus, for modulating the activity of the iron uptake machinery.

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Root iron mobilization genes are induced by iron deficiency downstream of an unknown signaling mechanism. The FER gene, encoding a basic helix-loop-helix domain protein and putative transcription factor, is required for induction of iron mobilization genes in roots of tomato (Lycopersicon esculentum). To study upstream regulatory events of FER action, we examined the control of FER gene and FER protein expression in response to iron nutritional status. We analyzed expression of the FER gene and FER protein in wild-type plants, in mutant plants with defects in iron uptake regulation, and in 35S transgenic plants that overexpressed the FER gene. An affinity-purified antiserum directed against FER epitopes was produced that recognized FER protein in plant protein extracts. We found that the FER gene and FER protein were consistently downregulated in roots after generous (100 mM, physiologically optimal) iron supply compared to low (0.1 mM) and sufficient (10 mM) iron supply. FER gene and FER protein expression were also occasionally down-regulated at sufficient compared to low iron supply. Analysis of FER protein expression in FER overexpression plants, as well as cellular protein localization studies, indicated that FER was down-regulated by high iron at the posttranscriptional level. The FER protein was targeted to plant nuclei and showed transcriptional activation in yeast (Saccharomyces cerevisiae). FER protein regulation in the iron accumulation mutant chloronerva indicated that FER protein expression was not directly controlled by signals derived from iron transport. We conclude that FER is able to affect transcription in the nucleus and its action is controlled by iron supply at multiple regulatory levels.