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    

Abstract

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.

The Arabidopsis Mediator subunit MED16 regulates iron homeostasis by associating with EIN3/EIL1 through subunit MED25

“…We tested if MED25 interacted with transcription factors involved in the regulation of iron homeostasis in Arabidopsis, i.e., FIT, PYE, bHLH038, bHLH039, EIN3 and EIL1 (Colangelo and Guerinot, 2004;Yuan et al, 2008;Long et al, 2010;Lingam et al, 2011). We found that MED25 interacted directly with EIN3 and EIL1, whereas YID1/ MED16 showed no interaction (Figure 5a).…”

“…The two transcription factors in ethylene signaling, EIN3 and EIL1, directly interact with FIT, affecting the level of FIT protein (Colangelo and Guerinot, 2004;Lingam et al, 2011;Sivitz et al, 2011). It was propose that a small number of FIT proteins are activated during iron-deficient conditions, as large portions of the FIT proteins remain inactive.…”

“…It was propose that a small number of FIT proteins are activated during iron-deficient conditions, as large portions of the FIT proteins remain inactive. However, neither active nor inactive FIT proteins were stable (Lingam et al, 2011). Iron deficiency could trigger ethylene signaling, leading to the activation of EIN3 and EIL1, which physically interact (a) (b) Figure 6.…”

“…The Mediator subunit MED16 participates in regulation of iron homeostasis by interacting with MED25. MED25 interacts with the transcription factors EIN3/EIL1, which then interact with and stabilize FIT (Fe-deficiency induced transcription factor) protein (Lingam et al, 2011). FIT, the key regulator of iron homeostasis, regulates the expression of downstream iron acquisition genes IRT1 and FRO2 to respond to iron deficiency.…”

“…Recently, two transcription factors ethylene-insensitive 3 (EIN3) and ethyleneinsensitive 3-like 1 (EIL1) in the ethylene signaling pathway were shown to be involved in the regulation of iron homeostasis. The EIN3/EIL1 transcription factors interact with FIT to maintain a normal amount of FIT because the FIT protein is unstable and easily degrades (Lingam et al, 2011;Sivitz et al, 2011). Furthermore, EIN3 and EIL1 are linked to ethylene signaling in the jasmonic acid (JA) signaling pathways.…”