Doctorate thesis of Montpellier University
Tuesday, june 22, 2021

at 2 PM –Campus de La Gaillarde- BPMP library room without audience in videoconference.

 Doctorale school : GAIA – Biodiversité, Agriculture, Alimentation, Environnement, Terre, Eau
Spéciality : BIDAP – Biologie, Interactions, Diversité Adaptative des Plantes
Montpellier university

Team: Mineral nutrition and oxidative stress

Study of the molecular mechanisms controlling iron homeostasis in plants


Jacqueline GRIMA-PETTENATI, Directrice de recherche INRAE, LRSV, Toulouse, Rapporteur
Sébastien THOMINE, Directeur de recherche, I2BC, Gif-sur-Yvette, Rapporteur
José GENTILHOMME, Maitre de conférence, IRHS, Angers, Examinateur
Loïc LEPINIEC, Directeur de recherche INRAE, IJPB, Versailles, Examinateur
Gabriel KROUK, Directeur de recherche CNRS, BPMP, Montpellier, Examinateur
Iron (Fe) is an essential micronutrient for plants. However, Fe availability for plant is low, in particular in neutral or alkaline soil, causing growth defects and yield loss. Excess Fe is also deleterious to plants because of its capacity to generate reactive oxygen species via the Fenton reaction. To maintain Fe homeostasis, and therefore prevent any deficiency or excess that could be detrimental, plants have evolved sophisticated transcriptional regulatory networks to tightly control Fe uptake, translocation, assimilation, and storage. In mammals, an integrated post-transcriptional mechanism couples the regulation of both Fe deficiency and Fe excess responses. Whether in plants an integrated control mechanism involving common players regulates responses both to deficiency and to excess was still to be determined. Using a combination of molecular, genetic, and biochemical approaches, we found that bHLH105/ILR3 (IVc bHLH), a previously reported transcriptional activator of responses to Fe deficiency in Arabidopsis thaliana, could also negatively regulate the expression of ferritin genes, which are markers of the plant’s response to Fe excess. We also found that ILR3 could directly repress the expression of several structural genes involved in the maintenance of Fe homeostasis and that ILR3 repressive activity was conferred by its dimerization with bHLH47/PYE. Due to the central roles of ILR3 in Fe homeostasis, we conducted Co-IP LC-MS/MS experiment to identify potential new actors involved in controlling iron homeostasis. Using this strategy, bHLH121 was identified as a ILR3-interacting transcription factor. Interaction studies showed that bHLH121 also interacts with the three closest homologs of ILR3 (i.e. bHLH34, bHLH104, and bHLH115). Loss-of-function of bHLH121 causes strong Fe-deficiency symptoms, reduces Fe contents, and alleviates expression of Fe deficiency-responsive, in particular transcriptional regulators (e.g. the PYE and the Ib bHLH bHLH38, bHLH39, bHLH100 and bHLH101 transcription factors, the BTS and BTSL1 ubiquitin ligases or the IMA1 and IMA2 regulatory peptides). ChIP-qPCR confirmed that bHLH121 could directly bind to the promoter of thesetranscriptional regulators but not to the one of FIT/bHLH29. We found that bHLH121 is expressed throughout the plant body and that its expression is not affected by Fe availability. By contrast, we showed that Fe availability affects the cellular localization of bHLH121 protein in roots. Altogether, these data demonstrated that bHLH121 is a key regulator of Fe homeostasis that acts upstream of Ib bHLHs and FIT in concert with ILR3 and its closest homologs. To study how bHLH121 and IVc bHLHs function collectively and efficiently to regulate their shared target genes, and thus Fe homeostasis, we generated the double mutants between bhlh121 and IVc bhlhs. We found that these double mutants displayed more severe growth defects, in agreement with the lower Fe accumulation, than those of single mutants under both Fe sufficient and deficient conditions. Expression analysis demonstrated also that the double mutants showed much more impaired Fe deficiency responses. Overexpression of bHLH34 and bHLH105 could partially complement the Fe-associated growth defects of bhlh121 mutant by activating the expression of both bHLH39 and FIT. Taken together these results indicate that bHLH121 and IVc bHLHs function coordinately to regulate Fe homeostasis.