Résumé:
STOP1 from the model plant Arabidopsis thaliana is a master gene for tolerance to low-pH and Al. STOP1 (Sensitive To Proton rhizotoxicity1) is a transcription factor (TF) responsible for tolerance not only to low-pH and Al but also to several other abiotic stresses. A. thaliana mutants lacking STOP1 activity are extremely sensitive to H+ and Al3+, with this being mainly due to a deficit in the STOP1-dependent expression of root efflux transporters of small metal-chelating organic acids. pH and metals post-transcriptionally regulate the abundance of the STOP1 protein. The mechanism by which acidic pH and Al stimulate the STOP1 signaling pathway remain elusive. STOP1 contains three predicted intrinsically disordered regions (IDRs) accounting for 55 % of the STOP1 sequence. The high conformational flexibility and multivalency of IDRs render them highly prone to undergo liquid-liquid phase separation (LLPS). In eukaryotes, including A. thaliana, TFs are enriched in IDRs. Transcriptional regulators are known to sub-compartmentalize via LLPS. Their condensation can either stimulate their activity by concentrating them with components of the transcription machinery or, on the contrary, inactivates them by sequestration. Importantly, phase-separation of certain IDR-containing proteins depends on the cellular pH. In silico analysis of the STOP1 sequence indicates that its three IDRs contain multiple droplet-promoting regions that could drive LLPS. Our working hypothesis is that STOP1 undergoes LLPS in response to a decrease in pH and that the resulting condensates trigger expression of the target gene(s). The aim of this project is to experimentally ascertain the disordered nature of STOP1 IDRs and to assess their ability to undergo pH-mediated LLPS both in vitro and in planta. The three IDRs will be expressed in E. coli and the resulting purified proteins will be characterized in vitro in terms of their conformational properties and phase-separating abilities. The ability of STOP1 to undergo pH- and Al-induced LLPS in planta will be assessed by confocal microscopy using STOP1-GFP fusions. STOP1 mutants will also be designed, transiently expressed in N.benthamiana and then assessed for their ability to undergo LLPS. This project, by shedding light on the potential phase-separating abilities of STOP1, will constitute a breakthrough in our understanding of the molecular mechanisms underlying the activity of this class of plant TFs with the potential to unveil a new regulatory mechanism.