Monday december 16 2024  at 2 pm

Organization in nanodomains and function in the regulation of water transport of Arabidopsis HIR2 protein

Doctoral school : GAIA – Biodiversité, Agriculture, Alimentation, Environnement, Terre, Eau
Spéciality : BIDAP – Biologie, Interactions, Diversité Adaptative des Plantes
University : Université Montpellier
Reasearch unit : IPSiM –  Institut for Plant Sciences of Montpellier

Team: Aqua

Jury:

François Chaumont, Professeur, Université catholique de Louvain – Rapporteur
Marie-Cécile Caillaud, Directrice de recherche, CNRS Lyon – Rapporteure
Yohann Boutté, Directeur de recherche, CNRS Bordeaux – Examinateur
Soazig Guyomarc’h, Professeure, Université de Montpellier – Examinatrice
Enric Zelazny, Chargé de recherche, CNRS Montpellier – Directeur de thèse

Abstract:

HIRs are plant-specific proteins belonging to the superfamily of SPFH domain-containing proteins that were proposed to play scaffolding functions in membranes. HIR2 isoform organizes in plasma membrane nanodomains that correspond to nanometric scale structures enriched in specific lipids and proteins acting as signaling/regulation platforms. So far, how plasma membrane nanodomains are formed and maintained in plant cells remains largely unknown. I investigated the mechanisms governing the trafficking and the organization into nanodomains of Arabidopsis HIR2 protein. We revealed that the mono S-acylation of HIR2 was required for HIR2 targeting to the plasma membrane of Arabidopsis cells, but not for HIR2 nanoclustering. Investigating the mechanisms implicated in the arrangement of HIR2 into nanodomains, we provided evidences that the lipid composition in sterols and very long chain fatty acids of the plasma membrane influences HIR2 organization. HIR2 was previously shown to oligomerize and we demonstrated here that the C-terminal part of HIR2 is required for self-assembly. Interestingly, we highlighted that the oligomerization of HIR2 was essential for its organization in nanodomains and for its functionality.

So far, the molecular functions of HIR proteins remain largely unknown. HIRs have been identified in the interactome of Arabidopsis PIP aquaporins, these later facilitating water and H₂O₂ diffusion across membranes. We investigated a putative role of HIR2 in the regulation of PIP proteins. We showed that HIR2 forms a complex with several PIP proteins. Additionally, we observed that HIR mutations induced an important decrease in the root hydraulic conductivity (Lp_r) in Arabidopsis, the Lp_r being largely controlled by PIP proteins. This reduction in the Lp_r is not due to an altered root architecture in hir mutants. Instead, our studies showed that the PIP-mediated water transport in roots was largely impaired in hir mutants. Investigating the reasons for this PIP deregulation, we observed by confocal microscopy that the overall subcellular localization of PIP2;1 remained unchanged in hir mutants. Additionally, PIP protein accumulation was unaffected in hir mutants. Interestingly super resolution microscopy revealed that HIR mutations had an impact on PIP2;1 organization within nanodomains. Further, functionality tests performed in yeast revealed that HIR2 enhanced the PIP2;1-mediated transport of H₂O₂, suggesting a positive regulatory role of HIR2 on PIPs. Phospho-proteomic analysis suggested that the phosphorylation level of some PIPs was lower in hir2 single mutant compared to wild-type plants. Since the phosphorylation of PIPs is crucial for their activity, this might explain, at least in part, the reduction in Lp_r. However, intriguingly, similar effects on PIP phosphorylation were not observed in a hir1hir2hir4 triple mutant.

Interestingly, we demonstrated that HIR2 could stimulate the apoplastic ROS accumulation in response to flg22. Moreover, Arabidopsis hir mutants showed delayed stomatal closure following flg22 stimulation. Using the Hyper7 H₂O₂ biosensor, we highlighted that HIRs are implicated in H₂O₂ diffusion and cytoplasmic H₂O₂ accumulation in stomatal guard cells. HIRs may regulate PIP activity to modulate H₂O₂ transport, which is crucial for plant immunity.

In conclusion, we propose that HIRs act as scaffolding proteins to recruit and/or stabilize PIP proteins in specific plasma membrane nanodomains to modulate PIP activity, which has an impact on plant hydraulics and immunity.

Key words: plasma membrane, nanodomains, HIR, oligomerization, S-acylation, PIP, water and H₂O₂ transport, root, stomata, Arabidopsis thaliana.