Doctorate thesis
Thursday, December 4, 2014
Roles of Arabidopsis thaliana guard cell aquaporins in water and hydrogen peroxyde
Olivier Rodrigues
BPMP, “Aquaporins” team
Jury :
Alain VAVASSEUR, DR CEA Cadarache Rapporteur
Jean COLCOMBET, CR Unité de Recherche en Génomique Végétale Rapporteur
Jean-Philippe REICHHELD, DR Laboratoire Génome et Développement des Plantes Examinateur
Bruno TOURAINE, Prof Université Montpellier 2 Examinateur
Nathalie LEONHARDT, DR CEA Cadarache Examinateur
Christophe MAUREL , DR Biochimie et Physiologie Moléculaire des Plantes Directeur de thèse
Lionel VERDOUCQ, CR Biochimie et Physiologie Moléculaire des Plantes CoDirecteur de thèse
Abstract :
Stomatal movements in response to changing environmental conditions are crucial for controlling the plant water status and protecting plants against pathogens. Abscisic acid (ABA) or Pathogen-Associated Molecular Patterns (PAMPs) such as the flg22 peptide, induce, via hydrogen peroxide (H2O2), signaling networks that lead to stomatal closure. Although stomatal movements involve marked water fluxes and changes in cell volume, the role of membrane water channels (aquaporins, AQP) has remained hypothetical. Functional assays in epidermal peels showed that Arabidopsis thaliana pip2;1 plants invalidated for plasma membrane AQP PIP2;1 have a defect in stomatal closure in response to ABA and flg22, but normal responses to dark, light or carbon dioxide. Guard cell protoplasts from wild-type plants showed a two-fold increase in osmotic water permeability (Pf) in response to ABA which was fully abrogated in pip2;1 plants. Plants expressing a H2O2 sensitive probe (HyPer) revealed an accumulation of H2O2 in guard cells of wild-type plants in response to ABA and flg22, which was abolished in pip2;1 plants. OST1 (SnRK2.6), a protein kinase closely linked to ABA receptors, was able to phosphorylate, in vitro and at Ser121, a cytosolic peptide of PIP2;1. SnRK2.6 also enhanced PIP2;1 water transport activity after co-expression in Xenopus oocytes. Expression in pip2;1 plants of a phosphomimetic (Ser121Asp) or a phosphodeficient form (Ser121Ala) of PIP2;1 allowed to demonstrate that the increase in guard cell protoplast Pf, the intracellular accumulation of H2O2 and stomatal closure induced by ABA and flg22 require the phosphorylation of PIP2;1 at Ser121. This work provides the first direct genetic and physiological evidence for aquaporin function in guard cells. We propose that PIP2;1 plays both a hydraulic and signaling role in guard cells, by facilitating the transport of water and H2O2 across the guard cell plasma membrane. PIP2;1 phosphorylation at Ser121, by SnRK2.6 in particular, would be required in this context.