Doctorate thesis

Thursday, December 5, 2013


Aquaporins of the Arabidopsis thaliana leaf: regulation and tissue hydraulics

Karine Prado
BPMP, “Aquaporins” team


Jury :
Mme Nathalie LEONHARDT, Directeur de recherche, CEA, Cadarache, Rapporteur
M. Enrico MARTINOIA, Professeur, Université de Zurich, Zurich, Examinateur
M. Christophe MAUREL, Directeur de recherche, CNRS, Montpellier, Directeur de thèse
Mme Catherine PERROT-RECHENMANN, Directeur de recherche, CNRS, Gif-sur-Yvette, Rapporteur
M. Thierry SIMONNEAU, Directeur de recherche, INRA, Montpellier, Examinateur
M. Bruno TOURAINE, Professeur, Université de Montpellier 2, Montpellier, Examinateur


Abstract :
The water status of leaves depends on the efficiency of water supply, from the vasculature to inner tissues. The present work investigates the genetic and molecular bases of water transport regulation by water channel aquaporins in leaves of Arabidopsis thaliana. Leaf hydraulic conductivity (K ros ) in Arabidopsis is higher in darkness than during the day. Knockout pip2;1 plants showed that the PIP2;1 aquaporin isoform can fully account for K ros responsiveness to darkness. Directed expression of PIP2;1 in veins of a pip2;1 mutant showed that living cells of veins form a major hydraulic resistance in leaves. Quantitative proteomic analyses and expression in pip2;1 of phosphomimetic and phosphorylation-deficient forms of PIP2;1 demonstrated that phosphorylation at sites Ser-280 and Ser-283 is necessary but not sufficient for K ros enhancement under darkness. A comprehensive analysis of time-dependent variations of K ros showed that K ros is under circadian control and immunological approaches using phosphospecific antibodies revealed that circadian rhythm was correlated with changes in C-terminal PIP2;1 phosphorylation rather than changes in protein abundance. Other stimuli than light, such as abscisic acid, hydrogen peroxide or sugars were found to alter K ros by as yet unknown molecular mechanisms. Finally, proteins able to interact with the C-terminal phospho-sites of PIP2;1 were investigated, and activation of PIP2;1 by 14-3-3 proteins of Arabidopsis was demonstrated using a co-expression assay in Xenopus laevis oocytes. The overall work establishes how the phosphorylation-dependent regulation of a single aquaporin isoform in leaf veins critically determines leaf hydraulics.
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