Doctorate thesis of National Institute of Further Education in Agricultural Science

Friday, September 19, 2014

Study of the mode of action of plant defensin AhPDF1.1b in the tolerance to zinc

Oriane Mith
BPMP, “Metal phytotoxicity” team

Jury :
Pierre BERTHOMIEU, PR, Montpellier SupAgro, Directeur de thèse
Laurence MARQUES, MCF, Université de Montpellier 2, CoDirecteur de thèse
Alain VAVASSEUR, DR, CEA, Rapporteur
Marc VALLS, PR, Université de Barcelone, Rapporteur
Jean Marie FRANCOIS, PR, CEA, Examinateur
Michel LEBRUN, PR, Université de Montpellier 2, Examinateur

Abstract :
Study of the mode of action of plant defensin AhPDF1.1b in zinc tolerance Plant defensins belong to the wide family of antimicrobial peptides implied in innate immunity. They are characterised by their richness in cysteines and disulphide bridges as well as their antifungal properties. However, other biological activities have also been described for some plant defensins. This work continues the study of one of these additional activities : the property to confer cellular tolerance to zinc excess when a defensin is expressed in yeast. Arabidopsis halleri defensin AhPDF1.1b is taken as a model to study the mode of action of defensins in this particular activity. Firstly, the possibility that recombinant AhPDF1.1b could chelate zinc was researched. This study has required the production of the recombinant protein. Its production in Pichia pastoris was set up. We showed by fluorimetry and ESI-MS in native conditions that it is indeed possible for the defensin AhPDF1.1b to fix several zinc atoms, particularly after reduction of the protein. However the stability of these complexes as well as zinc coordination remain to be elucidated. Secondly, a study of the cellular response of yeast to the expression of AhPDF1.1b under zinc excess was led. When it is expressed in yeast, AhPDF1.1b is not secreted but accumulated in the endoplasmic reticulum (ER) and in pre-vacuolar compartments, which led us to study the influence of AhPDF1.1b expression and zinc excess on ER functioning. It has been shown that the control of the ER quality, and especially the UPR “Unfolded Protein Response” and the ERAD “ER-associated degradation” boosted by the expression of AhPDF1.1b during a zinc overload what probably triggers the mechanisms of tolerance. Eventually, a transcriptomic analysis on yeast expressing AhPDF1.1b under zinc excess was developed. The expression of defensin alone does not disturb the gene expression profile. However, under zinc excess conditions, it allows a limitation of the deregulations of gene expression provoked by zinc overload. In the conditions of AhPDF1.1b expression and zinc excess, there is an induction of an hypoxic response and surprisingly, a great production of ergosterol. Although many things remain to be discovered concerning the mode of action of the defensin AhPDF1.1b, the results presented in this work are a first step towards its elucidation