CV Nicolas Rouhier

I am a biochemist interested in basic plant science processes with a particular research focus on structure-function analysis of oxidoreductases and on redox post-translational modifications of proteins.

After a PhD in plant physiology, molecular biology and biochemistry, I have been recruited in 2004 as an assistant professor at the Université of Nancy, obtained an habilitation (HDR) in 2007 before gettting a professor position in 2011 at the newly created Université of Lorraine. For our research projects, we employ biochemical, biophysical, spectroscopic and structural biology approaches with Populus trichocarpa and Arabidopsis thaliana as model organisms to explore (i) the roles of the antioxidant/detoxification systems in the physiology of plants subject to environmental constraints and (ii) the maturation and roles of iron-sulfur proteins in plant organelles.

• Redox post-translational modifications of proteins

In addition to performing structure-function analyses of thioredoxin (TRX) and glutaredoxin (GRX) family members which control most of the reversible oxidative modifications of protein cysteinyl residues, we are characterizing proteins of unknown function possessing one or several conserved CXXC motifs known to be particularly suited for disulfide bond formation but also other redox modifications. We hope to unravel new redox-regulated cellular processes or signaling pathways in poplar and more generally in plants which are controlled by redox reactions such as thiol-disulfide exchanges and understand how the functions of these proteins are controlled at the cellular level by TRX and GRX.

The same questions and strategies apply to sulfurtransferases, enzymes that catalyze the transfer of a sulfur atom from sulfur donors to nucleophilic sulfur acceptors (trans-persulfidation) by forming themselves persulfides. In this context, the intervention of the TRX and glutathione (GSH)/GRX reducing systems in the sulfur signaling/trafficking pathways is examined as both the TRX and GSH/GRX systems might modulate STR function and in particular be central to the formation of hydrogen sulfide, a molecule susceptible to trigger redox signalling cascades in several physiological situations.

Collaborators: C. Didierjean (Univ. Lorraine), JM. Herrmann (Kaiserslautern), S. Lemaire (CNRS – Sorbonne Université), A. Meyer (Bonn), B. Morgan (Saarbrücken), P. Rey (CEA Cadarache)

• Molecular analysis of late steps of Fe-S cluster maturation in organelles

The general objective here is to understand the functioning of the Fe-S cluster assembly machineries in organelles and more precisely the molecular mechanisms controlling the second step, i.e. the trafficking of preformed Fe-S clusters from scaffold proteins (made in the first step) to final acceptors. After focusing for some time on the capacity of several plant GRXs to assemble [2Fe-2S] centers into homodimers or into heterodimers with BOLA proteins, we extended our research interest to the functional characterization of the associated protein families (NFUs, SUFA/ISCAs, IBA57s, HCF101/INDH) assumed to participate in the transfer of these Fe-S clusters together with GRX and BOLA. This functional analysis will combine plant genetics and physiology, molecular and structural biology and biochemistry approaches to determine whether these proteins assemble different types of Fe-S clusters and have specific interaction partners and what are the physiological and metabolic consequences of deleting these genes for plant development and physiology. Particular emphasis is given to the maturation of the photosynthetic and respiratory electron transfer complexes.

Collaborators: J. Balk (JIC, Norwich), MK. Johnson (Athens, USA), O. Keech (Umea), A. Meyer (Bonn), U. Mühlenhoff (Marburg), C. Remacle (Liège), F. Vignols (BPMP Montpellier)

• Structure-Function analysis of glutathione transferases

Glutathione transferases (GSTs) constitute large multigenic families of more than 50 genes in terrestrial plants. They have three major biochemical functions. Those having a serinyl residue in their catalytic site (Ser-GSTs) possess GSH-conjugation activity. This catalytic activity is often described as required for xenobiotic detoxification. Those having a cysteinyl residue in their catalytic site (Cys-GSTs) possess the ability to catalyze the reverse reaction, i.e., the removal of glutathione from a number of structurally different heterocyclic molecules. The third function is the binding and/or transport of molecules/specialized metabolites without catalytic transformation, so-called ligandin function. It can be performed both by Ser-GSTs and Cys-GSTs. Although they are present in all kingdoms, the physiological functions of most plant GSTs remain elusive.

The current work is centered on the structure-function analysis of specific classes of glutathione transferases, focusing in particular on the isolation and identification of physiological substrates explaining their implication in biotic or abiotic stress responses. In particular, we are interested in GSTs regulated during poplar leaf infection by the rust fungus Melampsora larici-populina or regulated during iron starvation in A. thaliana.

The strategies used are to perform pull-down and affinity chromatographies, inhibition kinetics and thermal denaturation combining fluorescent probes and model ligands or plant extracts that are eventually separated by liquid ichromatography. We will not only focus on the previously characterized Cys-GSTs (lambda GSTs and glutathionyl hydroquinone reductases) but also on less explored or newly identified GST families. The nature (ligandin vs catalytic transformation) and the strength of the interactions will be validated by measuring binding constants using glutathionylated and non-glutathionylated molecules using biophycal approaches (ITC, Switchsense, fluorimetry). In parallel, the structure of GST alone or in complex with their ligand/substrate will be tentatively solved. Overall, we expect to decipher new intermediary steps of xenobiotic detoxification or of metabolic pathways requiring glutathione-conjugating or transport activities. New research questions have emerged recently from the observation that tau GST are able to bind porphyrins and hemes b in particular.

Collaborators: C. Didierjean (Univ. Lorraine), C. Dubos (BPMP, Montpellier), S. Lemaire (CNRS – Sorbonne Université)

Grants

-2019-2022 : Projet ANR-2018: MITOGLU: Dissecting the molecular interactions of mitochondrial glutaredoxin S15 in plants (coordinators N. Rouhier/A. Meyer)

-2018-2021 : Projet Lorraine Université Excellence, PEPS Mirabelle + : FeSprot: Maturation and structure-function analyses of iron-sulfur proteins (coordinator N. Rouhier)

– 2018-2021: Projet ANR 2017: MOBIFER: Dynamics of coumarin secretion by plant roots into the soil in the context of iron nutrition (coordinator C. Dubos)

– 2014-2017: Projet ANR 2013: Fe-S traffic: The cellular trafficking of iron-sulfur (Fe-S) clusters in plants (coordinator N. Rouhier)

– 2011-2013: Projet ANR 2010: FIRES: Functions of glutaredoxins in Iron and REdox sensing and Signaling in plants (coordinator N. Rouhier)

– 2009-2011: Projet INRA/FORMAS: The role of organellar glutaredoxins in iron-sulfur cluster biogenesis in plants: an interactomic study for the identification of protein partners (G. Wingsle (Umea Plant Science center, sweden)/ coordinator N. Rouhier)

– 2008-2010: Projet ANR Jeune Chercheur: The role of organellar CGFS containing glutaredoxins in iron-sulfur clusters assembly and biogenesis in plants (coordinator N. Rouhier)

– 2006-2008: Projet ANR, Réseau de Génomique végétale-Genoplante 2010: GNP05010P: Repair mechanisms of oxidized proteins in chloroplasts of Arabidopsis thaliana (coordinator Pascal Rey)