Team leader: Mélanie Morel-Rouhier

During the past decade, with the global objective of understanding how plants (using in particular the model tree Populus trichocarpa) and fungi (using in particular the white rot fungi Phanerochaete chrysosporium) can cope with and adapt to environmental constraints, our group studied the structure-function relationship of antioxidant enzymes belonging to the thioredoxin (TRX), glutaredoxin (GRX), thiol peroxidase (TPX) and methionine sulfoxide reductase (MSR) and glutathione transferase (GST) families. We provided details concerning the catalytic properties, substrate specificities, 3D structures and mode of interactions of these enzymes. Other novel projects and scientists involved are summarized below.

SULTRAF: Roles of sulfurtransferases in SULfur TRAFficking in plants – relationship with TRX and GRX systems
Actors: J. Couturier, N. Rouhier, A Moseler (post-doctoral researcher), D. Caubrière (PhD), M. Ziesel (CDD)

As a major sulfur donor molecule, cysteine is a key metabolite involved in the biosynthesis of many sulfur-containing cofactors or biomolecules (Fe-S cluster, biotin, thiamine, lipoic acid, molybdopterin and sulfur-containing bases in tRNA). The first hallmark step of sulfur mobilization is the formation of persulfides on a reactive catalytic cysteine of cysteine desulfurases (CDs). Considering the diversity of acceptor molecules, the trafficking of sulfur also implies the existence of carrier proteins, notably of the sulfurtransferase (STR) family. Most STRs catalyze the transfer of a sulfur atom from sulfur donors to nucleophilic sulfur acceptors by forming themselves persulfides. In this context, the intervention of the thioredoxin (TRX) and glutathione/glutaredoxin (GRX) reducing systems in the sulfur signaling/trafficking pathways will be examined as it might clearly modulate this process but it could also provide the required specificity to disseminate the signal from a single persulfide-generating enzyme to specific target proteins acting in this case as secondary carriers.

METOX: Identifying new Metal- or redOx-regulated functions in plants
Actors: N. Rouhier, L. de Bont, T. Dhalleine, J. Couturier, B. Pêtre, F. Zannini (ATER)

Within this project, we would like to identify and characterize proteins of unknown function possessing one or several conserved CXXC motifs which are particularly suited for disulfide bond formation and for the coordination of metals. From in silico genome analyses, we will select about 50 candidate proteins and perform a thorough biochemical and structural characterization of the corresponding recombinant proteins, before deciphering the biological roles of the most promising candidates using genetic approaches. We anticipate that the proposed approach will allow (i) unravelling new redox-regulated cellular processes or signaling pathways in poplar and more generally in plants which are controlled by redox reactions i.e., assisted by thiol-disulfide exchanges or by metalloproteins and (ii) understanding how the functions of these proteins are controlled at the cellular level.

FES: Molecular analysis of late steps of Fe-S cluster maturation in organelles
Actors: N. Rouhier, J. Couturier, J. Przybyla-Toscano (post-doctoral researcher), Loïck Christ (PhD), Esteban Guiot (M2)

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. This project will mainly focus on the functional characterization of two protein families (NFU and SUFA/ISCA) assumed to participate in the transfer of these Fe-S clusters together with GRX and BolA that we are still also characterizing. 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.

GLUTANAC: Biochemical and structural characterization of Glutathione Transferases with fished Natural Compounds
Actors: A. Hecker, M. Morel-Rouhier, R. Sormani, E. Gelhaye, N. Rouhier, J.M. Girardet, R. Bchini, E. Sylvestre-Gonon (ATER)

Until recently, most of the studies concerning metabolite modification via glutathione transferases (GSTs) have focused on their glutathionylation activity. However, several GST classes possess a cysteinyl residue in their catalytic site (Cys-GSTs) conferring them the ability to catalyze the reverse reaction, i.e. the removal of glutathione from a number of structurally different molecules. Although they are present in all kingdoms, the physiological functions of these enzymes remain often elusive. This project aims at understanding the role of these deglutathionylating enzymes by identifying the physiological substrates of both poplar and fungal isoforms using a strategy of ligand fishing in which these GSTs are used as baits (affinity chromatography, competition between fluorescent probes and ligands for binding onto the proteins). We will not only focus on the previously characterized Cys-GSTs (omega and,lambda GSTs as well as 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. In parallel, the structure of GST/ligand complexes will be tentatively solved. These aspects will provide invaluable information necessary for understanding the molecular and physiological functions of deglutathionylating GSTs. Overall, we expect to decipher new intermediary steps of metabolic pathways requiring glutathione-conjugating or transport activities.

LifAttack: Fungal resistance to toxic environment: towards new eco-friendly strategies to limit fungal attacks on plants and wood
Actors: M. Morel-Rouhier, R. Sormani, E. Gelhaye, K. Claudien (PhD), D. Noël (PhD)

Fungi are amazing organisms able to resist and rapidly adapt to hostile environments. This could have dramatic consequences in crop production or wood preservation. Since the adaptive capacity of a population is intimately correlated with the selective pressure exerted by its local niche, this project aims at delineating the molecular mechanisms explaining the sensibility/resistance phenotypes of fungi in presence of various natural plant extracts including wood. Based on our expertise on glutathione transferases, we will focus the analysis on the detoxification systems by coupling global non a priori approaches (transcriptomic analyses) to the functional characterization of proteins of interest (peroxidases, CYP450s, GSTs, dioxygenases). Besides the identification of extracts with antifungal properties, the results will help understanding fungal physiology, evolutionary history and adaptation, knowledge that are required for developing new eco-friendly strategies to limit fungal attacks on crops and wood material.

FunMic: Mimic the fungal systems to develop new biotechnological tools
Actors: E. Gelhaye, R. Sormani, N. Valette (post-doctoral researcher) M. Barbier (CDD)

From the expertise of the team in microbial polymers degradation, we plan to pursue the development of biotechnological innovations. The main aims are to mimic the fungal systems to (i) degrade synthetic or biological molecules for extraction or bioremediation purposes or to produce/identify/modify molecules of interest. All these projects are underway and are supported by industrial partners and/or SATT (Company involved in valorisation and in the transfer of technology). Two developed processes are at this moment evaluated for patent deposit.

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