Each year in France the building industry generates around 1,4 Mt of treated wood waste. Currently no recycling of this biomass is possible due to the toxicity of the compounds used for preservation, mainly alkaline copper quaternary and copper azole formulations. This project aims at identifying and developing a strategy using microorganisms and/or microbial enzymes as biocatalysts to remove toxic preservative compounds from wood waste in order to both (i) limit the impact of these molecules on environment and human health, and (ii) use these tons of wastes as new resource for industrial valorization of wood polymer and copper extraction. The working hypotheses are based on the fact that in natural environment, some fungal strains are highly resistant to fungicides used for wood preservation, and fungi and bacteria, either individually or in consortia of microorganisms, are efficient organisms for complex molecules breakdown and metal sequestration. We will work with the model fungus Phanerochaete chrysosporium since we have already shown that it is able to bypass the toxicity of copper/azole compounds. Moreover, 9 bacterial strains have been isolated from its mycosphere. These bacteria, which naturally cooperate with the fungus in a context of wood degradation and which exhibit interesting features regarding complex molecules and metal detoxification capabilities, will be tested as helper for wood decontamination. Indeed, it has been described many times in the literature that a consortium of microorganisms is more efficient than a single species in bioremediation mainly because of the induction of cryptic enzymatic pathways or complementary actions. The sub-objectives of the project aim at (i) evaluating the fate of copper and azoles in wood according to time after fungicide treatment by mapping the quantity and repartition of the compounds in various wood wastes (from 1 to 15 years after copper/azole treatment), (ii) determining the best conditions (medium, microbial strains, “age” of wood after treatment) for an efficient biological wood decontamination process, by developing a microcosm using a consortium of fungi and bacteria, (iii) identifying and characterizing the microbial enzymes and molecules (ie siderophores) directly involved in the decontamination process and (iiii) validating the upscaling potential of the process by testing the decontamination efficiency of the microorganisms and/or purified or semi-purified molecular actors in bioreactors. This will allow quantifying the remaining polymers and extractives in the decontaminated wood for subsequent chemical valorization. The scientific consortium is composed of molecular biologists, wood chemists and an industrial partner and will provide complementary skills to achieve the objectives. This project will provide fundamental information on the microbial detoxification systems but it will also give a proof of concept showing that microorganisms can be used to decontaminate wood waste. In the future this biological process could be developed at a larger scale to set up a process needed by industrials to valorize their wood wastes.

Forests play a major role in climate change mitigation. By performing photosynthesis, trees incorporate carbon from atmospheric CO2 into their tissues. Subsequently, dead plant debris (litterfalls, senescent fine roots) enrich the soil with organic carbon. In soils, once stabilised, organic carbon can be sequestered for up millennia. In addition to their carbon sequestration function, forests also play a positive role in the global carbon budget via substitution effects. Indeed, using wood instead of carbon-based fossil fuels, or materials that require a lot of these polluting energies, reduces the net CO2 emissions because the carbon of the wood was previously fixed by trees. However, optimising the carbon sinks role of forests is the subject of intense debates involving scientists and decision-makers: is it better to promote the effects of carbon substitution, or the effects of carbon sequestration? This project aims to solve this dilemma by optimising both biomass production and soil organic C sequestration. In practice, the CARTON project aims to (i) identify the characteristics of trees (notion of "functional traits") that are involved in rapid biomass growth, (ii) identify the functional traits that influence soil organic carbon sequestration, and (iii) evaluate to which extent these two groups of functional traits are compatible within the same tree species. To do this, we will study the functional traits of many species in sites where they were planted at the same time (the so-called "arboretum" sites). The link between growth and functional traits will be evaluated using a meta-analysis carried out at the global scale. The link between soil organic carbon storage and functional traits will be studied in 20 mature arboretums in contrasting contexts, along a European latitudinal gradient. Once these two groups of traits have been identified, we will study their relationships within tree species, using a database of functional traits. In particular, we will investigate what values optimise both growth in biomass and sequestration of organic carbon in soils. The final objective of this project is to indicate some species that optimise both carbon substitution (through a rapid growth in biomass) and carbon sequestration (through an efficient organic carbon storage in soils).

Several metabolic pathways and cellular processes in plants depend on the functioning of iron-sulfur (Fe-S) proteins, whose cofactors are assembled through dedicated assembly machineries present in the cytosol, plastids and mitochondria. To cite only a few examples, Fe- S proteins are present in the photosynthetic and respiratory electron transfer chains and they are needed for sulfur and nitrogen assimilation, or co-enzyme synthesis such as biotin and lipoic acid. In plants as in other organisms, the incorporation of Fe-S clusters into proteins requires first the de novo assembly of iron-sulfur clusters (ISCs) onto scaffold proteins and their transfer to acceptor proteins via the action of several maturation factors, and among those class II glutaredoxins (GRXs). The recent demonstration that GRXS15 coordinates an [2Fe-2S] cluster using glutathione molecules which can be transferred to an acceptor protein and that null mutants for the mitochondrial GRXS15 in Arabidopsis are embryo-lethal provided clear evidence that GRXS15 is an essential component of the ISC transfer machinery. This finding opens a new avenue towards molecular understanding of how mitochondrial Fe-S proteins are assembled. The proposed project aims primarily at dissecting the role of GRXS15 in the transfer of ISCs to target proteins in Arabidopsis. The biochemical, spectroscopic and structural analysis of GRXS15 holoforms obtained by in vitro anaerobic reconstitution will allow determining the oligomerisation status and nature of the assembled ISCs. On the basis of structural alignments and using targeted mutagenesis and combinatorial approaches, we will generate new knowledge on the structure-function relationship of this GRX and of proteins of the same class. The properties of these variants will be determined (i) by analysing their ability to bind an ISC and its lability, (ii) by finely examining protein-protein interactions with known or newly identified partner proteins, (iii) by performing heterologous expression in a yeast mutant deficient in mitochondrial Grx5 and (iv) by assessing their possible redox properties using in vitro activity assays with roGFP2 and oxidation sensitivity tests. Following earlier work, grxs15 null mutants partially rescued through expression of heterologous GRXs or mutated GRXS15 are expected to display distinct developmental and physiological phenotypes. Thus, it will be investigated whether these phenotypes are related to specific Fe-S enzyme defects and whether a bottleneck in mitochondrial ISC transfer is restricted to mitochondrial target proteins or whether it also affects cytosolic metalloenzymes. This could be because a yet unknown mitochondrial sulfur-containing compound is known to be exported for the maturation of cytosolic and nuclear proteins, and because synthesis of the molybdenum cofactor found in several cytosolic Fe-S enzymes requires a mitochondrial Fe-S enzyme.

Glutathione (gammaGlu-Cys-Gly, GSH) is a crucial metabolite in eukaryotes and many bacteria. In plants, glutathione deficiency leads to severe developmental defects. GSH binds covalently to diverse classes of endogenous or exogenous molecules. It can form a mixed-disulfide bond with protein cysteines, a redox post-translational modification termed S-glutathionylation which is favored under stress conditions. It can also be conjugated to electrophilic metabolites for the synthesis, recycling or intracellular distribution of specialized metabolites, reactions mainly catalyzed by glutathione transferases (GSTs). The Glutaclick project will address important but yet underexplored biological questions related to glutathione signaling and conjugation functions in a context of stress responses in the model photosynthetic eukaryote Chlamydomonas reinhardtii. This project will uncover the substrates of algal GSTs to unravel their specificities and thereby infer some of their functions. This project will have important repercussions in the way GSTs are studied notably by facilitating and clarifying their catalytic and functional role(s). GlutaClick will give also new insight into the importance and the role of S-glutathionylation in eukaryotes. By identifying a large set of target proteins including membrane proteins, an important class of proteins involved in crucial processes (bioenergetics, signaling, transport or intra/inter cellular communication), we expect to give a more complete picture of the processes under the control of this modification. The Glutaclick project will allow to define both the physiological conditions triggering this post-translational modification in vivo and how this modification is controlled and in particular by which glutaredoxins. It will also allow to analyze temporal and quantitative dynamics of the S-glutathionylation network and to unravel the underlying molecular mechanisms. Finally, qualitative and quantitative data will be used to initiate mathematical modelling of the glutathionylation network and identify key information about the chemical and physical features conferring glutathionylation specificity. We anticipate that many of the results obtained in the Glutaclick project will be relevant to other photosynthetic organisms. The data will therefore constitute a wealth of information for the plant scientist’s community and notably to initiate functional studies combining in vivo and in vitro approaches. Glutathione being found in most eukaryotes and many bacteria, the molecular mechanisms unraveled by our analyses will likely relevant to most signaling and conjugating functions of glutathione in both photosynthetic and non-photosynthetic organisms. Last but not least, this project will generate innovative chemical and biological tools that will be valuable for the scientific community to by-pass actual technological barriers limiting studies aiming at deciphering GSH roles.