Several metabolic pathways and cellular processes in plants depend on the functioning of iron-sulfur (Fe-S) proteins, whose cofactor is assembled through dedicated assembly machineries. To cite only a few examples, Fe-S proteins are needed for photosynthesis, respiration, sulfur and nitrogen assimilation, co-enzyme synthesis and by similarity with other eukaryotes, for DNA repair and replication or ribosome biogenesis. In plants as in other organisms, the incorporation of Fe-S clusters into proteins requires first the de novo assembly of Fe-S clusters onto scaffold proteins and then the transfer of these preformed clusters to acceptor proteins via the action of several chaperones and/or so-called carrier proteins. Using a combination of genetic, physiological, biochemical and structural approaches, the general objective of this research proposal is to understand precisely the molecular mechanisms controlling the second step, i.e., the delivery of Fe-S clusters from scaffold proteins to final acceptors, in the context of the chloroplastic and mitochondrial Fe-S cluster assembly machineries. Whereas the majority of the proteins required to assemble Fe-S clusters in the cells have likely been identified, the in vivo roles of many components, essentially those involved in Fe-S cluster trafficking, remain to be clarified. Owing to the fact that there are several dozens of Fe-S proteins but relatively few scaffold proteins in cells, carrier proteins are essential sentinels ensuring the correct and specific distribution of the different types of Fe-S clusters to acceptor client proteins. This project focuses on the Nfu and A-type carrier protein families which are assumed, from current working models, to be the major contributors for the trafficking of Fe-S clusters. Incidentally, in addition to providing improved knowledge on the global functioning of these biogenesis systems, the designed experimental program, which combines in vitro and in vivo approaches, should bring crucial information about the cellular network and regulatory mechanisms coordinating the concerted action of the different components.

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.

Sulfur represents an essential element for plant growth and development. As a major sulfur donor molecule, cysteine is a key metabolite involved in the biosynthesis of many sulfur-containing biomolecules through complex processes that are yet poorly described in plants. Among the processes requiring sulfur are the reactions leading to the maturation of iron-sulfur (Fe-S) clusters, to the synthesis of biotin, thiamine, lipoic acid, molybdopterin and sulfur-containing bases in tRNA (thionucleosides). In some of them, sulfur is provided by the degradation of an Fe-S cluster but other pathways need sulfur transfer reactions involving notably trans-sulfhydration reactions through the formation of persulfide groups on reactive cysteines. Since these sulfur-requiring pathways are found in several subcellular compartments, specific proteins dedicated to both sulfur mobilization and sulfur transfer should be present in many of them to carry out an efficient sulfur trafficking. The sulfur mobilization, catalyzed by the cysteine desulfurases (CDs), leads to the formation of a persulfide group at the level of an active site cysteine. Then, persulfide trafficking implies the existence of sulfur carrier proteins, so-called sulfurtransferases (STRs) which catalyze the transfer of a sulfur atom to nucleophilic acceptors. Despite its importance for plant physiology, the molecular mechanisms of sulfur trafficking remain mostly unclear and compared to nitrogen and phosphorus metabolism, the study of plant sulfur metabolism still lags behind, although more attention has been paid to this pathway recently. This research project aims at deciphering the molecular mechanisms of the sulfur delivery and trafficking systems in plants. We will develop an integrated approach combining in vitro and in vivo approaches in order to perform a functional analysis of proteins which are involved in sulfur mobilization (cysteine desulfurases) and trafficking (sulfurtransferases). Thus, we propose to study the roles of CDs and STRs from Arabidopsis thaliana, notably those specific to plants or that have not been characterized so far to answer to three major questions: 1. Do CD/STR couples act as central hubs for the mobilization and trafficking of sulfur in plants? 2. How do STRs differentiate and interact with sulfur donors and sulfur acceptors? 3. Are STRs and their physiological partners involved in H2S biogenesis and trans-sulfhydration signaling mechanisms? This project will improve our fundamental knowledge on sulfur metabolism and will help mapping sulfur transfer events across pathways of the biosynthesis of sulfur-containing compounds and providing insights into the hierarchy of sulfur incorporation into biomolecules in plants. Finally, the results will also provide fundamental knowledge on sulfur metabolism at the molecular level, which is essential for providing interesting clues necessary for developing applications in the long run. More generally, a better understanding of the metabolic pathways related to plant growth and productivity is essential to build a sustainable agriculture system able to satisfy the food demand without impacting environment in a context of the increase of world human population.

Soils and wastes contaminated with heavy metals are prone to create major problems because of their toxicity and their management is generally expensive. But this drawback can be turn into advantage if these solid matrices contain compounds of industrial interest. However, metal concentrations are generally too low for conventional mining and metallurgical recovery. Hence, new extraction and processing technologies must be developed to ensure production of strategic metals, while preserving soil functions, and improving soil and waste quality by decreasing their toxicity. These processes would provide a range of economic, social and environmental values from materials and lands of initial low value. AGROMINE is the conception of agro-metallurgical production chains based on the culture of hyperaccumulator plants on contaminated matrices (soils, wastes) or naturally rich in metals (ultramafic soils) to produce high value metal compounds. These chains are developed for nickel (Ni) and cobalt (Co), metals of high strategic importance. They are based on a previous work devoted to the synthesis process of ammonium and nickel sulfate double salt hexahydrate (ANSH) from the biomass of Alyssum murale. They combine agromining (or phytomining) and hydrometallurgy. • Agromining is an alternative treatment for contaminated soils and wastes, and an application of phytotechnologies to exploit secondary resources. On soils naturally rich in metals it generates incomes for farmers or managers and metal removal improves soil (or matrix) quality. Here the main innovation is the production of hyperaccumulator plants on constructed agrosystems. • Hydrometallurgy produces metals with a niche strategy, seeking forms of Ni and Co of strong industrial interest. Focus is put here on Ni and Co carboxylates, which is completely innovative, but attention will still be given on Ni and Co salts for surface treatment. The AGROMINE project involves 4 research teams of Nancy (LRGP and CRPG, LIEC, LSE of Labex Ressources 21) and two SMEs (Soléo Services and Microhumus), which have a long collaboration history. It also has strong connections with joint activities between Labex Ressources 21 and ERAMET, a major French nickel mining company. ERAMET has expressed its interest for the project by providing a support letter. The consortium maintains regular contacts with the main international actors of phytomining: Albania (UAT), Québec (INRS-ETE), China (SYSU), Australia (CMLR-UQ) and the United States (USDA). Work is organized in 1 management task and 5 scientific tasks, including: 1. Characterization of matrices, including soils, sediments and sludge: new agrosystems containing metal contaminated matrices will be designed, characterized and prepared to grow hyperaccumulators; 2. Selection of hyperaccumulators and control of metal bioavailability to identify the best Ni and/or Co hyperaccumulators for each environmental condition and metal recovery; 3. Implementation of agromining at platform scale with constructed agrosystems; 4. Hydrometallurgy for metal recovery from biomass and production of high-value compounds, based on our experience on the patented synthesis of a Ni salt (ANSH), and focus on the preparation of Co salts and Ni and Co carboxylates; 5. Life Cycle Assessment and economic evaluation of the agromining chain as well as its transfer to the end-users. AGROMINE is intended to produce economic and social value from low value material and land. It is not planned to supplant conventional mining technologies. Contrary to popular belief, our field data have shown that this process makes profit: agromining on 4 000 ha producing 200 kg Ni ha-1 converted in ANSH would give an economic benefit of c.a. € 6.15 million per year. The results obtained in AGROMINE would be of great importance for the two SMEs and for the ECONICK start-up, which is currently in an incubating process in Nancy.

Plastic pollution might lead to the degradation of soils, with major environmental and economical costs for agriculture. Considering the multiple facets of plastic pollution (contaminant cocktails including additives and non-intentionally added substances NIAS, added alone in mulching or closely entangled with residual organic matter in amendments), this project will take a lead in assessing the extent of this threat and propose ways to remediate it. With a novel methodology based on a back and forth collaboration between polymer chemistry and soil ecology we will explore several exposure scenarios of soil organisms to custom-made plastics, deciphering their toxicity in different environmental compartments (rhizosphere, microorganisms, mesofauna, plastisphere), their impacts on soil functions and on biogeochemical cycles, their dynamics and that of plastic-associated microorganisms and the physico-chemical and microbial retroactions of soils on plastics.