H2 is regarded as an attractive, clean energy carrier, but to date it cannot be produced using any “green” method. An option is to produce this gas by means of biological approaches, e.g using photosynthetic organisms that couple H2 production and water photolysis thanks to the biological catalysts of H2 production: hydrogenases. They are complex metalloenzymes which catalyze the reversible oxidation of H2 at a bimetallic (FeFe or NiFe) active site. The intense competition in the field of hydrogenase research is motivated in part by the potential use of these enzymes in biotechnological systems; therefore, research is more specifically focused on the inactivation of hydrogenases by O2, which greatly limits their applications. It can be envisaged to use rational protein engineering to render O2 tolerant the hydrogenases from photosynthetic organisms, on condition that the mechanism of O2-inactivation is understood. The spectacular results recently published by the partners of this project have stressed the need for maintaining efforts in studying the mechanism underlying O2-inactivation and O2-tolerance of NiFe hydrogenases. First, in Nature Chemical Biology in 2013, we challenged the commonly accepted mechanism of O2-inactivation, leaving open questions regarding the chemical structure of the inactive states. In PNAS and JACS in 2012, we have described a series of mutants of a sensitive enzyme whose tolerance to O2 was greatly increased by a mechanism which must be investigated further. Here, our goal is to use new biophysical methods and new biological systems to understand the mechanism O2-inactivation and O2-tolerance of NiFe hydrogenases, in order to eventually design O2-resistant NiFe hydrogenases that will be used for biological H2 production. The four teams of the two French labs and the three foreign participants involved in the project have already collaborated in a very prolific manner. They are experts in the field of metalloenzymes (particularly hydrogenases) and master the biochemical, spectroscopic, electrochemical and theoretical tools which they use for studying these enzymes. We will use the genetic system that has shown indispensable for routinely producing the sensitive NiFe enzyme from Desulfovibrio fructosovorans and tolerant mutants. We will develop the production, the purification and the physico-chemical studies of O2-tolerant hydrogenase from Salmonella enterica Typhimurium and E. coli. We will also capitalize on this expertise to develop an original and innovative field of research: the design and production of artificial, chimeric enzymes in which large structural motifs that are believed to be specific to naturally tolerant enzymes are transferred into the protein framework of the sensitive enzyme. We will develop and use new electrochemical methods, theoretical chemistry, and high resolution spectroscopic techniques to characterize and optimize the properties of these enzymes using the interdisciplinary approach that is the hallmark of the laboratory of bioenergetics and engineering of proteins in Marseille. Three distinct teams from this lab will collaborate on the project with their own approach (molecular biology, electrochemistry, EPR spectroscopy), and three foreign partners have been recruited to contribute their expertise (IR spectroscopy and theoretical chemistry). The large-scale production of the enzymes that are most promising in terms of applications will be optimized and carried out in the MIO laboratory, and then used in a ANR-funded network of researchers whose aim is to design biofuel cells (CAROUCELL ANR 2013 BIOME). The information obtained from these studies will benefit the rational engineering of the NiFe hydrogenases from photosynthetic organisms such as cyanobacteria, making it possible to design biological systems of production of H2 using light and water as source of energy.

Biodiversity is influenced by a wide range of external environmental pressures. Some direct impacts are easy to observe, whereas others are far less obvious and therefore more difficult to understand. In 2008, the EC estab-lished a framework for community action in the field of marine environmental policy regarding biodiversity: the Marine Strategy Framework Directive, MSFD, which defines a series of 11 qualitative descriptors of Good Envi-ronmental Status (GES). A range of specific pressures are treated within these separate descriptors but most also impact biodiversity in some way. A knowledge-based monitoring strategy is then recommended. Targets for ac-ceptable status, or “GES”, must be set according to the conditions relevant to each area. The overall aim of the Marine Directive is to achieve a sustainable balance between human needs and the natural environment across the European seas. Monitoring all aspects of biodiversity across the whole European seas is neither possible nor an acceptable use of resources. The ecological, socio-economical and patrimonial importance of the Mediter-ranenan coralligenous ecosystem and its variability all around the Mediterranean Sea give the highest priority to its knowledge, understanding and protection for a sustainable use. Moreover, as a coastal system, it is under endlessly increasing pressures. Its complexity and richness of the associated fauna are potential sources for numerous indicators. CIGESMED will design some new tools based on coralligenous for this perspective. Biologically mediated habitats are structured formations on a seafloor provided by marine living organisms alone (often engineer species) or incrusted by inorganic materials of biogenic origin. These habitats can be built by multi-layered aggregations of organisms to form meadows, colonies, banks or beds, mosaics and patches or accumulations of hard structural elements. These complex biological habitats offer a range of favorable hiding places, are vital reproduction and nursery areas, have protein-rich and easily accessible food resources for ben-thic invertebrates and fish, and are easily exploited by humans due to their high biodiversity and species richness. In the Mediterranean Sea, coralligenous and Posidonia meadows are the most important biologically me-diated habitats. They are the main milieu to generate the structural complexity and biodiversity, as well as for the functioning of these ecosystems. Posidonia meadows were extensively studied whereas coralligenous which is a typical hard bottom Mediterranean underwater seascape is comparatively less so. Feldmann (1937) was the first to unequivocally describe the algal composition of the coralligenous and identify the main calcareous algae mak-ing the coralligenous assemblages. Two main different morphologies are traditionally described: banks, over more or less horizontal substrates, and rims, in the outer parts of marine caves and vertical cliffs (Natura 2000 – sheet 1170.14, Barcelona Convention 2007, IV.3.1). This typical coastal benthic Mediterranean environment lies, de-pending of light conditions, from ca. 15 to 130 m depth. It expands in the circalittoral zone and also develops in the infralittoral zone. These assemblages are one of the most important hot-spots of species diversity in the Mediterranean, together with Posidonia oceanica meadows but which are by far more studied. As mentioned by a recent Mediterranean Action Plan-MAP’ working group13, even if an overall knowledge about its composition and distribution in the NW-Mediterranean basin, the distribution of coralligenous populations (lack of maps), their structuring, functioning and threats are important lacunae from the conservation point of view.

We propose to characterize the response of continental biosphere to large extraterrestrial impact craters, in particular the megafires they may have generated, associated with the melting of the Earth surface. Studying the impact glasses ejected at long distance, called tektite, will be instrumental in the project. Three tektite producing impacts occurring on forested tropical surfaces from Nicaragua, Ghana, and Indochina during the last 1.1 Myr will be investigated. Environmental response to impact, probed by pollen, charcoal and other proxies will be studied in various marine and lacustrine archives. Reaction of impact melt with biomass (elements such as C, P, S) will be traced using compositional variability of tektites explored in a systematic way, as well as nano-scale characterization of inclusions in tektites such as phosphates, metal, iron oxydes, and eventual carbon phases. This will bring new clues on tektite production processes and response of ecosystems to megafires.

Intensified drought is of a special concern in the Mediterranean region because scarce precipitation is already a threat to ecosystems. The response of Mediterranean vegetation to extreme water deficit conditions remains poorly understood because fully-controlled field experimentation to mimic such conditions is difficult and costly. A significant aspect of plant response to changing drought conditions is the alteration of the relation between primary and secondary metabolisms. On the one hand, increased drought stress is expected to trigger loss of primary productivity and greater sensitivity to disturbances. On the other hand, many species substantially develop their defence mechanisms such as their plant secondary metabolism (PSMs) under intense stress conditions. Both factors together determine the functioning of the ecosystem under the conditions expected later during the current century. SEC-PRIME² aims to use a combined experimental and modeling strategy to investigate drought impacts on the functioning of a Mediterranean forest ecosystem. Its originality is to focus on plant responses to rainfall restriction through the estimation of both plant primary metabolism (PPMs, e.g. plant productivity) and PSMs, as well as the trade-offs between them. PSMs may act as supplementary defences against abiotic factors (e.g. high temperature, high radiation levels, drought), which result in cell damage caused by cell oxidation, strongly scavenged by PSMs. In order to cope with drought episodes, plants may consequently allocate their resources to PSMs production rather than PPMs and thereby increase their survival likelihood in the ecosystem. As a result, modifications of carbon allocation in response to environmental constraints could impact the carbon cycle of the entire ecosystem. Moreover, PSMs are recalcitrant for microorganisms and, consequently, an increase of litter PSMs concentration could decrease litter mineralization rates. Thus, input of carbon into the soil and modified heterotrophic respiration would all impact the carbon exchange with the atmosphere and the amount of carbon stored in the ecosystem. SEC-PRIME² is structured in three main tasks. Task 1 will study the “Benefits and draw-backs from forest investment on PSMs under intensified drought at the tree level”. Additional investment in PSMs could result in increased defences for the plant with detrimental consequences for forest productivity. At the ecosystem level, task 2 will evaluate whether such secondary metabolites, in leaf litter, affect decomposer communities (i.e. soil microorganisms and fauna) and then litter decomposition, resulting in an indirect effect on nutrient availability for plants with the consequent feedback on PSMs and PPMs at the tree level. Finally, task 3 will use a generic ecosystem model (ORCHIDEE) to estimate the impact of plant investment in PSMs on the carbon budget, which will permit regional and longer-term scenario assessments of ecosystem structure and integrity. The main hypothesis of SEC-PRIME² is that a drier climate could lead to a shift from primary to secondary metabolism in leaves. Moreover, higher loads of PSMs in litter could induce a decrease of the litter mineralization rate as PSMs are considered as more recalcitrant material. The altered litter composition could modify the input of carbon into the soil impacting the carbon exchanges with the atmosphere and the carbon budget. The chosen forest system is Quercus pubescens forest in Southern France, which is assumed to be particularly responsive to climate change. Over a period of several years, we will experimentally reduce rainfall under otherwise natural conditions at the O3HP platform through a dynamic rain exclusion system. This type of study is essential to understand the mechanisms behind potential shifts in species composition and survival rate, and to develop quantitative tools to characterize the vulnerability of Q. pubescens ecosystems at the local and regional scale.

During the classic period (AD 250-950), the Maya interacted with many neighboring cultures, however, little is known about their interactions with more distant societies. Important quantities of iron ore mirrors and jade plaques, associated with the Maya elites, were thus found in Costa Rica some 1000 km away (AD 300-700). In the Maya area, Spondylus shells were often found in elite burials, but are absent from the Pacific coasts close to this area and abundant in the waters bordering Costa Rica. This project proposes to bring together archaeologists, archaeometers, traceologists and archaeozoologists to determine the areas of origin of these prestigious goods and to understand the nature and modalities of the relations between these regions. We will review all the available archaeological data on the studied objects in order to document their contexts and chronological frameworks. The study of their provenance will involve the chemical analyses of the stones and adhesives by a set of spectroscopic, chromatographic, datation and isotopic techniques. The manufacturing microtraces of stones will be characterized using traceology in order to understand the lapidary techniques and to determine whether they were imported or locally worked. Spondylus materials found in the Maya area will be submitted to malacological and isotopic analyses in order to distinguish their geographic sources. The iconographic and epigraphic study of the corpus of plaques and mirrors will enable us to document their origin, date and status in the Maya area. Finally, all data will be cross-referenced by being placed in geographic and chronological contexts in order to understand the distributions, productions, reuse and functions of these goods in the two regions. This project will enable us to rethink the old distinctions between what is called Mesoamerica and what is often considered as an "intermediate area" in order to define the entities composing them in a far more dynamic fashion.