Symbioses between arbuscular mycorrhizal (AM) fungi and plants have significant effects on plant biodiversity and carbon cycling, and are therefore of considerable agricultural importance. The discovery that strigolactones, which are secreted by plant roots, are root-recognition signals for AM fungi, raises important questions concerning the mechanisms by which strigolactone signalling is regulated and how these signals are perceived by AM fungi. Experimental evidence indicates that, prior to colonizing plant roots, AM fungi produce one or several diffusible molecules collectively called 'Myc factors'. The roles played by strigolactones and Myc factors are hypothesised to be equivalent to those played by flavonoids and bacterial Nod factors (NFs) in the establishment of the nitrogen fixing symbiosis between legumes and rhizobia, with Myc factors being responsible for recognition by host plants of their symbiotic partners. The mechanisms underlying early mycorrhizal signalling are not well understood, though by testing symbiotic mutants of Medicago truncatula affected in genes that control steps of NF signalling, it was discovered that the three so-called DMI (Doesn't Make Infections) genes also control steps of mycorrhizal signalling, thus defining a common symbiotic pathway. Given that there are many differences between the bacterial and fungal symbiosis, we can hypothesise that there are plant components specific to the AM symbiosis yet to be identified, both upstream and downstream of this pathway. Since projects are underway to characterise Myc factors, it is timely to build on the discovery of the common symbiotic pathway and exploit our pioneering expertise in both NF signalling, and characterisation of plant responses to Myc factors. The objectives of this project are to achieve breakthroughs in understanding whether strigolactones and Myc factor signalling are connected in a regulatory loop and how Myc factors produced by AM fungi activate their host plants for mycorrhization. The originality of the project is that it will use different approaches and tackle different steps of Myc factor signalling. The project is also highly original since we will dissect Myc factor signalling using active fractions, and pure Myc factors as soon as they are available. Throughout, we will focus on a single plant species, Medicago truncatula, on which most of the pioneering analysis of Myc factor signalling has been performed. This will allow us to simultaneously unravel the extent of specificity and overlap between Myc and Nod signalling. To achieve these ambitious aims we will unite the complementary expertise of two partners in a multidisciplinary approach involving molecular genetics, transcriptomics, proteomics and biochemistry. The project will benefit enormously from the unique pioneering experience of the partners in mycorrhizal and NF signalling. The project will also benefit from the recent, considerable development of genetic, genomic and transgenic approaches that have endorsed M. truncatula as a model, and are making functional genomics and cloning projects feasible in short periods of time. To identify new components that could intervene at different stages of the Myc signalling pathway, we will use genetics and transcriptomic approaches. The genetic approach is a forward screen for mycorrhization-deficient mutants that will be tested for responsiveness to Myc factors. Interesting new genes will be cloned by transcript-based cloning. The transcriptome approach will use Affymetrix chips carrying 50,000 M. truncatula sequences, combined with a plant genotype that has a supermycorrhization phenotype and increased responsiveness to mycorrhizal signals. To extend the Myc signalling pathway upstream and downstream of the common signalling pathway, we will target Myc factor perception and the point of divergence of the Nod and Myc signalling pathways at DMI3. Candidate Myc factor receptor genes will be selected from the LysM-RLK gene family of M. truncatula and tested by reverse genetics. Targets of DMI3 will be searched for by a proteomic approach combined with a truncated, constitutively-active form of DMI3. The transcriptomic approach will be combined with plant genetics to determine which genes are involved in the Myc factor-activated signalling pathway that leads to the stimulation of lateral root formation. Characterisation of selected genes will help elucidate the question of how symbiotic signalling intercepts the plant developmental pathway for root development. Finally, we will analyse the molecular dialogue between plant roots and AM fungi, including the use of bio-assays combined with biochemical analyses and relevant mutants to test the hypothesis of a regulation loop linking strigolactone and Myc factor production. Given the known hormonal role of strigolactones in plants, the consequences on plant development of a putative control of their production by Myc factors will also be examined.

The type I polyketide synthases (PKS) are giant, multifunctional enzymes. They are responsible for the biosynthesis of a full variety of natural compounds, the so-called polyketides. These compounds are involved in important biological processes such as the cell wall biogenesis or pathogenicity of various bacteria. Some polyketides also exhibit very interesting and widely used pharmacological properties (antifongal, antimicrobial, immunosuppressive, antitumoral') The polyketide synthases catalyse the elongation of a substrate using several catalytic domains and a wide range of starter and extender units. The number and the nature of the domains present on a PKS will determine which type of polyketide will be synthesized. Such a concept that relies both on molecular recognition and iterative processes may be called molecular programming. The aim of this project is to establish the molecular and structural bases that are responsible for the molecular programming of the PKS. To achieve this goal, three teams of the « Institut de Pharmacologie et de Biologie Structurale » decided to share their scientific expertise within a consortium. These teams have complementary skills in molecular biology and protein engineering (Team C. Guilhot), analytical and structural biochemistry (Team M. Daffé), and structural biophysics and biology (Team L. Mourey). A coherent set of PKS produced by Mycobacterium tuberculosis has been selected for this project. Deciphering the structure-function relationships of complex enzymatic machineries constitutes a fantastic challenge. It should be noticed that, to date, no structure of such a PKS has been solved. Several complementary strategies have been set up to study these enzymes. We propose to solve the structures of the full-length proteins using X-ray crystallography. To overcome eventual difficulties in crystallizing these large enzymes, we also intend to work on the various catalytic domains or fragments of the proteins, whose limits will be determined using several approaches. Thus, we will use a hybrid approach where structural information obtained at low resolution for the entire proteins will be combined with the high-resolution crystal structures of individual domains. The structural information will be combined with data deduced from complementary enzymology and molecular biology studies that comprise the elaboration of enzymatic assays and the study of ponctual mutants of the selected enzymes. This original and ambitious project will generate new and important results with respect to the structural organization and molecular programming of complex enzyme systems, paving the way for the rational modification of these enzymes. The complementarity and the synergy of the three implied teams are attested by a set of joint publications and contracts of collaboration. In addition, preliminary experiments and validations were realized on some of the selected PKS.

The CCAAT-box is a ubiquitous cis-acting regulatory element found in approximately 30% of eukaryotic promoters. The major protein complex binding this motif is the CCAAT-box binding complex (CBF), also called HAP (for Heme activator protein) in yeast and plants and nuclear factor-Y (NF-Y) in animals. The HAP complex was extensively studied in animal systems where it was shown to be composed of three subunits: HAP2 or NFY-A, HAP3 or NF-YB and HAP5 or NF-YC. In mammals the HAP complex is required to activate developmentally-regulated genes, in particular during the cell cycle and was shown to be a central regulator of cell proliferation and early development. In addition a major characteristic of NF-Y is its ability to interact with other key transcriptional regulators thereby controlling specific developmental pathways. In plants, although evidence for the importance of HAP protein complexes is gradually accumulating, much less is known about the structure and function of the HAP complex as well as of the individual transcription factors belonging to the CBF family. Like their homologues in mammalian systems, plant genes coding for HAPs can be classified in three groups, namely HAP2, HAP3 and HAP5. In contrast to their animal counterparts however, which are encoded by a single gene, the plant HAP genes have diversified and are encoded by small gene families of around 10 members. With so many individual HAP genes, plants have the potential to generate large numbers of HAP trimer combinations that have potentially been recruited into a wide range of processes including plant specific pathways. Indeed HAP genes have been implicated in early embryo development but also in flowering time control, drought tolerance, blue light and ABA signalling and chloroplast biogenesis. Our work provided the first evidence for a role of a HAP gene during the symbiotic interaction between soil bacteria known as Rhizobia and leguminous plants. This interaction results in the formation of a new organ called nodule on the root of its host plant and inside which atmospheric nitrogen is fixed for the benefit of the plant. We identified MtHAP2a by a transcriptome analysis as strongly and specifically expressed during early stages of nodule development and showed that, in mature nodules, Mt HAP2a expression is specifically restricted to the nodule meristematic zone. We established that MtHAP2a is by far the major HAP2 gene expressed in nodules. However several other MtHAP2 genes are also expressed in nodules. Using both RNAi and mutant analysis we have then shown that MtHAP2a plays a central role during both early symbiotic signalling and nodule meristem functioning and maintenance. This project aims at understanding the regulatory network by which MtHAP2a controls several important steps of the symbiotic rhizobium-legume interaction by indentifying both its partners inside protein complexes and the target genes whose expression is regulated by this transcription factor. For this purpose the present project is structured into three synergistic tasks. Task1: We will characterise the Medicago truncatula HAP genes expressed during nodulation. First an in situ expression analysis will unravel their tissular expression patterns. By looking for genes co-expressed with MtHAP2a we will identify HAP2 genes potentially playing complementary and/or additive roles, but also HAP3 and HAP5 genes coding for potential interactors inside symbiotic heterotrimeric HAP complexes. A functional analysis using RNAi and mutants will then reveal the potential role of co-expressed HAP genes. Task2: We aim at identifying and characterising the protein partners of MtHAP2a both the HAP3 and HAP5 partners and other transcriptional regulators. Screenings and tests will be performed using the yeast two hybrid system. For validation of interactors we will use a combination of bi-molecular fluorescence complementation (biFC), Fluorescence Resonance Energy Transfer technology (FRET), and co-localisation experiments using immunocytochemistry. Task 3: To identify target genes we will compare the transcriptome of wild type, Mthap2a-1 KO mutants and of plants overexpressing MtHAP2a using a Dexamethazone-inducible system. This will be complemented by PCR-assisted binding site selection (Selex) to determine the specific binding sites of this TF, combined with a bioinformatic approach. Validation of targets will be performed using electrophoretic mobility shift assays (EMSA) and immunoprecipitation (ChIP) assays. In addition to revealing the function of a nodal symbiotic regulator this project should, by understanding how MtHAP2a regulates nodule meristems, give insights into fundamental aspects of cell cycle control and cell differentiation during organogenesis. The present project should also enable us to show that, beyond nodulation, HAP2 proteins function in plants as regulatory HUBS connecting several signalling and developmental pathways.

Mass-independent isotope fractionation (MIF) effects in natural terrestrial environments were discovered twice during the 20th century - for the elements oxygen and sulfur. Both discoveries have opened a broad variety of applications, including physical chemistry studies, atmospheric chemistry, palaeoclimatology, biologic primary productivity assessment, Solar System origin and evolution, planetary atmospheres (Mars), and the origin and evolution of life in Earth's earliest environment. Recently mercury (Hg) was added to this shortlist when isotopic anomalies were observed for Hg's two odd isotopes, 199Hg and 201Hg in biological tissues. The objective of the MERCY project is to take Hg MIF beyond the initial discovery, and use it to address major outstanding scientific questions of societal and philosophical interest. We propose to use mercury MIF to better understand global Hg dynamics at different time scales, from the early Earth to Pleistocene and modern times, and in a context of global environmental change. Three main themes will be investigated: 1.The modern Hg cycle focusing on: emissions from coal burning, recent atmospheric Hg deposition in the Arctic from lichens, moss and peat, and recent Arctic Ocean Hg records from archived biological tissues. 2.Post-glacial atmospheric Hg deposition from up to 14,500 yr ombrotrophic peat deposits from the Pyrenees and Jura mountains, and minerotrophic peat deposits from the Candadian and Greenland (sub-) Arctic. 3.Palaeo-Hg dynamics spanning the 2.45 Ga 'great oxidation event' and the 635 Ma snowball Earth glaciation. The MERCY project places strong emphasis on the Arctic environment, because despite long-standing scientific and political interest, the reason for the high Hg levels in the Arctic ecosystem remains unknown, and recently climate change has entered the debate. Our preliminary work on the atmospheric and marine Arctic Hg cycle suggests that Hg MIF can put powerful constraints on key Hg sources and processes, such as anthropogenic Hg emission fingerprinting or marine Hg photo-reduction under the influence of climate change. Another point of focus is on the isotopic traceability of Hg emissions from coal burning. Coal burning currently represents 67% of all anthropogenic emissions (41% from Asia alone), and is likely going to increase in coming centuries. Recent evidence of variable Hg MIF signatures in coal suggest that theses can be traced upon atmospheric dispersion. In addition to modern, recent, and Pleistocene Hg records, we propose a journey into deep time and the origins of life on Earth. We will probe mercury MIF anomalies in ancient marine sediments as a proxy of atmospheric Hg chemistry, notably the dynamics of the Great Oxidation Event (GEO), when free O2 first accumulated in the atmosphere, and the last snowball Earth events, which spawned another oxidation event and the early origin of animals. By studying ancient Hg MIF dynamics during the 635 Ma global glaciation, we may be able to better understand our modern Hg MIF ' climate observations and those proposed on recent and post-glacial time scales. It is highly likely that MIF will be discovered in natural environments for other elements, and the understanding we gain by investigating mercury will accelerate the future application of other MIF processes and open up new horizons and opportunities. By tapping information from the isotopic dimension of Hg cycling, including revolutionary mass-independent effects, we expect a maximum scientific impact while supporting a socially relevant and urgently needed investigation at the frontier of isotope geosciences.

The presence of water (or hydrogen) in nominally-anhydrous mantle minerals, as well as its influence on the chemical and physical properties of these minerals, has been known for almost twenty years. Nevertheless, the total amount of water stored in the mantle is still very poorly constrained and this value is a crucial parameter in understanding the effects of water on the Earth’s dynamics. The poor knowledge of hydrogen concentration in the mantle comes partly from the fact that, strangely, the thermodynamics (solubility, diffusion coefficients, etc.) at mantle conditions of H in olivine, probably the most studied mantle silicate, and its high-pressure polymorphs is also poorly known. In this project we propose to explore this topic by associating experiments with theoretical works. Cutting-edge experiments will be performed, at both room and high pressure, in order to measure the self-diffusion coefficients of H in the three phases, forsterite, wadsleyite and ringwoodite. The pure-Mg end-member of olivine, forsterite, allows to run H self-diffusion experiments at room pressure. Fe-bearing samples would necessitate more time-consuming high-pressure annealing. On the other hand, experiments on wadsleyite and ringwoodite, have to be performed at high pressure, in a large-volume multi-anvil apparatus. H concentration will be measured using infrared spectroscopy, with spectra collected after various annealing durations that will then permit to calculate the hydrogen self-diffusion coefficients. These IR measurements will be obtained on an infrared microscope available in our laboratory and also at the infrared synchrotron facility “Soleil” in Orsay for the shorter profiles. In parallel, computer simulations, atomistic and ab initio, will be used in order to investigate the microscopic processes involved in H diffusion and related point defects in forsterite first, and then wadsleyite and ringwoodite. Atomistic calculations will be done with the code GULP. Because this method is computationally fast with today’s calculators, we will be able to explore a wide range of H-related defect configurations and diffusion paths. Calculations using a quantum approach will be performed with the code SIESTA. These are much more computationally demanding but also much more precise (or exact) than the atomistic approach. They will run on national facility supercomputers and be applied to specific key points found from the GULP calculations. Results of both theoretical and experimental approaches will be compared and will allow to propose a coherent point-defect model explaining H defects and migration in forsterite. We will later investigate if this model also applies, with some adjustments, to Fe-bearing olivine. Theoretical calculations will also be applied to the understanding of the effect of hydrogen on the mobility (and related defects) of other major cations, Mg, Si. The ultimate goal of this project is then to model the upper-mantle electrical conductivitiy profile, linked to H diffusivity via the Nernst-Einstein equation, using our data. We will then compare this model with magnetotelluric measurements. It will follow an estimate of the total amount of water present in the upper mantle, a fundamental result for modelling and understanding mantle dynamics. This project has been already submitted in February 2008 to the programme “Blanc” and was selected in the reserve list.