At the heart of bioprocesses the activity and the physiological state of microorganisms are variables still difficult to assess. Most of the information is obtained from delayed off-line measurements and remains insufficient for the development of real time control strategies to optimize the potential of micro-organisms and design high performance processes. On-line quantification of the physiological state of cells is paramount for the understanding and improvement of cell metabolism and thus to control pathways of interest. The main objective of SPECTRE is to develop an on-line system able to monitor the physiological state of microorganisms during fermentation or cell cultures. SPECTRE is partly in continuity with the work developped during the previous ANR FASST program (Programme ANR-06-BIOE-003-01-FASST : Fermentation Alcoolique d'hydrolysats lingo-cellulosiques et obtention de Souches adaptées aux Stress Technologiques). During FASST, advanced methods for the determination of yeast strain viability state were developed. In association with off-line data, on-line dielectric spectroscopy was able to track variations of cell cytoplasm conductivity and microscopy image analysis showed that cell size distribution and cell optical properties were strongly correlated with yeast cell viability. The results of the program have been positively evaluated by the ANR and ADEME expert boards. Dielectric spectroscopy (DS) has been operational for the last ten years. This technique is now routinely used in a number of cell culture and fermentation processes for the determination of biomass concentration. However, it can also give access to informations dependent on the biomass state, but has to be completed by additional techniques to access the value of biologically significant variables. The determination of total cell volume, viability, and cell size are required to calculate the membrane capacitance Cm, representative of the cell enveloppe state, and the cytoplasmic conductivity si, a marker of water and ion exchanges between cells and their environment. Off-line measurements, on samples taken during fermentation or cell cultures, give a differed access to the information provided by the DS and are not suitable for online control. The proposed SPECTRE project is based primarily on : - the study of a coupling of two innovative technologies - spatially resolved optical spectroscopy (SRS) and dielectric spectroscopy (DS) - for the online determination of cell physiological marker variables (size, membrane capacitance, intracellular conductivity...). - the implementation of associated measurement (quantitative microscopy, flow cytometry, optical density, fluorescence, ...) which will allow - the validation of the information collected by DS and SRS, and - the selection of the most relevant additional physical variables (and their associated measurement techniques) eventually able to further improve the robustness of the physiological state evaluation of the cultivated populations. The project will lead to the development of generic tools allowing the real-time control of the physiological state of microbial populations. SPECTRE connects six academic teams expert in Bioprocess Engineering and an SME, leader in the SRS domain and in the associated data analysis techniques. Each team will use cell models chosen both for their established academic and industrial interest.

Gene flow has long been considered to take place within species only but we now realize that it often occurs between species as well. We still don’t know, however, how much gene flow effectively affects the genome of hybridizing species in the late stage of speciation. Such hybridization may be a source of adaptive genetic variation via the transfer of adaptations from the genome of one species to another, a phenomenon called “adaptive introgression”. While there are a few known prominent examples, its overall importance for adaptation is still largely unknown. In this project, we address the following main questions: i) how much of the genome is affected by introgression and ii) what proportion of introgression is adaptive? We have selected the Iberian wall lizard species complex because they have accumulated substantial genomic divergence; in spite of strong barriers to gene flow, nuclear and mitochondrial introgression still occurs; a transcriptome from our model and a reference genome from a close relative are available and we know their distribution, ecology and climatic niches. Last, we already have over 1000 tissue samples so sampling will be limited to additional locations specifically targeted for this project. To achieve this, we will use whole-genome sequencing to quantify the proportion of the genome affected by admixture. We will then quantify which proportion of introgressed genome is better explained by positive selection. To do so, instead of trying to pinpoint which genes have been experienced adaptive introgression, we will develop a theoretical study using simulations to establish the neutral variance in admixture rates among loci then estimate which proportion of admixture events cannot be explained by neutral processes (see Task 4). To overcome some of the limits of purely genomic approaches, we also propose an ecological test of the adaptation hypothesis based on candidate genes for climatic adaptation (mitochondrial DNA and the nuclear genes of the OXPHOS chain) in populations living in contrasted climatic conditions (Task 5). We will sample several pairs of populations within each species, each pair being composed of one population located in highly suitable climatic areas and the other in areas where climatic conditions resemble the climatic niche of a hybridizing (donor) species. Finding more loci that have been subjected to introgression in areas that resemble more the climatic conditions of the “donor” species would support the role of adaptive introgression. Tasks 1 & 2 We will model the current realized climatic niche in all lineages. We will then sample populations in locations (2 per species) of high climatic suitability for the focal species and in the heart of their distribution and in locations (2 per species) where climatic suitability is higher for the other species that hybridizes with the focal species. Task 3 We will obtain WGS data from 3 individuals in each sampled population (6 per species, 6 species). Task 4 We will establish by simulation the neutral variance in introgression levels between nuclear loci in the absence of selection. This should give us the limits of the variation that can be reached between loci in terms of introgression level in absence of selection and allow developing methodological tools to identify loci that have been subject to adaptive introgression. Task 5 We will identify introgressed genomic regions using already published methods then apply results from task 4 to test our idea that the proportion of loci affected by adaptive introgression (the proportion of high-frequency introgressed alleles that cannot be explained by neutral processes) is higher in areas where climatic conditions are closer to the climatic niche of the species which “gave” its genes through introgression, both for the whole genome data and for the OXPHOS genes and mtDNA.

In Western countries, diseases related to foods represent a major issue in public poliy. Overweight and obesity are increasing at an alarming rate in the world and in Europe more particularly. Obesity is one of the most serious public health problems because it increases significantly the risk of many chronic diseases such as cardiovascular disease and type 2 diabete. Nutrition is a major health determinant and is one of the key priorities in public health policy, especially in Europe. The comsumption of cereals-based foods with low glycemic indexes, high micronutrients and fibers contents are highly recommended. The target of this work, is to provide new solutions for cereal based foods: the knowledge and understanding on the in vivo fate will be used to define structural features to gain in foods. The objective of this proposal is to use new genetic resources and to assess the role of the role of viscosity on gastric emptying and the kinetic aspects of starch digestion. The digestibility of starch in foods varies widely and can be affected by high content of viscous soluble dietary fiber constituents and relatively high amylose / amylopectine ratios. Amylose content also influences some functional properties of starches like swelling power, solubility, in vitro glycemic index and viscosity. Thus, the strategy of this work is based upon the complementarity of the research teams and upon the integration of various scientific disciplines, from genetics of wheat grain to the human subject while passing by in vitro and animal studies. Natural biodiversity present in a core collection of bread wheat (Clermont-Ferrand) will be examined in order to bring out new wheat varieties containing high amylose contents. These varieties will be selected by the use of molecular and biochemical markers, by phenotyping using a new experimental device based upon image analysis of seeds sections, by biochemical analyses and by nutritional investigations. Viscosity of the ingested meal, gastric function in vivo and the nutritive impact of cereal products with high amylose content will be evaluated using an artificial stomach, a porcine model and a human panel.

Nitrate is the major mineral anion in cultivated plants and is essential for their N-nutrition. Although its uptake from the medium is energetically costly, numerous abiotic stresses (mechanic shocks, salinity, medium acidification...) induce a net root NO3- excretion into the medium. The physiological significance of this excretion remains obscure. It is nevertheless aknowledged that anion excretion is a major determinant for the control of electric polarization (and related signalization pathways) and osmotic potential in plant cells. For instance in guard cells, anion excretion leads in fine to stomata closure. The molecular knowledge of transport systems responsible for cellular efflux of NO3- in plants is scarce. One of the 3 partners of this project identified NAXT1 (NitrAte eXcretion Transporter 1), the first NO3- efflux transporter at the plasma membrane of plant cells (Segonzac et al., 2007, Plant Cell). NAXT1 is responsible for the massive root NO3- excretion to the external medium, triggered by acidification stresses. In Arabidopsis, the NAXT family encompasses 7 closely related genes whose expression is detected in roots at different levels. Upon salt stress, NAXT2 (another member) was found to be involved in NO3- excretion in the root stele and its translocation to shoots. Recently, one of the partners recently observed that NAXT1 and NAXT2 are also expressed in leaves, at the level of guard cells that constitute stomata. A first stomata-related phenotype was uncovered in salt stress conditions for a NAXT2 KO mutant (enhanced leaf transpiration rate compared to wild type plants). Also, NAXT1 and NAXT2 gene expression is regulated by the stress hormone ABA and salt stress. These first observations are of importance because stomata constitute an essential control point for plant tolerance to agronomicaly important stresses such as salinity and drought. For these reasons, it is proposed in the present project to study the role of NAXT transporters in stomatal movements in Arabidopsis, with the aim to uncover new plant tolerance factors to these stresses. In line with this proposal, the composition of our consortium allows to associate recognized expertise in (1) electrophysiological analyses of transport systems [Montpellier, BPMP], (2) anion channel and stomatal activity [Cadarache, IBEB-LEMS], and (3) Integrative biology of plants under environmental constraints [Montpellier, LEPSE]. First, the expression of NAXT genes will be searched for in guard cell RNAs extracted from plants submitted or not to treatments of interest (salt and water stress, ABA). Transport properties of selected NAXT members (expressed in stomata) will be characterized electrophysiologically after expression in Xenopus oocytes, and their expression profile in stomata will be documented at the gene and protein levels (in NAXT:GFP plants) in response to stresses and ABA. Then, NAXT underexpressing mutants will be phenotyped in response to stresses on their stomatal activity (aperture measurements, electrophysiology in situ) and by an integrative approach (tolerance to stresses in terms of growth/development, leaf transpiration rate) using a phenotyping platform (Phenopsis) developped by one of the partners. Beyond the deciphering of the biological role of the NAXT family, it is anticipated that this project will allow a better understanding of the beneficial role of nitrate supply observed at an agronomic level to plants under major abiotic stresses limiting production and thereby, to uncover new determinants of their tolerance.