Increased frequency and intensity of extreme climatic events are predicted in Europe, including severe droughts and intense precipitation events. Such changes will influence plant physiology and soil microbial activity, inducing changes in ecosystem functioning such as primary productivity, nutrient cycling and carbon balance. Thus, an improved mechanistic comprehension of ecosystem responses to altered precipitation patterns is required to adequately assess responses to future climatic conditions. This must be conducted within a research framework that can address couplings between biogeochemical cycles by integrating the plant and soil players and their interactive effects on soil nitrogen cycling. The effects of contrasting precipitation patterns will be addressed in an integrated, multidisciplinary approach combining state-of-the-art molecular microbiology techniques with stable isotopes approaches and biogeochemical methods. The overall goal is to understand the temporal and spatial couplings between i) precipitation patterns, ii) the structure and activity of the soil microbial community and the associated soil nitrogen transformations, and iii) plant water and nitrogen uptake, and how these couplings affect the stability of ecosystems functions. The proposed work will i) investigate which active microbial groups are most involved in the response of the plant-microbial system to precipitation patterns, ii) carry out temporally resolved investigations of precipitation patterns impacts on the outcome of plant-microbial competition for nitrogen and the coupling between plant water uptake depth and soil microbial activity, iii) assess the coupling between the stability of soil microbial community and of major functions that it performs, in response to altered precipitation patterns. The outcome of this research should enable valuable insights into the future implications of changes in European summer climate for soil nitrogen availability, and provide crucial information for the development of mitigation and adaptation strategies.

Nous avons récemment contribué à la première caractérisation sur le plasmalemme végétal de microdomaines membranaires de compositions protéique et lipidique distinctes de celles de la membrane plasmique (Mongrand et al. 2004). Ces microdomaines participent chez l'animal à de nombreux processus physiologiques mais, à ce jour, on ignore tout de leur(s) rôle(s) dans la physiologie de la plante. Le cœur de notre projet consistera à caractériser l'implication de microdomaines de la membrane plasmique dans la réponse de la cellule végétale à des éliciteurs de réaction de défense, en utilisant des approches complémentaires. Nous déterminerons par spectrométrie de masse la composition protéique globale des microdomaines et leur éventuelle modification lors du traitement de suspensions cellulaires par des éliciteurs. Les protéines susceptibles d'intervenir très précocement dans la transduction du signal d'élicitation, feront l'objet d'une analyse détaillée (suivi de l'expression génique et analyse fonctionnelle). Nous attendons de cette étude l'identification de nouveaux acteurs membranaires des voies de signalisation déclenchées par un éliciteur de réaction de défense. En complément, nous investirons dans une approche permettant de visualiser in vivo la présence de microdomaines sur la membrane plasmique et leur dynamique en réponse à différents stimuli. Nous développerons des marqueurs protéiques de ces domaines couplés à la GFP, et des sondes lipidiques permettant de repérer les domaines membranaires riches en stérols et en sphingolipides. Nous pourrons ainsi visualiser la compartimentation de la membrane plasmique en microdomaines et suivre la dynamique spatiotemporelle de ces microdomaines en réponse à un traitement éliciteur. Au delà des résultats attendus, ces travaux permettront de générer des outils utilisables par la communauté scientifique pour étudier le rôle de ce type de structures dans différentes situations physiologiques. Afin de démontrer le rôle joué par la « structure microdomaine » dans la signalisation, nous développerons des stratégies destinées à déstructurer ces microdomaines au moyen d'agents pharmacologiques. Cette déstructuration sera suivie à la fois par imagerie et par voie biochimique, et ses effets sur les différentes étapes de la transduction du signal d'élicitation seront analysés. Ces approches apporteront des informations sur la structuration des microdomaines et son influence sur la réponse des cellules à l'élicitation. Au delà de la seule caractérisation des microdomaines, nous étudierons la dynamique de la membrane plasmique dans son ensemble et son rôle au cours de la confrontation cellule végétale/éliciteur en associant une approche d'imagerie descriptive à une approche fonctionnelle. Nous étudierons en particulier, ce qui n'a a priori jamais été réalisé chez les plantes, l'effet de molécules capables de bloquer le trafic endosomal sur les différentes étapes de la transduction du signal d'élicitation.

Upon insertion in or next to coding regions, Transposable Elements (TEs) can impact gene expression and function in various ways, from contributing to proteic sequences to changing gene regulatory patterns. TE-generated variations significantly expand the functional potentialities of host genomes, bearing important consequences for the generation of phenotypic diversity. TEs themselves are subject to transcriptional and epigenetic regulations in response to developmental cues and external stimuli. As a consequence, host genes adjacent to TE insertions can be placed under the control of these TE responses, leading to reprogramming of batteries of genes as part of the organism's response to specific stimuli, hence a recent definition of TEs as "distributed genomic control modules". This is particularly true for LTR-retrotransposons, whose Long Terminal Repeats (LTRs) represent small autonomous promoter/regulatory capsules that can be functional at different genomic positions. In the last few years, a wealth of data from human and animal studies showed that LTRs mediate the coordinated regulation of numerous adjacent genes in response to developmental cues, via the production of LTR/gene chimeric co-transcripts driven from the LTR and extending into flanking sequences. LTRs may thus represent intermediates or "sensors" of specific stimuli, able to translate and redirect these messages towards adjacent cellular functions. This new and open field of research is still largely unchartered in plants, although large plant genomes are highly enriched in TEs (up to 80%), with LTR-retrotransposons being by far the predominant type. The objectives of this proposal aim at evaluating the functional and evolutionary importance of LTR-mediated reprogramming of plant gene expression in response to stress. The proposal builds up on very recent preliminary bioinformatic data reporting an abundance of LTR/gene co-transcripts produced by tobacco in stress conditions, and notably from the LTR of the best known plant retrotransposon, the stress-activated tobacco Tnt1 element, previously isolated and characterized by one partner of this project. These preliminary data provide a very strong base to support the hypothesis that activation of the Tnt1 LTR by stress, in addition to driving Tnt1 amplification, may also be involved in modulating the expression of several tobacco genes in stress conditions. In this project, we will evaluate the impact of LTR-driven co-transcripts on adjacent genes expression changes, and the precise correlation of these changes with the tobacco stress responses. We will notably evaluate the quantitative importance of LTR/gene co-transcripts produced in tobacco in various developmental and stress conditions and their molecular impact targeted genes, as well as their evolutionary importance. These studies will allow a global insight on the modifications of gene expression and/or function generated in tobacco by transcriptional activation of retrotransposon LTRs in response to stress. In addition, we will expand the significance of our studies by monitoring changes in the production (and possibly the impact) of LTR/gene co-transcripts in conditions where the retrotransposon transcriptional activation is affected, firstly by artificially altering the tobacco defense response, and secondly by artificially altering Tnt1 transcripts levels. To this aim, we will use transgenic tobacco lines in which the early signal transduction pathways elicited by microbial stress (such as fungal elicitins also known to activate Tnt1 expression) are defective, and transgenic tobacco lines expressing double-stranded and antisense Tnt1 transcripts designed to silence Tnt1 expression via the RNAi pathways. These complementary analyses will confirm that LTR-mediated changes in adjacent gene expression are directly correlated to the retrotransposon transcriptional activation and to the host stress response. The project represents a direct, original and timely valorization of previous competences established in the proposed partnership in the field of plant stress response and its fine interplay with retrotransposon activity. We expect that it will rapidly generate a wealth of experimental data from which will emerge, for the first time, a large-scale picture of the co-optation of retrotransposon LTR promoters by plant host genomes. This will allow a major breakthrough on the understanding of the biological impact of retrotransposons on plant host genomes, which may be of key significance for plant adaptability to environmental changes, as plants can not move to avoid these changes and have thus evolved complex and highly coordinated responses to biotic and abiotic challenges.