High ambient temperatures due to climate change impact plant growth and survival. Recent data indicate that chromatin modification is an essential process of gene expression reprogramming during plant response to elevated growth temperature. Histone deacetylases (HDAC) that regulate histone acetylation levels were shown to play important roles in plant adaptation to environment. In addition, HDACs are also involved in deacetylation of non-histone proteins such as metabolic enzymes and transcription factors to control their activity. Our preliminary data indicate that Arabidopsis HDAC members play distinct roles in plant response to high ambient temperature. In addition, we found that high ambient temperature alters plant cell redox environment and that cellular redox environment regulates HDAC subcellular localization and deacetylase activity. The objective of this proposal is to elucidate the molecular mechanisms of interplay between cellular redox and chromatin modification that regulate plant response to high ambient temperature. In particular, the project aims to elucidate how cellular redox environment regulates HDAC activity to control lysine acetylation of histone and non-histone proteins involved in either epigenetic regulation of gene expression or metabolism and/or signaling during plant response to high ambient temperature, and to identify and study implicated redox regulators. We propose to first identify redox post-translational modifications of Arabidopsis HDACs under normal and high ambient temperature conditions by using biochemical and mass spectrometry approaches. Then, we will examine effects of redox modifications on HDAC enzymatic activity, subcellular localization, and function in plant response to high ambient temperature thanks to plants expressing tagged HDAC proteins in wild-type and mutated versions of redox-sensitive residues. Then, we plan to identify and validate redox regulators involved in modifications of the HDACs by both biochemical and genetic approaches and to study the role of redox regulators in plant response to high temperature. Next, we will study the effects of redox modifications on HDAC epigenetic function in terms of chromatin structure, genome-wide histone modifications, DNA methylation and gene expression by high throughput sequencing and cell biology approaches. Finally, we will investigate the effect of redox modifications on HDAC functions in regulating lysine acetylation of non-histone proteins in order to identify HDAC-regulated key metabolic enzymes and signaling proteins involved in plant response to high ambient temperature. This project aiming to elucidate redox-epigenetics-metabolism networks in plants will deepen current understanding of how plants adapt or resist to a changing environment. More specifically, the project will decipher the molecular basis of HDAC regulation in response to stress and how thiol modifications modulate their functions. Noteworthy, this study will reveal the function of HDAC-dependent lysine acetylation in regulating activity of non-histone proteins which is largely unknown at present time. We believe that the results obtained from this project will lead to establish a general link and reveal the molecular mechanisms of interplay between redox signaling, epigenetic regulation and plant adaptation to environment. The REPHARE project assembles complementary expertise in the fields of epigenetic regulation and redox signaling from the two partners who have already built a solid basis which will lead the project to success.

Horizontal Transfers (HTs) refers to the movement of genetic material between distantly related species by mechanisms other than sexual reproduction. In prokaryotes, HTs are well known to be a source of new adaptive traits such as the spreading of genes implicated in antibiotic resistance. Over the last decade, it has become increasingly obvious that in eukaryotes too such as plants, HTs can also lead to major evolutionary leaps and very fast adaptation to new ecological niches that would not be possible only by standard genetic mutations. However, little is known about their rate, evolutionary significance and the biotic interaction promoting HT between plants. While host-parasite relationships appear to be a major pathway for horizontal transfers, an increasing number of HT cases reported in recent years involve species that do not share intimate cell-to-cell contact. This, raises the question alternative HT pathway between species not involved in tight physical contact and the possible involvement of vectors. With the advent of next and third generation sequencing technologies and the substantial increase of genomic data in plants, it becomes feasible to assess the extent and routes of HT at an unprecedented larger-scale. While these sequencing technologies have enabled a massive increase in genomic data from plants and other organisms, new challenges must be addressed not only in terms of bioinformatics analysis of large datasets, but also for the automatic and accurate HT characterization. To date, no studies have been conducted on a genome-wide scale and for a large number of plant species. Indeed, most previous HT studies have focused on specific genes or only on certain transposable elements (TEs) classes and on a limited number of plant species. The EXOTICA project seeks to tackle those challenges by providing innovative comparative genomic approaches for HTs characterization that could handle big genomic data in order to determine the extent, possible routes and the nature of biological interactions promoting HTs in plants. We have recently developed novel bioinformatics approaches that seek to characterize HTs at the whole genome scale and without any prior knowledge on genome annotation. Thanks to these tools, we will characterize HTs on an unprecedented scale by comparing thousands of publicly available genomes and by de novo sequencing of several plant species from a natural ecosystem: the Massane forest located in southern France. EXOTICA will set up the basis for a full and comprehensive understanding of HT routes and the type of biotic interactions promoting HT between non-parasitic plants. We will also test the hypothesis of the implication of vector such as bacteria, fungi or insect in plant-to-plant DNA transfer.

Developing an integrative research allowing to elucidate both the links between genotype, phenotype and fitness and the genomic bases of the variation form a major challenge in our understanding of speciation and the emergence of biodiversity. In DiversiFly, I propose to implement such approach by generating, analyzing and comparing phenotypic (morphometry, coloration, odor), genomic and ecological data on Ophrys orchids. This genus displays one of the highest rates of diversification and hybrid zones formed in the wild by some of its species makes Ophrys a promising model to better understand the causes of adaptive radiations: phenomena of intense and rapid diversification in response to ecological selection pressures. Through its approaches, DiversiFly aims at combining studies led at different evolutionary scales, working on endemic and threatened species. In a first part, the parallel study of two hybrid zones will allow us to determine what are the phenotypic traits (morphology, coloration, odor) predominantly involved in adaptation and reproductive isolation within the O. insectifera clade. Through cline analyses, outlier research and association studies, we will then confront phenotypic and genomic data (transcriptomes and GBS data) in order to look for the genomic bases of traits of interest. The links between phenotypes and individual fitness will be evaluated based on life history traits related to reproductive success and survival. As all individuals will be marked, phenotyping and fitness will be evaluated each year over the four years of the project to investigate their stability. In a second part, we will link micro and ‘macro-’ evolutionary scales by working on the whole genus Ophrys. We will use floral transcriptomes on species, representative of the diversity of the genus in order to identify genes involved in shaping floral phenotype, so particular of the Ophrys. Following a comparative approach, we will base our study on both sequence variation and gene levels of expression. Our results will have a significant impact in the field of ecology and evolutionary biology, contributing to the emergence of a new plant model, with a complex genome, to study speciation and evolutionary diversification. They will also contribute to fields such as genomics, conservation biology, systematics and taxonomy. We also believe that with its flowers mimicking insects, Ophrys forms an excellent model for scientific communication and towards general audience.

Inter-species transfer of mobile elements is now recognized as an important factor in eukaryotic genome evolution. Yet the dynamics, control and short-term impacts of this process are poorly known because difficult to study. A major limitation to investigate these is the low frequency at which transfer presumably occurs and the need of a tractable system to study the different steps involved and their control. The T-DNA is a mobile element that is transferred from Agrobacterium tumefaciens bacteria to many dicot plants, including Arabidopsis thaliana. Interestingly, many wild and domesticated dicots contain relics of T-DNA in their genome indicating that the T-DNA transfer can modify the genome permanently. Hence, the interaction between A. tumefaciens, T-DNA and A. thaliana offers a unique opportunity to study, under controlled conditions, a natural process of DNA transfer since its arrival into the cell, in real-time, the control of its expression, as well as its impact on host-pathogen interaction and genome rearrangements. The MOBIL_DNA project will investigate the fate and regulation of wild T-DNA in the host plant and its impact on host cells as well as on pathogen cells and lifestyle. By accessing single-cell and single-molecule resolution, the MOBIL_DNA project proposes to unravel novel plant molecular processes to cope with T-DNAs (and more generally foreign DNA) upon arrival in the cell. This project, at the interface of plant-pathogen interactions, and foreign DNA regulation is possible because of complementary expertise of the 3 partners, their published data and preliminary data they acquired. The MOBIL_DNA consortium combines expertise in A. tumefaciens lifestyle (Partner1 Faure team at I2BC, Gif-sur-Yvette), DNA and RNA real-time imaging in plants (Partner2 Pontvianne team at LGDP, Perpignan) and regulation of mobile elements in plants (Partner3 Déléris team at I2BC, Gif-sur-Yvette). This project will also benefit of infrastructure and expertise of I2BC and LGDP sequencing, microscopy and bioinformatics platforms. Mobil_DNA is organized in 3 work packages (WPs). In WP1, we will determine the co-transcriptomic landscape of the A. thaliana-A. tumefaciens interaction at two key stages of infection, with a focus on processes involved in T-DNA transfer, host response to foreign DNA, control of genomic variability and pathogen lifestyle on roots and tumors. Single cell transcriptomics will further resolve the heterogeneity of the plant cell populations in the tumor to reveal genome responses and regulatory pathways associated with T-DNA expression or lack thereof, at the cellular level. In WP2, we will use pioneering live-imaging of DNA and RNA to characterize the localization and fate of pathogenic T-DNA in the plant cells, with sub-tissular and sub-cellular resolution, and relate this information to T-DNA transcriptional status and Single Cell transcriptomics. In WP3, we aim to understand the mechanisms controlling T-DNA expression (post-transcriptional / transcriptional silencing), as these are likely to be critical for plant defense, and how they could be dampened by bacteria. In this somatic context of insertion, we aim to reveal the regulation of armed T-DNA, global changes of epigenomic landscape in the plant tumor, and crosstalks between regulations of T-DNA and host transposable elements (TE). Somatic transpositions and other genome rearrangements in the plant and bacterial genome will be assessed. The contribution of the proliferative tumor state to mobile element activation and to bacterial abundance /gene expression patterns will be tested using appropriate mutants and tools. Overall, this work will lead to major discoveries on the spatial-temporal dynamics of T-DNA horizontal transfer with unprecedented resolution and on T-DNA control and impacts, providing valuable information for genome evolution studies, and for improving efficiency of T-DNA-based genome engineering and plant protection.

In multicellular eukaryotes, repression of gene transcription by Polycomb group (PcG) proteins is fundamental to cell fate determination and developmental transitions. Cis DNA motifs, called polycomb responsive elements (PREs), allow the recruitment of PcG proteins to their target genes, on which they deposit repressive epigenetic marks such as histone H3 lysine 27 trimethylation (H3K27me3). However, the understanding of the mechanisms controlling PcG recruitment is only fragmentary as certain genes do contain PREs in their promoter sequences and yet are ubiquitously transcribed implying that they are not targeted by, or refractory to PcG-mediated silencing. This raises the interesting possibility that unknown mechanisms actively protect certain genes from PcG-mediated silencing. We recently discovered that Plant Mobile Domain (PMD) proteins MAINTENANCE OF MERISTEMS (MAIN) and MAIN-LIKE1 (MAIL1) act in a same complex that is required for proper transcription of many genes in Arabidopsis thaliana. Unpublished data indicate that their evolutionary conserved paralog MAIN-LIKE2 (MAIL2) also regulates expression of several hundred genes. Our preliminary results strongly suggest that these PMD proteins secure the transcription of distinct sets of genes by antagonizing PcG-mediated silencing. The PolyPMD project combines genetics, genomics and biochemistry to unravel the mode of action of the PMD proteins in counteracting PcG-mediated gene repression. This will provide important new insights into a conserved mechanism of primary importance for the development of all multicellular organisms.