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Trees are long-lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via down regulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula 9 Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit.We compared the growth and drought response of two RNAi-ddm1 lines to wild-type (WT) trees under well-watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi-ddm1 lines were more tolerant to drought-induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi-ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways ; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone-related stress responses, and the maintenance of genome integrity through repression of transposable elements.

As fixed and long living organisms subjected to repeated environmental stresses, trees have developed mechanisms such as phenotypic plasticity that help them to cope with fluctuating environmental conditions. Here, we tested the role DNA methylation as a hub of integration, linking plasticity and physiological response to water deficit in the shoot apical meristem of the model tree poplar (Populus). Using a reverse genetic approach, we compared hypomethylated RNAi-ddm1lines to wild-type trees for drought tolerance. An integrative analysis was realized with phytohormone balance, methylomes, transcriptomes and mobilomes.Hypomethylated lines were more tolerant when subjected to moderate water deficit and were intrinsically more tolerant to drought-induced cavitation. The alteration of theDDM1machinery induced variation in DNA methylation in a cytosine context dependent manner, both in genes and transposable elements. Hypomethylated lines subjected to water deficit showed altered expression of genes involved in phytohormone pathways, such as salicylic acid and modified hormonal balance. Several transposable elements showed stress- and/or line-specific patterns of reactivation, and we could detect copy number variations for two of them in stressedddm1lines.Overall, our data highlight two major roles for DNA methylation in the shoot apical meristem: control of stress response and plasticity through transduction of hormone signaling and maintenance of genome integrity through the control of transposable elements.

International audience; Genetic and epigenetic variations are commonly known to underlie phenotypic plastic responses to environmental cues. however, the role of epigenetic variation in plastic responses harboring ecological significance in nature remains to be assessed. The shade avoidance response (SAR) of plants is one of the most prevalent examples of phenotypic plasticity. It is a phenotypic syndrome including stem elongation and multiple other traits. Its ecological significance is widely acknowledged, and it can be adaptive in the presence of competition for light. Underlying genes and pathways were identified, but evidence for its epigenetic basis remains scarce. We used a proven and accessible approach at the population level and compared global DNA methylation between plants exposed to regular light and three different magnitudes of shade in seven highlyinbred lines of snapdragon plants (Antirrhinum majus) grown in a greenhouse. Our results brought evidence of a strong SAR syndrome for which magnitude did not vary between lines. They also brought evidence that its magnitude was not associated with the global DNA methylation percentage for five of the six traits under study. The magnitude of stem elongation was significantly associated with global DNA demethylation. We discuss the limits of this approach and why caution must be taken with such results. In-depth approaches at the DNA sequence level will be necessary to better understand the molecular basis of the SAR syndrome

International audience; In a context of climate change and forest decline, a better understanding of the sources of tree flexibility involved in phenotypic plasticity and adaptation is needed. These last years, the role of epigenetics in the response to environmental variations has been established in several model plants at the genotype level but little is known at the level of natural populations grown in pedoclimatic sites. Here, we focused on three French natural populations of black poplar, a key pioneer tree from watersheds, planted in common garden and subjected to controlled variations of water availability. We estimated common genetic parameters such as narrow-sense heritability (h(2)), phenotypic differentiation index (P-ST), and the overall genetic differentiation index (F-ST) from genome-wide SNPs to evaluate the extent of epigenetic variations. Indeed, global DNA methylation levels from individuals exposed to drought or irrigated in a common garden were used. We found that the three populations were not distinguished by their levels of DNA methylation. However, a moderate drought was associated to a significant decrease in DNA methylation in the populations. Narrow-sense heritability and P-ST estimates of DNA methylation were similar to those found for biomass productivity. Heritability and P-ST were higher when trees were subjected to drought than in control condition. Negative genetic correlations between global DNA methylation and height or biomass were detected in drought condition only. Altogether, our data highlight that global DNA methylation acts as a genetic marker of natural population differentiation under drought stress in a pedoclimatic context.

International audience; Trees have a long lifespan and must continually adapt to environmental pressures, notably in the context of climate change. Epigenetic mechanisms are doubtless involved in phenotypic plasticity and in stress memory; however, little evidence of the role of epigenetic processes is available for trees growing in fields. Here, we analyzed the possible involvement of epigenetic mechanisms in the winter-dormant shoot apical meristem of Populus × euramericana clones in memory of the growing conditions faced during the vegetative period. We aimed to estimate the range of genetic and environmentally induced variations in global DNA methylation and to evaluate their correlation with changes in biomass production, identify differentially methylated regions (DMRs), and characterize common DMRs between experiments. We showed that the variations in global DNA methylation between conditions were genotype dependent and correlated with biomass production capacity. Microarray chip analysis allowed detection of DMRs 6 months after the stressful summer period. The 161 DMRs identified as common to three independent experiments most notably targeted abiotic stress and developmental response genes. Results are consistent with a winter-dormant shoot apical meristem epigenetic memory of stressful environmental conditions that occurred during the preceding summer period. This memory may facilitate tree acclimation.

The effects of polysaccharide elicitors such as chitin, pectin, and dextran on the production of phenylpropanoids (phenolics and flavonoids) and naphtodianthrones (hypericin and pseudohypericin) inHypericum perforatumshoot cultures were studied. Nonenzymatic antioxidant properties (NEAOP) and peroxidase (POD) activity were also observed in shoot extracts. The activities of phenylalanine ammonia lyase (PAL) and chalcone-flavanone isomerase (CHFI) were monitored to estimate channeling in phenylpropanoid/flavonoid pathways of elicited shoot cultures. A significant suppression of the production of total phenolics and flavonoids was observed in elicited shoots from day 14 to day 21 of postelicitation. This inhibition of phenylpropanoid production was probably due to the decrease in CHFI activity in elicited shoots. Pectin and dextran promoted accumulation of naphtodianthrones, particularly pseudohypericin, within 21 days of postelicitation. The enhanced accumulation of naphtodianthrones was positively correlated with an increase of PAL activity in elicited shoots. All tested elicitors induced NEAOP at day 7, while chitin and pectin showed increase in POD activity within the entire period of postelicitation. The POD activity was in significantly positive correlation with flavonoid and hypericin contents, suggesting a strong perturbation of the cell redox system and activation of defense responses in polysaccharide-elicitedH. perforatumshoot cultures.

International audience; Sugar beet (Beta vulgaris altissima) is a biennial root crop with an absolute requirement for cold exposure to bolt and flower, a process called vernalization. Global DNA methylation variations have been reported during vernalization in several plants. However, few genes targeted by DNA methylation during vernalization have been described. The objectives of this study were to identify differentially methylated regions and to study their involvement in bolting induction and tolerance. Restriction landmark genome scanning was applied to DNA from shoot apical meristems of sugar beet genotypes, providing a direct quantitative epigenetic assessment of several CG methylated genes without prior knowledge of gene sequence. Several differentially methylated regions exhibiting variations of gene-body DNA methylation and expression during cold exposure and/or between genotypes were identified, including an AROGENATE DEHYDRATASE and two RNA METHYLCYTOSINE TRANSFERASE sequences. One RNA METHYLCYTOSINE TRANSFERASE sequence displayed gene-body hypermethylation and activation of expression, while the other was hypomethylated and inhibited by cold exposure. Global RNA methylation and phenolic compound levels changed during cold exposure in a genotype-dependent way. The use of methyl RNA immunoprecipitation of total RNA and reverse transcriptionPCR analysis revealed mRNA methylation in a vernalized bolting-resistant genotype for the FLOWERING LOCUS 1 gene, a repressor of flowering. Finally, Arabidopsis mutants for RNA METHYLCYTOSINE TRANSFERASE and AROGENATE DEHYDRATASE were shown to exhibit, under different environmental conditions, early or late bolting phenotypes, respectively. Overall, the data identified functional targets of DNA methylation during vernalization in sugar beet, and it is proposed that RNA methylation and phenolic compounds play a role in the floral transition.

National audience; Global climate changes in progress will impact tree productivity in the northern hemisphere. The understanding of physiological as well as genetic/molecular processes controlling plants response to abiotic stress will help to improve plant breeding. Recently, epigenetic mechanisms such as DNA methylation have been shown to participate to the control of plant development and their adaptation to environment through modifications of chromatin compaction and gene expression profiles. Phenotypic plasticity defines as the different phenotypes for a given genotype in distinct environments is a key process for plant to adapt to their changing environment. This is particularly relevant for perennialplants such as trees that are exposed to repeated fluctuations of their living conditions. In this context, drought is a significant threat to forest health and agro-ecosystem productivity. With the availability of its genome and its important natural genetic and phenotypic variations, Populus became a model tree.The aim of our project is to develop an integrative approach to identify ecophysiological and molecular bases, particularly DNA methylation, involved in phenotypic plasticity. Contrary to animals, plants exhibit a continuous development and their germline arise from very specific somatic tissue: the shoot apical meristem. We focus our effort to the study the role of DNA methylation in shoot apical meristem using various epigenomic approaches (MeDIP-SEQ, MeDIP-CHIP, WGBS) in parallel to transcriptomics and phenotyping. Our experimental designs include collection of genotypes and are established in controlled conditions such as in greenhouse and nursery as well as in field plantations. We have also characterized the first hypomethylated tree (DDM1-RNAi poplars) in different environments.Altogether, our results demonstrate the role of DNA methylation in memorizing environmental influence and identify the corresponding genes network in relation to phenotypic plasticity.

DNA methylation is involved in the control of plant development and adaptation to the environment through modifications of chromatin compaction and gene expression. In poplar (Populus trichocarpa), a perennial plant, variations in DNA methylation have been reported between genotypes and tissues or in response to drought. Nevertheless, the relationships between gene-body DNA methylation, gene expression and chromatin compaction still need clarification. Here, DNA methylation was mapped in the noncondensed chromatin fraction from P. trichocarpa shoot apical meristematic cells, the center of plant morphogenesis, where DNA methylation variations could influence the developmental trajectory. DNase I was used to isolate the noncondensed chromatin fraction. Methylated sequences were immunoprecipitated, sequenced using Illumina/Solexa technology and mapped on the v2.0 poplar genome. Bisulfite sequencing of candidate sequences was used to confirm mapping data and to assess cytosine contexts and methylation levels. While the methylated DNase I hypersensitive site fraction covered 1.9% of the poplar genome, it contained sequences corresponding to 74% of poplar gene models, mostly exons. The level and cytosine context of gene-body DNA methylation varied with the structural characteristics of the genes. Taken together, our data show that DNA methylation is widespread and variable among genes in open chromatin of meristematic cells, in agreement with a role in their developmental trajectory.

National audience; Recent findings suggest that epigenetic processes such as DNA methylation are involved in water-stress acclimation. Poplars are increasingly used for tree research as model organisms and poplar RNAi lines represent powerful tools to address physiological questions. Here, three RNAi poplar lines inhibited for the DECREASED DNA METHYLATION 1 gene (DDM1, involved in the maintenance of DNA methylation profile) were compared to the control genotype (Populus tremula × Populus alba). A drought-rewatering experiment was conducted to evaluate the role of DNA methylation in phenotypic plasticity. In vitro plantlets of the four genotypes were propagated and transferred to a greenhouse where they were grown under controlled conditions of temperature, light and photoperiod. At the onset of the drought experiment, half of the plants were subjected to a controlled and moderate water deficit while the other half was maintained at field capacity. After three weeks of water depletion, waterstressed plants were re-watered for one week before being harvested. Results related to growth behaviour, photosynthetic capacities, water-use efficiency, xylem vulnerability to cavitation and xylem anatomy will be presented. This phenotypic characterization will drive future work in epigenomic such as the identification of ‘Differentially Methylated Regions’ and their relationships with corresponding gene expression profiles.

International audience; An epigenetic control of vernalization has been demonstrated in annual plants such as Arabidopsis and cereals, but the situation remains unclear in biennial plants such as sugar beet that has an absolute requirement for vernalization. The role of DNA methylation in flowering induction and the identification of corresponding target loci also need to be clarified. In this context, sugar beet (Beta vulgaris altissima) genotypes differing in bolting tolerance were submitted to various bolting conditions such as different temperatures and/or methylating drugs. DNA hypomethylating treatment was not sufficient to induce bolting while DNA hypermethylation treatment inhibits and delays bolting. Vernalizing and devernalizing temperatures were shown to affect bolting as well as DNA methylation levels in the shoot apical meristem. In addition, a negative correlation was established between bolting and DNA methylation. Genotypes considered as resistant or sensitive to bolting could also be distinguished by their DNA methylation levels. Finally, sugar beet homologues of the Arabidopsis vernalization genes FLC and VIN3 exhibited distinct DNA methylation marks during vernalization independently to the variations of global DNA methylation. These vernalization genes also displayed differences in mRNA accumulation and methylation profiles between genotypes resistant or sensitive to bolting. Taken together, the data suggest that the time course and amplitude of DNA methylation variations are critical points for the induction of sugar beet bolting and represent an epigenetic component of the genotypic bolting tolerance, opening up new perspectives for sugar beet breeding.

International audience; The first step of Agrobacterium tumefaciens/plant interaction corresponds to the activation of a transduction pathway of the bacterium by plant exudate. Phenolic compounds rapidly secreted by wounded plant cells induce the expression of bacterial virulence (vir) genes; however, little is known about their biosynthesis in plant. Here we show that inoculation of an Agrobacterium tumefaciens virulent strain on orthodiphenol-O-methyltransferases-suppressed tobacco plants leads to significantly smaller tumors compared to control plants. These transgenic plants are inhibited for caffeic acid O-methyltransferase class I or II (OMT; EC 2.1.1.6) and/or caffeoyl-coenzyme A O-methyltransferase (CCoAOMT; EC 2.1.1.104) that are involved in monolignol biosynthesis. The significant decrease of tumor size could be suppressed by the pre-activation of bacterial virulence, before inoculation, using acetosyringone a known vir inducer. Total soluble phenolic amounts and cell wall composition analyzed by FT-IR analysis did not show significant differences between transgenic and control plants. The potential of phenolic extracts from control and OMT-suppressed plants to induce virulence was evaluated using an Agrobacterium tumefaciens reporter strain carrying a vir::LacZ gene fusion plasmid. Lower vir-inducing activities were recorded for plants that show inhibition to caffeic acid O-methyltransferase activity. HPLC analysis confirmed that the levels of several phenolic compounds were differently affected by wounding and/or by bacterial inoculation. Statistical correlations were established between tumor sizes, vir-inducing activities, O-methyltransferases proteins accumulations and the levels of various soluble phenolic compounds such as acetosyringone. These results demonstrate the role of the O-methyltransferases of the phenylpropanoid pathway in the early production of soluble Agrobacterium tumefaciens vir inducers.

Several reports on annual plants have already shown the involvement of epigenetic modifiers such as DNA methylation in their adaptation to abiotic stresses. Nevertheless, the genotypic variations of epigenetic modifiers, their possible correlations with morphological traits and the impact of water deficit have not been described for perennial plants. Six genotypes of Populus deltoides × P. nigra were subjected or not to a moderate water deficit treatment. Various morphological traits such as the height of the plants, their biomass and the total leaf area were measured to characterize the productivity in both conditions. Levels of DNA methylation, histone acetylation and the activities and isoform accumulation of the corresponding enzymes were measured at the shoot apex, the site of morphogenesis. Genotypic variation was observed for the morphological traits and the epigenetic variables and correlations were established among them. Genotypic variation for DNA methylation was detected in hybrid poplars. A positive correlation was demonstrated between DNA methylation percentage and productivity under well watered conditions. While there was a general decrease of growth for all genotypes in response to a moderate water deficit, genotypic dependant variations of DNA methylation were found suggesting different strategies among hybrids., Plusieurs études sur des plantes annuelles ont déjà montré l’implication des modifications épigénétiques telles que la méthylation de l’ADN dans la plasticité de leurs réponses aux contraintes abiotiques. Néanmoins, les variations génotypiques de ces modifications épigénétiques, leur possible corrélation avec des variables de croissance et l’impact d’un déficit hydrique n’ont pas été décrits sur une plante pérenne. Six génotypes de Populus deltoïdes × P. nigra ont été soumis ou non à un déficit hydrique modéré et plusieurs variables de croissance ont été mesurées afin de caractériser leur productivité. Les niveaux de méthylation de l’ADN, d’acétylation des histones, les activités enzymatiques et l’accumulation des isoformes correspondantes ont été mesurés sur des apex caulinaires, site de la morphogenèse. Des variations génotypiques ont été observées pour les variables de croissance et épigénétiques. Une corrélation positive a été mise en évidence entre la méthylation de l’ADN et la productivité en condition hydrique favorable. Bien qu’il y ait une diminution générale de la croissance de tous les génotypes en réponse à un déficit hydrique modéré, des variations génotype-dépendant de la méthylation de l’ADN ont été trouvées suggérant différentes stratégies entre hybrides.