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17 Pág., Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown. By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) and two demethylases DEMETER-LIKE (DML) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor. We compared the ectomycorrhizas formed between transgenic and wild-type (WT) trees and analyzed their methylomes and transcriptomes. The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi-ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX-dml and RNAi-ddm1) and 120 genes/1441 TEs in the fungal genome suggesting a host-induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes (cis and trans effects) in common with 17 being differentially methylated (cis). Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate-mediated pathways, and immune response but also terpenoid metabolism., MDS and JV received PhD grants from the MRES and Conseil Régional Région Centre Val de Loire, respectively. The LBLGC and the LEE/CNRGH benefit from the support of the ANR EPITREE (ANR-17-CE32-0009-01, https://www6.inrae.fr/epitree-project_eng/) to SM and JT. We thank RTP3E CNRS and IHPE for the access to the bioinformatic platform (http://galaxy.univ-perp.fr/, Christian Chaparro, France). CVF, AK, FM, FG, and LF benefit from the support of the Genomic Science Program (project ‘Plant-Microbe Interface’), US Department of Energy, Office of Science, Biological and Environmental Research under the contract DE-AC05-00OR22725 and the Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems (ARBRE; grant ANR-11-LABX-0002-01). The work (proposal: 10.46936/10.25585/60001022) conducted by the US Department of Energy JGI (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy operated under Contract no. DE-AC02-05CH11231.

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.

Through its role in the regulation of gene expression, DNA methylation can participate in the control of specialized metabolite production. We have investigated the link between DNA methylation and anthocyanin accumulation in grapevine using the hypomethylating drug, zebularine and Gamay Teinturier cell suspensions. In this model, zebularine increased anthocyanin accumulation in the light, and induced its production in the dark. To unravel the underlying mechanisms, cell transcriptome, metabolic content, and DNA methylation were analyzed. The up-regulation of stress-related genes, as well as a decrease in cell viability, revealed that zebularine affected cell integrity. Concomitantly, the global DNA methylation level was only slightly decreased in the light and not modified in the dark. However, locus-specific analyses demonstrated a decrease in DNA methylation at a few selected loci, including a CACTA DNA transposon and a small region upstream from the UFGT gene, coding for the UDP glucose:flavonoid-3-O-glucosyltransferase, known to be critical for anthocyanin biosynthesis. Moreover, this decrease was correlated with an increase in UFGT expression and in anthocyanin content. In conclusion, our data suggest that UFGT expression could be regulated through DNA methylation in Gamay Teinturier, although the functional link between changes in DNA methylation and UFGT transcription still needs to be demonstrated.

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; Ongoing global changes affect ecosystems and open up new opportunities for biological invasion. The ability of invasive species to rapidly adapt to new environments represents a relevant model for studying short‐term adaptation mechanisms. The aquatic invasive plant, Ludwigia grandiflora subsp. hexapetala, is classified as harmful in European rivers. In French wet meadows, this species has shown a rapid transition from aquatic to terrestrial environments with emergence of two distinct morphotypes in 5 years. To understand the heritable mechanisms involved in adjustment to such a new environment, we investigate both genetic and epigenetic as possible sources of flexibility involved in this fast terrestrial transition. We found a low overall genetic differentiation between the two morphotypes arguing against the possibility that terrestrial morphotype emerged from a new adaptive genetic capacity. Artificial hypomethylation was induced on both morphotypes to assess the epigenetic hypothesis. We analyzed global DNA methylation, morphological changes, phytohormones and metabolite profiles of both morphotype responses in both aquatic and terrestrial conditions in shoot and root tissues. Hypomethylation significantly affected morphological variables, phytohormone levels and the amount of some metabolites. The effects of hypomethylation depended on morphotypes, conditions and plant tissues, which highlighted differences among the morphotypes and their plasticity. Using a correlative integrative approach, we showed that hypomethylation of the aquatic morphotype mimicked the characteristics of the terrestrial morphotype. Our data suggest that DNA methylation rather than a new adaptive genetic capacity is playing a key role in L. grandiflora subsp. hexapetala plasticity during its rapid aquatic to terrestrial transition.

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.

Hypericum perforatum cell suspensions were evaluated for their growth, phenylpropanoid and naphtodianthrone productions, and antioxidant activity after treatments with polysaccharide elicitors chitin (CHI), pectin (PEC) and dextran (DEX). Polysaccharide elicitation of cell cultures showed a considerable improving effect on phenylpropanoid production (phenolics, flavonoids, flavanols and anthocyanins) during the post-elicitation period. Even that enhanced phenylpropanoid production was related to growth suppression of CHI elicited cells, PEC and DEX were presented as effective elicitors without loss of cell biomass. Phenylpropanoid accumulation in elicited cells was correlated to markedly higher enzyme activities of phenylalanine ammonia lyase and chalcone-flavanone isomerase. Cell cultures were found to respond rapidly towards the applied elicitors through an early stimulation of both naphtodiantrones, hypericin (HYP) and pseudohypericin (PHYP). The CHI was proposed as a much more prominent elicitor in stimulation of HYP and PHYP levels in cell suspensions. All tested polysaccharide elicitors stimulated non-enzymatic antioxidant properties in cell extracts indicating the potential role of phenolic compounds as effective antioxidants. With regards to the antioxidant enzymes, an up-regulation of peroxidase and catalase activities was observed in PEC and DEX elicited cells, whereas CHI did not give any stimulatory effect on enzymatic activities. These findings suggest the involvement of an efficient antioxidant defense system in the adaptive response of cells to polysaccharide elicitation. Altogether, these results indicated that H. perforatum cells elicited with polysaccharides represent a promising experimental system for enhanced production of phenylpropanoids and naphtodianthrones.

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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.

International audience; The adaptive capacity of long-lived organisms such as trees to the predicted climate changes, including severe and successive drought episodes, will depend on the presence of genetic diversity and phenotypic plasticity. Here, the involvement of epigenetic mechanisms in phenotypic plasticity toward soil water availability was examined in Populus×euramericana. This work aimed at characterizing (i) the transcriptome plasticity, (ii) the genome-wide plasticity of DNA methylation, and (iii) the function of genes affected by a drought-rewatering cycle in the shoot apical meristem. Using microarray chips, differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were identified for each water regime. The rewatering condition was associated with the highest variations of both gene expression and DNA methylation. Changes in methylation were observed particularly in the body of expressed genes and to a lesser extent in transposable elements. Together, DEGs and DMRs were significantly enriched in genes related to phytohormone metabolism or signaling pathways. Altogether, shoot apical meristem responses to changes in water availability involved coordinated variations in DNA methylation, as well as in gene expression, with a specific targeting of genes involved in hormone pathways, a factor that may enable phenotypic plasticity.

International audience; In the context of climate changes, clarifying the causes underlying tree phenotypic plasticity and adaptation is crucial. Studies suggest a role of epigenetic mechanisms in response to external stimuli, raising the question whether such processes can promote acclimation of trees exposed to adverse climate conditions. Recently, we revealed an environmental epigenetic footprint in the shoot apical meristem (SAM) which could partially be transmitted mitotically, for several months, up until the winter-dormant bud in field conditions. Here, we extended our previous analysis to the leaves of the same P. deltoidesxP. nigra clones. We aimed at estimating the range of developmentally, genetically, and environmentally induced variations on DNA methylation. We showed that only the first leaves emerging from the SAM displayed variations of DNA methylation under changing water conditions. We also found that these variations are genotype- and pedoclimatic site-dependent. Altogether, our data raised questions and perspectives on the direct acquisition, the maintenance of environmentally induced DNA methylation changes, and their mitotic transmission from the SAM to the first emerging leaves.

International audience; A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.

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.

International audience; Hypericum perforatum is a well known medicinal plant. The main pharmacological properties are due to the presence of naphtodianthrones such as hypericin and pseudohypericin. Unfortunately the levels of these compounds vary under different environmental conditions. Elicitation of in vitro cultures is a useful approach to enhance and extend production of desirable products. Therefore, the effects of salicylic acid were characterized on different explants of H. perforatum L. (cells, calli and shoots) cultured in vitro. It appears at first that salicylic acid did not affect growth and development of these explants. In addition, the production of both hypericin and pseudohypericin has doubled in elicited cell suspension cultures but not in the two other cultures. Furthermore, phenylpropanoids that are among the most frequently observed metabolites affected upon treatment of in vitro culture material with elicitors, were produced and the enzymatic activities of phenylalanine ammonia lyase and of chalcone isomerase were stimulated upon elicitation. These effects were dependant of the type of in vitro culture, the concentration of salicylic acid and the duration post-elicitation. The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures. In the absence of glands considered as the sites of naphtodianthrones biosynthesis, cells and calli were capable of producing these compounds. This implies that salicylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin. Consequently, the regulation of this process is more complex than cited in the literature involving the responsibility of only Hyp-1 gene, encoding a hypericin biosynthetic enzyme, cloned and characterized from H. perforatum.

Hypericum perforatum L. cell suspensions were evaluated for their viability, growth, dark gland formation and ability to produce phenylpropanoids and naphtodiantrones after elicitation with different jasmonic acid (JA) concentrations. Phenolic compounds were analyzed by high performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization mass spectrometry (ESI-MS). The activities of two key enzymes of the phenylpropanoid/flavonoid pathways, phenylalanine ammonia lyase (PAL) and chalcone isomerase (CHI) were also monitored to estimate general channeling in the different metabolic pathways. A 6-fold increase of phenolic compounds, flavanols and flavonols after JA elicitation was observed in cells. In contrast, anthocyanins were in lower amounts in JA treated cells suggesting a modification of the channeling in the phenylpropanoid pathway. Similar accumulations with maxima after 4 days of elicitation were found for naphtodianthrones (2.4-fold) such as hypericin and pseudohypericin in cells. At least a 6–8-fold increase of PAL and CHI activities was observed in JA elicited cells confirming a strong activation of the phenylpropanoid pathway. JA elicitation increased production of phenylpropanoids and naphtodianthrones in H. perforatum cell suspension without differentiation of dark glands under 16 h photoperiod.

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.

In order to evaluate the permanent chromatin remodeling in plant allowing their high developmental plasticity, three sugarbeet cell lines (Beta vulgaris L. altissima) originating from the same mother plant and exhibiting graduate states of differentiation were analyzed. Cell differentiation has been estimated by the cell redox state characterized by 36 biochemical parameters as reactive oxygen species steady-state levels, peroxidation product contents and enzymatic or non-enzymatic protective systems. Chromatin remodeling has been estimated by the measurement of levels of DNA methylation, histone acetylation and corresponding enzyme activities that were shown to differ between cell lines. Furthermore, distinct loci related to proteins involved in cell cycle, gene expression regulation and cell redox state were shown by restriction landmark genome scanning or bisulfite sequencing to display differential methylation states in relation to the morphogenic capacity of the lines. DNA methylating, demethylating and/or histone acetylating treatments allowed to generate a collection of sugarbeet cell lines differing by their phenotypes (from organogenic to dedifferentiated), methylcytosine percentages (from 15.0 to 43.5%) and acetylated histone ratios (from 0.37 to 0.52). Correlations between methylcytosine or acetylated histone contents and levels of various parameters (23 or 7, respectively, out of 36) of the cell redox state could be established. These data lead to the identification of biomarkers of sugarbeet morphogenesis in vitro under epigenetic regulation and provide evidence for a connection between plant morphogenesis in vitro, cell redox state and epigenetic mechanisms.

In plants organogenesis, cell differentiation and dedifferentiation are fundamental processes allowing high developmental plasticity. Such plasticity involved epigenetic mechanisms but limited knowledge is available concerning quantitative aspects. Three sugarbeet (Beta vulgaris L. altissima) cell lines originating from the same mother plant and exhibiting graduate states of morphogenesis were used to assess whether these differences could be related or not to changes in DNA methylation levels. Methylcytosine percentages from 18.3 to 28.8% and distinct levels of DNA methyltransferase (EC 2.1.1.37) activities were shown in the three cell lines. The lowest methylcytosine percentage was associated to organogenesis. In order to test the plasticity of these cell lines, various treatments causing DNA hypo or hypermethylation were performed at different times and concentrations. In this collection of treated lines with+/-10% of methylcytosine percentages, loss of organogenic properties and cell dedifferentiation were observed. As cell wall formation fits well with cell differentiation state, the lignification process was further investigated in treated and untreated lines as a biochemical marker of the phenotypic changes. For example, peroxidase specific activities (EC 1.11.1.7) varied from 0.7 to 0.02 pkat mg(-1) of protein in organogenic and dedifferentiated lines, respectively. A negative relationship between peroxidase activities, incorporation of cell wall-bound phenolic compounds as ferulate and sinapate derivatives and methylcytosine percentages was obtained. This is the first biochemical evidence that phenotypic changes in plant cells induced by DNA hypo- or hypermethylating treatments are correlated in a linear relationship to modifications of the cell wall differentiation state.

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.

International audience; During differentiation, in vitro organogenesis calls for the adjustment of the gene expression program toward a new fate. The role of epigenetic mechanisms including DNA methylation is suggested but little is known about the loci affected by DNA methylation changes, particularly in agronomic plants for witch in vitro technologies are useful such as sugar beet. Here, three pairs of organogenic and non-organogenic in vitro cell lines originating from different sugar beet (Beta vulgaris altissima) cultivars were used to assess the dynamics of DNA methylation at the global or genic levels during shoot or root regeneration. The restriction landmark genome scanning for methylation approach was applied to provide a direct quantitative epigenetic assessment of several CG methylated genes without prior knowledge of gene sequence that is particularly adapted for studies on crop plants without a fully sequenced genome. The cloned sequences had putative roles in cell proliferation, differentiation or unknown functions and displayed organ-specific DNA polymorphism for methylation and changes in expression during in vitro organogenesis. Among them, a potential ubiquitin extension protein 6 (UBI6) was shown, in different cultivars, to exhibit repeatable variations of DNA methylation and gene expression during shoot regeneration. In addition, abnormal development and callogenesis were observed in a T-DNA insertion mutant (ubi6) for a homologous sequence in Arabidopsis. Our data showed that DNA methylation is changed in an organ-specific way for genes exhibiting variations of expression and playing potential role during organogenesis. These epialleles could be conserved between parental lines opening perspectives for molecular markers.

Plant development is influenced by changes in the levels and types of sugars produced metabolically. The normal (N), habituated organogenic (HO) and habituated nonorganogenic (HNO) sugar beet cell lines originate from the same mother plant but exhibit distinct levels of morphogenesis and differentiation, and contain different levels of simple carbohydrates. We aim to elucidate whether differences in the abundance and activity of enzymes involved in carbohydrate metabolism and sugar sensing/signalling help explain the different carbohydrate profiles and differentiation states of the cell lines. Using 13C NMR spectroscopy to analyze cultures of the cell lines over 28 days, we found that N cells accumulated sucrose; HO cells sucrose, glucose and fructose; and HNO cells glucose and fructose. Of three invertase isoforms, the activity of cell wall invertase (CWI) was highest in all the cell lines, and CWI activity was greatest in HNO line. The specific accumulation of intracellular carbohydrates during subculture correlated strongly with CWI activity but less so with the vacuolar and cytoplasmic invertase isoforms, or with sucrose synthase activity. Cell lines showed differences in how sugars regulated invertase and sucrose synthase activity. The role of sugar sensing in the regulation of CWI activity was investigated in the cell lines using glucose and sucrose, as well as carbohydrate analogues such as mannitol, 2-O-deoxyglucose and 3-O-methylglucose. Differences in the regulation of CWI activity by carbohydrates across the three cell lines suggest that CWI can be fine-tuned according to the specific carbohydrate requirements of each line during growth. Differences in sugar signalling pathways across the cell lines were explored using glucose and sucrose in the presence of inhibitors of protein kinases or phosphatases. Taken together, our findings suggest that specific regulation of CWI activity plays an important role in determining the intracellular carbohydrate levels of sugar beet cell lines, and possibly their differentiation state as well., Na razvoj biljke utječu promjene razine i tipa proizvedenog šećera. Iako sve stanične linije šećerne repe, i to normalna, prilagođena organogena i prilagođena neorganogena potječu od iste biljke, na različitim su stupnjevima diferencijacije i morfogeneze te sadrže različitu količinu jednostavnih ugljikohidrata. Svrha je rada bila utvrditi može li se razlikama u količini i aktivnosti enzima, koji sudjeluju u metabolizmu šećera te onih u detekciji i prijenosu signala šećera, objasniti razlika u stupnju diferencijacije staničnih linija te u udjelu ugljikohidrata u njima. Primjenom 13C NMR spektroskopije analizirane su stanične linije tijekom 28 dana kultivacije i ustanovljeno je da normalne stanične linije akumuliraju saharozu, prilagođena organogena stanična linija saharozu, glukozu i fruktozu, a prilagođena neorganogena stanična linija šećerne repe glukozu i fruktozu. Od 3 izoformna oblika invertaze najveća je aktivnost invertaze stanične stijenke u sve 3 stanične linije, osobito u prilagođenih neorganogenih stanica. Specifičnost akumulacije ugljikohidrata u stanici tijekom kultivacije uvelike je ovisila o aktivnosti invertaze stanične stijenke, a manje o onoj vakuolarne i citoplazmatske invertaze te saharoza sintetaze. Šećeri su različito regulirali aktivnost invertaze stanične stijenke i saharoza sintetaze, ovisno o tipu stanične linije. Njihova je uloga istražena pomoću glukoze i saharoze te njihovih analoga, kao što su manitol, 2-O-deoksiglukoza i 3-O-metilglukoza. Utvrđeno je da se aktivnost invertaze stanične stijenke može fino regulirati pomoću ugljikohidrata, ovisno o potrebi staničnih linija za izvorom šećera tijekom rasta. Razlike u putovima prijenosa signala šećera u staničnih linija istražene su pomoću glukoze i saharoze u prisutnosti inhibitora protein kinaza i fosfataza. Na kraju, rezultati autora potvrđuju da specifična regulacija aktivnosti invertaze stanične stijenke ima važnu ulogu u određivanju razine ugljikohidrata u staničnim linijama šećerne repe, a možda i stupnju diferencijacije stanica.

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.

International audience; 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 x 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 (Gourcilleau et al. 2010). In order to confirm these data and to test the long term stability of these epigenetic modifications, a new set of eight hybrids was analysed for global DNA methylation and productivity nine months after being subjected or not to a water deficit. Finally, the relationship established between global DNA methylation in shoot apex and productivity in Populus deltoides x P. nigra hybrids was investigated in three populations of P. nigra.

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.