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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.

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.

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.

Keywords: methylcytosine / shoot apex / vegetative development / water deficit / productivity Abstract • 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. Mots-clés : Résumé -Méthylation de l'ADN et acétylation des histones : variations génotypiques chez des peupliers hybrides, impact d'un déficit hydrique et relations avec la productivité.• Plusieurs études sur des plantes annuelles ont déjà montré l'implication des modifications épi-génétiques 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 corréla-tion 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 défi-cit hydrique modéré, des variations génotype-dépendant de la méthylation de l'ADN ont été trouvées suggérant différentes stratégies entre hybrides.

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.

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.