A strategy for studying epigenetic diversity in natural populations: proof of concept in poplar and oak.

In the last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulfite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, taking into account the structural complexity of genomes, their size, and their content in unique coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by sequencing capture bisulfite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

DNA hypomethylation of the host tree impairs interaction with mutualistic ectomycorrhizal fungus.

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.

RNAi suppression of DNA methylation affects the drought stress response and genome integrity in transgenic poplar.

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 downregulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula × 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.

Deciphering the Epigenetic Alphabet Involved in Transgenerational Stress Memory in Crops.

Although epigenetic modifications have been intensely investigated over the last decade due to their role in crop adaptation to rapid climate change, it is unclear which epigenetic changes are heritable and therefore transmitted to their progeny. The identification of epigenetic marks that are transmitted to the next generations is of primary importance for their use in breeding and for the development of new cultivars with a broad-spectrum of tolerance/resistance to abiotic and biotic stresses. In this review, we discuss general aspects of plant responses to environmental stresses and provide an overview of recent findings on the role of transgenerational epigenetic modifications in crops. In addition, we take the opportunity to describe the aims of EPI-CATCH, an international COST action consortium composed by researchers from 28 countries. The aim of this COST action launched in 2020 is: (1) to define standardized pipelines and methods used in the study of epigenetic mechanisms in plants, (2) update, share, and exchange findings in epigenetic responses to environmental stresses in plants, (3) develop new concepts and frontiers in plant epigenetics and epigenomics, (4) enhance dissemination, communication, and transfer of knowledge in plant epigenetics and epigenomics.

Hypomethylation of the aquatic invasive plant, Ludwigia grandiflora subsp. hexapetala mimics the adaptive transition into the terrestrial morphotype.

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.

Developmental Methylome of the Medicinal Plant Catharanthus roseus Unravels the Tissue-Specific Control of the Monoterpene Indole Alkaloid Pathway by DNA Methylation.

Catharanthus roseus produces a wide spectrum of monoterpene indole alkaloids (MIAs). MIA biosynthesis requires a tightly coordinated pathway involving more than 30 enzymatic steps that are spatio-temporally and environmentally regulated so that some MIAs specifically accumulate in restricted plant parts. The first regulatory layer involves a complex network of transcription factors from the basic Helix Loop Helix (bHLH) or AP2 families. In the present manuscript, we investigated whether an additional epigenetic layer could control the organ-, developmental- and environmental-specificity of MIA accumulation. We used Whole-Genome Bisulfite Sequencing (WGBS) together with RNA-seq to identify differentially methylated and expressed genes among nine samples reflecting different plant organs and experimental conditions. Tissue specific gene expression was associated with specific methylation signatures depending on cytosine contexts and gene parts. Some genes encoding key enzymatic steps from the MIA pathway were found to be simultaneously differentially expressed and methylated in agreement with the corresponding MIA accumulation. In addition, we found that transcription factors were strikingly concerned by DNA methylation variations. Altogether, our integrative analysis supports an epigenetic regulation of specialized metabolisms in plants and more likely targeting transcription factors which in turn may control the expression of enzyme-encoding genes.

Winter-dormant shoot apical meristem in poplar trees shows environmental epigenetic memory.

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.

Changes in the epigenome and transcriptome of the poplar shoot apical meristem in response to water availability affect preferentially hormone pathways.

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.

Chilling-responsive DEMETER-LIKE DNA demethylase mediates in poplar bud break.

Annual dormancy-growth cycle is a developmental and physiological process essential for the survival of deciduous trees in temperate and boreal forests. Seasonal control of shoot growth in woody perennials requires specific genetic programmes responding to environmental signals. The environmental-controlled mechanisms that regulate the shift between winter dormancy and the growth-promoting genetic programmes are still unknown. Here, we show that dynamics in genomic DNA methylation levels are involved in the regulation of dormancy-growth cycle in poplar. The reactivation of growth in the apical shoot during bud break process in spring is preceded by a progressive reduction of genomic DNA methylation in apex tissue. The induction in apex tissue of a chilling-dependent poplar DEMETER-LIKE 10 (PtaDML10) DNA demethylase precedes shoot growth reactivation. Transgenic poplars showing downregulation of PtaDML8/10 caused delayed bud break. Genome-wide transcriptome and methylome analysis and data mining revealed that the gene targets of DEMETER-LIKE-dependent DNA demethylation are genetically associated with bud break. These data point to a chilling-dependent DEMETER-like DNA demethylase mechanisms being involved in the shift from winter dormancy to a condition that precedes shoot apical vegetative growth in poplar.

Epigenomics and bolting tolerance in sugar beet genotypes.

In sugar beet (Beta vulgaris altissima), bolting tolerance is an essential agronomic trait reflecting the bolting response of genotypes after vernalization. Genes involved in induction of sugar beet bolting have now been identified, and evidence suggests that epigenetic factors are involved in their control. Indeed, the time course and amplitude of DNA methylation variations in the shoot apical meristem have been shown to be critical in inducing sugar beet bolting, and a few functional targets of DNA methylation during vernalization have been identified. However, molecular mechanisms controlling bolting tolerance levels among genotypes are still poorly understood. Here, gene expression and DNA methylation profiles were compared in shoot apical meristems of three bolting-resistant and three bolting-sensitive genotypes after vernalization. Using Cot fractionation followed by 454 sequencing of the isolated low-copy DNA, 6231 contigs were obtained that were used along with public sugar beet DNA sequences to design custom Agilent microarrays for expression (56k) and methylation (244k) analyses. A total of 169 differentially expressed genes and 111 differentially methylated regions were identified between resistant and sensitive vernalized genotypes. Fourteen sequences were both differentially expressed and differentially methylated, with a negative correlation between their methylation and expression levels. Genes involved in cold perception, phytohormone signalling, and flowering induction were over-represented and collectively represent an integrative gene network from environmental perception to bolting induction. Altogether, the data suggest that the genotype-dependent control of DNA methylation and expression of an integrative gene network participate in bolting tolerance in sugar beet, opening up perspectives for crop improvement.

In search of markers for somatic embryo maturation in hybrid larch (Larix × eurolepis): global DNA methylation and proteomic analyses.

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.

Effects of polysaccharide elicitors on secondary metabolite production and antioxidant response in Hypericum perforatum L. shoot cultures.

The effects of polysaccharide elicitors such as chitin, pectin, and dextran on the production of phenylpropanoids (phenolics and flavonoids) and naphtodianthrones (hypericin and pseudohypericin) in Hypericum perforatum shoot 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-elicited H. perforatum shoot cultures.

Poplars with a PtDDM1-RNAi transgene have reduced DNA methylation and show aberrant post-dormancy morphology.

The Arabidopsis thaliana DDM1 (Decreased DNA Methylation) gene is necessary for the maintenance of DNA methylation and heterochromatin assembly. In Arabidopsis, ddm1 mutants exhibit strong but delayed morphological phenotypes. We used RNA interference (RNAi) to suppress transcripts of two orthologous DDM1 paralogs in Populus trichocarpa and examined effects on whole plant phenotypes during perennial growth and seasonal dormancy. The RNAi-PtDDM1 transgenic poplars showed a wide range of DDM1 transcript suppression; the most strongly suppressed line had 37.5 % of the expression of the non-transgenic control. Genomic cytosine methylation (mC %) was 11.1 % in the non-transgenic control, compared with 9.1 % for the transgenic event with lowest mC %, a reduction of 18.1 %. An evaluation of greenhouse growth directly after acclimation of in vitro grown plants showed no developmental or growth rate abnormalities associated with the decrease in PtDDM1 expression. However, after a dormancy cycle and growth outdoors, a mottled leaf phenotype appeared in some of the transgenic insertion events that had strongly reduced PtDDM1 expression and DNA methylation. The phenotypic consequences of reduced DDM1 activity and DNA methylation appears to increase with cumulative plant propagation and growth.

Methylome of DNase I sensitive chromatin in Populus trichocarpa shoot apical meristematic cells: a simplified approach revealing characteristics of gene-body DNA methylation in open chromatin 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.

Identification of differentially methylated regions during vernalization revealed a role for RNA methyltransferases in bolting.

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 transcription-PCR 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.

Exploring the crop epigenome: a comparison of DNA methylation profiling techniques.

Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

RNAseq based variant dataset in a black poplar association panel.

OBJECTIVE: Black poplar (Populus nigra L.) is a species native to Eurasia with a wide distribution area. It is an ecologically important species from riparian ecosystems, that is used as a parent of interspecific (P. deltoides x P. nigra) cultivated poplar hybrids. Variant detection from transcriptomics sequences of 241 P. nigra individuals, sampled in natural populations from 11 river catchments (in four European countries) is described here. These data provide new valuable resources for population structure analysis, population genomics and genome-wide association studies. DATA DESCRIPTION: We generated transcriptomics data from a mixture of young differentiating xylem and cambium tissues of 480 Populus nigra trees sampled in a common garden experiment located at Orléans (France), corresponding to 241 genotypes (2 clonal replicates per genotype, at maximum) by using RNAseq technology. We launched on the resulting sequences an in-silico pipeline that allowed us to obtain 878,957 biallelic polymorphisms without missing data. More than 99% of these positions are annotated and 98.8% are located on the 19 chromosomes of the P. trichocarpa reference genome. The raw RNAseq sequences are available at the NCBI Sequence Read Archive SPR188754 and the variant dataset at the Recherche Data Gouv repository under https://doi.org/10.15454/8DQXK5 .

Genic DNA methylation changes during in vitro organogenesis: organ specificity and conservation between parental lines of epialleles.

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.

From Methylome to Integrative Analysis of Tissue Specificity.

DNA methylation is the most studied epigenetic mark in both plants and animals. The gold standard for assaying genome-wide DNA methylation at single-base resolution is whole-genome bisulfite sequencing (WGBS). Here, we describe an improved procedure for WGBS and original bioinformatic workflows applied to unravel tissue-specific variations of the methylome in relation to gene expression and accumulation of secondary metabolites in the medicinal plant Catharanthus roseus.

Zebularine, a DNA Methylation Inhibitor, Activates Anthocyanin Accumulation in Grapevine Cells.

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.