Terroir Influence on Polyphenol Metabolism from Grape Canes: A Spatial Metabolomic Study at Parcel Scale.

The composition of bioactive polyphenols from grape canes, an important viticultural byproduct, was shown to be varietal-dependent; however, the influence of soil-related terroir factors remains unexplored. Using spatial metabolomics and correlation-based networks, we investigated how continuous changes in soil features and topography may impact the polyphenol composition in grape canes. Soil properties, topography, and grape cane extracts were analyzed at georeferenced points over 3 consecutive years, followed by UPLC-DAD-MS-based metabolomic analysis targeting 42 metabolites. Principal component analyses on intra-vintage metabolomic data presented a good reproducibility in relation to geographic coordinates. A correlation-driven approach was used to explore the combined influence of soil and topographic variables on metabolomic responses. As a result, a metabolic cluster including flavonoids was correlated with elevation and curvature. Spatial metabolomics driven by correlation-based networks represents a powerful approach to spatialize field-omics data and may serve as new field-phenotyping tool in precision agriculture.

Alternative splicing creates a pseudo-strictosidine ß-d-glucosidase modulating alkaloid synthesis in Catharanthus roseus.

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by ß-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine ß-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks ß-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of ß-glucosidase multimerization, an organization common to many defensive GH1 members.

An Erwinia amylovora inducible promoter for improvement of apple fire blight resistance.

KEY MESSAGE: pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes. Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be upregulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-constitutive regulatory sequences, respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results does not confirmed pKFDV02 as a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.

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.

A BAHD acyltransferase catalyzing 19-O-acetylation of tabersonine derivatives in roots of Catharanthus roseus enables combinatorial synthesis of monoterpene indole alkaloids.

While the characterization of the biosynthetic pathway of monoterpene indole alkaloids (MIAs) in leaves of Catharanthus roseus is now reaching completion, only two enzymes from the root counterpart dedicated to tabersonine metabolism have been identified to date, namely tabersonine 19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT). Albeit the recombinant MAT catalyzes MIA acetylation at low efficiency in vitro, we demonstrated that MAT was inactive when expressed in yeast and in planta, suggesting an alternative function for this enzyme. Therefore, through transcriptomic analysis of periwinkle adventitious roots, several other BAHD acyltransferase candidates were identified based on the correlation of their expression profile with T19H and found to localize in small genomic clusters. Only one, named tabersonine derivative 19-O-acetyltransferase (TAT) was able to acetylate the 19-hydroxytabersonine derivatives from roots, such as minovincinine and hörhammericine, following expression in yeast. Kinetic studies also showed that the recombinant TAT was specific for root MIAs and displayed an up to 200-fold higher catalytic efficiency than MAT. In addition, gene expression analysis, protein subcellular localization and heterologous expression in Nicotiana benthamiana were in agreement with the prominent role of TAT in acetylation of root-specific MIAs, thereby redefining the molecular determinants of the root MIA biosynthetic pathway. Finally, identification of TAT provided a convenient tool for metabolic engineering of MIAs in yeast enabling efficiently mixing different biosynthetic modules spatially separated in the whole plant. This combinatorial synthesis associating several enzymes from Catharanthus roseus resulted in the conversion of tabersonine in tailor-made MIAs bearing both leaf and root-type decorations.

Two Tabersonine 6,7-Epoxidases Initiate Lochnericine-Derived Alkaloid Biosynthesis in Catharanthus roseus.

Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.

The epigenetic regulation of HsMar1, a human DNA transposon.

BACKGROUND: Both classes of transposable elements (DNA and RNA) are tightly regulated at the transcriptional level leading to the inactivation of transposition via epigenetic mechanisms. Due to the high copies number of these elements, the hypothesis has emerged that their regulation can coordinate a regulatory network of genes. Herein, we investigated whether transposition regulation of HsMar1, a human DNA transposon, differs in presence or absence of endogenous HsMar1 copies. In the case where HsMar1 transposition is regulated, the number of repetitive DNA sequences issued by HsMar1 and distributed in the human genome makes HsMar1 a good candidate to regulate neighboring gene expression by epigenetic mechanisms. RESULTS: A recombinant active HsMar1 copy was inserted in HeLa (human) and CHO (hamster) cells and its genomic excision monitored. We show that HsMar1 excision is blocked in HeLa cells, whereas CHO cells are competent to promote HsMar1 excision. We demonstrate that de novo HsMar1 insertions in HeLa cells (human) undergo rapid silencing by cytosine methylation and apposition of H3K9me3 marks, whereas de novo HsMar1 insertions in CHO cells (hamster) are not repressed and enriched in H3K4me3 modifications. The overall analysis of HsMar1 endogenous copies in HeLa cells indicates that neither full-length endogenous inactive copies nor their Inverted Terminal Repeats seem to be specifically silenced, and are, in contrast, devoid of epigenetic marks. Finally, the setmar gene, derived from HsMar1, presents H3K4me3 modifications as expected for a human housekeeping gene. CONCLUSIONS: Our work highlights that de novo and old HsMar1 are not similarly regulated by epigenetic mechanisms. Old HsMar1 are generally detected as lacking epigenetic marks, irrespective their localisation relative to the genes. Considering the putative existence of a network associating HsMar1 old copies and SETMAR, two non-mutually exclusive hypotheses are proposed: active and inactive HsMar1 copies are not similarly regulated or/and regulations concern only few loci (and few genes) that cannot be detected at the whole genome level.

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

Virus-induced gene silencing of the two squalene synthase isoforms of apple tree (Malus × domestica L.) negatively impacts phytosterol biosynthesis, plastid pigmentation and leaf growth.

MAIN CONCLUSION: The use of a VIGS approach to silence the newly characterized apple tree SQS isoforms points out the biological function of phytosterols in plastid pigmentation and leaf development. Triterpenoids are beneficial health compounds highly accumulated in apple; however, their metabolic regulation is poorly understood. Squalene synthase (SQS) is a key branch point enzyme involved in both phytosterol and triterpene biosynthesis. In this study, two SQS isoforms were identified in apple tree genome. Both isoforms are located at the endoplasmic reticulum surface and were demonstrated to be functional SQS enzymes using an in vitro activity assay. MdSQS1 and MdSQS2 display specificities in their expression profiles with respect to plant organs and environmental constraints. This indicates a possible preferential involvement of each isoform in phytosterol and/or triterpene metabolic pathways as further argued using RNAseq meta-transcriptomic analyses. Finally, a virus-induced gene silencing (VIGS) approach was used to silence MdSQS1 and MdSQS2. The concomitant down-regulation of both MdSQS isoforms strongly affected phytosterol synthesis without alteration in triterpene accumulation, since triterpene-specific oxidosqualene synthases were found to be up-regulated to compensate metabolic flux reduction. Phytosterol deficiencies in silenced plants clearly disturbed chloroplast pigmentation and led to abnormal development impacting leaf division rather than elongation or differentiation. In conclusion, beyond the characterization of two SQS isoforms in apple tree, this work brings clues for a specific involvement of each isoform in phytosterol and triterpene pathways and emphasizes the biological function of phytosterols in development and chloroplast integrity. Our report also opens the door to metabolism studies in Malus domestica using the apple latent spherical virus-based VIGS method.

Class II Cytochrome P450 Reductase Governs the Biosynthesis of Alkaloids.

Expansion of the biosynthesis of plant specialized metabolites notably results from the massive recruitment of cytochrome P450s that catalyze multiple types of conversion of biosynthetic intermediates. For catalysis, P450s require a two-electron transfer catalyzed by shared cytochrome P450 oxidoreductases (CPRs), making these auxiliary proteins an essential component of specialized metabolism. CPR isoforms usually group into two distinct classes with different proposed roles, namely involvement in primary and basal specialized metabolisms for class I and inducible specialized metabolism for class II. By studying the role of CPRs in the biosynthesis of monoterpene indole alkaloids, we provide compelling evidence of an operational specialization of CPR isoforms in Catharanthus roseus (Madagascar periwinkle). Global analyses of gene expression correlation combined with transcript localization in specific leaf tissues and gene-silencing experiments of both classes of CPR all point to the strict requirement of class II CPRs for monoterpene indole alkaloid biosynthesis with a minimal or null role of class I. Direct assays of interaction and reduction of P450s in vitro, however, showed that both classes of CPR performed equally well. Such high specialization of class II CPRs in planta highlights the evolutionary strategy that ensures an efficient reduction of P450s in specialized metabolism.

The N-Glycan cluster from Xanthomonas campestris pv. campestris: a toolbox for sequential plant N-glycan processing.

N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N2M3FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the ß-xylosidase NixI (GH3), which is involved in the cleavage of the ß-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.

Hybrid histidine kinases in pathogenic fungi.

Histidine kinases (HK) sense and transduce via phosphorylation events many intra- and extracellular signals in bacteria, archaea, slime moulds and plants. HK are also widespread in the fungal kingdom, but their precise roles in the regulation of physiological processes remain largely obscure. Expanding genomic resources have recently given the opportunity to identify uncharacterised HK family members in yeasts and moulds and now allow proposing a complex classification of Basidiomycota, Ascomycota and lower fungi HK. A growing number of genetic approaches have progressively provided new insight into the role of several groups of HK in prominent fungal pathogens. In particular, a series of studies have revealed that members of group III HK, which occur in the highest number of fungal species and contain a unique N-terminus region consisting of multiple HAMP domain repeats, regulate morphogenesis and virulence in various human, plant and insect pathogenic fungi. This research field is further supported by recent shape-function studies providing clear correlation between structural properties and signalling states in group III HK. Since HK are absent in mammals, these represent interesting fungal target for the discovery of new antifungal drugs.

An additional Meyerozyma guilliermondii IMH3 gene confers mycophenolic acid resistance in fungal CTG clade species.

The fungal CTG clade comprises a number of well-known yeasts that impact human health or with high biotechnological potential. To further extend the set of molecular tools dedicated to these microorganisms, the initial focus of this study was to develop a mycophenolic acid (MPA) resistance cassette. Surprisingly, while we were carrying out preliminary susceptibility testing experiments in a set of yeast species, Meyerozyma guilliermondii, although not being a MPA producer, was found to be primarily resistant toward this drug, whereas a series of nine related species were susceptible to MPA. Using comparative and functional genomic approaches, we demonstrated that all MPA-susceptible CTG clade species display a single gene, referred to as IMH3.1, encoding the MPA target inosine monophosphate dehydrogenase (IMPDH) and that MPA resistance relies on the presence in the M. guilliermondii genome of an additional IMPDH-encoding gene (IMH3.2). The M. guilliermondii IMH3.2 gene displays marked differences compared to IMH3.1 including the lack of intron, a roughly 160-fold higher transcription level and a serine residue at position 251. Placed under the control of the M. guilliermondii actin 1 gene promoter, IMH3.2 was successfully used to transform Lodderomyces elongisporus, Clavispora lusitaniae, Scheffersomyces stipitis and Candida parapsilosis.

Characterization of a second secologanin synthase isoform producing both secologanin and secoxyloganin allows enhanced de novo assembly of a Catharanthus roseus transcriptome.

BACKGROUND: Transcriptome sequencing offers a great resource for the study of non-model plants such as Catharanthus roseus, which produces valuable monoterpenoid indole alkaloids (MIAs) via a complex biosynthetic pathway whose characterization is still undergoing. Transcriptome databases dedicated to this plant were recently developed by several consortia to uncover new biosynthetic genes. However, the identification of missing steps in MIA biosynthesis based on these large datasets may be limited by the erroneous assembly of close transcripts and isoforms, even with the multiple available transcriptomes. RESULTS: Secologanin synthases (SLS) are P450 enzymes that catalyze an unusual ring-opening reaction of loganin in the biosynthesis of the MIA precursor secologanin. We report here the identification and characterization in C. roseus of a new isoform of SLS, SLS2, sharing 97 % nucleotide sequence identity with the previously characterized SLS1. We also discovered that both isoforms further oxidize secologanin into secoxyloganin. SLS2 had however a different expression profile, being the major isoform in aerial organs that constitute the main site of MIA accumulation. Unfortunately, we were unable to find a current C. roseus transcriptome database containing simultaneously well reconstructed sequences of SLS isoforms and accurate expression levels. After a pair of close mRNA encoding tabersonine 16-hydroxylase (T16H1 and T16H2), this is the second example of improperly assembled transcripts from the MIA pathway in the public transcriptome databases. To construct a more complete transcriptome resource for C. roseus, we re-processed previously published transcriptome data by combining new single assemblies. Care was particularly taken during clustering and filtering steps to remove redundant contigs but not transcripts encoding potential isoforms by monitoring quality reconstruction of MIA genes and specific SLS and T16H isoforms. The new consensus transcriptome allowed a precise estimation of abundance of SLS and T16H isoforms, similar to qPCR measurements. CONCLUSIONS: The C. roseus consensus transcriptome can now be used for characterization of new genes of the MIA pathway. Furthermore, additional isoforms of genes encoding distinct MIA biosynthetic enzymes isoforms could be predicted suggesting the existence of a higher level of complexity in the synthesis of MIA, raising the question of the evolutionary events behind what seems like redundancy.

The Rauvolfia tetraphylla genome suggests multiple distinct biosynthetic routes for yohimbane monoterpene indole alkaloids.

Monoterpene indole alkaloids (MIAs) are a structurally diverse family of specialized metabolites mainly produced in Gentianales to cope with environmental challenges. Due to their pharmacological properties, the biosynthetic modalities of several MIA types have been elucidated but not that of the yohimbanes. Here, we combine metabolomics, proteomics, transcriptomics and genome sequencing of Rauvolfia tetraphylla with machine learning to discover the unexpected multiple actors of this natural product synthesis. We identify a medium chain dehydrogenase/reductase (MDR) that produces a mixture of four diastereomers of yohimbanes including the well-known yohimbine and rauwolscine. In addition to this multifunctional yohimbane synthase (YOS), an MDR synthesizing mainly heteroyohimbanes and the short chain dehydrogenase vitrosamine synthase also display a yohimbane synthase side activity. Lastly, we establish that the combination of geissoschizine synthase with at least three other MDRs also produces a yohimbane mixture thus shedding light on the complex mechanisms evolved for the synthesis of these plant bioactives.

RNA-seq Analysis of Monoterpene Indole Alkaloid Biosynthetic Pathway Elucidation in Catharanthus roseus.

Increased affordability and availability of high-throughput next-generation sequencing (NGS) technologies have resulted in an explosion of available RNA-seq data, igniting a variety of data-mining methodologies, valuable for plant-specialized biosynthetic pathway discovery. When combined with traditional homology-based annotations, these methods can facilitate short-listing candidate genes for downstream functional validation screenings. Genes related to common pathways often display homogenous expression patterns across different tissue types and experimental conditions. Here, we describe bioinformatic protocols for exploiting such coexpression to shortlist candidate genes of the well-described monoterpene indole alkaloid (MIA) pathway of Catharanthus roseus. These methods aim to inspire researchers to utilize this publicly available RNA-seq treasure trove to guide their own endeavors in the characterization of missing steps in plant metabolic pathways.

Chromosome-scale genomes throw light on plant drug biosynthesis.

Recent chromosome-scale genomes from prominent medicinal plants have provided unprecedented insight into the architecture and evolution of some prominent metabolic pathways. These new technologies also facilitate the identification of plant drug biosynthetic genes and would likely accelerate the development of new bioengineering procedures to secure the supply of important plant-derived pharmaceuticals.

Predicting Monoterpene Indole Alkaloid-Related Genes from Expression Data with Artificial Neural Networks.

Elucidation of biological pathways leading to specialized metabolites remains a complex task. It is however a mandatory step to allow bioproduction into heterologous hosts. Many steps have already been identified using conventional approaches, enlarging the space of known possible chemical steps. In the recent past years, identification of missing steps has been fueled by the generation of genomic and transcriptomic data for nonmodel species. The analysis of gene expression profiles has revealed that in many cases, genes encoding enzymes involved in the same biosynthetic pathways are coexpressed across different tissue types and environmental conditions. Hence, coexpressed studies, either in the form of differential gene expression, gene coexpression network, or unsupervised clustering methods, have helped deciphering missing steps to complete knowledge on biosynthetic pathways. Already identified biosynthetic steps can be used as baits to capture the remaining unknown steps. The present protocol shows how supervised machine learning in the form of artificial neural networks (ANNs) can efficiently classify genes as specialized metabolism related or not according to their expression levels. Using Catharanthus roseus as an example, we show that ANN trained on a minimal set of bait genes results in many true positives (correctly predicted genes) while keeping false positives low (containing possible candidate genes).

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.