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Abstract 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.

Specialized metabolites possess diverse interesting biological activities and some cardenolides- and monoterpene indole alkaloids- (MIAs) derived pharmaceuticals are currently used to treat human diseases such as cancers or hypertension. While these two families of biocompounds are produced by specific subfamilies of Apocynaceae, one member of this medicinal plant family, the succulent tree Pachypodium lamerei Drake (also known as Madagascar palm), does not produce such specialized metabolites. To explore the evolutionary paths that have led to the emergence and loss of cardenolide and MIA biosynthesis in Apocynaceae, we sequenced and assembled the P. lamerei genome by combining Oxford Nanopore Technologies long-reads and Illumina short-reads. Phylogenomics revealed that, among the Apocynaceae whose genomes have been sequenced, the Madagascar palm is so far the species closest to the common ancestor between MIA producers/non-MIA producers. Transposable elements, constituting 72.48% of the genome, emerge as potential key players in shaping genomic architecture and influencing specialized metabolic pathways. The absence of crucial MIA biosynthetic genes such as strictosidine synthase in P. lamerei and non-Rauvolfioideae species hints at a transposon-mediated mechanism behind gene loss. Phylogenetic analysis not only showcases the evolutionary divergence of specialized metabolite biosynthesis within Apocynaceae but also underscores the role of transposable elements in this intricate process. Moreover, we shed light on the low conservation of enzymes involved in the final stages of MIA biosynthesis in the distinct MIA-producing plant families, inferring independent gains of these specialized enzymes along the evolution of these medicinal plant clades. Overall, this study marks a leap forward in understanding the genomic dynamics underpinning the evolution of specialized metabolites biosynthesis in the Apocynaceae family, with transposons emerging as potential architects of genomics restructuring and gene loss.

Synthetic biology is constantly making progress for producing compounds on demand. Recently, Yocum and collaborators have developed an outstanding approach based on the anchoring of biosynthetic enzymes to the peroxisomal membrane. This allowed access to an untapped resource of acetyl-CoA and stimulated the synthesis of a valuable polyketide.

Summary The pharmaceutical industry faces a growing demand and recurrent shortages in many anticancer plant drugs given their extensive use in human chemotherapy. Efficient alternative strategies of supply of these natural products such as bioproduction by microorganisms are needed to ensure stable and massive manufacturing. Here, we developed and optimized yeast cell factories efficiently converting tabersonine to vindoline, a precursor of the major anticancer alkaloids vinblastine and vincristine. First, fine‐tuning of heterologous gene copies restrained side metabolites synthesis towards vindoline production. Tabersonine to vindoline bioconversion was further enhanced through a rational medium optimization (pH, composition) and a sequential feeding strategy. Finally, a vindoline titre of 266 mg l−1 (88% yield) was reached in an optimized fed‐batch bioreactor. This precursor‐directed synthesis of vindoline thus paves the way towards future industrial bioproduction through the valorization of abundant tabersonine resources.

The Madagascar periwinkle, Catharanthus roseus, belongs to the Apocynaceae family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the C. roseus genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads. This more contiguous assembly consists of 173 scaffolds with a total length of 581.128 Mb and an N50 of 12.241 Mb. Using publicly available RNAseq data, 21,061 protein coding genes were predicted and functionally annotated. A total of 42.87% of the genome was annotated as transposable elements, most of them being long-terminal repeats. Together with the increasing access to MIA-producing plant genomes, this updated version should ease evolutionary studies leading to a better understanding of MIA biosynthetic pathway evolution.

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.

Camptothecin is a clinically important monoterpene indole alkaloid (MIAs) used for treating various cancers. Currently, the production of this biopharmaceutical hinges on its extraction from camptothecin-producing plants, leading to high market prices and supply bottlenecks. While synthetic biology combined with metabolic approaches could represent an attractive alternative approach to manufacturing, it requires firstly a complete biosynthetic pathway elucidation, which is, unfortunately, severely missing in species naturally accumulating camptothecin. This knowledge gap can be attributed to the lack of high-quality genomic resources of medicinal plant species. In such a perspective, Yamazaki and colleagues produced the first described and experimentally validated chromosome-level plant genome assembly of Ophiorrhiza pumila, a prominent source plant of camptothecin for the pharmaceutical industry. More specifically, they have developed a method incorporating Illumina reads, PacBio single-molecule reads, optical mapping and Hi-C sequencing, followed by the experimental validation of contig orientation within scaffolds, using fluorescence in situ hybridization (FISH) analysis. This relevant strategy resulted in the most contiguous and complete de novo plant reference genome described to date, which can streamline the sequencing of new plant genomes. Further mining approaches, including integrative omics analysis, phylogenetics, gene cluster evaluation and comparative genomics were successfully used to puzzle out the evolutionary origins of MIA metabolism and revealed a short-list of high confidence MIA biosynthetic genes for functional validation.

Terpenoids, such as squalene, are valuable compounds for cosmetic and drug industries, the supply of which is often limited by natural sources. Alternative production strategies have been investigated for decades but remain challenging due to low yields.

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.

The biosynthetic pathway of preakuammicine, a monoterpene precursor of the anti-cancer agent vinblastine, has remained largely unexplored. Here, the authors provide transcriptomic and biochemical data to identify two enzymes that, in tandem, convert strictosidine to akuammicine, the stable shunt product of preakuammicine.

The lesser periwinkle Vinca minor accumulates numerous monoterpene indole alkaloids (MIAs) including the vasodilator vincamine. While the biosynthetic pathway of MIAs has been largely elucidated in other Apocynaceae such as Catharanthus roseus, the counterpart in V. minor remains mostly unknown, especially for reactions leading to MIAs specific to this plant. As a consequence, we generated a comprehensive V. minor transcriptome elaborated from eight distinct samples including roots, old and young leaves exposed to low or high light exposure conditions. This optimized resource exhibits an improved completeness compared to already published ones. Through homology-based searches using C. roseus genes as bait, we predicted candidate genes for all common steps of the MIA pathway as illustrated by the cloning of a tabersonine/vincadifformine 16-O-methyltransferase (Vm16OMT) isoform. The functional validation of this enzyme revealed its capacity of methylating 16-hydroxylated derivatives of tabersonine, vincadifformine and lochnericine with a Km 0.94 ± 0.06 µM for 16-hydroxytabersonine. Furthermore, by combining expression of fusions with yellow fluorescent proteins and interaction assays, we established that Vm16OMT is located in the cytosol and forms homodimers. Finally, a gene co-expression network was performed to identify candidate genes of the missing V. minor biosynthetic steps to guide MIA pathway elucidation.

Cytokinins (CKs) and ethylene (ET) are among the most ancient organic chemicals on Earth. A wide range of organisms including plants, algae, fungi, amoebae, and bacteria use these substances as signaling molecules to regulate cellular processes. Because of their ancestral origin and ubiquitous occurrence, CKs and ET are also considered to be ideal molecules for inter-kingdom communication. Their signal transduction pathways were first historically deciphered in plants and are related to the two-component systems, using histidine kinases as primary sensors. Paradoxically, although CKs and ET serve as signaling molecules in different kingdoms, it has been supposed for a long time that the canonical CK and ET signaling pathways are restricted to terrestrial plants. These considerations have now been called into question following the identification over recent years of genes encoding CK and ET receptor homologs in many other lineages within the tree of life. These advances shed new light on the dissemination and evolution of these hormones as both intra- and inter-specific communication molecules in prokaryotic and eukaryotic organisms.

The stereochemistry of the plant heteroyohimbine alkaloids is a key factor determining their diverse biological activities. Here, the authors carry out structural, localization and genetic experiments to understand the mechanism of stereoselectivity for three heteroyohimbine synthases and to identify their function in vivo.

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.

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis.

Grape accumulates numerous polyphenols with abundant health benefit and organoleptic properties that in planta act as key components of the plant defense system against diseases. Considerable advances have been made in the chemical characterization of wine metabolites particularly volatile and polyphenolic compounds. However, the metabotyping (metabolite-phenotype characterization) of grape varieties, from polyphenolic-rich vineyard by-product is unprecedented. As this composition might result from the complex interaction between genotype, environment and viticultural practices, a field experiment was setting up with uniform pedo-climatic factors and viticultural practices of growing vines to favor the genetic determinism of polyphenol expression. As a result, UPLC-MS-based targeted metabolomic analyses of grape stems from 8 Vitis vinifera L. cultivars allowed the determination of 42 polyphenols related to phenolic acids, flavonoids, procyanidins, and stilbenoids as resveratrol oligomers (degree of oligomerization 1–4). Using a partial least-square discriminant analysis approach, grape stem chemical profiles were discriminated according to their genotypic origin showing that polyphenol profile express a varietal signature. Furthermore, hierarchical clustering highlights various degree of polyphenol similarity between grape varieties that were in agreement with the genetic distance using clustering analyses of 22 microsatellite DNA markers. Metabolite correlation network suggested that several polyphenol subclasses were differently controlled. The present polyphenol metabotyping approach coupled to multivariate statistical analyses might assist grape selection programs to improve metabolites with both health-benefit potential and plant defense traits.

Cytokinin signaling is a key regulatory pathway of many aspects in plant development and environmental stresses. Herein, we initiated the identification and functional characterization of the five CHASE-containing histidine kinases (CHK) in the economically important Malus domestica species. These cytokinin receptors named MdCHK2, MdCHK3a/MdCHK3b, and MdCHK4a/MdCHK4b by homology with Arabidopsis AHK clearly displayed three distinct profiles. The three groups exhibited architectural variations, especially in the N-terminal part including the cytokinin sensing domain. Using a yeast complementation assay, we showed that MdCHK2 perceives a broad spectrum of cytokinins with a substantial sensitivity whereas both MdCHK4 homologs exhibit a narrow spectrum. Both MdCHK3 homologs perceived some cytokinins but surprisingly they exhibited a basal constitutive activity. Interaction studies revealed that MdCHK2, MdCHK4a, and MdCHK4b homodimerized whereas MdCHK3a and MdCHK3b did not. Finally, qPCR analysis and bioinformatics approach pointed out contrasted expression patterns among the three MdCHK groups as well as distinct sets of co-expressed genes. Our study characterized for the first time the five cytokinin receptors in apple tree and provided a framework for their further functional studies.

ABSTRACT Histidine kinases (HKs) are among the most prominent sensing proteins studied in the kingdom Fungi. Their distribution and biological functions in early diverging fungi (EDF), however, remain elusive. We have taken advantage of recent genomic resources to elucidate whether relationships between the occurrence of specific HKs in some EDF and their respective habitat/lifestyle could be established. This led to the unexpected discovery of fungal HKs that share a high degree of similarity with receptors for plant hormones (ethylene and cytokinin). Importantly, these phytohormone receptor homologs are found not only in EDF that behave as plant root symbionts or endophytes but also in EDF species that colonize decaying plant material. We hypothesize that these particular sensing proteins promoted the interaction of EDF with plants, leading to the conquest of land by these ancestral fungi.

The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite there was a significant increment in the amount of β1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and β1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1 null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with an atypical role for O-linked mannans.

Previous works have shown the existence of protein partnerships belonging to a MultiStep Phosphorelay (MSP) in Populus putatively involved in osmosensing. This study is focused on the identification of a histidine-aspartate kinase, HK1b, paralog of HK1a. The characterization of HK1b showed its ability to homo- and hetero-dimerize and to interact with a few Histidine-containing Phosphotransfer (HPt) proteins, suggesting a preferential partnership in poplar MSP linked to drought perception. Furthermore, determinants for interaction specificity between HK1a/1b and HPts were studied by mutagenesis analysis, identifying amino acids involved in this specificity. The HK1b expression analysis in different poplar organs revealed its co-expression with three HPts, reinforcing the hypothesis of partnership participation in the MSP in planta. Moreover, HK1b was shown to act as an osmosensor with kinase activity in a functional complementation assay of an osmosensor deficient yeast strain. These results revealed that HK1b showed a different behaviour for canonical phosphorylation of histidine and aspartate residues. These phosphorylation modularities of canonical amino acids could explain the improved osmosensor performances observed in yeast. As conserved duplicates reflect the selective pressures imposed by the environmental requirements on the species, our results emphasize the importance of HK1 gene duplication in poplar adaptation to drought stress.

The Madagascar periwinkle, Catharanthus roseus, belongs to the Apocynaceae family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the C. roseus genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads. This more contiguous assembly consists of 173 scaffolds with a total length of 581.128 Mb and an N50 of 12.241 Mb. Using publicly available RNAseq data, 21,061 protein coding genes were predicted and functionally annotated. A total of 42.87% of the genome was annotated as transposable elements, most of them being long-terminal repeats. Together with the increasing access to MIA-producing plant genomes, this updated version should ease evolutionary studies leading to a better understanding of MIA biosynthetic pathway evolution.