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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.

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.

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.

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.

Vinca minor, also known as the lesser periwinkle, is a well-known species from the Apocynaceae, native to central and southern Europe. This plant synthesizes monoterpene indole alkaloids, which are a class of specialized metabolites displaying a wide range of bioactive- and pharmacologically important properties. Within the almost 50 monoterpene indole alkaloids it produces, V. minor mainly accumulates vincamine, which is commercially used as a nootropic. Using a combination of Oxford Nanopore Technologies long read- and Illumina short-read sequencing, a 679,098 Mb V. minor genome was assembled into 296 scaffolds with an N50 scaffold length of 6 Mb, and encoding 29,624 genes. These genes were functionally annotated and used in a comparative genomic analysis to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. Furthermore, homology-based monoterpene indole alkaloid gene predictions together with a metabolic analysis across 4 different V. minor tissue types guided the identification of candidate monoterpene indole alkaloid genes. These candidates were finally used to identify monoterpene indole alkaloid gene clusters, which combined with synteny analysis allowed for the discovery of a functionally validated vincadifformine-16-hydroxylase, reinforcing the potential of this dataset for monoterpene indole alkaloids gene discovery. It is expected that access to these resources will facilitate the elucidation of unknown monoterpene indole alkaloid biosynthetic routes with the potential of transferring these pathways to heterologous expression systems for large-scale monoterpene indole alkaloid production.

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2 C -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.

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.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

Isopentenyl diphosphate isomerase (IDI) is a key enzyme of the isoprenoid pathway, catalyzing the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors of all isoprenoids. In plants, several subcellular compartments, including cytosol/ER, peroxisomes, mitochondria and plastids, are involved in isoprenoid biosynthesis. Here, we report on the unique triple targeting of two Catharanthus roseus IDI isoforms encoded by a single gene (CrIDI1). The triple localization of CrIDI1 in mitochondria, plastids and peroxisomes is explained by alternative transcription initiation of CrIDI1, by the specificity of a bifunctional N-terminal mitochondria/plastid transit peptide and by the presence of a C-terminal peroxisomal targeting signal. Moreover, bimolecular fluorescence complementation assays revealed self-interactions suggesting that the IDI likely acts as a multimer in vivo.

Isopentenyl diphosphate isomerases (IDI) catalyze the interconversion of the two isoprenoid universal C5 units, isopentenyl diphosphate and dimethylally diphosphate, to allow the biosynthesis of the large variety of isoprenoids including both primary and specialized metabolites. This isomerisation is usually performed by two distinct IDI isoforms located either in plastids/peroxisomes or mitochondria/peroxisomes as recently established in Arabidopsis thaliana mainly accumulating primary isoprenoids. By contrast, almost nothing is known in plants accumulating specialized isoprenoids. Here we report the cloning and functional validation of an IDI encoding cDNA (CrIDI1) from Catharanthus roseus that produces high amount of monoterpenoid indole alkaloids. The corresponding gene is expressed in all organs including roots, flowers and young leaves where transcripts have been detected in internal phloem parenchyma and epidermis. The CrIDI1 gene also produces long and short transcripts giving rise to corresponding proteins with and without a N-terminal transit peptide (TP), respectively. Expression of green fluorescent protein fusions revealed that the long isoform is targeted to both plastids and mitochondria with an apparent similar efficiency. Deletion/fusion experiments established that the first 18-residues of the N-terminal TP are solely responsible of the mitochondria targeting while the entire 77-residue long TP is needed for an additional plastid localization. The short isoform is targeted to peroxisomes in agreement with the presence of peroxisome targeting sequence at its C-terminal end. This complex plastid/mitochondria/peroxisomes triple targeting occurring in C. roseus producing specialized isoprenoid secondary metabolites is somehow different from the situation observed in A. thaliana mainly producing housekeeping isoprenoid metabolites.

The monoterpene indole alkaloids (MIAs) from Madagascar periwinkle (Catharanthus roseus) are secondary metabolites of high interest due to their therapeutical values. Secologanin, the monoterpenoid moiety incorporated into MIAs, is derived from the plastidial methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have cloned a cDNA encoding hydroxymethylbutenyl diphosphate synthase (HDS), a MEP pathway enzyme, and generated antibodies to investigate the distribution of transcripts and protein in MIA-producing aerial tissues. Consistent with our earlier work, transcripts for the genes encoding the so-called early steps in monoterpenoid biosynthesis (ESMB) enzymes (HDS, others MEP pathway enzymes and geraniol 10-hydroxylase) were preferentially co-localized to internal phloem associated parenchyma (IPAP) cells. By contrast, transcripts for the enzyme catalysing the last biosynthetic step to secologanin, secologanin synthase, were found in the epidermis. A coordinated response of ESMB genes was also observed in cell cultures stimulated to synthesise MIAs by hormone treatment, whereas no changes in SLS expression were detected under the same experimental conditions. Immunocytolabelling studies with the HDS-specific serum demonstrated the localisation of HDS to the plastid stroma and revealed that HDS proteins were most abundant in IPAP cells but could also be found in other cell types, including epidermal and mesophyll cells. Besides showing the existence of post-transcriptional mechanisms regulating the levels of HDS in C. roseus cells, our results support that intercellular translocation likely plays an important role during monoterpene-secoiridoid assembly.