Audrey Oudin

Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus

Two periwinkle cDNAs (crdxr and crmecs) encoding enzymes of the non-mevalonate terpenoid pathway were characterized using reverse transcription-PCR strategy based on the design of degenerated oligonucleotides. The deduced amino acid sequence of crdxr is homologue to 1-deoxy-D-xylulose 5-phosphate reductoisomerases. Crmecs represents the first plant cDNA encoding a protein similar to the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase from Escherichia coli. Expression of crdxr and crmecs genes was up-regulated in periwinkle cells producing monoterpenoid indole alkaloids. Involvement of the 2C-methyl-D-erythritol 4-phosphate pathway in alkaloid biosynthesis is discussed.

1-Deoxy-D-xylulose 5-phosphate synthase from periwinkle: cDNA identification and induced gene expression in terpenoid indole alkaloid-producing cells.

Abstract Terpenoid indole alkaloids (TIAs) arise from the indole and the monoterpene pathways. The latter route derives from isopentenyl diphosphate (IPP). We report on the isolation and characterization of a cDNA ( crdxs ) encoding for 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) in Catharanthus roseus suspension cultures. The enzyme catalyses the formation of the precursor of the non-mevalonate pathway leading to IPP biosynthesis. Expression in Escherichia coli of the truncated DXPS lacking the putative plastid transit peptide revealed that this pseudomature protein was active. crdxs mRNA were detected only in TIA producing-cells and the accumulation of transcripts was found to be associated with TIA production. This result corroborates recent studies obtained with a labelled precursor, which have given evidence that the non-mevalonate pathway is involved in the biosynthesis of the precursor of TIAs secologanin.

Spatial distribution and hormonal regulation of gene products from methyl erythritol phosphate and monoterpene-secoiridoid pathways in Catharanthus roseus

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.

Optimization of the transient transformation of Catharanthus roseus cells by particle bombardment and its application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and geraniol 10-hydroxylase.

The monoterpene indole alkaloids (MIA) synthesized in Catharanthus roseus are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits a complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize MIA biosynthetic enzymes within C. roseus cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol 10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation of HDS within plastids of C. roseus cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids, i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both necessary and sufficient to drive the ER anchoring.

The iridoid pathway in Catharanthus roseus alkaloid biosynthesis

The Apocynaceae Catharanthus roseus accumulates a number of monoterpene indole alkaloids (MIAs) that originate from the coupling of the indole and the iridoid pathways. The latter pathway is usually considered as limiting for MIA biosynthesis, but evidence is now strong that the precursors tryptamine (from the indole pathway) and secologanin (from the iridoid pathway) have to be provided within the cells in a concerted manner for sustained MIA synthesis. Secologanin is formed from isopentenyl diphosphate (IPP) in a number of steps, some of which are still unknown. However significant progress has been obtained recently with the characterisation of cDNAs encoding secologanin synthase and the two constituents of geraniol 10-hydroxylase (G10H). IPP itself is formed through both the plastidial methyl-erythritol phosphate (MEP) pathway and the cytosolic mevalonate (MVA) pathway. The MEP pathway comprises 7 steps of which 4 have been identified at the molecular level in C. roseus. This pathway plays a major role in the production of MIAs, but there is now evidence that the MVA pathway serves as a minor source of precursors for iridoid biosynthesis and/or contributes (through protein prenylation) to a fine regulation of the MEP gene expression. G10H is one of the key enzymes of the MIA pathway and the up-regulation of the gene activity concomitantly with an increase in G10H activity and MIA production have been reported with various hormones and elicitors. Since regulatory genes encoding transcription factors acting on several genes of the MEP and terpenoid pathways are beginning to be characterised, metabolic engineering of the iridoid pathway could be a promising approach to control the metabolite flux towards secologanin and MIA production through biotechnological applications in the future.

The subcellular organization of strictosidine biosynthesis in Catharanthus roseus epidermis highlights several trans‐tonoplast translocations of intermediate metabolites

Catharanthus roseus synthesizes a wide range of valuable monoterpene indole alkaloids, some of which have recently been recognized as functioning in plant defence mechanisms. More specifically, in aerial organ epidermal cells, vacuole‐accumulated strictosidine displays a dual fate, being either the precursor of all monoterpene indole alkaloids after export from the vacuole, or the substrate for a defence mechanism based on the massive protein cross‐linking, which occurs subsequent to organelle membrane disruption during biotic attacks. Such a mechanism relies on a physical separation between the vacuolar strictosidine‐synthesizing enzyme and the nucleus‐targeted enzyme catalyzing its activation through deglucosylation. In the present study, we carried out the spatial characterization of this mechanism by a cellular and subcellular study of three enzymes catalyzing the synthesis of the two strictosidine precursors (i.e. tryptamine and secologanin). Using RNA in situ hybridization, we demonstrated that loganic acid O‐methyltransferase transcript, catalysing the penultimate step of secologanin synthesis, is specifically localized in the epidermis. A combination of green fluorescent protein imaging, bimolecular fluorescence complementation assays and yeast two‐hybrid analysis enabled us to establish that both loganic acid O‐methyltransferase and the tryptamine‐producing enzyme, tryptophan decarboxylase, form homodimers in the cytosol, thereby preventing their passive diffusion to the nucleus. We also showed that the cytochrome P450 secologanin synthase is anchored to the endoplasmic reticulum via a N‐teminal helix, thus allowing the production of secologanin on the cytosolic side of the endoplasmic reticulum membrane. Consequently, secologanin and tryptamine must be transported to the vacuole to achieve strictosidine biosynthesis, demonstrating the importance of trans‐tonoplast translocation events during these metabolic processes.

A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ dependent manner in Catharanthus roseus

A newly identified cytochrome P450 isoform initiates the synthesis of valuable alkaloids in leaves of Catharanthus roseus by hydroxylating tabersonine. Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.

A single gene encodes isopentenyl diphosphate isomerase isoforms targeted to plastids, mitochondria and peroxisomes in Catharanthus roseus

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.

CaaX-prenyltransferases are essential for expression of genes involvedin the early stages of monoterpenoid biosynthetic pathwayin Catharanthus roseus cells

CaaX-prenyltransferases (CaaX-PTases) catalyse the covalent attachment of isoprenyl groups to conserved cysteine residues located at the C-terminal CaaX motif of a protein substrate. This post-translational modification is required for the function and/or subcellular localization of some transcription factors and components of signal transduction and membrane trafficking machinery. CaaX-PTases, including protein farnesyltransferase (PFT) and type-I protein geranylgeranyltransferase (PGGT-I), are heterodimeric enzymes composed of a common α subunit and a specific β subunit. We have established RNA interference cell lines targeting the β subunits of PFT and PGGT-I, respectively, in the Catharanthus roseus C20D cell line, which synthesizes monoterpenoid indole alkaloids in response to auxin depletion from the culture medium. In both types of RNAi cell lines, expression of a subset of genes involved in the early stage of monoterpenoid biosynthetic pathway (ESMB genes), including the MEP pathway, is strongly decreased. The role of CaaX-PTases in ESMB gene regulation was confirmed by using the general prenyltransferase inhibitor s-perillyl alcohol (SP) and the specific PFT inhibitor Manumycin A on the wild type line. Furthermore, supplementation of SP inhibited cells with monoterpenoid intermediates downstream of the steps encoded by the ESMB genes restores monoterpenoid indole alkaloids biosynthesis. We conclude that protein targets for both PFT and PGGT-I are required for the expression of ESMB genes and monoterpenoid biosynthesis in C. roseus, this represents a non previously described role for protein prenyltransferase in plants.

Biosynthetic Origin of E-Resveratrol Accumulation in Grape Canes during Postharvest Storage

Grape canes are vineyard waste products containing valuable phytochemicals of medicine and agriculture interest. Grape canes storage is critical for the accumulation of these bioactive compounds. In the present study, we investigated the changes in stilbenoid phytochemical composition during grape cane storage and the influence of the temperature on final concentrations. A strong increase in the concentration of the monomer E-resveratrol (approximately 40-fold) was observed during the first 6 weeks of storage at 20 °C in eight different grape varieties without any change in oligomer concentrations. The E-resveratrol accumulation was temperature-dependent with an optimal range at 15-20 °C. A 2 h heat-shock treatment aiming at protein denaturation inhibited E-resveratrol accumulation. The constitutive expression of key genes involved in the stilbene precursor biosynthesis along with an induction of stilbene synthase (STS) expression during the first weeks of storage contribute to a de novo biosynthesis of E-resveratrol in pruned wood grapes.