Marc Rideau

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.

Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media.

Catharanthus roseus cells were cultured in three types of media. These media were: a low sucrose subculture medium and two high sucrose media, each of which differed in their mineral and hormonal contents. The kinetics of tryptophan decarboxylase activity and the accumulations of tryptophan, tryptamine, ajmalicine and serpentine were different in each series but no correlation between maximum enzyme activity and alkaloid contents was observed. Ajmalicine and serpentine productions were unaffected by addition of Trp to the media, whereas addition of secologanin enhanced alkaloid production. The results seem to imply that the terpenoid pathway is the limiting factor in alkaloid production in C. roseus cells.

Cytokinin-enhanced accumulation of indole alkaloids in Catharanthus roseus cell cultures - The factors affecting the cytokinin response.

SummaryCytokinins were found to stimulate the alkaloid synthesis induced by removing auxin from the medium of a cell line of Catharanthus roseus. Diluting the mineral salts of the culture medium decreased the alkaloid production but increased the “sensitivity” of the cells. Addition of high levels of Ca2+, Mg2+ or Sr2+ to B5 media in which the mineral salts were diluted to 5–40%, increased the alkaloid production. The latter effect is related only partially to enhanced osmotic potential.

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.

Cytokinins and ethylene stimulate indole alkaloid accumulation in cell suspension cultures of Catharanthus roseus by two distinct mechanisms

Abstract The interactions between cytokinins and ethylene on alkaloid accumulation in a periwinkle cell line have been examined. It was found that (a) either exogenously-applied cytokinins or ethylene (the latter through ethephon degradation) greatly enhanced ajmalicine accumulation in cells subcultured in a 2,4-dichlorophenoxyacetic acid-free medium; (b) the enhancing effect of cytokinin was not mediated by enhancement of endogenous ethylene production contrary to what is found in some plant models; (c) the responses to exogenous cytokinin and ethylene were additive and showed a different pattern of expression. It may be concluded that cytokinin and ethylene can up-regulate the alkaloid production in a periwinkle cells through independent pathways when added exogenously to the cultures.

Metabolic changes and alkaloid production in habituated and non-habituated cells of Catharanthus roseus grown in hormone-free medium. Comparing hormone-deprived non-habituated cells with habituated cells

Summary Habituated cells of Catharanthus roseus (Apocynaceae) selected from an auxin-dependent line accumulated alkaloid in higher amounts than did the non-habituated cells grown in their maintenance medium. Ajmalicine and serpentine contents could be enhanced in both cell lines by feeding the indole alkaloid precursor secologanin or low concentrations of IAA or NAA. However the production remained low in the habituated cells as compared to that of non-autotrophic cells grown in a hormone-free production medium. By comparing the evolution of pH values, the levels of some cellular components and the activities of strictosidine synthase and tryptophan decarboxylase in habituated cells and non-habituated cells grown either in their maintenance medium (with auxin) or in a hormone-free medium, we present evidence that enhancement of alkaloid production in auxin depleted cells might be due to a combination of several factors including a better coordination between tryptamine and secologanine supplies and an increase of vacuolar accumulation capacities.

Alcaloïdes dihydrofuroquinoleiques de quelques rutaceae: Isolement, structure, proprietes biologiques

Abstract Six dihydrofuroquinoline quaternary bases have been isolated from Choisya ternata , Ptelea trifoliata and Ruta graveolens . Their structures have been determined by spectrometry and for some, the absolute configuration is reported. Some of the six isolated alkaloids have a prominent cytotoxic action on plant and animal tumor cells, and an inhibitory effect on Gram-positive bacteria.

Synthesis and biological evaluation with plant cells of new fosmidomycin analogues containing a benzoxazolone or oxazolopyridinone ring.

Fosmidomycin, 3-(N-formyl-N-hydroxyamido) propylphosphonic acid sodium salt, is an efficient inhibitor of 1-deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway notably present in Plasmodium species. We have synthesized a new series of analogues of fosmidomycin, containing a benzoxazolone, benzoxazolethione or oxazolopyridinone ring. As the MEP pathway is involved in the biosynthesis of all isoprenoids, accumulation of ajmalicine in Catharanthus roseus cells was chosen as a marker of monoterpenoid indole alkaloid (MIA) production. None of the twelve studied phosphonic esters 3 and phosphonic acids 4 affected periwinkle cell growth, but some of them (3c, 3e, 3g and 3h) showed a significant inhibition of ajmalicine accumulation: 45–85% at 125 μM. Surprisingly, this effect disappeared by conversion of 3c and 3g into the corresponding acids 4c and 4g, respectively.

Histidine-containing phosphotransfer domain extinction by RNA interference turns off a cytokinin signalling circuitry in Catharanthus roseus suspension cells

We previously reported that cytokinins (CK) induce the fast and specific transcription of CrRR1, a gene encoding a type A response regulator in Catharanthus roseus cell cultures. Here, we characterized the CrHPt1 gene that encodes a histidine‐containing phosphotransfer domain. CrHPt1 was silenced through RNA interference (RNAi) to test its possible implication in the CK signalling pathway. In transgenic lines stably transformed with an intron‐spliced construct, the degradation of CrHPt1 transcripts abolishes the CK inductive effect on CrRR1 transcription. These result give a new in vivo functional argument for the crucial role of HPt proteins in the CK signalling pathway leading to the expression of the genes encoding type A response regulators. They also show that RNAi is a powerful strategy to turn off the CK signalling circuitry.