In Catharanthus roseus cell cultures, cytokinins (CK) improve monoterpenoid indole alkaloids (MIAs) accumulation. This metabolite production is correlated with an increase of transcripts corresponding to genes encoding both elements of the CK-signaling pathway and enzymes implicated in MIAs biosynthesis. However, it has not been demonstrated that the CK signal, leading to MIAs accumulation, comes through components identified as belonging to the CK-signaling pathway. In this work, we addressed this question, by transgenesis, using an inducible RNAi system targeting element of CK-signaling. In transgenic lines, the up-regulation by CK of two genes involved in MIA biosynthesis was abolished. These results demonstrate a relationship between the CK-signaling and the MIAs biosynthetic pathways.

Calcium-mediated signalling is ubiquitous in both animals and plants. Changes in cytoplasmic free Ca2+ concentration couple diverse arrays of stimuli to their specific responses, the specificity of the stimulus being determined by integrated actions between multiple Ca2+ mobilization pathways. In this work, a pharmacological approach is reported, aimed at deciphering the role of calcium as a second messenger in the transduction pathway leading to the inhibitory effect of 2,4-dichlorophenoxyacetic acid (2,4-D), in regulating monoterpene indole alkaloid (MIA) biosynthesis in Catharanthus roseus cells. It is demonstrated here that auxin-dependent MIA biosynthesis is differentially regulated by two distinct calcium release components from internal stores in C. roseus showing pharmacological profiles similar to those displayed by animal RyR and IP3 channels. MIA biosynthesis is stimulated by caffeine (Ca2+-release activator through RyR channels) and by heparin and TMB8 (Ca2+-release inhibitors of IP3 channels) whereas MIA biosynthesis is inhibited by mastoparan (Ca2+-release activator of IP3 channels) and by ruthenium red and DHBP (Ca2+-release inhibitors of RyR channels). Furthermore, calcium, as 2,4-D, acts on MIA biosynthesis by regulating the monoterpene moiety of the MIA biosynthesis pathway since calcium channel modulators preferentially modulate g10h expression, the gene encoding the enzyme of the secoiridoid monoterpene pathway, that is the major target of 2,4-D action. In addition, the simultaneous use of caffeine (an activator of RyR channel in animals) and TMB8 (an inhibitor of the IP3 channel) in 2,4-D treated cells triggers a synergistic effect on MIA accumulation. This finding suggests an opposite and co-ordinated action of multiple Ca2+-release pathways in 2,4-D signal transduction, adding a new level of complexity to calcium signalling in plants and questioning the existence of RyR and IP3 channels in plants.

In periwinkle cell suspensions, amounts of gibberellic acid ranging from 10 ( - 10) M to 10 ( - 7) M significantly antagonized, in a dose-dependant manner, the stimulation of ajmalicine biosynthesis by cytokinins (CKs). This inhibitory effect was strictly correlated with the abolition of the expression of two genes encoding enzymes of the monoterpenoid indole alkaloid (MIA) biosynthetic pathway and was normally upregulated after CK treatments. Moreover, low concentrations of the gibberellin biosynthesis inhibitor paclobutrazol could reverse the inhibitory effects of low auxin levels on ajmalicine accumulation in the cells. On the other hand, gibberellic acid could not affect the expression of two type-A response regulators considered to be CK primary response genes in periwinkle cells. The antagonistic effects of gibberellins and cytokinins on MIA biosynthesis and their possible impact on elements of the signal transduction are discussed.

In periwinkle cell suspensions, the accurate quantification of gene expression through real-time RT-PCR showed that two type-A response regulators ( RR-A ), considered primary cytokinin (CK)-responsive genes, were differentially regulated after CK treatment. Specific inhibition of phospholipase D (PLD)-dependent phosphatidic acid (PA) production by primary alcohols reduced significantly the transcript level of one gene in response to CK, although the other gene was unaffected. Moreover, this inhibitory effect on gene transcript level could be antagonized by exogenous supply of PA. These results suggest that PA, likely released from the membrane by PLD activity, could operate in the early steps of CK signalling in periwinkle cells.

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.

In the past two decades, hairy root research for the production of important secondary metabolites has received a lot of attention. The addition of knowledge to overcome the limiting culture parameters of the regulation of the metabolic pathway by specific molecules and the development of novel tools for metabolic engineering now offer new possibilities to improve the hairy root technique for the production of metabolites. Furthermore, engineering hairy roots for the production of animal proteins of therapeutic interest in confined and controlled in vitro conditions is seen as one of the exciting spin-offs of the technology. Recent progress made in the scale-up of the hairy root cultures has paved the way for industrial exploitation of this system. This review highlights some of the significant progress made in the past three years and discusses the potential implications of that research.

In the past two decades, hairy root research for the production of important secondary metabolites has received a lot of attention. The addition of knowledge to overcome the limiting culture parameters of the regulation of the metabolic pathway by specific molecules and the development of novel tools for metabolic engineering now offer new possibilities to improve the hairy root technique for the production of metabolites. Furthermore, engineering hairy roots for the production of animal proteins of therapeutic interest in confined and controlled in vitro conditions is seen as one of the exciting spin-offs of the technology. Recent progress made in the scale-up of the hairy root cultures has paved the way for industrial exploitation of this system. This review highlights some of the significant progress made in the past three years and discusses the potential implications of that research.

Signalling pathways involving histidine kinase receptors (HKRs) are widely used by prokaryotes and fungi to regulate a large palette of biological processes. In plants, HKRs are known to be implicated in cytokinin, ethylene, and osmosensing transduction pathways. In this work, a full length cDNA named CRCIK was isolated from the tropical species CATHARANTHUS ROSEUS (L.) G. Don. It encodes a 1205 amino acid protein that belongs to the hybrid HKR family. The deduced amino acid sequence shows the highest homology with AtHK1, an osmosensing HKR in ARABIDOPSIS THALIANA. In return, CrCIK protein shares very low identity with the other 10 ARABIDOPSIS HKRs. Southern blot analysis indicates that the CRCIK corresponding gene is either present in multiple copies or has very close homologues in the genome of the tropical periwinkle. The gene is widely expressed in the plant. In C. ROSEUS C20D cell suspension, it is slightly induced after exposure to low temperature, pointing to a putative role in cold-shock signal transduction.

High stability of the production of secondary metabolites is an interesting characteristic of hairy root cultures. For 25 years, hairy roots have been investigated as a biological system for the production of valuable compounds from medicinal plants. A better understanding of the molecular mechanism of hairy root development, which is based on the transfer of Agrobacterium rhizogenes T-DNA into the plant genome, has facilitated its increasing use in metabolic engineering. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for the pharmaceutical industry. In addition, hairy roots offer promise for phytoremediation because of their abundant neoplastic root proliferation. Recent progress in the scaling-up of hairy root cultures is making this system an attractive tool for industrial processes.

Substituted 3-[2-(diethoxyphosphoryl)propyl]oxazolo[4,5-b]pyridine-2(3H)-ones were obtained (R=aryl, vinyl, carbonyl chains) via reactions catalyzed with palladium. These new fosmidomycin analogues inhibited the accumulation of indole alkaloids in periwinkle cells cultures (Catharanthus roseus).

Identification of molecular markers of monoterpenoid indole alkaloid (MIA) accumulation in cell-suspension cultures of Madagascar periwinkle (Catharanthus roseus (L.) G. Don) was performed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the protein patterns from alkaloid-producing and non-producing cells showed the specific occurrence of a 28 kDa polypeptide restricted to cells accumulating MIAs. The polypeptide was purified by preparative two-dimensional gel electrophoresis, digested with trypsin, and microsequenced by the Edman degradation method. Cloning of the corresponding cDNA revealed that the protein which has been named CrPS (Catharanthus roseus Protein S) is a member of the alpha/beta hydrolase superfamily. Time-course expression studies by northern blot analysis confirmed that CrPS gene expression was associated with MIA accumulation in cell suspension cultures. In the whole plant, multicellular compartmentation is required for alkaloid biosynthesis. In situ mRNA hybridization on developing leaves revealed that CrPS mRNA and transcripts encoding the first enzymes of the MIA pathway were co-localized in internal phloem parenchyma cells. The possible implication of the alkaloid-accumulation associated protein CrPS in the signal transduction pathway leading to MIA production is discussed.

The Madagascar periwinkle Catharanthus roseus accumulates a number of terpenoid indole alkaloids, some of which have high therapeutic interest. The biotechnological approach with cells in vitro remains an alternative to the field culture of periwinkle for the production of such compounds. We previously reported that two phytohormones, cytokinin and ethylene, remarkably enhanced the accumulation of alkaloids in periwinkle cell suspensions. In this work, we investigated the effects of these hormones on the regulation of several genes of the indole alkaloid biosynthetic pathway. We show that cytokinin and/or ethylene greatly enhanced the expression of the geraniol 10-hydroxylase gene. When given together, these hormones also increased the expression of three genes belonging to the methyl-erythritol pathway. These results make it possible to consider elements of cytokinin and ethylene signalling pathways as tools for improving terpenoid indole alkaloid production through metabolic engineering.

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.

1,3 Dipolar cycloadditions of nitrile oxides generated in situ in the presence of a variety of olefins provided 4,5-dihydroisoxazoles. The whole procedure could be performed in a practical and efficient one-pot operation. The products are of excellent purity (95%) and are isolated in 60-83% yields. Some of them enhanced the accumulation of indole alkaloids in periwinkle cell cultures.

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.

In higher plants, isopentenyl diphosphate (IPP) is synthesised both from the plastidic 2-C-methyl-d-erythritol 4-phosphate (MEP) and from the cytosolic mevalonate (MVA) pathways. Primary metabolites, such as phytol group of chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and gibberellins (GAs) are derived directly from the MEP pathway. Many secondary metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus, are also synthesised from this source of IPP. Using Northern blot and in situ hybridisation experiments, we show that three MEP pathway genes (1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MECS)) and the gene encoding geraniol 10-hydroxylase (G10H), a cytochrome P450 monooxygenase involved in the first committed step in the formation of iridoid monoterpenoids display identical cell-specific expression patterns. The co-localisation of these four transcripts to internal phloem parenchyma of young aerial organs of C. roseus adds a new level of complexity to the multicellular nature of MIA biosynthesis. We predict the translocation of pathway intermediates from the internal phloem parenchyma to the epidermis and, ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the translocation of intermediates from the phloem parenchyma is probably also required during the biosynthesis of hormones and photosynthetic primary metabolites derived from the MEP pathway.

A full length cDNA (designated CrETR1) was isolated by polymerase chain reaction amplification of a cDNA library from periwinkle (Catharanthus roseus) cell cultures. CrETR1 cDNA encodes a polypeptide of 740 amino acids with a predicted molecular mass of 82 kDa. The deduced protein contains a hydrophobic ethylene-binding transmembrane region, a GAF domain, a third domain homologous to the histidine protein kinase domain of the prokaryotic two-component systems, and a fourth carboxyl-terminal domain homologous to the receiver domain of the response regulators, as found in the A. thaliana ethylene receptor ETR1. CrETR1 transcripts are strongly accumulated in petals and ovaries of C. roseus young plants whereas no significant changes are detected in cell cultures submitted to various stress or hormonal (including ethylene) treatments. The amount of the monoterpene indole alkaloid ajmalicine in the cells treated by ethylene is reduced after addition of inhibitors of histidine kinases showing a possible involvement of CrETR1 protein in the ethylene-related signalling pathway leading to alkaloid biosynthesis enhancement in C. roseus cell cultures.