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.

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.

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).

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.

Callus cultures of Choisya ternata have been prepared by different strategies: aggregate clones, subclones and protoclones obtained from well-established strains; protoclones obtained from mesophyll tissue; cultures transformed by Agrobacterium tumefaciens. All of them show high variability in their dihydrofuroquinoline alkaloid production. As compared to the alkaloid content of the whole plant, one alkaloid (platydesminium) could be obtained in higher amounts in some lines, but it was impossible to get high-balfourodinium accumulating lines. Moreover balfourodinium-producing capacities were lower in transformed cells as compared to those of normal cell lines.

Protoclones and aggregate clones have been prepared from 5 callus strains of C. ternata chosen for their dihydrofuroquinoline-accumulating capacities in a population of well-established strains. The results show that it is possible to obtain cell lines which accumulate higher alkaloid contents than the highest alkaloid-producing strain; the probability of obtaining a high-producing clone is greater if a high-producing strain is chosen as the parent strain for cloning. Compared to the alkaloid content of the whole plant, one alkaloid (platydesminium) could be obtained in greater amounts in some clones, but another alkaloid (balfourodinium) was always found in lesser quantities, even in the clones which accumulated most alkaloids. Aggregate clones were easier to obtain than protoclones and alkaloid production was rather unstable in all the clones. The protoplast-cloning procedure was not more efficient than the aggregate-cloning procedure for the selection of high-alkaloid producing lines.