Marc-André Fliniaux

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only.

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciens rol ABC. The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW). This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.

Hairy root induction of Papaver somniferum var. album , a difficult-to-transform plant, by A. rhizogenes LBA 9402

Two strains of Agrobacterium rhizogenes (15834, LBA 9402) and one Agrobacterium tumefaciens strain [GV 3101 (PMP90RK, p35SGUS-2)] and four culture media were tested and compared for their ability to induce hairy root formation on wounded Papaver somniferum L. hypocotyls. Five weeks after the infection with A. rhizogenes LBA 9402, hairy roots appeared on 80% of the hypocotyls maintained in the hormone-free liquid medium. Six hairy-root cultures were established. Transformation was confirmed by polymerase chain reaction analysis. One clone was analysed for its alkaloid production. The total alkaloid content was higher in the transformed roots (0.46±0.06% DW) than in the untransformed roots (0.32±0.05% DW). The transformed roots accumulated three times more codeine (0.18±0.02% DW) than intact roots (0.05±0% DW). Moreover, morphine (0.255±0.03% DW) and sanguinarine (0.014±0% DW) were found in the liquid culture medium.

The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots

The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied. The effects of nitrate and ammonium concentrations on these cultures were investigated. A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content. The alkaloid production obtained with 15.8 mM of NO3- and 20.5 mM of NH4+ was 1.2-1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO3- and 20.5 mM of NH4+). The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio. When nitrate or ammonium concentrations were raised, that ratio also was increased 2-3-fold. The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.

Simultaneous analysis of l-hyoscyamine, l-scopolamine and dl-tropic acid in plant material by reversed-phase high-performance liquid chromatography

Abstract A sensitive reversed-phase high-performance liquid chromatographic (HPLC) procedure for the analysis of the main parasympatholytic tropane alkaloids in plant material is described. It uses an acidic aqueous acetonitrile mobile phase and UV detection at 204 nm. It allows a good simultaneous separation of l-hyoscyamine, l-scopolamine and tropic acid, their acidic precursor. The detection limits are 20 ng for the alkaloids and 5 ng for tropic acid. A simple and rapid method, very convenient for HPLC analysis, is also described for the preparation of purified alkaloid extracts. The procedure was applied to the evaluation of the alkaloid content of Datura leaves. The results are in good correlation with those obtained with a tropic acid derivatives- specific enzyme immunoassay.

Immobilization of Nicotiana tabacum plant cell suspensions within calcium alginate gel beads for the production of enhanced amounts of scopolin.

Scopolin-producing cells of Nicotiana tabacum were immobilized within Ca-alginate gel beads. Free cell suspensions accumulated scopolin within cytoplasmic compartments and cell disruption was necessary to recover scopolin. On the contrary, immobilized plant cells excreted considerable amounts of scopolin. Scopolin diffused throughout the gel matrix and reached the culture media. A large fraction of produced scopolin could then be recovered from the culture medium without disrupting cells. Immobilized N. tabacum cells produced more scopolin than free cell suspensions did (3.8 mg/g fresh weight biomass [into the culture media] versus 0.2 mg/g fresh weight biomass [intracellular]). Variation of the immobilization conditions revealed a marked influence on the behavior of N. tabacum plant cells: production of scopolin and enhanced excretion, cell growth, and morphological aspect of plant cell colonies. This excretion phenomenon could be used advantageously at an industrial production level.

Glucosylation of butyric acid by cell suspension culture of Nicotiana plumbaginifolia

Abstract Suspension cells of Nicotiana plumbaginifolia rapidly absorbed exogenously applied butyric acid and converted it into a new product within one day. The conversion product was found to be 6- O -butyryl- d -glucose. This molecule could be of great interest because of its capacity to transport and release butyric acid in vivo .

Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures.

Abstract. Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (15NH4)2SO4 and K15NO3. By using two-dimensional 15N-1H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of 15N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional 15N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid γ-aminobutyric acid in the one-dimensional 15N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into γ-aminobutyric acid, N-acetylputrescine. The occurrence of 15N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen.

Chiral specificity of the degradation of nicotine by Nicotiana plumbaginifolia cell suspension cultures

Abstract The kinetics of nicotine degradation in a cell suspension culture of Nicotiana plumbaginifolia has been examined. It is shown by GC and chiral HPLC that when (R,S)-nicotine is presented, (R)-nicotine is more rapidly degraded than the natural isomer, (S)-nicotine. Conversely, (R)-nornicotine accumulates in the culture medium to a greater extent than (S)-nornicotine, indicating that the latter undergoes more rapid further metabolism to unidentified products. The demethylation of the analogue of nicotine, (R,S)-1-methyl-2-phenylpyrrolidine, was found to be competitive with the demethylation of (R,S)-nicotine.

Evaluation of the relation between the endogenous scopoletin and scopolin level of some solanaceous and papaver cell suspensions and their ability to bioconvert scopoletin to scopolin

Abstract Endogenous scopolin and its aglycon scopoletin were tested in various plant cell suspensions by HPLC. The results indicate that the levels of these compounds varied greatly from one plant cell suspension to another (10 −3 –5 mg/g dry mass). Both scopoletin and scopolin were detected in the Nicotiana tabacum L. cell suspensions while only scopolin was detected in the Duboisia myoporoides R.Br., Solanum aviculare Forst. and Papaver somniferum L. cells. The identity of this scopolin was confirmed by NMR and MS data. Neither of these compounds was accumulated in the Nicotiana plumbaginifolia Viv. cell suspensions. Moreover, all the cell suspensions, except N. tabacum could bioconvert exogenous scopoletin to scopolin. The yield of the bioconversion varied from one cell suspension to another (21–92%). No correlation could be established between the capacity of the cells to bioconvert scopoletin to scopolin and the endogenous scopolin level.

Ability of a Nicotiana plumbaginifolia cell suspension to demethylate nicotine into nornicotine

Abstract A cell suspension of Nicotiana plumbaginifolia which does not accumulate tobacco alkaloids was found to keep the ability to demethylate nicotine into nornicotine. The highest bioconversion yield was 53.2%. The influence of some environmental factors upon the reaction has been studied. In particular, it appears that light enhances the catalytic activity of the cells which leads to the hypothesis that this metabolic step of tobacco alkaloids is bound to photodependent systems.