Enhanced accumulation of atropine in Atropa belladonna transformed by Rac GTPase gene isolated from Scoparia dulcis.

Leaf tissues of Atropa belladonna were transformed by Sdrac2, a Rac GTPase gene, that is isolated from Scoparia dulcis, and the change in atropine concentration of the transformants was examined. Re-differentiated A. belladonna overexpressing Sdrac2 accumulated considerable concentration of atropine in the leaf tissues, whereas the leaves of plants transformed by an empty vector accumulated only a very low concentration of the compound. A. belladonna transformed by CASdrac2, a modified Sdrac2 of which translate was expected to bind guanosine triphosphate (GTP) permanently, accumulated very high concentrations of atropine (approximately 2.4-fold excess to those found in the wild-type plant in its natural habitat). In sharp contrast, the atropine concentration in transformed A. belladonna prepared with negatively modified Sdrac2, DNSdrac2, expected to bind guanosine diphosphate instead of GTP, was very low. These results suggested that Rac GTPases play an important role in the regulation of secondary metabolism in plant cells and that overexpression of the gene(s) may be capable of enhancing the production of natural products accumulated in higher plant cells.

Chromium-induced tropane alkaloid production and H6H gene expression in Atropa belladonna L. (Solanaceae) in vitro-propagated plantlets.

Hyoscyamine and scopolamine tropane alkaloids found in several solanaceous plants are anticholinergic drugs. Hyoscyamine 6ß-hydroxylase (H6H) catalyzes two consecutive oxidation reactions. The first reaction is the hydroxylation of hyoscyamine to 6ß-hydroxyhyoscyamine and the second is epoxidation of 6ß-hydroxyhyoscyamine yielding scopolamine that is the final metabolite in the tropane alkaloid biosynthetic pathway. The effects of trivalent chromium as KCr (SO4)(2) on the production of tropane alkaloids and the expression of hyoscyamine 6ß-hydroxylase gene (h6h) were studied in micro-propagated Atropa belladonna L. plantlets. The results showed that chromium treatment decreased the growth parameters (weights and lengths of the plantlets) and chlorophyll contents and increased proline contents. Moreover, semiquantitave RT-PCR analysis showed that the transcript level of H6H increased under chromium treatment. This treatment also increased hyoscyamine and scopolamine contents as shown by HPLC analysis. Changes of scopolamine contents correlate with the expression levels of h6h gene under different concentrations of chromium.

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.