Identification of Tropane Alkaloid Chemotypes and Genotypes in Hyoscyamus niger L.

Abstract

Tropane alkaloids (TA) are compounds widespread in the Solanaceae family. The genera Atropa, Brugmansia, Hyoscyamus, and Scopolia, produce the pharmaceuticals hyoscyamine (Hy) and scopolamine (Sc), which are valued for their antimuscarinic and anticholinergic actions. The enzyme hyoscyamine 6β-hydroxylase (H6H) [electrical conductivity (EC) 1.14.11.11] catalyzes both the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine and the epoxidation of the latter, leading to scopolamine (Hashimoto et al. 1993). During the examination of three genes in the TA biosynthetic pathway, the first committed step, the path branch point, and the final step in 13 accessions of Hyoscyamus niger from North America and Europe, genetic variations were found to be absent except in the h6h gene locus (GenBank: D26583.1). Quantification of TA showed average concentrations of 26 to 520 μg/g of dry leaf tissue among the accessions. From a monohybrid cross of the expected (Pennsylvania accession Ames 3103, aa) and novel (Netherlands accession PI 641691, bb) genotypes, the F2 population (n = 104) leaf and root tissues were extracted, analyzed for Hy and Sc contents, and compared with the h6h genotypes (aa, ab, bb). The polymorphism showed Mendelian inheritance. The presence of the polymorphic gene bb showed a marginally significantly greater concentration of hyoscyamine in the leaf tissue (P = 0.0675) and significantly greater concentration in root tissue (P = 0.0436), along with increased concentration of scopolamine in the root tissue (P = 0.0494) compared with the aa genotype. The increase in overall TA in the root tissue of the genotype bb was accompanied by a reduction in scopolamine in the foliar tissue. The 694-bp b amplicon has been sequenced for comparison with the expected 550-bp a amplicon and can be a useful enzymatic variant for TA metabolic engineering.