Characteristic alatoid 'cineole cassette' monoterpene synthase present in Nicotiana noctiflora.

Nicotiana species of the section Alatae emit a characteristic floral scent comprising the' cineole cassette' monoterpenes 1,8-cineole, limonene, myrcene, ß-pinene, α-pinene, sabinene and α-terpineol. All previously isolated 'cineole cassette'-monoterpene synthase genes are multi product enzymes that synthesize the seven compounds of the 'cineole cassette'. Interestingly, so far this 'alatoid' trait was only shared with the eponymous species Nicotiana suaveolens of the sister section Suaveolentes. To determine the origin of the 'cineole cassette' monoterpene phenotype other potential parent species of section Noctiflorae or Petunoides as well as of the distantly related section Trigonophyllae were analysed. A monoterpene synthase producing the set of 'cineole cassette' compounds was isolated from N. noctiflorae. N. obtusifolia emitted solely 1,8-cineole and no monoterpenes were found in floral scents of N. petunoides and N. palmeri. Interestingly, the phylogenetic analysis clustered the new gene of N. noctiflora closely to the terpineol synthase genes of e.g. N. alata rather than to cineole synthase genes of e.g. N. forgetiana.

Synthesis of 'cineole cassette' monoterpenes in Nicotiana section Alatae: gene isolation, expression, functional characterization and phylogenetic analysis.

The scent bouquets of flowers of Nicotiana species, particularly those of section Alatae, are rich in monoterpenes, including 1,8-cineole, limonene, ß-myrcene, α- and ß-pinene, sabinene, and α-terpineol. New terpene synthase genes were isolated from flowers of Nicotiana bonariensis, N. forgetiana, N. longiflora, and N. mutabilis. The recombinant enzymes synthesize simultaneously the characteristic 'cineole cassette' monoterpenes with 1,8-cineole as the dominant volatile product. Interestingly, amino acid sequence comparison and phylogenetic tree construction clustered the newly isolated cineole synthases (CIN) of section Alatae together with the catalytically similar CIN of N. suaveolens of section Suaveolentes, thus suggesting a common ancestor. These CIN genes of N. bonariensis, N. forgetiana, N. longiflora, and N. mutabilis are distinct from the terpineol synthases (TERs) of the taxonomically related N. alata and N. langsdorfii (both Alatae), thus indicating gene diversification of monoterpene synthases in section Alatae. Furthermore, the presence of CINs in species of the American section Alatae supports the hypothesis that one parent of the Australian section Suaveolentes was a member of the present section Alatae. Amino acid sequences of the Nicotiana CINs and TERs were compared to identify relevant amino acids of the cyclization reaction from α-terpineol to 1,8-cineole.

Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds.

During the last years halogenated compounds have drawn a lot of attention. Metabolites with one or more halogen atoms are often more active than their non-halogenated derivatives like indole-3-acetic acid (IAA) and 4-Cl-IAA. Within this work, bacterial flavin-dependent tryptophan halogenase genes were inserted into Brassica rapa ssp. pekinensis (Chinese cabbage) with the aim to produce novel halogenated indole compounds. It was investigated which tryptophan-derived indole metabolites, such as indole glucosinolates or potential degradation products can be synthesized by the transgenic root cultures. In vivo and in vitro activity of halogenases heterologously produced was shown and the production of chlorinated tryptophan in transgenic root lines was confirmed. Furthermore, chlorinated indole-3-acetonitrile (Cl-IAN) was detected. Other tryptophan-derived indole metabolites, such as IAA or indole glucosinolates were not found in the transgenic roots in a chlorinated form. The influence of altered growth conditions on the amount of produced chlorinated compounds was evaluated. We found an increase in Cl-IAN production at low temperatures (8 °C), but otherwise no significant changes were observed. Furthermore, we were able to regenerate the wild type and transgenic root cultures to adult plants, of which the latter still produced chlorinated metabolites. Therefore, we conclude that the genetic information had been stably integrated. The transgenic plants showed a slightly altered phenotype compared to plants grown from seeds since they also still expressed the rol genes. By this approach we were able to generate various stably transformed plant materials from which it was possible to isolate chlorinated tryptophan and Cl-IAN.