Functional expression of tropinone reductase I (trI) and hyoscyamine-6beta-hydroxylase (h6h) from Hyoscyamus niger in Nicotiana tabacum

The cDNAs from Hyoscyamus niger, encoding two enzymes of tropane-alkaloid biosynthesis, tropinone reductase I (TR-I) and hyoscyamine-6beta-hydroxylase (H6H), have been simultaneously introduced into Nicotiana tabacum using particle bombardment and expressed under the control of the CaMV 35S promoter. Southern and Northern analyses confirmed integration and transcript accumulation for both trl and h6h. Fertile tobacco plants expressing both transgenes were regenerated and detached leaves of these plants were fed with tropinone and hyoscyamine, the substrates, respectively, of TR-I and H6H. Besides the expected TR-I and H6H reaction products, acetylated forms of tropine were also generated in these experiments, indicating that the expression of alkaloid-pathway enzymes in a transgenic background can produce unexpected substances. In addition, leaves of the transgenic plants showed in most cases higher nicotine content than leaves of control plants. Nicotine levels were approximately 3- to 13-fold higher in both the parental transgenic lines and in T1 progeny expressing functional TR-I and H6H, compared to levels in wild-type plants and in transgenic plants carrying the nptII transgene alone. In addition, nicotine related compounds such as anatabine, nornicotine, bipyridine, anabasine, and myosmine were identified in transgenic tobacco lines and below detection limit in wild-type plants, suggesting changes in the activity of the enzymes in the nicotine biosynthetic pathway in the transgenic background.