Non-functionalization of two CYP82E nicotine N-demethylase genes abolishes nornicotine formation in Nicotiana langsdorffii.

Nornicotine is formed from nicotine by nicotine N-demethylase, a CYP82E family monooxygenase, and accumulates to high levels in some tobacco (Nicotiana tabacum) cultivars and many wild Nicotiana species. Nicotiana langsdorffii does not form nornicotine, whereas the closely related species N. alata accumulates this alkaloid abundantly. We show here that the two nicotine N-demethylase genes in N. langsdorffii have been inactivated by different molecular mechanisms. We identified four N. alata CYP82E genes that encode functional nicotine N-demethylases. In N. langsdorffii, however, one CYP82E gene encoding a functional enzyme was not expressed at all, whereas the other was weakly expressed but contained a one-nucleotide deletion in the first exon, yielding a truncated protein. Expression analysis of interspecific F(1) hybrids between N. alata and N. langsdorffii indicated that cis-acting polymorphisms abolish expression of the otherwise functional CYP82E gene in N. langsdorffii. Segregation analysis of tobacco alkaloids and individual CYP82E alleles in F(2) progeny revealed that duplicated CYP82E genes in both species are genetically linked, and provide genetic evidence that CYP82E genes are solely responsible for nornicotine formation in these wild Nicotiana species.

Root-to-shoot translocation of alkaloids is dominantly suppressed in Nicotiana alata.

In tobacco (Nicotiana tabacum), nicotine and related pyridine alkaloids are produced in the root, and then transported to the aerial parts where these toxic chemicals function as part of chemical defense against insect herbivory. Although a few tobacco transporters have been recently reported to take up nicotine into the vacuole from the cytoplasm or into the cytoplasm from the apoplast, it is not known how the long-range translocation of tobacco alkaloids between organs is controlled. Nicotiana langsdorffii and N. alata are closely related species of diploid Nicotiana section Alatae, but the latter does not accumulate tobacco alkaloids in the leaf. We show here that N. alata does synthesize alkaloids in the root, but lacks the capacity to mobilize the root-borne alkaloids to the aerial parts. Interspecific grafting experiments between N. alata and N. langsdorffii indicate that roots of N. alata are unable to translocate alkaloids to their shoot system. Interestingly, genetic studies involving interspecific hybrids between N. alata and N. langsdorffii and their self-crossed or back-crossed progeny showed that the non-translocation phenotype is dominant over the translocation phenotype. These results indicate that a mechanism to retain tobacco alkaloids within the root organ has evolved in N. alata, which may represent an interesting strategy to control the distribution of secondary products within a whole plant.