title: Non-functionalization of two CYP82E nicotine N-demethylase genes abolishes nornicotine formation in Nicotiana langsdorffii.
abstract: 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.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara, 630-0192 Japan.
authors.forename: Phattharaporn
authors.initials: P
authors.lastname: Pakdeechanuan
authors.forename: Seddon
authors.initials: S
authors.lastname: Teoh
authors.forename: Tsubasa
authors.initials: T
authors.lastname: Shoji
authors.forename: Takashi
authors.initials: T
authors.lastname: Hashimoto
mesh_terms: D000470:Alkaloids / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D005787:Gene Frequency; D005838:Genotype; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D009538:Nicotine / Q000031:analogs & derivatives* / Q000378:metabolism; D010641:Phenotype; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D017384:Sequence Deletion; D013045:Species Specificity; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D015533:Transcriptional Activation
chemical_list: D000470:Alkaloids; D010940:Plant Proteins; D009538:Nicotine; C005311:nornicotine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C516919:nicotine demethylase, tobacco