Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene

In most tobacco (Nicotiana tabacum L.) plants, nornicotine is a relatively minor alkaloid, comprising about 2–5% of the total pyridine alkaloid pool in the mature leaf. Changes in gene expression at an unstable locus, however, can give rise to plants that produce high levels of nornicotine, specifically during leaf senescence and curing. Minimizing the nornicotine content in tobacco is highly desirable, because this compound serves as the direct precursor in the synthesis of N′-nitrosonornicotine, a potent carcinogen in laboratory animals. Nornicotine is likely produced almost entirely via the N-demethylation of nicotine, in a process called nicotine conversion that is catalyzed by the enzyme nicotine N-demethylase (NND). Previous studies have identified CYP82E4 as the specific NND gene responsible for the unstable conversion phenomenon, and CYP82E5v2 as a putative minor NND gene. Here, by discovery and characterization of CYP82E10, a tobacco NND gene, is reported. PCR amplification studies showed that CYP82E10 originated from the N. sylvestris ancestral parent of modern tobacco. Using a chemical mutagenesis strategy, knockout mutations were induced and identified in all three tobacco NND genes. By generating a series of mutant NND genotypes, the relative contribution of each NND gene toward the nornicotine content of the plant was assessed. Plants possessing knockout mutations in all three genes displayed nornicotine phenotypes that were much lower (∼0.5% of total alkaloid content) than that found in conventional tobacco cultivars. The introduction of these mutations into commercial breeding lines promises to be a viable strategy for reducing the levels of one of the best characterized animal carcinogens found in tobacco products.