RNA interference (RNAi)-induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves.

Technologies for reducing the levels of tobacco product constituents that may contribute to unwanted health effects are desired. Target compounds include tobacco-specific nitrosamines (TSNAs), a class of compounds generated through the nitrosation of pyridine alkaloids during the curing and processing of tobacco. Studies have reported the TSNA N'-nitrosonornicotine (NNN) to be carcinogenic in laboratory animals.

Isolation and characterization of the cytochrome P450 gene CYP82E5v2 that mediates nicotine to nornicotine conversion in the green leaves of tobacco.

In the species of genus Nicotiana, nicotine to nornicotine conversion is mediated by closely related nicotine N-demethylase (NND) proteins that are encoded by the CYP82E subfamily of cytochrome P450 genes. The diverse number and transcriptional regulation of the NND genes have created large variations in the time and rate of nornicotine production in various Nicotiana species. In tobacco, previous studies have identified the senescence-inducible CYP82E4 gene as an important factor controlling nicotine conversion.

Inactivation of the cytochrome P450 gene CYP82E2 by degenerative mutations was a key event in the evolution of the alkaloid profile of modern tobacco.

The alkaloid profile of cultivated tobacco (Nicotiana tabacum) is different from that of its two progenitors, Nicotiana sylvestris and Nicotiana tomentosiformis, in that tobacco accumulates nicotine as the most abundant alkaloid, while its ancestors convert nicotine to nornicotine in the senescing leaf. The nicotine-retaining phenotype of tobacco is thought to have evolved through the inactivation of the conversion loci inherited from its two progenitors. Here, the genetic changes associated with the inactivation of the conversion locus derived from N.

Genetic engineering of Nicotiana tabacum for reduced nornicotine content.

Unlabelled: Nornicotine is an undesirable secondary alkaloid in cultivated tobacco, because it serves as a precursor to N'-nitrosonornicotine (NNN), a tobacco-specific nitrosamine with suspected carcinogenic properties. Nornicotine is produced through the oxidative N-demethylation of nicotine by a nicotine N-demethylase enzyme during the senescence and curing of tobacco leaves. While the nornicotine content of most commercial burley tobacco is low, a process termed "conversion" can bestow considerably increased nornicotine levels in a portion of the plants within the population.

Functional analysis of nicotine demethylase genes reveals insights into the evolution of modern tobacco.

Tobacco (Nicotiana tabacum L.) is a natural allotetraploid derived from the interspecific hybridization between ancestral Nicotiana sylvestris and Nicotiana tomentosiformis. The majority of cultivated tobacco differs from both of its progenitor species in that tobacco typically contains nicotine as the primary alkaloid, in contrast to its two progenitors that accumulate nornicotine in the senescing leaves.