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. sylvestris were investigated. Candidate genes were isolated from a N. sylvestris senescing leaf cDNA library and characterized by heterologous gene expression in yeast, site-directed mutagenesis and quantitative real-time polymerase chain reaction. A cytochrome P450 gene, designated NsylCYP82E2, was isolated from N. sylvestris. Located on the chromosomal fragment defined by the N. sylvestris conversion locus, NsylCYP82E2 confers high nicotine N-demethylase (NND) activity in the senescing leaves of N. sylvestris, but the gene is inactivated by two degenerative mutations in tobacco. Collectively with previously published data, these results show that inactivation of NND genes by degenerative mutations and/or transcriptional suppression played a key role in the evolution of the alkaloid profile of modern tobacco.