BackgroundNicotiana rustica (Aztec tobacco), like common tobacco (Nicotiana tabacum), is an allotetraploid formed through a recent hybridization event; however, it originated from completely different progenitor species. Here, we report the comparative genome analysis of wild type N. rustica (5 Gb; 2n = 4x = 48) with its three putative diploid progenitors (2.3–3 Gb; 2n = 2x =24), Nicotiana undulata, Nicotiana paniculata and Nicotiana knightiana.ResultsIn total, 41% of N. rustica genome originated from the paternal donor (N. undulata), while 59% originated from the maternal donor (N. paniculata/N. knightiana). Chloroplast genome and gene analyses indicated that N. knightiana is more closely related to N. rustica than N. paniculata. Gene clustering revealed 14,623 ortholog groups common to other Nicotiana species and 207 unique to N. rustica. Genome sequence analysis indicated that N. knightiana is more closely related to N. rustica than N. paniculata, and that the higher nicotine content of N. rustica leaves is the result of the progenitor genomes combination and of a more active transport of nicotine to the shoot.ConclusionsThe availability of four new Nicotiana genome sequences provide insights into how speciation impacts plant metabolism, and in particular alkaloid transport and accumulation, and will contribute to better understanding the evolution of Nicotiana species.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5241-5) contains supplementary material, which is available to authorized users.
Substantial quantities of the carcinogenic tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (1; NNK) are still found in the mainstream smoke of tobacco exhaustively extracted with water, indicating the presence of an insoluble, matrix-bound form. Soluble and matrix-bound concentrations of 1 in tobacco were determined by applying a new method using sequential aqueous extraction at room temperature and at 130 °C. On average, 77% and 53% of the total content of 1 were matrix-bound in air-cured (Burley type) and flue-cured tobaccos, respectively. Thermal release of 1 from its matrix-bound form above ca. 200 °C can account for a large fraction of its concentration in cigarette mainstream smoke. An already matrix-bound alkaloid precursor of matrix-bound 1 was identified in vascular tissue of green leaf midribs. The incubation of vascular cell-wall preparations with the lignin precursor coniferyl alcohol and isotopically labeled nicotine or pseudooxynicotine (2) led to the formation of labeled matrix-bound 1 after nitrosation, suggesting that incorporation of nicotine or its oxidized product 2 during lignin polymerization is the origin of the formation of matrix-bound 1.
In the tobacco plant, nicotine N-demethylase enzymes (NND) belonging to the cytochrome P450 family catalyse the conversion of nicotine to nornicotine, the precursor of the carcinogenic tobacco-specific N-nitrosamine, N-nitrosonornicotine. To date three demethylase genes, namely CYP82E4, CYP82E5 and CYP82E10, have been shown to be involved in this process, while the related CYP82E2 and CYP82E3 genes are not functional. We have identified a further gene named CYP82E21 encoding a putative nicotine N-demethylase closely related to the CYP82E genes. The CYP82E21 gene was found in all Nicotiana tabacum cultivars analysed and originates from the tobacco ancestor Nicotiana tomentosiformis. We show that, in contrast to all other previously characterized NND genes, CYP82E21 is not expressed in green or senescent leaves, but in flowers, more specifically in ovaries. The nicotine N-demethylase activity of CYP82E21 was confirmed by ectopic expression of the coding sequence in a tobacco line lacking functional CYP82E4, CYP82E5 and CYP82E10 genes, resulting in an eightfold increase of nicotine demethylation compared to the control plants. Furthermore, nornicotine formation can be reduced in ovaries by introducing a CYP82E21-specific RNAi construct. Together, our results demonstrate that the CYP82E21 gene encodes a functional ovary-specific nicotine N-demethylase.
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