Determination and Inheritance of Nicotine to Nornicotine Conversion in Tobacco

Griffith, R.B.; Valleau, W. D.; Stokes, G. W.

doi:10.1126/science.121.3140.343

Cited by 28 publications

(15 citation statements)

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“…The principal interest in these works was the kind and amount of alkaloids found in each species. The findings of these independent investigations were in good agreement: [ 1] all observed wild species contained one or more alkaloids, [2] generally, a single alkaloid was found to predominate in a given species, · [3] nicotine or nornicotine occurred as the main alkaloid in most species, but a few species produce anabasine as the principal alkaloid, [ 4] total-alkaloid levels were found to be highly variable, and [5] no clearly defined associations between phylogenetic position and the kinds or amounts of alkaloids was evident. Various techniques for isolation and analysis of alkaloids were used in these investigations.…”

Section: Introductionsupporting

confidence: 69%

Alkaloid Composition of the Nicotiana Species

Sisson

Severson

1990

Contributions to Tobacco &Amp; Nicotine Research

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SUMMARYPlants from 64 Nicotiana species were sampled in the greenhouse in 1983 and 1984 and from the field in 1985 and 1986 for the purpose of developing a chemical profile of each species. Mature green leaves were harvested at flowering, freeze-dried and ground to pass a 1 mm screen prior to chemical analysis. In this study we report the type and amounts of nicotinoid alkaloids. Alkaloid values were determined by glass -capillary gas chromatography and were averaged over the two years for each environment. Both total alkaloids (mg g-1 dry weight) and the distribution (percentage composition) of nicotine, nornicotine, anabasine, and anatabine were highly correlated between years for greenhouse and field samples. Greenhouse and field data were highly correlated, although total-alkaloid levels were significantly higher from field-grown plants. All of the Nicotiana species tested contained a measurable alkaloid fraction (at least 10 !lg g-1 ) . There was a wide range in total-alkaloid levels with nearly a

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Section: Introductionsupporting

confidence: 69%

Alkaloid Composition of the Nicotiana Species

Sisson

Severson

1990

Contributions to Tobacco &Amp; Nicotine Research

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“…The pioneering work of Griffith et al (8) followed by reports by Burk and Jeffrey (9) and Mann et al (10) demonstrated that the high nornicotine phenotype in converter tobacco plants is controlled by a single dominant genetic locus. Because nicotine N-demethylase, the enzyme that mediates the final step in nornicotine biosynthesis ( Fig.…”

mentioning

confidence: 99%

Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase

Siminszky

Gavilano

Bowen

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

155

163

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Nornicotine is a secondary tobacco alkaloid that is produced by the N-demethylation of nicotine. Nornicotine production and accumulation in tobacco are undesirable because nornicotine serves as the precursor in the synthesis of the well characterized carcinogen N-nitrosonornicotine during the curing and processing of tobacco. Although nornicotine is typically a minor alkaloid in tobacco plants, in many tobacco populations a high percentage of individuals can be found that convert a substantial proportion of the nicotine to nornicotine during leaf senescence and curing. We used a microarray-based strategy to identify genes that are differentially regulated between closely related tobacco lines that accumulate either nicotine (nonconverters) or nornicotine (converters) as the predominant alkaloid in the cured leaf. These experiments led to the identification of a small number of closely related cytochrome P450 genes, designated the CYP82E2 family, whose collective transcript levels were consistently higher in converter versus nonconverter tobacco lines. RNA interference-induced silencing of the CYP82E2 gene family suppressed the synthesis of nornicotine in strong converter plants to levels similar to that observed in nonconverter individuals. Although each of the six identified members of the P450 family share >90% nucleotide sequence identity, sense expression of three selected isoforms revealed that only one (CYP82E4v1) was involved in the conversion of nicotine to nornicotine. Yeast expression analysis revealed that CYP82E4v1 functions as a nicotine demethylase. Identification of the gene(s) responsible for nicotine demethylation provides a potentially powerful tool toward efforts to minimize nornicotine levels, and thereby N-nitrosonornicotine formation, in tobacco products.NЈ-nitrosonornicotine ͉ N-demethylation ͉ tobacco ͉ alkaloid ͉ tobacco-specific nitrosamines

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“…In theory, either of the two progenitor species could have donated the conversion locus to tobacco, because nicotine to nornicotine conversion occurs in both N. sylvestris and N. tomentosiformis at one developmental stage. Classical genetic studies conducted by Mann et al (8) demonstrated that the unstable conversion locus found in converter tobacco is allelic to the green leaf conversion locus of N. tomentosiformis and inherited as a dominant gene (8,9,23). The observation that the unstable conversion locus was originated from green-leaf converter N. tomentosiformis was surprising, because the reversion of the conversion locus to the ancestral type in tobacco confers senescing leaf conversion phenotype characteristic to N. sylvestris.…”

mentioning

confidence: 99%

Functional Analysis of Nicotine Demethylase Genes Reveals Insights into the Evolution of Modern Tobacco

Gavilano

Coleman

Bowen

et al. 2007

Journal of Biological Chemistry

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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. However, most, if not all, tobacco cultivars possess an unstable mutation, commonly referred to as the conversion locus, that when activated mediates the conversion of a large percentage of nicotine to nornicotine in the senescing leaf. We have recently identified CYP82E4, a tobacco nicotine N-demethylase gene whose expression was highly induced during senescence in plants that have converted, and CYP82E3, a closely related homolog that exhibited no nicotine N-demethylase activity. In this study, domain swapping and site-directed mutagenesis studies identified a single amino acid change that fully restored nicotine N-demethylase activity to CYP82E3. An examination of the N. tomentosiformis orthologs of CYP82E3 and CYP82E4 revealed that both are functional nicotine N-demethylase genes in N. tomentosiformis. Collectively, our results suggest that a single base pair mutation in CYP82E3 and transcriptional suppression of CYP82E4 played important roles in the evolution of the alkaloid profile characteristic of modern tobacco.

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Determination and Inheritance of Nicotine to Nornicotine Conversion in Tobacco

Cited by 28 publications

References 1 publication

Alkaloid Composition of the Nicotiana Species

Alkaloid Composition of the Nicotiana Species

Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase

Functional Analysis of Nicotine Demethylase Genes Reveals Insights into the Evolution of Modern Tobacco

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