Enantioselective demethylation of nicotine as a mechanism for variable nornicotine composition in tobacco leaf.

Nicotine and its N-demethylation product nornicotine are two important alkaloids in Nicotiana tabacum L. (tobacco). Both nicotine and nornicotine have two stereoisomers that differ from each other at 2'-C position on the pyrrolidine ring. (S)-Nicotine is the predominant form in the tobacco leaf, whereas the (R)-enantiomer only accounts for ∼0.2% of the total nicotine pool. Despite considerable past efforts, a comprehensive understanding of the factors responsible for generating an elevated and variable enantiomer fraction of nornicotine (EF(nnic) of 0.04 to 0.75) from the consistently low EF observed for nicotine has been lacking. The objective of this study was to determine potential roles of enantioselective demethylation in the formation of the nornicotine EF. Recombinant CYP82E4, CYP82E5v2, and CYP82E10, three known tobacco nicotine demethylases, were expressed in yeast and assayed for their enantioselectivities in vitro. Recombinant CYP82E4, CYP82E5v2, and CYP82E10 demethylated (R)-nicotine 3-, 10-, and 10-fold faster than (S)-nicotine, respectively. The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta. Collectively, our studies suggest that an enantioselective mechanism facilitates the maintenance of a reduced (R)-nicotine pool and, depending on the relative abundances of the three nicotine demethylase enzymes, can confer a high (R)-enantiomer percentage within the nornicotine fraction of the leaf.

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. NNN is formed via the nitrosation of nornicotine, a secondary alkaloid produced through enzymatic N-demethylation of nicotine. Strategies to lower nornicotine levels in tobacco (Nicotiana tabacum L.) could lead to a corresponding decrease in NNN accumulation in cured leaves. The major nicotine demethylase gene of tobacco has recently been isolated. In this study, a large-scale field trial was conducted to evaluate transgenic lines of burley tobacco carrying an RNA interference (RNAi) construct designed to inhibit the expression of this gene. Selected transgenic lines exhibited a six-fold decrease in nornicotine content relative to untransformed controls. Analysis of cured leaves revealed a commensurate decrease in NNN and total TSNAs. The inhibition of nicotine demethylase activity is an effective means of decreasing significantly the level of a key defined animal carcinogen present in tobacco products.

CYP82E4-mediated nicotine to nornicotine conversion in tobacco is regulated by a senescence-specific signaling pathway.

Nicotine to nornicotine conversion in tobacco (Nicotiana tabacum L.) is regulated by an unstable converter locus which in its activated state gives rise to a high nornicotine, low nicotine phenotype in the senescing leaves. In plants that carry the high nornicotine trait, nicotine conversion is primarily catalyzed by a cytochrome P450 protein, designated CYP82E4 whose transcription is strongly upregulated during leaf senescence. To further investigate the regulation of CYP82E4 expression, we examined the spatiotemporal distribution and the stress- and signaling molecule-elicited expression patterns of CYP82E4 using alkaloid analysis and a fusion construct between the 2.2 kb upstream regulatory region of CYP82E4 and the beta-glucurodinase (GUS) gene. Histochemical and fluorometric analyses of GUS expression revealed that the CYP82E4 promoter confers high levels of expression in the senescing leaves and flowers, and in the green stems of young and mature plants, but only very low activity was detected in the roots. In the leaves, GUS activity was strongly correlated with the progression of senescence. Treatments of leaf tissue with various signaling molecules including abscisic acid, ethylene, jasmonic acid, salicylic acid and yeast extract; and stresses, such as drought, wounding and tobacco mosaic virus infection did not enhance nicotine conversion or GUS activity in the green leaves, but an increase in CYP82E4 expression was observed in response to ethylene- or tobacco mosaic virus-induced senescence. These results suggest that the expression of CYP82E4 is senescence-specific in the leaves and the use of the CYP82E4 promoter could provide a valuable tool for regulating gene expression in the senescing leaves.

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. Nornicotine is an undesirable alkaloid in tobacco, because it serves as a precursor for N'-nitrosonornicotine, a potent carcinogen in laboratory animals. The objective of this study was to investigate the possible catalytic roles of additional NND genes in shaping the alkaloid profile of tobacco. A PCR-based strategy using primers complementary to conserved regions of CYP82E genes yielded a cDNA, designated CYP82E5v2, which conferred NND activity in heterologous expression studies using yeast as a host. PCR amplification of CYP82E5v2 orthologs revealed that of the two progenitor species of tobacco, CYP82E5v2 was donated by the N. tomentosiformis parent. A comparison of CYP82E4 and CYP82E5v2 expression using qualitative real-time PCR analysis demonstrated that the transcription of CYP82E5v2 was higher in the green leaves of all tobacco genotypes tested, while the expression of CYP82E4 dominated in the senescing leaves of converter tobacco. These results suggest that differentially regulated NND genes regulate nornicotine production in the green and senescing leaves of tobacco and provide tools to reduce nornicotine levels in tobacco leaves.

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.

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. 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.

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. Previously, we isolated a nicotine N-demethylase gene, designated CYP82E4, and demonstrated that RNAi-induced silencing of CYP82E4 and its close homologues is an effective means for suppressing nicotine to nornicotine conversion. In this study, we used real-time polymerase chain reaction to confirm the central role of CYP82E4 in nicotine N-demethylation by demonstrating that the transcript accumulation of CYP82E4 is enhanced as much as 80-fold in converter vs nonconverter tobacco. We also show the design of an optimized RNAi construct (82E4Ri298) that suppressed nicotine to nornicotine conversion from 98% to as low as 0.8% in a strong converter tobacco line, a rate of nornicotine production that is about 3.6-fold lower than typically detected in commercial varieties. Southern blot analysis showed that a single copy of the RNAi transgene was as effective in suppressing nornicotine accumulation as multiple copies. Greenhouse-grown transgenic plants transformed with the RNAi construct were morphologically indistinguishable from the empty vector or wild-type controls. These results demonstrate that the genetic transformation of tobacco with the 82E4Ri298 construct is an effective strategy for reducing nornicotine and ultimately NNN levels in tobacco. KEYWORDS: Alkaloid; cytochrome P450; gene silencing; nicotine N-demethylase; N'-nitrosonornicotine; plant genetic engineering; metabolic engineering; Nicotiana tabacum L.; real-time PCR; RNA interference; tobacco-specific nitrosamines.

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

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.