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

Abstract

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. 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. Pyridine alkaloids, such as nicotine, nornicotine, anabasine, and anatabine, have been detected in numerous species of the Nicotiana genus (1Sisson V.A. Severson R.F. Beitr. Tabakforsch. 1990; 14: 327-339Google Scholar) where they are thought to function as herbivore deterrents (2Metcalf R.L. Metcalf R.A. Destructive and Useful Insects.5th Ed. McGraw-Hill Inc., New York1992Google Scholar). The production of pyridine alkaloids is elicited by herbivore attacks (3Baldwin I.T. Ohnmeiss T.E. J. Chem. Ecol. 1993; 19: 1143-1153Crossref PubMed Scopus (64) Google Scholar, 4Baldwin I.T. Oecologia. 1988; 75: 367-370Crossref PubMed Scopus (95) Google Scholar) through a jasmonic acid-induced signaling pathway (5Halitschke R. Baldwin I.T. Plant J. 2003; 36: 794-807Crossref PubMed Scopus (292) Google Scholar, 6Baldwin I.T. Schmelz E.A. Ohnmeiss T.E. J. Chem. Ecol. 1994; 20: 2139-2157Crossref PubMed Scopus (207) Google Scholar). Several studies have demonstrated that the alkaloid composition within the Nicotiana genus is species-specific and highly variable, and in the majority of the species a single alkaloid predominates (1Sisson V.A. Severson R.F. Beitr. Tabakforsch. 1990; 14: 327-339Google Scholar, 7Saitoh F. Noma M. Kawashima N. Phytochemistry. 1985; 24: 477-480Crossref Scopus (199) Google Scholar). For example, Nicotiana tomentosiformis primarily accumulates nornicotine, whereas in Nicotiana glauca anabasine is the major alkaloid (1Sisson V.A. Severson R.F. Beitr. Tabakforsch. 1990; 14: 327-339Google Scholar). Interestingly, the alkaloid profiles of some Nicotiana are different in green versus senescing (yellow) leaves. For instance, in the green leaves of Nicotiana sylvestris nicotine is the primary alkaloid, but a substantial portion of nicotine is converted into nornicotine once the leaves senesce through air-curing (1Sisson V.A. Severson R.F. Beitr. Tabakforsch. 1990; 14: 327-339Google Scholar). In cultivated tobacco alkaloid composition can vary because of the presence of an unstable locus, termed the “converter locus” (8Mann T.J. Weybrew J.A. Matzinger D.F. Hall J.L. Crop Sci. 1964; 4: 349-353Crossref Google Scholar). In commercial tobacco leaves that harbor an inactive converter locus, the alkaloid pool typically contains about 95% nicotine and 3% nornicotine (7Saitoh F. Noma M. Kawashima N. Phytochemistry. 1985; 24: 477-480Crossref Scopus (199) Google Scholar). These plants are frequently referred to as “nonconverters.” In some of the individuals, however, the converter locus becomes activated, and a large portion of nicotine is metabolized to nornicotine (9Burk L.G. Jeffrey R.N. Tob. Sci. 1958; 2: 139-141Google Scholar) giving rise to plants termed “converters.” Similar to N. sylvestris, nicotine to nornicotine metabolism in converter tobacco is induced during leaf senescence (10Wernsman E.A. Matzinger D.F. Tob. Sci. 1968; 12: 226-228Google Scholar). The accumulation of nornicotine in converter plants is undesirable, because nornicotine is a biochemical precursor of N′-nitrosonornicotine, a tobacco-specific nitrosamine that has been shown to exhibit carcinogenic properties in laboratory animals (11Hecht S.S. Chem. Res. Toxicol. 1998; 11: 559-603Crossref PubMed Scopus (989) Google Scholar, 12Hecht S.S. Nat. Rev. Cancer. 2003; 3: 733-744Crossref PubMed Scopus (1122) Google Scholar, 13Hecht S.S. Hoffmann D. Cancer Surv. 1989; 8: 273-294PubMed Google Scholar). In addition, nornicotine has been recently recognized as an elicitor of protein glycation (14Dickerson T.J. Janda K.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15084-15088Crossref PubMed Scopus (56) Google Scholar) and other biochemical processes that are detrimental to human health (15Katz J. Caudle R.M. Bhattacharyya I. Stewart C.M. Cohen D.M. J. Periodontol. 2005; 76: 1171-1174Crossref PubMed Scopus (75) Google Scholar, 16Brogan A.P. Dickerson T.J. Boldt G.E. Janda K.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10433-10438Crossref PubMed Scopus (24) Google Scholar). Nicotiana tabacum is a natural allotetraploid derived from the interspecific hybridization of ancestral N. sylvestris and N. tomentosiformis (17Murad L. Lim K.Y. Christopodulou V. Matyasek R. Lichtenstein C.P. Kovarik A. Leitch A.R. Am. J. Bot. 2002; 89: 921-928Crossref PubMed Scopus (99) Google Scholar, 18Yukawa M. Tsudzuki T. Sugiura M. Mol. Genet. Genomics. 2006; 275: 367-373Crossref PubMed Scopus (77) Google Scholar, 19Ren N. Timko M.P. Genome. 2001; 44: 559-571Crossref PubMed Google Scholar, 20Riechers D.E. Timko M.P. Plant Mol. Biol. 1999; 41: 387-401Crossref PubMed Scopus (85) Google Scholar, 21Goodspeed T.H. Verdoorn F. The Genus Nicotiana. 16. Chronica Botanica, Waltham, MA1954: 1-536Google Scholar, 22Bogani P. Lio P. Intrieri M.C. Buiatti M. Mol. Phylogenet. Evol. 1997; 7: 62-70Crossref PubMed Scopus (34) Google Scholar), species that convert nicotine to nornicotine in senescing and green leaves, respectively (1Sisson V.A. Severson R.F. Beitr. Tabakforsch. 1990; 14: 327-339Google Scholar). As a result, the emergence of the nicotine-accumulating (nonconverter) phenotype of N. tabacum from its two progenitors would have required the inactivation of nornicotine-accumulating traits originating from both ancestral parents. Mann et al. (8Mann T.J. Weybrew J.A. Matzinger D.F. Hall J.L. Crop Sci. 1964; 4: 349-353Crossref Google Scholar) suggested that the nicotine-accumulating phenotype of nonconverter tobacco was developed by the successive inactivation of the two dominant conversion loci donated by the nornicotine-containing parental species, but to date such loci have not been identified. Another interesting question relates to the origin of the unstable conversion locus present in converter tobacco. 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. (8Mann T.J. Weybrew J.A. Matzinger D.F. Hall J.L. Crop Sci. 1964; 4: 349-353Crossref Google Scholar) 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 (8Mann T.J. Weybrew J.A. Matzinger D.F. Hall J.L. Crop Sci. 1964; 4: 349-353Crossref Google Scholar, 9Burk L.G. Jeffrey R.N. Tob. Sci. 1958; 2: 139-141Google Scholar, 23Griffith R.B. Valleau W.D. Stokes G.W. Science. 1955; 121: 343-344Crossref PubMed Scopus (16) Google Scholar). 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. The apparent inconsistency between the time of nornicotine production and the origin of the unstable conversion locus in tobacco suggested that evolution of the alkaloid profile of modern tobacco involved complex genetic changes. We have previously isolated a closely related gene family, designated the CYP82E2 gene subfamily, whose collective transcript accumulation was up-regulated in converter versus nonconverter tobacco (24Siminszky B. Gavilano L. Bowen S.W. Dewey R.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14919-14924Crossref PubMed Scopus (149) Google Scholar). Of these cDNAs, CYP82E4 was shown to mediate the metabolic conversion of nicotine to nornicotine when overexpressed in tobacco and yeast (24Siminszky B. Gavilano L. Bowen S.W. Dewey R.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14919-14924Crossref PubMed Scopus (149) Google Scholar). Expression analysis of CYP82E4 revealed a >80-fold increase of transcript accumulation in the cured leaves of converter versus nonconverter plants demonstrating that CYP82E4 plays a central role in mediating nicotine N-demethylation in senescing leaves of tobacco (25Gavilano L.B. Coleman L.P. Burnley L. Bowman M.L. Kalengamaliro N.E. Hayes A. Bush L.P. Siminszky B. J. Agric. Food Chem.,. 2006; 54: 9071-9078Crossref PubMed Scopus (49) Google Scholar). To distinguish between orthologous P450 genes from tobacco and N. tomentosiformis, an Ntab or Ntom prefix is attached, respectively, to the name of the genes in the remaining part of the paper (e.g. NtabCYP82E4 or NtomCYP82E4). Because not all members of the NtabCYP82E2 gene subfamily exhibited nicotine N-demethylase (NND) 2The abbreviations used are: NND, nicotine N-demethylase; RT, real time. activity (24Siminszky B. Gavilano L. Bowen S.W. Dewey R.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14919-14924Crossref PubMed Scopus (149) Google Scholar), the original goal of this study was to define the structural elements responsible for NND activity in the closely related P450 isoforms of the NtabCYP82E2 gene subfamily. Our results showed that a single base amino acid substitution in the coding region of another closely related isoform, NtabCYP82E3, is sufficient to confer NND activity to the encoded protein. DNA sequence and gene expression data also demonstrated that N. tomentosiformis possesses two active NND genes, in the green leaves and up-regulated during We that during the evolution of modern tobacco, was by a mutation, and NtabCYP82E4 was by an unstable mutation in the transcriptional suppression of the Plant converter tobacco was by an interspecific between N. tomentosiformis and tobacco tobacco by of of the to the In green-leaf converter and used in the the conversion locus was to by the and whose no for the green-leaf conversion (8Mann T.J. Weybrew J.A. Matzinger D.F. Hall J.L. Crop Sci. 1964; 4: 349-353Crossref Google Scholar). tobacco cultivars in the study (nonconverter) and for in a and to a a For tobacco leaves in for at and in they and of the by the and NtabCYP82E4 that between the two and the NtabCYP82E4 the into the yeast expression and the to yeast as D. A. R.A. Res. PubMed Scopus Google Scholar). and of yeast as previously D. B. A. P. PubMed Google Scholar). for at in of and in a of The of and of the was a of nicotine to nornicotine was by and the of nicotine a mutagenesis was conducted the site-directed mutagenesis as by the and DNA of the CYP82E3 and CYP82E4 DNA was by a leaf in of the in a of leaf of and in a The used for the of the P450 are in DNA was in of at for at for and at for For the of the CYP82E3 a was an and into the DNA sequence of the by the F. S. A.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the T.J. Res. 1994; PubMed Scopus Google and DNA of the used in this to to of CYP82E3 for to the sequence at of the CYP82E3 to to of the CYP82E4 to of the CYP82E4 to for expression analysis of the CYP82E3 orthologs by for expression analysis of the CYP82E4 orthologs by in a of and was from leaves the the was was of the of the and DNA of the P450 as for the of the P450 The of the used in the are in of and as previously D. B. A. P. PubMed Google Scholar). protein by the protein used in the study and for and by activity versus and the to the data by between by analysis of the transcript of the CYP82E3 and CYP82E4 was 1998; 24: Google Scholar). of the P450 was used as the for a The of of and of The of the P450 and are in was to the for of for for for by at for in a a of the as an P450 was the to the The of was by analysis as previously 1997; PubMed Scopus Google Scholar). DNA sequence of the was by the into the and of The NND to the have demonstrated previously that a of P450 designated the CYP82E2 gene subfamily, a major role in the conversion of nicotine to nornicotine (24Siminszky B. Gavilano L. Bowen S.W. Dewey R.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14919-14924Crossref PubMed Scopus (149) Google Scholar). We isolated members of the CYP82E2 gene subfamily, of one isoform, was shown to an NtabCYP82E3, the closely related homolog to 95% amino acid sequence NtabCYP82E4 but no NND activity when in yeast or tobacco (24Siminszky B. Gavilano L. Bowen S.W. Dewey R.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14919-14924Crossref PubMed Scopus (149) Google Scholar). The amino acid sequence different NND activity between and NtabCYP82E4 an to structural elements that NND activity to Our was that one or of the amino between and NtabCYP82E4 important for NND activity in To of the coding region that NND activity to domain a that a from two protein and the of that the expression of traits (e.g. encoded in the The of sequence between and NtabCYP82E4 to and in the in both these as the and coding region of NtabCYP82E4 the gene of NtabCYP82E3, the and respectively The into the yeast expression and the used to the yeast In yeast used to the NND activity of all The results showed that and NND whereas not nicotine that an required for NND activity is between the and of NtabCYP82E4 To this the of NtabCYP82E4 the region from and the encoded protein for NND activity. in the protein the nicotine N-demethylation the that the of NtabCYP82E4 amino for NND activity. To define amino acid for NND site-directed mutagenesis The region of NtabCYP82E4 amino of are to The amino encoded within the region of the NtabCYP82E4 and genes to a mutation for important amino all amino acid in the of the coding region for the of the NtabCYP82E4 and in isolated from yeast in the single or exhibited no NND activity. In NND activity was apparent when a the protein was These results that the the amino acid mutation, was to confer NND activity to the To the substitution was sufficient to NND activity to NtabCYP82E3, the single mutation in the yeast for nicotine The results of the showed that the nicotine N-demethylation a mutation in the NtabCYP82E4 coding region to that the is for the NND As the substitution in NtabCYP82E4 in a of NND activity as by the yeast these results showed that the substitution is both and sufficient to NND activity to N. tomentosiformis CYP82E3 a observation that a single substitution confers NND activity to to the that the inactivation of by the mutation have one of the genetic responsible for the evolution of N. tabacum from its nicotine To this to the CYP82E3 orthologs from N. tomentosiformis and N. sylvestris DNA detected when DNA isolated from N. tomentosiformis was used as that in tobacco CYP82E3 originated from the N. tomentosiformis ancestral not DNA sequence analysis of a amino acid at to our that a functional NND gene In to the mutation, a substitution was also detected in The of the sequence of is expression of the in however, that the is that also an NND activity to that by the and the encoded amino of the orthologous CYP82E3 genes isolated from tobacco and N. is is in a of and for in a a NND in N. the genes involved in nicotine N-demethylation in N. tomentosiformis, to also a NND protein. was a NND because NtabCYP82E4 has been shown previously to mediate the nicotine to nornicotine conversion in senescing tobacco leaves (24Siminszky B. Gavilano L. Bowen S.W. Dewey R.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14919-14924Crossref PubMed Scopus (149) Google Scholar). To the role of in nicotine was isolated from N. tomentosiformis and the sequence of the gene was that of NtabCYP82E4 derived from tobacco revealed two between and NtabCYP82E4 at and where and and NtabCYP82E4 showed and respectively The for a amino acid in tobacco and a in N. tomentosiformis The of the sequence of is and the encoded amino in the orthologous CYP82E4 genes isolated from tobacco and N. is is at not change in the amino acid is The at not change in the amino acid in a Similar to also exhibited NND activity when in yeast To of and for nicotine the of the two isolated from yeast the or the conversion of nicotine to nornicotine, whereas the not both exhibited nicotine, the and of about and that of respectively and the of the converter phenotype between N. tomentosiformis and tobacco is the accumulation of nornicotine in the leaf of N. tomentosiformis. To a between and the green-leaf converter phenotype of N. tomentosiformis, our sequence analysis of the CYP82E3 and CYP82E4 orthologs to nonconverter of the and the green-leaf converter tobacco was developed by an interspecific between N. tomentosiformis and by to the (8Mann T.J. Weybrew J.A. Matzinger D.F. Hall J.L. Crop Sci. 1964; 4: 349-353Crossref Google Scholar). was the dominant green-leaf converter originating from N. tomentosiformis. plants of the N. tomentosiformis the DNA the green-leaf converter locus the genes are the green-leaf converter one would to these genes within the DNA sequence analysis of the CYP82E3 and CYP82E4 orthologs showed that both and present in and As the and NtabCYP82E4 genes detected in all tobacco and and These results suggest that the presence of the and genes NND activity in the green leaves of tobacco. the at also in the origin of the CYP82E3 in the for or in an of was when the was used to the from the and plants or when the was used and N. tomentosiformis DNA In no DNA was detected when the These results that the CYP82E3 found in originated from N. tomentosiformis. Expression of the CYP82E3 and CYP82E4 in and of the N. tomentosiformis orthologs of CYP82E3 and CYP82E4 in the suggest that the NND genes responsible for mediating the green-leaf converter phenotype to N. tomentosiformis and To the roles of the CYP82E3 and CYP82E4 orthologs in nornicotine the transcript accumulation of and in green and senescing leaves of tobacco and respectively, an and NtabCYP82E4 in the green leaves of our however, NtabCYP82E4 was induced in the cured leaves. of expression was in cured leaves of analysis of the expression of CYP82E3 and CYP82E4 orthologs in the green and cured leaves of and are for in the leaves of plants are for in the leaves of plants are for in the leaves of plants in a accumulation of and was different in Expression of was in the green leaves but during senescence transcript accumulation of in the green leaves of plants was about that of NtabCYP82E4 in the tobacco was about that of of the was by DNA sequence analysis of derived from In all the the DNA sequence to the that the not These results that both and are in the green leaves of the expression of the of originating from N. tomentosiformis is by senescence in the to the in NtabCYP82E4 is induced in converter tobacco the expression of both and NtabCYP82E4 in green leaves is that of N. tomentosiformis swapping and site-directed mutagenesis to the structural for the activity of These studies revealed that a single amino acid substitution was and sufficient to confer NND activity to acid of isolated from and of or at the to of not that the presence of an amino acid at this is important for P450 the in is to functional in The observation that a single amino acid mutation could NND activity to and the that NND genes are for the converter loci by Mann et al. (8Mann T.J. Weybrew J.A. Matzinger D.F. Hall J.L. Crop Sci. 1964; 4: 349-353Crossref Google Scholar) to sequence the CYP82E3 from N. tomentosiformis. of the DNA sequence of and its N. tomentosiformis revealed that a and a amino acid substitution and expression of the in yeast demonstrated that encoded a functional NND In nicotine metabolism demonstrated that also NND activity The observation that and in the the that NND genes the converter loci by genetic studies and and The that the green-leaf converter the gene in to the that and are an observation the of other closely related P450 isoforms that are in S. R. DNA Biol. 19: PubMed Scopus Google Scholar). Because of the genetic the NND genes in N. tomentosiformis, nicotine conversion to inherited as a single as by (8Mann T.J. Weybrew J.A. Matzinger D.F. Hall J.L. Crop Sci. 1964; 4: 349-353Crossref Google Scholar, 9Burk L.G. Jeffrey R.N. Tob. Sci. 1958; 2: 139-141Google Scholar, 23Griffith R.B. Valleau W.D. Stokes G.W. Science. 1955; 121: 343-344Crossref PubMed Scopus (16) Google Scholar). Expression analysis of the CYP82E3 and CYP82E4 orthologs demonstrated that is highly in the green leaves and is highly induced during leaf in the expression profiles of the NND genes are between N. tomentosiformis and the expression of in and NtabCYP82E4 in converter tobacco the that NtabCYP82E4 the unstable locus, derived from N. tomentosiformis, that is responsible for the genetic conversion of tobacco Collectively, these results the in In N. tomentosiformis at two active and are the region by the These genes by the of an ancestral NND gene by sequence the of both progenitor species donated a of conversion to tobacco. Because the conversion loci and ancestral tobacco as a green-leaf converter the evolution of tobacco, at conversion loci the alkaloid composition of the These a or large mutation, at the N. locus, whose to that of the CYP82E4 and the mutation at the locus originating from N. tomentosiformis. In to the an unstable mutation in the N. CYP82E4 locus to the inactivation of this NND gene by transcriptional Similar to the of was also These and other conversion loci rise to modern nonconverter tobacco, and the transcriptional of the NtabCYP82E4 locus played a major role in the emergence of converter tobacco. also the by the conversion locus derived from the green-leaf converter N. tomentosiformis the converter phenotype to converter tobacco. Because the locus donated by N. tomentosiformis both the and genes, the inactivation of in tobacco nornicotine production to the senescing leaves. Our can to the in the nicotine alkaloid profiles of other Nicotiana et al. (1Sisson V.A. Severson R.F. Beitr. Tabakforsch. 1990; 14: 327-339Google Scholar) numerous species of the Nicotiana genus as nicotine, green leaf nornicotine, or nornicotine is that nicotine possess a CYP82E3 and CYP82E4, green leaf nornicotine a functional CYP82E3 or CYP82E4, and nornicotine a functional CYP82E4 and a CYP82E3. The the evolution of nicotine-accumulating tobacco an nicotine and nornicotine tobacco plants could have been by at the of tobacco However, and Matzinger (10Wernsman E.A. Matzinger D.F. Tob. Sci. 1968; 12: 226-228Google Scholar) this that could not have nicotine-accumulating tobacco because of the presence of the two conversion loci donated by the N. sylvestris and N. tomentosiformis. our results that a of was required for the evolution of nicotine-accumulating tobacco, the of these plants by to tobacco could have also through natural because nicotine activity to nornicotine in I. R. Agric. Biol. Chem. 1968; Scopus Google Scholar, Bowen J. Google Scholar, P. R. Nat. 1997; 11: Scopus Google Scholar). is that the of nicotine and nornicotine are and nicotine N-demethylation is a function in tobacco. Interestingly, in the of the Nicotiana genus nornicotine as a precursor for a that activity nicotine J. D. J. J. Severson R. V. J. Chem. Ecol. 1989; PubMed Scopus Google Scholar, Baldwin I.T. PubMed Scopus Google Scholar, J. 1964; Scopus Google Scholar). of however, the for nornicotine production no in these species J. D. J. J. Severson R. V. J. Chem. Ecol. 1989; PubMed Scopus Google Scholar). Similar to members of the an progenitor of Nicotiana could have from nornicotine but the of the could have to the of NND function in tobacco. We for the and tobacco We also and for of the yeast expression and the yeast We for