BR112012017565B1 - TOBACCO PRODUCTS, METHODS FOR REDUCE THE CARCINOGENIC POTENTIAL OF A TOBACCO PRODUCT, FOR REDUCE THE LEVEL OF NORNICOTINE, OR FOR REDUCE THE RATE OF CONVERSION OF NICOTINE TO NORNICOTINE, IN A TOBACCO PLANT OR PLANT PART, AND FOR IDENTIFYING A TOBACCO PLANT WITH LOW LEVELS OF NORNICOTINE, ISOLATED POLYNUCLEOTIDE, AND ISOLATED POLYPEPTIDE - Google Patents

Abstract

TOBACCO PLANT OR PLANT PART, SEED, TOBACCO PRODUCT, METHODS FOR REDUCE THE CARCINOGENIC POTENTIAL OF A TOBACCO PRODUCT, FOR REDUCE THE LEVEL OF NORNICOTINE, OR FOR REDUCE THE RATE OF CONVERSION OF NICOTINE TO NORNICOTINE, IN A TOBACCO PLANT OR PLANT PART AND FOR IDENTIFYING A TOBACCO PLANT WITH LOW LEVELS OF NORNICOTINE, ISOLATED POLYNUCLEOTIDE, ISOLATED POLYPEPTIDE AND MUTATION IN A GENE ENCODING A CYP82E10 NICOTINE DEMETHYLASE Compositions and methods for reducing the level of nornicotine and N'-nitrosonornicotine (NNN) in tobacco plants and plant parts are provided. The compositions include isolated polynucleotides and polypeptides for root-specific CYP82E10 nicotine demethylases and variants thereof, which participate in the metabolic conversion of nicotine to nornicotine in these plants. Compositions of the invention also include tobacco plants or parts of tobacco plants, including a mutation in a gene encoding CYP82E10 nicotine demethylase, the mutation causing reduced expression or function of CYP82E10 nicotine demethylase. Seeds of these tobacco plants, or progenies thereof, and tobacco products prepared from the tobacco plants of the invention, or from parts of plants or (...).

Description

FIELD OF INVENTION

[0001] The invention relates to compositions and methods for minimizing the synthesis of nornicotine, and therefore of its metabolite N'-nitrosonornicotine, in tobacco plants and plant parts, particularly relating to compositions and methods for inhibiting the expression or function of a root-specific nicotine demethylase in combination with a green leaf and a senescence-induced nicotine demethylase.

BACKGROUND OF THE INVENTION

[0002] The predominant alkaloid found in commercial tobacco varieties is nicotine, which typically represents 90 to 95% of the total alkaloid pool. The remaining alkaloid fraction is composed primarily of three additional pyridine alkaloids: nornicotine, anabasine, and anatabine. Nornicotine is generated directly from nicotine through the activity of the enzyme nicotine N-demethylase. Nornicotine typically represents less than 5% of the total pyridine alkaloid pool, but through a process called "conversion," tobacco plants that initially produce very low amounts of nornicotine give rise to progeny that metabolically "convert" a large percentage of leaf nicotine to nornicotine. In tobacco plants capable of gene conversion (termed “converters”), most nornicotine production occurs during senescence and curing of the mature leaf (Wernsman and Matzinger (1968) Tob. Sci. 12:226-228). Burley tobaccos are particularly prone to gene conversion, with high rates reaching 20% per generation observed in some cultivars.

[0003] During curing and processing of tobacco leaf, the nornicotine portion is metabolized to the compound N-nitrosonornicotine (NNN), a tobacco-specific nitrosamine (TSNA) that has been shown to be carcinogenic in laboratory animals (Hecht and Hoffmann (1990) Cancer Surveys 8:273-294; Hoffmann et al. (1994) J. Toxicol. Environ. Health 41:1-52; Hecht (1998) Chem. Res. Toxicol. 11:559-603). In oven-cured tobacco, TSNAs have been shown to be predominantly formed by the reaction of alkaloids with the minute amounts of nitrogen oxide present in the flue gases generated by the direct-fired heating systems found in traditional curing houses (Peele and Gentry (1999) “Formation of Tobacco-specific Nitrosamines in Flue-cured Tobacco,” CORESTA Meeting, Agro-Phyto Groups, Suzhou, China). By retrofitting these curing houses with heat exchangers, it was possible to almost completely eliminate the mixing of flue gases with the curing air and significantly reduce the formation of TSNAs in tobaccos cured in this manner (Boyette and Hamm (2001) Rec. Adv. Tob. Sci. 27:17-22.). However, in air-cured Burley tobaccos, TSNA formation occurs primarily through the reaction of tobacco alkaloids with nitrite, a process catalyzed by leaf microbes (Bush et al. (2001) Rec. Adv. Tob. Sci. 27:23-46). To date, attempts to reduce TSNAs by modifying curing conditions while maintaining acceptable quality standards have not proven effective in air-cured tobaccos.

[0004] In addition to serving as a precursor to NNN, recent research indicates that nornicotine found in tobacco products may have other negative health consequences. Dickerson and Jea (2002) Proc. Natl. Acad. Sci. USA 99: 1508415088 demonstrated that nornicotine causes aberrant glycation of proteins within the cell. Nornicotine-modified protein concentrations were observed to be much higher in the plasma of smokers than in nonsmokers. This same research also demonstrated that nornicotine can covalently modify commonly prescribed steroid drugs such as prednisone. Such modifications have the potential to alter both the efficacy and toxicity of these drugs. Furthermore, other research has demonstrated a link between nornicotine found in tobacco products and age-related macular degeneration, birth defects, and periodontal disease (Brogan et al. (2005) Proc. Natl. Acad. Sci. USA 102: 10433-10438; Katz et al. (2005) J. Periodontol. 76: 1171-1174).

[0005] In Burley tobaccos, a positive correlation was found between leaf nornicotine content and the amount of NNN that accumulates in the cured product (Bush et al. (2001) Rec. Adv. Tob. Sci, 27:23-46; Shi et al. (2000) Tob. Chem. Res. Conf. 54:Abstract 27). Therefore, strategies that can effectively reduce leaf nornicotine content would not only help to alleviate the potential health harms caused by nornicotine itself, as described above, but would also simultaneously reduce NNN levels. This correlation was reinforced in recent research by Lewis et al. (2008) Plant Biotech. J. 6: 346–354, who demonstrated that reducing nornicotine levels using a transgenic RNAi construct targeting the CYP82E4v2 gene, which encodes senescence-induced nicotine demethylase, resulted in simultaneous reductions in the NNN content of cured leaves. Although this research demonstrated that transgenic technologies can be used to significantly reduce the nornicotine and NNN content of tobacco, issues of both public perception and intellectual property significantly hinder the commercialization of products derived from transgenic plants.

[0006] Therefore, there is a great need to develop means capable of effectively minimizing the accumulation of nornicotine in tobacco without relying on the use of GMOs.

SUMMARY OF THE INVENTION

[0007] Compositions and methods for minimizing the nornicotine content in tobacco plants and plant parts are provided. The compositions include an isolated root-specific cytochrome P450 polynucleotide designated CYP82E10 polynucleotide as set forth in SEQ ID NO:1 and the nicotine demethylase CYP82E10 polypeptide encoded thereby as set forth in SEQ ID NO:2, as well as variants and fragments thereof, including, but not limited to, polypeptides containing the sequence set forth in SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12 or 13, as well as polynucleotides encoding the polypeptide set forth in SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12 or 13. The CYP82E10 polypeptide of the invention is a nicotine demethylase that participates in the metabolic conversion of nicotine to nornicotine in the roots of tobacco plants. The isolated polynucleotides of the invention also include a polynucleotide containing the sequence indicated in SEQ ID NO: 3 or 4, as well as variants and fragments thereof. The compositions of the invention also include tobacco plants or parts of tobacco plants that contain a mutation in a gene encoding a nicotine demethylase CYP82E10, the mutation causing reduced expression or function of the nicotine demethylase CYP82E10. In some embodiments, the tobacco plants of the invention further include a mutation in a gene encoding the CYP82E4 nicotine demethylase and/or a mutation in a gene encoding the CYP82E5 nicotine demethylase, the mutation within these genes causing reduced expression or function of the CYP82E4 or CYP82E5 nicotine demethylase. Seeds of these tobacco plants, or their progeny, and tobacco products prepared from the tobacco plants of the invention, or from parts of plants or progeny thereof, are also provided.

[0008] Methods for reducing the level of nornicotine, or for reducing the rate of conversion of nicotine to nornicotine, in a tobacco plant or plant part are also provided. The methods consist of introducing into the genome of a tobacco plant a mutation within at least one allele of each of at least three nicotine demethylase genes, wherein the mutation causes reduced expression of the nicotine demethylase gene, and wherein the first of these nicotine demethylase genes encodes a root-specific nicotine demethylase that participates in the metabolic conversion of nicotine to nornicotine in a tobacco plant or plant part. In some embodiments, the root-specific nicotine demethylase is CYP82E10 or a variant thereof. In other embodiments, these methods involve introducing into the genome of a tobacco plant a mutation within at least one allele of the nicotine demethylase gene encoding CYP82E10 or a variant thereof, and a mutation within at least one allele of a nicotine demethylase encoding CYP82E4 or a variant thereof, and/or a nicotine demethylase encoding CYP82E5 or a variant thereof. Methods for identifying a tobacco plant with low nornicotine levels are also provided, wherein the plant or plant part is examined for the presence of a mutation in a gene encoding CYP82E10 or a variant thereof, alone or in combination with an examination for the presence of a mutation in a gene encoding CYP82E4 or a variant thereof, and/or the presence of a mutation in a gene encoding CYP82E5 or a variant thereof.

[0009] The following embodiments are contained in the present invention. 1. A tobacco plant or part of a plant containing a mutation in a gene encoding nicotine demethylase CYP82E10, wherein this mutation causes reduced expression or function of nicotine demethylase CYP82E10. 2. The tobacco plant or part of a plant according to claim 1, wherein the nicotine demethylase CYP82E10 is selected from the group consisting of the sequence indicated in SEQ ID NO: 2, 5, 6, 7, 8 and 9. 3. The tobacco plant or plant part of claim 1 or 2, wherein said mutation causes a modification of the nicotine demethylase CYP82E10 that occurs at a position selected from the group consisting of amino acid residues 79, 107, 381, 419, and any combination thereof, such numbering being in accordance with SEQ ID NO:2.4. The tobacco plant or plant part of claim 3, wherein said mutation is selected from the group consisting of: a) a serine substitution at the glycinated residue at position 79; b) a serine substitution at the prolineated residue at position 107; c) a serine substitution at the prolineated residue at position 381; d) a serine substitution at the prolineated residue at position 419; and e) any combination thereof. 5. The tobacco plant or plant part of any one of claims 1 to 4, further comprising a mutation in a gene encoding CYP82E4 nicotine demethylase, wherein said mutation causes reduced expression or function of CYP82E4 nicotine demethylase. 6. The tobacco plant or plant part of claim 5, wherein said CYP82E4 nicotine demethylase is selected from the sequence indicated in SEQ ID NO: 14, 15, 16, 17, 18, 19 and 20. 7. The tobacco plant or plant part of claim 5 or 6, wherein said mutation causes a modification of CYP82E4 nicotine demethylase that occurs at a position selected from the group consisting of amino acid residues 329, 364, 376, 381, and 458, such numbering being in accordance with SEQ ID NO:14.8. The tobacco plant or plant part of claim 7, wherein said mutation is selected from the group consisting of: a) a stop codon substitution at the tryptophan residue of position 329; b) an asparagine substitution at the delysine residue of position 364; c) a methionine substitution at the devaline residue of position 376; d) a serine substitution at the proline residue of position 381; e) a serine substitution at the proline residue of position 458; and f) any combination thereof. 9. The tobacco plant or plant part of any one of claims 1 to 8, further comprising a mutation in a gene encoding CYP82E5 nicotine demethylase, such mutation causing reduced expression or function of CYP82E5 nicotine demethylase. 10. The tobacco plant or plant part of claim 9, wherein such CYP82E5 nicotine demethylase is selected from the sequence indicated in SEQ ID NO:26, 27, 28, 29, 30, 31 and 32. 11. The tobacco plant or plant part of claim 9 or 10, wherein such mutation causes a modification of CYP82E5 nicotine demethylase that occurs at a position selected from the group consisting of amino acid residues 422 and 449, such numbering being in accordance with SEQ ID NO:26. The tobacco plant or plant part of claim 11, wherein said mutation is selected from the group consisting of: a) a stop codon substituted at the tryptophan residue at position 422; b) a leucine substituted at the proline residue at position 449; and c) any combination thereof. 13. The tobacco plant or plant part of claim 12, comprising a mutation in the nicotine demethylase gene CYP82E10 and the nicotine demethylase gene CYP82E4. 14. The tobacco plant or plant part of claim 13, wherein said tobacco plant or plant part is homozygous for said mutation. The tobacco plant or plant part of claim 14, wherein the nicotine demethylase CYP82E10 includes a mutation at position 381, the CYP82E4 nicotine demethylase includes the mutation at position 329, and the CYP82E5 nicotine demethylase includes the mutation at position 422, such numbering being in accordance with SEQ ID NO:2, 14, and 26, respectively. The tobacco plant or plant part of claim 15, wherein said mutation is selected from the group consisting of: a) a serine substitution at the proline residue of position 381; b) a stop codon substitution at the tryptophan residue of position 329; c) a stop codon substitution at the tryptophan residue of position 422; and d) any combination thereof. The tobacco plant or plant part of any one of claims 13 to 16, wherein said plant or plant part has less than 1.5% conversion of nicotine to nornicotine. 18. The tobacco plant or plant part of claim 17, wherein said plant or plant part has no more than 0.5% conversion of nicotine to nornicotine. 19. A seed of the tobacco plant of any one of claims 1 to 18, or a progeny thereof. 20. A tobacco product prepared from a tobacco plant, or a part or progeny of the plant, of any one of claims 1 to 19. 21. A method of reducing the carcinogenic potential of a tobacco product, the method comprising preparing the tobacco product from a tobacco plant, or a part or progeny of the plant, of any one of claims 1 to 18. 22. A method for reducing the level of nornicotine, or reducing the rate of conversion of nicotine to nornicotine, in a tobacco plant or part of a plant, the method comprising introducing into the genome of said plant a mutation within at least one allele of each of at least three nicotine demethylase genes, said mutation reducing the expression of the nicotine demethylase gene, and a first of said nicotine demethylase genes encoding a root-specific nicotine demethylase that participates in the metabolic conversion of nicotine to nornicotine in a tobacco plant or part of a plant. The method of claim 22, wherein the root-specific nicotine demethylase is nicotine demethylase CYP82E10 containing an amino acid sequence selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID NO: 2, 5, 6, 7, 8, 9, or 10; and b) an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, 5, 6, 7, 8, 9, or 10. 24. The method of claim 23, wherein said amino acid sequence for said CYP82E10 nicotine demethylase has a substitution at an amino acid residue at a position selected from the group consisting of residues 79, 107, 381, 419, and any combination thereof, the numbering being in accordance with SEQ ID NO:2. 25. The method of claim 24, wherein the substitution at position 79, 107, 381, or 419 is a serine residue. 26. The method of any one of claims 22-25, wherein a second of the nicotine demethylase genes encodes a CYP82E4 nicotine demethylase. 27. The method of claim 26, wherein the CYP82E4 nicotine demethylase includes an amino acid sequence selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID NO:14, 15, 16, 17, 18, 19, 20, or 21; and b) an amino acid sequence that has at least 98% sequence identity to the sequence set forth in SEQ ID NO:14, 15, 16, 17, 18, 19, 20, or 21. 28. The method of claim 27, wherein the amino acid sequence for the CYP82E4 nicotine demethylase has a substitution at an amino acid residue at a position selected from the group consisting of residues 329, 364, 381, 458, and any combination thereof, the numbering being in accordance with SEQ ID NO:14.29. The method of claim 28, wherein the substitution at position 329 is a stop codon, the substitution at position 364 is an asparagine residue, the substitution at position 381 is a serine residue, the substitution at position 458 is a serine residue, or any combination thereof. 30. The method of any one of claims 22-29, wherein one-third of the nicotine demethylase genes encode a CYP82E5 nicotine demethylase. 31. The method of claim 30, wherein the CYP82E5 nicotine demethylase includes an amino acid sequence selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID NO:26, 27, 28, 29, 30, 31, or 32; and b) an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO: 26, 27, 28, 29, 30, 31, or 32. 32. The method of claim 31, wherein the amino acid sequence for CYP82E5 nicotine demethylase has a substitution at an amino acid residue at a position selected from the group consisting of residues 422 and 449, and any combination thereof, the numbering being in accordance with SEQ ID NO: 26. 33. The method of claim 32, wherein the substitution at position 422 is a stop codon, the substitution at position 449 is a leucine residue, or any combination thereof. 34. The method of any one of claims 22 to 33, wherein said plant or plant part is homozygous for said mutation. The method of any one of claims 22 to 34, wherein the introduction includes a cultivation protocol. 36. The method of any one of claims 22 to 35, wherein the plant is a Burley, Virginia, oven-cured, air-cured, fire-cured, Oriental tobacco plant, or a dark tobacco plant. 37. The tobacco plant or part of a tobacco plant according to any one of claims 1 to 19, wherein said tobacco plant is a Burley, Virginia, oven-cured, air-cured, fire-cured, Oriental tobacco plant, or a dark tobacco plant. 38. A method of identifying a tobacco plant with low levels of nornicotine, the method comprising examining a DNA sample from a tobacco plant of interest for the presence of a mutation in SEQ ID NO: 1 or 3. 39. The method according to claim 38, wherein said tobacco plant is a non-converter plant. 40. The method of claim 38 or 39, wherein the screening is performed using a sequence selected from the group consisting of SEQ ID NOS:1, 3, 35, 36, 37, and 38. 41. The method of any one of claims 38 to 40, further comprising screening the DNA sample, or another DNA sample from the tobacco plant of interest, for the presence of a mutation in SEQ ID NO:14, a mutation in SEQ ID NO:26, or a mutation in both SEQ ID NO:14 and SEQ ID NO:26. 42. An isolated polynucleotide containing a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence containing SEQ ID NO: 1, 3, or 4; b) a nucleotide sequence containing a fragment of at least 20 consecutive nucleotides from SEQ ID NO: 1, 3, or 4; c) a nucleotide sequence that has at least 97% sequence identity to the entire sequence indicated in SEQ ID NO: 1, such polynucleotide encoding a polypeptide that participates in the metabolic conversion of nicotine to nornicotine in a plant; d) a nucleotide sequence that encodes a polypeptide selected from the group consisting of SEQ ID NOS: 2 and 5-13, or a fragment thereof containing at least 115 contiguous residues; e) a nucleotide sequence that encodes a polypeptide that has at least 98% sequence identity to the indicated sequence in SEQ ID NO:2, 5, 6, 7, 8, 9, 10, 11, 12 or 13; and f) a nucleotide sequence that is complementary to the sequence according to any one of the previous items (a) to (e). 43. An isolated polypeptide containing an amino acid sequence selected from the group consisting of: a) an amino acid sequence set forth in SEQ ID NO:2, 5, 6, 7, 8, 9, 10, 11, 12 or 13; b) an amino acid sequence that is at least 98% identical to an amino acid sequence set forth in SEQ ID NO:2, 5, 6, 7, 8, 9, 10, 11, 12, or 13; and c) an amino acid sequence that is a fragment of the amino acid sequence set forth in SEQ ID NO: 2, 5, 6, 7, 8, 9, 10, 11, 12, or 13, wherein such fragment includes at least 115 contiguous residues of the amino acid sequence of SEQ ID NO: 2, 5, 6, 7, 8, 9, 10, 11, 12, or 13.44. A tobacco plant or plant part that is homozygous for a mutation in a gene encoding CYP82E10 nicotine demethylase, a gene encoding CYP82E4 nicotine demethylase, and a gene encoding CYP82E5 nicotine demethylase, wherein such mutation causes reduced expression or function of CYP82E10, CYP82E4, and CYP82E5 nicotine demethylase, wherein CYP82E10 nicotine demethylase includes a mutation at position 381, CYP82E4 nicotine demethylase includes a mutation at position 329, and CYP82E5 nicotine demethylase includes a mutation at position 422, wherein such numbering is in accordance with SEQ ID NO:2, 14, and 26, respectively.45. A mutation in a gene encoding a nicotine demethylase CYP82E10, which causes reduced expression or function of nicotine demethylase CYP82E10.46. A plant that has a mutation in a CYP82E10 gene that inhibits nicotine demethylase activity in roots, a mutation in a CYP82E4v2 gene that inhibits nicotine demethylase activity in senescent leaves, and a mutation in a CYP83E5v2 gene that inhibits nicotine demethylase activity in green leaves.

BRIEF DESCRIPTION OF THE FIGURES

[0010] Figure 1A-C illustrates the DNA (SEQ ID NO:4) and predicted protein sequences of the CYP82E10 nicotine demethylase gene. Protein-coding sequences are in capital letters and the 5' and 3' flanking sequences are in lowercase. The intron sequence (SEQ ID NO:3) is in lowercase italic. Nucleotide sequence numbers are indicated on the left and protein sequence numbers are indicated on the right. The nucleotide sequence corresponding to the PCR primers used to specifically amplify exon 1 for a mutation screen is underlined (not indicated in bold), with sequences underlined in bold indicating the exon 2-specific primer site. Individual nucleotide and amino acid residues shown to undergo changes in a mutation screen (Table 2) are underlined and in bold.

[0011] Figure 2A-C illustrates an alignment of genomic sequences for CYP82E10 (SEQ ID NO:4), CYP82E5v2 (SEQ ID NO:38), and CYP82E4v2 (SEQ ID NO:37). Protein coding sequences are in capital letters; 5' and 3' untranslated regions are indicated in lowercase; and intron sequences are illustrated in lowercase italics. Positions of shared sequence identities are indicated with a shaded background.

[0012] Figure 3 illustrates thin layer chromatography data of nicotine demethylase activities of microsomal membranes from yeast cells expressing CYP82E10 and CYP82E10 and having the Pro381Ser (P381S) mutation from plant 1041. CPM means counts per minute.

[0013] Figures 4A and 4B illustrate the mean percent nicotine conversion for Burley tobacco plants with varying mutant combinations at the CYP82E4v2, CYP82E5v2, and CYP82E10 loci. Means with different letters are significantly different at the P < 0.05 level.

DESCRIPTION OF SEQUENCES IN THE SEQUENCE LIST

[0014] The list below presents the sequence information for the Sequence List. Standard notation for amino acid substitutions is used. Thus, for example, CYP82E10 P419S indicates that the protein variant has a serine substitution at the proline residue at position 419, with the numbering referring to the wild-type sequence, in this case, the CYP82E10 sequence shown in SEQ ID NO:2. As another example, CYP82E4 P38L indicates that the protein variant has a leucine substitution at the proline residue at position 38, with the numbering referring to the wild-type sequence, in this case, the CYP82E4 sequence shown in SEQ ID NO:14. As another example, CYP82E5 P72L indicates that the protein variant has a leucine substitution at the proline residue at position 72, with the numbering referring to the wild-type sequence, in this case, the CYP82E5 sequence indicated in SEQ ID NO:26.

[0015] SEQ ID NO:1 indicates a coding sequence for CYP82E10.

[0016] SEQ ID NO:2 indicates the amino acid sequence for aCYP82E10.

[0017] SEQ ID NO:3 indicates the nucleotide sequence of an intron of the CYP82E10 gene.

[0018] SEQ ID NO:4 indicates the genomic sequence for aCYP82E10.

[0019] SEQ ID NO:5 indicates the amino acid sequence for aCYP82E10 L148F.

[0020] SEQ ID NO:6 indicates the amino acid sequence for aCYP82E10 G172R.

[0021] SEQ ID NO:7 indicates the amino acid sequence for aCYP82E10 A344T.

[0022] SEQ ID NO:8 indicates the amino acid sequence for aCYP82E10 A410T.

[0023] SEQ ID NO:9 indicates the amino acid sequence for aCYP82E10 R417H.

[0024] SEQ ID NO:10 indicates the amino acid sequence for aCYP82E10 P419S.

[0025] SEQ ID NO:11 indicates the amino acid sequence for aCYP82E10 G79S.

[0026] SEQ ID NO:12 indicates the amino acid sequence for aCYP82E10 P107S.

[0027] SEQ ID NO:13 indicates the amino acid sequence for aCYP82E10 P381S.

[0028] SEQ ID NO:14 indicates the amino acid sequence for aCYP82E4.

[0029] SEQ ID NO:15 indicates the amino acid sequence for aCYP82E4 P38L.

[0030] SEQ ID NO:16 indicates the amino acid sequence for aCYP82E4 D171N.

[0031] SEQ ID NO:17 indicates the amino acid sequence for aCYP82E4 E201K.

[0032] SEQ ID NO:18 indicates the amino acid sequence for aCYP82E4 R169Q.

[0033] SEQ ID NO:19 indicates the amino acid sequence for aCYP82E4 G459R.

[0034] SEQ ID NO:20 indicates the amino acid sequence for aCYP82E4 T427I.

[0035] SEQ ID NO:21 indicates the amino acid sequence for aCYP82E4 V376M.

[0036] SEQ ID NO:22 indicates the amino acid sequence for aCYP82E4 W329Stop.

[0037] SEQ ID NO:23 indicates the amino acid sequence for aCYP82E4 K364N.

[0038] SEQ ID NO:24 indicates the amino acid sequence for aCYP82E4 P381S.

[0039] SEQ ID NO:25 indicates the amino acid sequence for aCYP82E4 P458S.

[0040] SEQ ID NO:26 indicates the amino acid sequence for aCYP82E5.

[0041] SEQ ID NO:27 indicates the amino acid sequence for aCYP82E5 P72L.

[0042] SEQ ID NO:28 indicates the amino acid sequence for aCYP82E5 L143F.

[0043] SEQ ID NO:29 indicates the amino acid sequence for aCYP82E5 S174L.

[0044] SEQ ID NO:30 indicates the amino acid sequence for aCYP82E5 M224I.

[0045] SEQ ID NO:31 indicates the amino acid sequence for aCYP82E5 P235S.

[0046] SEQ ID NO:32 indicates the amino acid sequence for aCYP82E5 A410V.

[0047] SEQ ID NO:33 indicates the amino acid sequence for aCYP82E5 W422Stop.

[0048] SEQ ID NO:34 indicates the amino acid sequence for aCYP82E5 P449L.

[0049] SEQ ID NO:35 indicates a forward primer sequence for exon 1 of CYP82E10.

[0050] SEQ ID NO:36 indicates a reverse primer sequence for exon 1 of CYP82E10.

[0051] SEQ ID NO:37 indicates a forward primer sequence for exon 2 of CYP82E10.

[0052] SEQ ID NO:37 indicates a reverse primer sequence for exon 2 of CYP82E10.

[0053] SEQ ID NO:38 indicates the genomic sequence for aCYP82E4v2.

[0054] SEQ ID NO:39 indicates the genomic sequence for aCYP82E5v2.

DEFINITIONS

[0055] The present invention includes compositions and methods for inhibiting the expression or function of root-specific nicotine demethylase polypeptides that participate in the metabolic conversion of nicotine to nornicotine in the roots of a plant, especially in plants of the genus Nicotiana, including tobacco plants of various commercial varieties.

[0056] As used herein, “inhibit,” “inhibition,” and “inhibitor” are defined as any method known in the art or described herein that reduces the expression or function of a gene product of interest (e.g., the target gene product), in this case, a nicotine demethylase such as the root-specific nicotine demethylase of the invention. It is recognized that nicotine demethylase polypeptides can be inhibited by any suitable method known in the art, including sense and antisense suppression, RNAi suppression, knockout approaches such as mutagenesis, among others. Of particular interest are methods that produce knock-out, or knock-down, of the expression and/or function of these root-specific nicotine demethylases, especially mutagenic approaches that allow the selection of favorable mutations in the nicotine demethylase gene CYP82E10.

[0057] “Favorable mutation” means a mutation that causes a substitution, insertion, deletion, or truncation of the CYP82E10 polypeptide such that nicotine demethylase activity is inhibited. In some embodiments, nicotine demethylase activity is inhibited by at least 25%, 30%, 35, 40%, 45, 50%, 55%, or 60% compared to the activity of the wild-type CYP82E10 polypeptide under the same test conditions. In other embodiments, nicotine demethylase activity is inhibited by at least 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In preferred embodiments, the favorable mutation enables complete inhibition (i.e., 100% inhibition), and nicotine demethylase activity is knocked out (i.e., its activity cannot be measured).

[0058] The term “inhibitor” may be used in the context of a comparison between two plants, e.g., a genetically altered plant and a wild-type plant. The comparison may be between plants, e.g., a wild-type plant and one lacking a DNA sequence capable of producing root-specific nicotine demethylase that converts nicotine to nornicotine. Inhibition of the expression or function of a target gene product may also occur in the context of a comparison between plant cells, organelles, organs, tissues, or plant parts within the same plant or between different plants, including comparisons between developmental or evolutionary stages within the same plant or plant part or between plants or plant parts.

[0059] The term “inhibitor” may indicate any relative reduction in the function or production of a gene product of interest, in this case, a root-specific nicotine demethylase, including the complete elimination of the function or production of that gene product. When levels of a gene product are compared, such comparison is preferably made between organisms with similar genetic backgrounds. Preferably, a similar genetic background is a background in which the organisms being compared share 50% or more, preferably 75% or more, and ideally 90% or more, sequence identity with respect to the nuclear genetic material. A similar genetic background is a background in which the organisms being compared are isogenic plants, except in the case of any genetic material originally introduced using plant transformation techniques or mutation generated by human intervention. Measurement of the level or quantity of a gene product may be performed by any suitable method, examples of which include, but are not limited to, comparison of mRNA transcript levels, protein or peptide levels, and/or phenotypes, particularly the conversion of nicotine to nornicotine. As used herein, mRNA transcripts may be processed or unprocessed mRNA transcripts, and polypeptides or peptides may be polypeptides or peptides with or without any post-translational modification.

[0060] As used herein, the term "variant" indicates a substantially similar sequence. A variant may have different functions or substantially similar function as a wild-type polypeptide of interest. For nicotine demethylase, substantially similar function has at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 60%, 50%, 25%, or 15% of the wild-type enzyme's function of converting nicotine to nornicotine under the same conditions or in a near-isogenic line. A wild-type CYP82E10 is indicated in SEQ ID NO:2. A wild-type CYP82E4 is indicated in SEQ ID NO:14. A wild-type CYP82E5 is indicated in SEQ ID NO:26. Exemplary variants of wild-type CYP82E10 of the present invention include polypeptides containing the sequence set forth in SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, or 13. The variant set forth in SEQ ID NO:10 (CYP82E10 P419S) has the advantage of possessing a favorable mutation that causes the enzyme to have only about 25% of the nicotine demethylase activity of the wild-type CYP82E10 polypeptide. The variants indicated in SEQ ID NOs: 11 (CYP82E10 G79S), 12 (CYP82E10 with P107S) and 13(CYP82E10 with P381S) have the advantage of possessing favorable mutations that cause their nicotine demethylase activity to be knocked out (i.e., 100% inhibition and therefore a non-functional polypeptide). Similarly, exemplary variants of wild-type CYP82E4 include polypeptides containing the sequence indicated in SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. The variant indicated in SEQ ID NO:21 (CYP82E4 V376M) has the advantage of possessing a favorable mutation that causes the enzyme to have only about 50% of the nicotine demethylase activity of the wild-type CYP82E4 polypeptide. The variants indicated in SEQ ID NOs: 22 (CYP82E4 W329Stop), 23 (CYP82E4 K364N), 24 (CYP82E4 P381S) and 25 (CYP82E4 P458S) have the advantage of possessing favorable mutations that cause their nicotine demethylase activity to be knocked out (i.e., 100% inhibition). Similarly, exemplary variants of wild-type CYP82E4 include polypeptides containing the sequence indicated in SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, or 34. The variant indicated in SEQ ID NO: 34 (CYP82E5 P449L) has the advantage of having a favorable mutation that causes inhibition of its nicotine demethylase activity, and the variant indicated in SEQ ID NO: 33 has the advantage of having a favorable mutation that causes knockout (i.e., 100% inhibition) of its nicotine demethylase activity.

[0061] As used herein, a “variant polynucleotide” or a “variant polypeptide” denotes a nucleic acid or amino acid sequence that is not wild-type.

[0062] A variant may have one addition, deletion, or substitution; two or fewer additions, deletions, or substitutions; three or fewer additions, deletions, or substitutions; four or fewer additions, deletions, or substitutions; or five or fewer additions, deletions, or substitutions. A mutation includes additions, deletions, and substitutions. These deletions or additions may occur at the C-terminus, the N-terminus, or both the C- and N-terminus. Fusion polypeptides or epitope-tagged polypeptides are also part of the present invention. "Silent" nucleotide mutations do not modify the encoded amino acid at a given position. Amino acid substitutions may be conservative. A conservative substitution is a modification to an amino acid that belongs to the same amino acid family as the original amino acid. The family is defined by the side chain of the individual amino acids. An amino acid family may have basic, acidic, uncharged polar, or nonpolar side chains. See Alberts et al. (1994) Molecular biology of the cell (3rd ed., pp. 56-57, Garle Publishing Inc., New York, New York), incorporated herein by reference as indicated in its entirety. The deletion, substitution, or addition may be made at an amino acid of another member of the CYP82E family at that same position. As used herein, a "fragment" denotes a portion of a polynucleotide or a portion of a polypeptide and hence the protein thus encoded.

[0063] As used herein, the term “plant parts” refers to plant cells, plant protoplasts, plant cell tissue cultures from which an entire plant can be regenerated, plant callus, plant clods, and plant cells that are intact in plants or plant parts such as embryos, pollen, anthers, ovules, seeds, leaves, flowers, trunks, branches, fruits, roots, root tips, among others. Progenies, variants, and mutants of regenerated plants are also part of the scope of the present invention, as long as they contain the introduced polynucleotides of the invention. As used herein, “tobacco plant material” represents any portion of a plant part or any combination of plant parts.

DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention relates to a novel nicotine demethylase gene, CYP82E10 (genomic sequence indicated in SEQ ID NO:4) and its nicotine demethylase CYP82E10 (SEQ ID NO:2) that participate in the root-specific conversion of nicotine to nornicotine in the roots of tobacco plants, as well as their use to reduce or minimize the conversion of nicotine to nornicotine and therefore reduce nornicotine levels in tobacco plants and plant parts. By “root-specific” it is understood that it is preferentially expressed within the roots of tobacco plants, and not in other plant organs such as leaves or seeds. By introducing selected favorable mutations within such a root-specific nicotine demethylase or variants thereof that possess nicotine demethylase activity, in combination with one or more selected favorable mutations within a gene encoding a green leaf nicotine demethylase (e.g., CYP82E5 set forth in SEQ ID NO:26) or variant thereof that possesses nicotine demethylase activity, or in combination with one or more selected favorable mutations within a gene encoding a senescence-induced nicotine demethylase (e.g., CYP82E4 set forth in SEQ ID NO:14) or variant thereof that possesses nicotine demethylase activity, it is possible to produce non-transgenic tobacco plants with minimal conversion of nicotine to nornicotine, with the conversion rate being less than about 1.5%, preferably less than about 1%.

[0065] Reducing nornicotine levels in tobacco is highly desirable because this alkaloid serves as a precursor to the well-documented carcinogen N’-nitrosonornicotine (NNN). Two genes encoding proteins with nicotine demethylase activity in tobacco have previously been identified and named CYP82E4v2 and CYP82E5v2. The CYP82E4 polypeptide (SEQ ID NO:14) is a senescence-induced nicotine demethylase. The CYP82E4v2 gene (including the coding and intron regions), its role in nornicotine production in tobacco plants, and methods for inhibiting its expression and function are described in U.S. Patent Application No. 11/580,765, published as U.S. Patent Application Publication No. 2008/0202541 A1. The CYP82E5 polypeptide (SEQ ID NO:26) is a green leaf nicotine demethylase (i.e., its predominant expression is in green leaves). The CYP82E4 gene (including the coding and intron regions), its role in nornicotine production in tobacco plants, and methods for inhibiting its expression and function are described in U.S. Patent Application No. 12/269,531, published as U.S. Patent Application Publication No. 2009/0205072 A1. The contents of both U.S. patent applications and their respective publications are incorporated herein by reference in their entirety.

[0066] Plants homozygous for favorable mutant alleles cyp82e4v2 and cyp82e5v2 (e.g., mutant alleles that knock down, or knock out, the expression of the respective nicotine demethylase genes) can therefore still metabolize more than 2% of their nicotine to nornicotine, representing levels of nornicotine that can still trigger substantial NNN formation. The discovery of the nicotine demethylase gene CYP82E10 provides a further avenue to minimize the rate of nicotine to nornicotine conversion in tobacco plants and thus further reduce nornicotine, and consequently NNN, levels in tobacco plants and plant materials derived therefrom. Combining favorable mutant alleles cyp82e10 with favorable mutant alleles cyp82e4v2 and cyp82e5v2 results in tobacco plants that have more than a threefold reduction in nornicotine, compared to that observed in tobacco plants that have only the cyp82e4v2 mutation or this mutation and the cyp82e5v2 mutation together. In one embodiment, the present invention provides a homozygous triple mutant combination of nicotine demethylase genes (cyp82e4v2, cyp82e5v2, and cyp82e10) that results in non-transgenic tobacco plants that produce very low levels of nornicotine, comparable to those that could only be achieved through transgenic gene knockout approaches such as those described in U.S. Patent Application Publication Nos. 2008/0202541 A1 and 2009/0205072 A1.

Nicotine demethylase polynucleotides and polypeptides and variants and fragments thereof

[0067] The compositions of the present invention include the CYP82E10 polypeptide, as well as variants and fragments thereof. These nicotine demethylase polypeptides and polypeptides participate in the metabolic conversion of nicotine to nornicotine in plants, including commercial varieties of tobacco plants. In particular, the compositions of the invention include isolated polypeptides containing the amino acid sequences indicated in SEQ ID NOs:2 and 5-13, isolated polynucleotides containing the nucleotide sequences indicated in SEQ ID NOs:1, 3 and 4, and isolated polynucleotides encoding the amino acid sequence of SEQ ID NOs:2 and 5-13. The polynucleotides of the present invention can be used to inhibit the expression of nicotine demethylase polypeptides or variants thereof that participate in the metabolic conversion of nicotine to nornicotine in plants, especially tobacco plants. Some of the polynucleotides of the invention contain mutations that inhibit the nicotine demethylase activity of wild-type nicotine demethylase. Inhibition of the polypeptides of the present invention is effective in reducing nornicotine levels in tobacco lines in which genetic conversion occurs in less than 30%, 50%, 70%, or 90% of the population, such as in greenhouse-cured tobaccos. Inhibition of the polypeptides of the present invention is effective in reducing nornicotine levels in tobacco populations in which genetic conversion occurs in at least 90%, 80%, 70%, 60%, or 50% of the plant population. The population preferably contains more than about 25, 50, 100, 500, 1,000, 5,000, or 25,000 plants, with ideally at least about 10%, 25%, 50%, 75%, 95%, or 100% of the plants containing a polypeptide of the present invention.

[0068] The nicotine demethylase polynucleotides and encoded polypeptides of the present invention include a novel cytochrome P450 gene, designated the CYP82E10 nicotine demethylase gene, which has been shown for the first time to play a role in the metabolic conversion of nicotine to nornicotine in the roots of tobacco plants. Transgenic approaches such as sense, antisense, and RNAi suppression can be used to knock down expression of this nicotine demethylase, in a manner similar to that described for the nicotine demethylases CYP82E4 and CYP82E5, as described in U.S. Patent Application Publication Nos. 2008/0202541 A1 and 2009/0205072 A1, the contents of which are incorporated herein by reference in their entirety. The preferred approach is to introduce one or more favorable mutations into this gene, as this approach has the advantage of producing non-transgenic tobacco plants that have reduced rates of nicotine-to-nornicotine conversion and, therefore, reduced levels of nornicotine and NNN. These approaches include, but are not limited to, mutagenesis, among others, as described elsewhere in this document.

[0069] The invention encompasses isolated or substantially purified polynucleotide or protein compositions of the present invention. An “isolated” or “purified” polynucleotide or protein, or a biologically active portion thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein, as occurs in the naturally occurring environment. Therefore, an isolated or purified polynucleotide or protein is substantially free of other cellular materials or culture media when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Ideally, an “isolated” polynucleotide is free of the sequences (ideally, protein-coding sequences) that naturally flank the polynucleotide (e.g., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of the nucleotide sequence that naturally flanks the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Protein that is substantially free of cellular material includes protein preparations with less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) contaminating protein. When the protein of the invention or a biologically active portion thereof is produced recombinantly, ideally the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) chemical precursors or chemicals other than those of the protein of interest.

[0070] Fragments of the polynucleotides and polypeptides thus encoded are also encompassed by the present invention. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and thus participate in the metabolic conversion of nicotine to nornicotine in a plant. Alternatively, fragments of a polynucleotide that are useful as hybridization probes or PCR primers generally do not encode protein fragments that retain biological activity. In addition, fragments of the described nucleotide sequences include those that can be assembled into recombinant constructs for use in gene silencing with any method known in the art, including, but not limited to, sense/co-suppression, antisense suppression, double-stranded RNA (dsRNA) interference, hairpin RNA interference and intron-containing hairpin RNA interference, amplicon-mediated ribozyme interference, ribozymes, and small interfering RNA or micro RNA, as described in the art and later herein. Therefore, fragments of a nucleotide sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 70 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, 250 nucleotides, 300 nucleotides, up to the full length of the polynucleotide encoding the proteins of the invention, depending on the desired result. In one aspect, the fragments of a nucleotide sequence can be fragments of between 100 and about 350 nucleotides, between 100 and about 325 nucleotides, between 100 and about 300 nucleotides, between about 125 and about 300 nucleotides, between about 125 and about 275 nucleotides in length, between about 200 and about 320 contiguous nucleotides, between about 200 and about 420 contiguous nucleotides in length, between about 250 and about 450 contiguous nucleotides in length. Another embodiment includes a recombinant nucleic acid molecule of between about 300 and about 450 contiguous nucleotides in length.

[0071] A fragment of a nicotine demethylase polynucleotide of the present invention that encodes a biologically active portion of a CYP82E10 polypeptide of the present invention will encode at least 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 contiguous amino acids, or up to the total number of amino acids present in the full length of a nicotine demethylase polypeptide of the invention (e.g., 517 amino acids for SEQ ID NOs:2 and 5-13). The biologically active portion of a nicotine demethylase polypeptide can be prepared by isolating a portion of one of the CYP82E10 polynucleotides of the present invention, expressing the encoded portion of the CYP82E10 polypeptide (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the CYP82E10 polypeptide, e.g., the ability to promote the conversion of nicotine to nornicotine, using assays known in the art and those provided later herein.

[0072] Polynucleotides that are fragments of a CYP82E10 nucleotide sequence of the present invention contain at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, or 1700 contiguous nucleotides, or up to the number of nucleotides present in the full length of a CYP82E10 polynucleotide as described herein (e.g., 1551 for SEQ ID NO: 1; 2636 for SEQ ID NO: 4). The polynucleotides that are fragments of a CYP82E10 nucleotide sequence of the present invention contain fragments of about 20 to about 1700 contiguous nucleotides, from about 50 to about 1600 contiguous nucleotides, from about 75 to about 1500 contiguous nucleotides, from about 100 to about 1400 nucleotides, from about 150 to about 1300 contiguous nucleotides, from about 150 to about 1200 contiguous nucleotides, from about 175 to about 1100 contiguous nucleotides, about 200 to about 1000 contiguous nucleotides, about 225 to about 900 nucleotides contiguous, about 500 to about 1600 contiguous nucleotides, about 775 to about 1700 contiguous nucleotides, about 1000 to about 1700 contiguous nucleotides, or from about 300 to about 800 contiguous nucleotides of a CYP82E10 polynucleotide as described herein. In one aspect, the polynucleotide fragments contain a polynucleotide sequence containing the polynucleotide sequence of the nucleotide located at about position 700 to about position 1250 of the CYP82E10 coding sequence, about position 700 to about position 1250 of a CYP82E10 genomic sequence, about position 10 to about position 900 of a CYP82E10 intron sequence, or about position 100 to about position 800 of a CYP82E10 intron sequence.

[0073] Variants of the polynucleotides and polypeptides thus encoded are also encompassed by the present invention. Naturally occurring variants include those variants that share substantial sequence identity with the CYP82E10 polynucleotides and polypeptides described herein, as defined later herein. In another embodiment, naturally occurring variants also share substantial functional identity with the CYP82E10 polynucleotides described herein. The compositions and methods of the invention may be used to achieve the expression or function of any naturally occurring CYP82E10 that shares substantial sequence identity with the CYP82E10 polypeptides described. These CYP82E10 polypeptides may possess the relevant nicotine demethylase activity, e.g., participation in the metabolic conversion of nicotine to nornicotine in plants, or may not. These variants may be the result of, for example, genetic polymorphism or human manipulation as occurs with breeding and selection, including mutagenesis approaches. Biologically active variants of a CYP82E10 protein of the invention, e.g., variants of the polypeptide indicated in SEQ ID NO:2 and 5-13, should possess at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the wild-type protein, as determined by the sequence alignment programs and parameters described in other sections of this document, and may be characterized by their functional participation in the metabolic conversion of nicotine to nornicotine in plants, or by the absence thereof. A biologically active variant of a protein of the invention may differ from that protein by as few as 1 to 15 amino acid residues, as few as 10, as few as 9, as few as 8, as few as 7, as few as 6, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. A biologically inactive variant of a protein of the invention may differ from that protein by as few as 1 to 15 amino acid residues, as few as 10, as few as 9, as few as 8, as few as 7, as few as 6, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue.

[0074] Variants of a particular polynucleotide of the present invention include those naturally occurring polynucleotides that encode a CYP82E10 polypeptide that participates in the metabolic conversion of nicotine to nornicotine in plant roots. Such polynucleotide variants may provide for the deletion and/or addition of one or more nucleotides at one or more sites within the polynucleotide described herein and/or a substitution of one or more nucleotides at one or more sites of the native polynucleotide. Due to the degeneracy of the genetic code, conservative variants for polynucleotides include those sequences that encode the amino acid sequence of one of the CYP82E10 polypeptides of the invention. Naturally occurring variants such as these can be identified using known molecular biology techniques such as, for example, polymerase chain reaction (PCR) and hybridization techniques, as known in the art and described herein. Polynucleotide variants also include synthetically derived polynucleotides, such as those generated, for example, using site-directed mutagenesis, but which continue to share substantial sequence identity with the naturally occurring sequences described herein and therefore can be used in the methods of the invention to inhibit the expression or function of a nicotine demethylase that participates in the metabolic conversion of nicotine to nornicotine, including the nicotine demethylase polypeptides set forth in SEQ ID NOS:2, 5, 6, 7, 8, 9, and 10. Generally, variants of a particular polynucleotide of the invention, for example, the polynucleotide sequence of SEQ ID NO:3 or the polynucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:2 and 5-13, should have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the particular polynucleotide, as determined by sequence alignment programs and parameters described elsewhere in this document.

[0075] Variants of a particular polynucleotide of the present invention (also called reference polynucleotides) can also be assessed by comparing the percent sequence identity between the polypeptide encoded by the reference polynucleotide and the polypeptide encoded by a variant polynucleotide. The percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. When any pair of polynucleotides of the invention is evaluated by comparing the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0076] Furthermore, the polynucleotides of the invention can be used to isolate corresponding root-specific nicotine demethylase sequences, especially CYP82E10 sequences, from other members of the genus Nicotiana. PCR, hybridization, and other similar methods can be used to identify these sequences based on their sequence homologies to the sequences indicated herein. Sequences isolated based on their sequence identity to the nucleotide sequences indicated herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologous to the sequences described.

[0077] According to the present invention, “orthologs” are genes derived from a common ancestral gene and that are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequence and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. Functions of orthologs are typically highly conserved among species. Therefore, isolated polynucleotides encoding a nicotine demethylase polypeptide that participates in the metabolic conversion of nicotine to nornicotine and that hybridize under stringent conditions to the CYP82E10 sequence described herein, or variants or fragments thereof, are encompassed by the present invention. These sequences can be used in the methods of the present invention to inhibit the expression of nicotine demethylase polypeptides that participate in the metabolic conversion of nicotine to nornicotine in plants.

[0078] Using PCR, oligonucleotide primers can be designed for use in PCR reactions to amplify the corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York). Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfe, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfe, eds. (1999) PCR Methods Manual (Academic Press, New York). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, among others.

[0079] Hybridization techniques contemplate the use of all or part of a known polynucleotide as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic mDNA fragments or cmDNA fragments (e.g., genomic or cDNA libraries) from a chosen organism.

[0080] Hybridization may be performed under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” are meant conditions under which a probe will hybridize to its target sequence to a detectably higher degree than other sequences (e.g., at least twice as much as background). Stringent conditions are sequence-dependent and will be different under different circumstances. By controlling the stringency of the hybridization and/or wash conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow for some mismatch in the sequences, such that lower degrees of similarity are detected (heterologous probing). Typically, the probe is less than about 1000 nucleotides in length, ideally less than 500 nucleotides in length.

[0081] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and at a temperature of at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Exemplary low-stringency conditions include hybridization with a buffer solution containing 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and washing in 1X to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50-55°C. Exemplary moderate-stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C and washing in 0.5X to 1X SSC at 55–60°C. Exemplary high-stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C and washing in 0.1X SSC at 60–65°C. Optionally, wash buffers may contain about 0.1% to about 1% SDS. Hybridization duration is generally less than about 24 hours, typically about 4 to about 12 hours. The wash time should be at least long enough to reach equilibrium.

[0082] In a specific embodiment, stringency conditions include hybridization in a solution containing 5X SSC, 0.5% SDS, 5X Denhardt's, 0.45 ug/ul Poly A RNA, 0.45 ug/ul calf thymus DNA, and 50% formamide at 42°C, as well as at least one post-hybridization wash in a solution containing from about 0.01X SSC to about 1X SSC. The duration of hybridization is from about 14 to about 16 hours.

[0083] Specificity is typically a function of post-hybridization washes, with the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6(log M) + 0.41(%GC) - 0.61(% form.) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form. is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1°C for every 1% mismatch; therefore, Tm, hybridization, and/or wash conditions can be adjusted to hybridize sequences of the desired identity. For example, if sequences with >90% identity are sought, Tm can be reduced to 10°C. Generally, stringent conditions are selected to be about 5°C below the thermal melting point (Tm) for the given sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions may utilize hybridization and/or washing 1, 2, 3, or 4°C below the thermal melting point (Tm); Moderately stringent conditions may utilize hybridization and/or washing 6, 7, 8, 9, or 10°C below the thermal melting point (Tm); low-stringency conditions may utilize hybridization and/or washing 11, 12, 13, 14, 15, or 20°C below the thermal melting point (Tm). Using the equation, hybridization, and wash compositions, and the desired Tm, those skilled in the art will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatch results in a Tm of less than 45°C (aqueous solution) or 32°C (formamide solution), it is ideal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to nucleic acid hybridization is found in Thiessen (1993) Laboratory Toxiques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chap. 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chap. 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0084] Hybridization probes can be genomic mDNA fragments, cmDNA fragments, RmDNA fragments, or other oligonucleotides, and can be labeled with a detectable group such as 32P or any other detectable marker. For example, hybridization probes can be created by labeling synthetic oligonucleotides based on the CYP82E10 polynucleotide sequences of the present invention. Methods for preparing hybridization probes and for constructing cDNA and genomic libraries are generally known in the art and are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0085] For example, the CYP82E10 polynucleotide sequences described herein, or one or more portions thereof, can be used as probes capable of specifically hybridizing to the corresponding root-specific nicotine demethylase polynucleotides and messenger RNAs. To achieve specific hybridization under a variety of conditions, these probes include sequences that are unique among CYP82E10 polynucleotide sequences or unique to one of the CYP82E10 polynucleotide sequences, including regions 5' upstream of the coding sequence and regions 3' downstream of the coding sequence, and an intron region (e.g., SEQ ID NO:3), ideally at least about 10 contiguous nucleotides in length, more ideally at least about 20 contiguous nucleotides in length, more ideally at least about 50 contiguous nucleotides in length, more ideally at least about 75 contiguous nucleotides in length, and most ideally at least about 100 contiguous nucleotides in length. These probes can be used to amplify the corresponding CYP82E10 polynucleotides. This technique can be used to isolate additional coding sequences or mutations from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include identification by hybridization of DNA libraries on plates (either plates or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0086] As used herein, with respect to sequence relationships between two or more polynucleotides or polypeptides, the term “reference sequence” is a defined sequence used as a basis for sequence comparison. The reference sequence may be a subset or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence or a complete cDNA or gene sequence.

[0087] As used herein, the term “comparison window” refers to a specified, contiguous segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may contain additions or deletions (e.g., gaps), unlike the reference sequence (which contains no additions or deletions), for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and may optionally be 30, 40, 50, 100, or more. Those skilled in the art understand that to avoid high similarity to a reference sequence due to the inclusion of gaps in the polynucleotide sequence, a gap penalty is typically introduced and subtracted from the number of matches.

[0088] Methods of sequence alignment by comparison are well known in the art. Therefore, the determination of percent sequence identity between any two sequences can be achieved using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the local alignment search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:24442448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified after Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0089] The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. Nucleotide BLAST searches can be performed with the program BLASTN, score = 100, word length = 12, to obtain a nucleotide sequence homologous to a nucleotide sequence encoding the protein of the invention. Protein BLAST searches can be performed with the program BLASTX, score = 50, word length = 3, to obtain an amino acid sequence homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When using BLAST, gapped BLAST, or PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used (See www.ncbi.nlna.nih.gov). Alignment can also be performed manually by inspection.

[0090] In some embodiments, the sequence identity/similarity values provided herein are calculated using the BLASTX (Altschul et al. (1997) supra), Clustal W (Higgins et al. (1994) Nucleic Acids Res. 22:46734680), and GAP (University of Wisconsin Genetic Computing Group software package) algorithms with predefined parameters. The present invention also encompasses the use of any program equivalent thereto for the analysis and comparison of nucleic acid and protein sequences. By "equivalent program" is meant any sequence comparison program that, for any two sequences in question, generates an alignment with identical combinations of nucleotides or amino acid residues and an identical percent sequence identity when compared to the corresponding alignment generated by BLASTX, Clustal W, or GAP.

[0091] For purposes of the foregoing discussion of nucleotide and polypeptide sequence variants encompassed by the present invention, the term "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues of the two sequences that are the same when aligned by maximum match over a specified comparison window. When percent sequence identity is used in reference to proteins, it is recognized that residue positions that are not identical typically differ by conservative amino acid substitutions, wherein the amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not modify the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are identified as having "sequence similarity" or "similarity." Methods for malleability of this adjustment are well known to those skilled in the art. Generally, it is necessary to score a conservative substitution as a partial rather than a complete mismatch, thus increasing the percentage of sequence identity. Thus, for example, when an identical amino acid receives a score of 1 and a non-conservative substitution receives a score of zero, the conservative substitution receives a score between zero and 1. The score for conservative substitutions is calculated, for example, as implemented in the PC/GENE program (Intelligenetics, Mountain View, California).

[0092] The term “percent sequence identity” as used herein means the value determined by comparing two ideally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (e.g., gaps), unlike the reference sequence (which contains no additions or deletions), for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of adjusted positions, dividing the number of adjusted positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity.

[0093] Therefore, CYP82E10 polynucleotide and polypeptide sequences can be identified using the sequences provided herein. These methods consist of obtaining a polynucleotide or polypeptide sequence with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 1, 3, or 4, or a complement or fragment thereof, or a polypeptide sequence of SEQ ID NO: 2 or 5-13. A preferred embodiment includes a polypeptide that corresponds to SEQ ID NO:2 and that has a serine at position 79, 107, or 381 of the CYP82E10 polypeptide, the numbering corresponding to SEQ ID NO:2.

Methods for inhibiting the expression or function of nicotine demethylase

[0094] Methods for reducing the concentration, content, and/or activity of a CYP82E10 polypeptide of the present invention in a Nicotiana plant or plant part, especially in root tissue, are provided. Many methods can be used, alone or in combination, to reduce or eliminate the activity of the CYP82E10 polypeptide of the present invention (SEQ ID NO:2), as well as variants thereof that retain nicotine demethylase activity (e.g., SEQ ID NOs:7, 8, 9, and 10). Furthermore, combinations of methods can be used to reduce or eliminate the activity of two or more different nicotine demethylases, especially the root-specific nicotine demethylase CYP82E10 and one or both of the green leaf nicotine demethylases CYP82E5 and senescence-induced CYP82E4. In a particular embodiment, CYP82E5 is a polypeptide with at least one amino acid mutation in the sequence SEQ ID NO: 26 that negatively affects the conversion to green leaves, and CYP82E4 has the sequence indicated in SEQ ID NO: 14 with at least one amino acid mutation that negatively affects the conversion to senescent leaves.

[0095] According to the present invention, the expression of a nicotine demethylase CYP82E10 of the present invention is inhibited if the protein level of the CYP82E10 polypeptide is statistically lower than the protein level of the same CYP82E10 polypeptide in a plant that has not been genetically modified or mutagenized to inhibit the expression of that CYP82E10 polypeptide, and these plants were grown and harvested using the same protocols. In particular embodiments of the invention, the protein level of the CYP82E10 polypeptide in a plant modified according to the invention is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the protein level of the same CYP82E10 polypeptide in a plant that is not mutant or that has not been genetically modified to inhibit the expression of that CYP82E10 polypeptide and that has been grown and harvested using the same protocols. The expression level of the CYP82E10 polypeptide can be measured directly, for example, by performing an assay to measure the level of the CYP82E10 transcript or CYP82E10 polypeptide expressed in the tobacco plant or plant part, or indirectly, for example, by measuring the conversion of nicotine to nornicotine in the tobacco plant or plant part. Methods for monitoring the expression level of a protein are known in the art, including, but not limited to, Northern blot analysis and RNA differentiation assays. Methods for determining the activity of a CYP82E10 target polypeptide in the conversion of nicotine to nornicotine are known in the art and described later in this document, including, but not limited to, alkaloid analysis using gas chromatography.

[0096] The present invention provides methods for reducing the level of nornicotine, or for reducing the rate of conversion of nicotine to nornicotine, in a tobacco plant or plant part. The methods consist of introducing into the genome of a tobacco plant a mutation within at least one allele of each of at least three nicotine demethylase genes, wherein the mutation causes reduced expression of the nicotine demethylase gene, and wherein the first of these nicotine demethylase genes encodes a root-specific nicotine demethylase that participates in the metabolic conversion of nicotine to nornicotine in a tobacco plant or plant part. In some embodiments, the root-specific nicotine demethylase is CYP82E10 or a variant thereof. In other embodiments, these methods consist of introducing into the genome of a tobacco plant a mutation within at least one allele of the nicotine demethylase gene encoding CYP82E10 or a variant thereof, and a mutation within at least one allele of a nicotine demethylase encoding CYP82E4 or a variant thereof, and/or a nicotine demethylase encoding CYP82E5 or a variant thereof.

[0097] A number of approaches have been used to combine mutations in a plant, including sexual crossing. A plant possessing a favorable mutation in a CYP82E10 gene that inhibits nicotine demethylase activity in roots can be crossed with a plant possessing a favorable mutation in the CYP82E4v2 gene that inhibits nicotine demethylase activity in senescent leaves, or it can be crossed with a plant possessing a favorable mutation in the CYP83E5v2 gene that inhibits nicotine demethylase activity in green leaves to produce a plant with reduced conversion of nicotine to nornicotine. In preferred embodiments, the crosses are made to introduce a favorable mutation within a CYP82E10, CYP82E4v2, and CYP82E5v2 gene from the same plant. Thus, a plant with a favorable mutation in the CYP82E10 gene that inhibits nicotine demethylase activity in roots is crossed with a plant with a favorable mutation in the CYP82E4v2 gene that inhibits nicotine demethylase activity in senescent leaves and a favorable mutation in the CYP83E5v2 gene that inhibits nicotine demethylase activity in green leaves. Alternatively, a plant with a favorable mutation in the CYP82E4v2 gene that inhibits nicotine demethylase activity in senescent leaves is crossed with a plant with a favorable mutation in the CYP82E10 gene that inhibits nicotine demethylase activity in roots and a favorable mutation in the CYP83E5v2 gene that inhibits nicotine demethylase activity in green leaves. In another configuration, a plant carrying a favorable mutation in the CYP82E5v2 gene that inhibits nicotine demethylase activity in green leaves is crossed with a plant carrying a favorable mutation in the CYP82E10 gene that inhibits nicotine demethylase activity in roots and a favorable mutation in the CYP83E4v2 gene that inhibits nicotine demethylase activity in senescent leaves. By introducing a favorable mutation into each of these nicotine demethylase genes, it is possible to produce a plant with reduced rates of nicotine-to-nornicotine conversion, with conversion levels below approximately 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%.

[0098] In an even more preferred embodiment, a plant having one or more favorable mutations causing a modification of the CYP82E10 polypeptide at position 79, 107, 381, or 419 (the numbering being in accordance with SEQ ID NO:2) can be crossed with a plant having one or more favorable mutations causing a modification of the CYP82E4 polypeptide at position 329, 364, 376, 381, or 458 and/or having one or more favorable mutations causing a modification of the CYP82E5 polypeptide at position 422 or 449 to produce a plant having conversion levels of less than 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%. An especially preferred nicotine to nornicotine conversion level can be between 0.05% and 0.4%, between 0.1% and 0.2%, or between 0.1% and 0.2%. to 0.6%, between 0.1% and 0.3%, between 0.1% and 0.5%, between 0.1% and 0.4%, between 0.1% and 0.7%, between 0.1% and 1.0%, between 0.1% and 1.1%, between 0.1% and 1.2%, between 0.1% and 1.3%, between 0.1% and 1.4%, or between 0.1% and 1.5%. Any mutation of a polynucleotide of the present invention that causes a truncation of the CYP82E10, CYP83E4, or CYP83E5 polypeptide prior to a hemobinding pattern will inhibit the enzyme and can be used in a cross described above. The domains of cytochrome P450 proteins are known in the art. See, for example, Xu et al. (2007) Physiologia Plantarum 129:307-319, incorporated herein by reference. By crossing plants that have a non-functional or inhibited CYP82E10 gene with plants that have a non-functional or inhibited CYP82E4v2 gene, a non-functional or inhibited CYP82E5v2 gene, or non-functional or inhibited CYP82E4v2 and CYP82E5v2 genes, nornicotine levels can be reduced in a tobacco plant.

[0099] The activity of a nicotine demethylase polypeptide CYP82E10, CYP82E4, or CYP82E5 in converting nicotine to nornicotine in a tobacco plant or plant part is inhibited according to the present invention if this conversion activity is statistically lower than the conversion activity of the same nicotine demethylase polypeptide in a tobacco plant or plant part that has not been genetically modified to inhibit the conversion activity of this nicotine demethylase polypeptide and that has been grown and harvested using the same protocols. In particular embodiments, the activity of a nicotine demethylase polypeptide in transforming nicotine into nornicotine in a tobacco plant or plant part modified according to the invention is inhibited if the activity is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, or less than 1% of the conversion activity of the same nicotine demethylase polypeptide in a tobacco plant that has not been genetically modified to inhibit the expression of that nicotine demethylase polypeptide and that has been grown and harvested using the same protocols. The activity of a nicotine demethylase polypeptide in converting nicotine to nornicotine in a tobacco plant or part of a plant is eliminated according to the invention when it is not detectable by the assay methods described elsewhere in this document. Methods for determining the activity of a nicotine demethylase polypeptide in converting nicotine to nornicotine in a tobacco plant using gas chromatography are described in the examples presented below.

[0100] In some embodiments, a favorable mutation is introduced into a tobacco plant or plant part using a mutagenesis approach, wherein the introduced mutation is selected from methods known to those skilled in the art, including, but not limited to, Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. A plant or plant part altered or modified by the above embodiments is grown under plant-forming conditions for a period sufficient to modulate the concentration and/or activity of the polypeptides of the present invention in the plant. Plant-forming conditions are known in the art and are briefly explained in several sections herein.

[0101] A modified tobacco plant containing a favorable mutation in a nicotine demethylase described herein has a reduced level of nicotine to nornicotine conversion. In particular embodiments, the conversion of nicotine to nornicotine in a tobacco plant or plant part modified according to the invention is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, or less than 1% of the conversion in a tobacco plant that has not been genetically modified to inhibit expression of such nicotine demethylase polypeptide and that has been grown and harvested using the same protocols. In some embodiments, the modified tobacco plant is a converting tobacco plant. In other embodiments, the modified tobacco plant is a non-converting tobacco plant. In some configurations, the modified tobacco plant has a conversion rate lower than the rate observed in non-converting commercial tobacco plants.

[0102] According to the present invention, changes in the levels, proportions, activities or distributions of the CYP82E10 polypeptides of the present invention or, further, changes in the phenotype of the tobacco plant or plant part, especially reduced accumulation of nornicotine and its carcinogenic metabolite, NNN, can be measured by comparing an experimental plant or plant part with a control plant or plant part, provided that the experimental plant or plant part and the control plant or plant part have been grown and/or harvested using the same protocols. As used herein, an experimental plant or plant part is a plant in which genetic alteration, for example by mutagenesis, has been affected by the nicotine demethylase polypeptide of interest or, further, a tobacco plant or plant part derived from a tobacco plant or plant part altered in this way and that contains the alteration. A control plant or plant part provides a reference point for measuring changes in the phenotype of the experimental plant or plant part. Measurement of phenotypic changes in a plant or plant part can be made at any time, including during development, senescence, or after the plant has cured. In other configurations, phenotypic changes can be measured in plants grown under any conditions, including plants grown in a growth chamber, greenhouse, or field. In one configuration, phenotypic changes can be measured by determining the rate of conversion of nicotine to nornicotine. In a preferred configuration, conversion can be measured by dividing the percentage of nornicotine (as a percentage of total tissue weight) by the sum of the percentages of nicotine and nornicotine (as percentages of total tissue weight) and multiplying by 100.

[0103] According to the present invention, a control plant or plant part may consist of a wild-type tobacco plant or plant part, e.g., of the same genotype as the starting material for the genetic alteration that gave rise to the experimental plant or plant part. A control plant or plant part may also consist of a tobacco plant or plant part of the same genotype as the starting material but that has been transformed with a null construct (e.g., with a construct that produces no known effects on the trait of interest, such as a construct containing a selectable marker gene). In all such cases, the experimental plant or plant part and the control plant or plant part are grown and harvested using the same protocols.

[0104] In some embodiments, the activity of a nicotine demethylase polypeptide of the present invention can be reduced or eliminated by disrupting the gene encoding the nicotine demethylase polypeptide. The invention encompasses mutagenized plants that carry mutations in nicotine demethylase genes, the mutations of which reduce the expression of the nicotine demethylase gene or inhibit the activity of an encoded nicotine demethylase polypeptide of the present invention.

[0105] In other embodiments, the activity of a nicotine demethylase polypeptide of the present invention is reduced or eliminated by disrupting the gene encoding the nicotine demethylase polypeptide. The gene encoding the nicotine demethylase polypeptide can be disrupted by any method known in the art, for example, by transposon tagging or by mutagenizing plants using random or site-directed mutagenesis and selecting for plants that possess reduced nicotine demethylase activity or mutations in CYP82E10, alone or in combination with mutations in CYP82E4 or CYP82E5.

[0106] Transposon tagging can be used to reduce or eliminate the activity of one or more CYP82E10 nicotine demethylase polypeptides of the present invention. Transposon tagging consists of inserting a transposon into an endogenous nicotine demethylase gene to reduce or eliminate the expression of the nicotine demethylase polypeptide.

[0107] Methods for transposon-tagging specific genes in plants are well known in the art. See, for example, Maes et al. (1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti (1999) FEMS Micerobiol. Lett. 179:53-59; Meissner et al. (2000) Plant J. 22:265-274; Phogat et al. (2000) J. Biosci. 25:57-63; Walbot (2000) Curr. Opin. Plant Biol. 2:103-107; Gai et al. (2000) Nucleic Acids Res. 28:94-9b; Fitzmaurice et al. (1999) Genetics 153:1919-1928).

[0108] Additional methods for reducing or eliminating the expression of endogenous genes in plants are also known in the art and can be applied similarly to the instant invention. These methods include other forms of mutagenesis using mutagenic or carcinogenic compounds, including ethyl methanesulfonate-induced mutagenesis, knockout mutagenesis, and fast neutron knockout mutagenesis used in a reverse genetic direction (with PCR) to identify plant lines in which the endogenous gene has been eliminated. As examples of these methods, see Ohshima et al. (1998) Virology 213:472-481; Okubara et al. (1994) Genetics 137:867-874; and Quesada et al. (2000) Genetics 154:421-4315; each of which is incorporated herein by reference. Furthermore, a rapid and automatable method for the identification of chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denatured HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention. See McCallum et al (2000) Nat. Biotechnol. 18:455-457, incorporated herein by reference.

[0109] Mutations that impact gene expression or interfere with the function of the encoded nicotine demethylase protein can be determined using methods that are well known in the art. Mutations inserted into gene exons typically result in null mutants. Mutations at conserved residues can be especially effective in inhibiting the metabolic function of the encoded protein. Conserved residues of plant nicotine demethylase polypeptides suitable for mutagenesis to eliminate the activity of a nicotine demethylase polypeptide in transforming nicotine to nornicotine in a tobacco plant or plant part have been described. See Figure 1A-C of U.S. Patent Application Publication No. 2009/0205072 A1, incorporated herein by reference in its entirety, where residues that differ from other P450 polypeptides are shaded gray. Conserved residues are those not shaded gray at each position. These mutants can be isolated according to known procedures.

[0110] In another embodiment of this invention, dominant mutants can be used to trigger RNA silencing due to genetic inversion and recombination of a duplicated genetic locus. See, e.g., Kusaba et al. (2003) Plant Cell 15:1455-1467.

[0111] In another embodiment of the invention, the compositions of the invention can be used in identification methods to identify non-converter plants that can be used in breeding programs. Thus, the nucleotide sequences of the invention can be used to identify wild-type germplasm for non-converter plants that have a stable mutation in the CYP82E10 gene identified herein. These non-converter plants identified by the methods of the invention can be used to develop breeding lines.

[0112] In addition to the nucleotide sequences encoding the CYP82E10 polypeptides described herein, compositions of the invention include an intron sequence within the CYP82E10 gene sequence that can be used in identification methods. Although not restricted by any mechanism of action, the CYP82E10 genes may represent the only members of the cytochrome P450 family that participate in the metabolic conversion of nicotine to nornicotine in tobacco roots. For certain applications, it would be useful to have ways to diagnostically differentiate this specific member of the cytochrome P450 gene family from the rest of the closely related sequences within that family. For example, it is possible that within naturally occurring tobacco germplasm (or in mutagenized populations) there are accessions in which this gene is naturally dysfunctional and may therefore be valuable as a permanently non-converting resource. These dysfunctional sequences may include those encoding the polypeptides indicated in SEQ ID NO: 11, 12, or 13. A method to specifically test for these genotypes (e.g., deletion mutants, rearrangements, among others) could be a powerful tool. The present invention includes primers designed to specifically amplify exon 1 and exon 2 of CYP82E10, with one of the two primer pairs corresponding to the intron between the exons. Examples of primers useful for amplifying CYP82E10 exons include SEQ ID NO: 35 with SEQ ID NO: 36 and SEQ ID NO: 37 with SEQ ID NO: 38. The same primers can be used for sequence analysis of the products.

[0113] Since the intron regions of genes are typically less conserved than the exons, it can be anticipated that the use of an intron-specific probe would allow better distinction between the gene(s) corresponding to the CYP82E10 gene and the other members of the CYP82E family. The use of a CYP82E10 intron-specific probe, and/or the PCR primers used to generate products, provide powerful tools in assays to determine whether any naturally occurring or mutagenized tobacco plant has deletions or rearrangements that may render the gene inactive. Such a plant can then be used in breeding programs to create tobacco lines that are incapable of conversion.

Tobacco plants and parts of plants and products with reduced nornicotine and NNN content

[0114] The CYP82E10 polynucleotides of the invention, as well as variants and fragments thereof, can be used in the methods of the present invention to inhibit the expression or function of CYP82E10 nicotine demethylases that participate in the metabolic conversion of nicotine to nornicotine in a plant. In this way, favorable mutations can be introduced into a CYP82E10 gene of interest. The methods of the invention do not depend on a particular method for introducing a favorable mutation into the CYP82E10 nicotine demethylase gene.

[0115] The compositions and methods of the invention can be used to reduce the nornicotine content, especially in the leaves and stems, of any plant of the genus Nicotiana, including, but not limited to, the following species: acuminata, affinis, alata, attenuate, bigelovii, clevelandii, excelsior, forgetiana, glauca, glutinosa, langsdorffii, longiflora, obtusifolia, palmeri, paniculata, plumbaginifolia, qudrivalvis, repanda, rustica, suaveolens, sylvestris, tabacum, tomentosa, trigonophylla and x sanderae. The present invention may also be practiced using any variety of a plant of the genus Nicotiana, including, but not limited to: Nicotiana acuminata multiflora, Nicotiana alata grandiflora, Nicotiana bigelovii quadrivalvis, Nicotiana bigelovii wallacei, Nicotiana obtusifolia obtusifolia, Nicotiana obtusifolia plameri, Nicotiana quadrivalvis bigelovii, Nicotiana quadrivalvis quadrivalvis, Nicotiana quadrivalvis wallacei, and Nicotiana trigonophylla palmeri, as well as varieties commonly known as greenhouse or bright varieties, Burley varieties, dark varieties, and Oriental/Turkish varieties. In some embodiments, a tobacco plant of interest is a Burley, Virginia, greenhouse-cured, air-cured, fire-cured, Oriental, or dark tobacco plant.

[0116] The tobacco plants and varieties described herein are suitable for traditional planting and harvesting techniques, such as cultivation in rich or unfertilized soils, bagging of the leaves or not bagging, as well as covering or not covering. The harvested leaves and stems can be used in any traditional tobacco product, including, but not limited to, pipe, cigar, and cigarette tobacco, as well as chewing tobacco in any form, including leaf tobacco, ground tobacco, or cut tobacco.

[0117] Therefore, the present invention provides a tobacco plant or plant part that contains a mutation in a gene encoding the nicotine demethylase CYP82E10, which mutation causes reduced expression or function of these nicotine demethylases CYP82E10, in addition to a reduced amount of nornicotine and N'-nitrosonornicotine. As used herein, the term "a reduced amount" or "a reduced level" refers to an amount of nornicotine and/or N'-nitrosonornicotine in a plant or plant parts of the present invention, or a tobacco product thereof, that is lower than that which would be found in a plant of the genus Nicotiana or in plant parts or in tobacco products of the same tobacco variety, processed (i.e., grown and harvested) in the same manner, and that has not been genetically modified to have reduced nornicotine and/or N'-nitrosonornicotine. The amount of nornicotine can be reduced from about 10% to more than about 90%, including more than about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, and about 80%. The conversion of nicotine to nornicotine can be less than 0.3%, less than 0.5%, less than 0.7%, between 0.1% and 0.5%, between 0.1% and 0.4%, between 0.1% and 0.7%, or between 0.1% and 1.0% in plants, plant parts, and products of the present invention, more specifically in plants and plant parts with mutations in CYP82E10, CYP82E4v2, and CYP825v2.

[0118] The term “tobacco products” as used herein includes, but is not limited to, smoking materials (e.g., any cigarette, including a thin cigar, a non-ventilated or recess-ventilated filter cigarette, a cigar, a pipe), smokeless products (e.g., snuff, chewing tobacco, biodegradable inserts (e.g., gum, lozenges, dissolving strips)). See, for example, U.S. Patent 2005/0019448, incorporated herein by reference. The present invention also encompasses a series of blended tobacco products that can be made by combining conventional tobacco with different amounts of the low nornicotine and/or N’-nitrosonornicotine tobaccos described herein. In other embodiments, the plant or plant part of the genus Nicotiana as described above is cured tobacco.

[0119] In some embodiments of the present invention, the tobacco product reduces the carcinogenic potential of tobacco smoke that is directly inhaled through the consumption of a tobacco product such as cigars, cigarettes, or pipes, or inhaled as sidestream smoke (e.g., by an individual inhaling tobacco smoke generated by another individual consuming a tobacco product such as cigars, cigarettes, or pipes). The cured tobacco described herein can be used to prepare a tobacco product, especially one that undergoes chemical modifications due to heat, including a reduced amount of nornicotine and/or N'-nitrosonornicotine in the smoke stream that is directly inhaled or inhaled as sidestream smoke. Likewise, the tobacco products of the invention can be useful in the preparation of smokeless tobacco products, such as chewing tobacco, snuff, among others.

[0120] Tobacco products derived from the tobacco plants of the present invention therefore find use in methods of reducing the carcinogenic potential of such tobacco products, as well as reducing human exposure to the carcinogenic nitrosamine NNN, especially in the case of individuals who are users of such tobacco products. The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

[0121] The citations mentioned in the following discussion are provided at the end of the experimental section.

Background

[0122] The knowledge that CYP82E4v2 represents the nicotine demethylase locus responsible for the high nornicotine accumulation observed in converter plants (Siminszky et al., 2005) opened the door for non-transgenic and transgenic approaches that seek to overcome the conversion problem and lower the nornicotine content of senescent and cured leaves. Specifically, it has been possible for researchers to generate tobacco populations with prior exposure to chemical mutagens and to select individuals with non-functional alleles at the CYP82E4v2 locus. In fact, three independent groups have already generated non-converter tobacco lines based on this strategy (Dewey et al., 2007; Xu et al., 2007b; Julio et al., 2008).

[0123] As indicated previously, a tobacco plant designated 775 was identified from an EMS-mutagenized population of the Burley line DH98-325-6 and shown to possess a knock-out mutation within the CYP82E4v2 gene (Dewey et al., 2007). In the summer of 2008, plants homozygous for the 775 mutation were grown at the Upper Coastal Plains Research Station in Rocky Mount, North Carolina, USA, and air-cured according to standard industry procedures. Alkaloid analysis of these materials was performed using the LC protocol described by Jack et al. (2007). As illustrated in Table 1, plants possessing the 775 mutation had an average nicotine-to-nornicotine conversion of 2.6%. In contrast, a conversion greater than 60% was observed in the parent line DH98-325-6, a strong converter genotype. Nearly identical results were obtained by Julio et al. (2008), who reported conversion percentages ranging from 2.82 to 3.37 in plants homozygous for a cyp82e4v2 knock-out mutant within the strong-converting Burley genotype BB16NN (the mother's conversion rates ranged from 68% to 98%). Therefore, debilitating mutations in CYP82E4v2 alone appear to be effective in eliminating the problems arising from unstable genetic phenomena associated with the generation of converting plants. Table 1. Alkaloid profiles for experimental materials evaluated in a 2008 field experiment. Percentage values represent an average. aThe number in parentheses indicates the total number of plants analyzed.bNumbering relative to the start codon of the cDNA sequence.cThe percentages were calculated based on the dry weight of the tobacco.dThe percentage of nicotine conversion is equal to [% nornicotine/ (% nornicotine + % nicotine)] X 100.

[0124] Although the use of tobacco plants harboring the 775 mutation, or comparable mutations, in CYP82E4v2 may be an effective way to eliminate the introduction of converter plants into tobacco populations, a low but significant amount of nornicotine remains in these plants. Because conversion rates of nicotine to nornicotine as low as 0.45% were observed in transgenic plants expressing an RNAi-based construct directed against CYP82E4v2 (Lewis et al., 2008), it was evident that at least one other gene with high DNA sequence homology to CYP82E4v2 must be responsible for the majority of the nornicotine synthesis observed in both non-converter plants and converter plants harboring an inactive CYP82E4v2 gene. This possibility was reinforced by the discovery of CYP82E5v2, a gene that shares 92.7% DNA sequence identity with CYP82E4v2, which has also been shown to encode the functional nicotine demethylase enzyme (Dewey et al., 2007; Gavilano and Siminszky, 2007). The nicotine demethylase gene CYP82E5v2 is expressed at low levels in green tobacco leaves of similar converting and non-converting plants, unlike CYP82E4v2, which is expressed at very high levels but only in the leaves of converting plants during senescence and outdoor curing.

[0125] As outlined in Dewey et al. (2007), identification of an EMS-mutagenized tobacco population DH98-325-6 resulted in the identification of one individual (plant 1013) that possesses a knock-out mutation in CYP82E5v2. To determine the impact of the non-functional cyp82e5v2 allele on nornicotine accumulation, crosses were made that combined the mutations from plants 775 and 1013. Molecular genotyping of several F2 individuals derived from the F1 progeny of the initial cross resulted in the identification of nine individuals that were homozygous for both mutations (e4e4/e5e5). These nine plants were also included in the 2008 field trial. Regardless of the fact that CYP82E5v2 has been shown to encode a functional nicotine demethylase enzyme (Dewey et al., 2007; Gavilano and Siminszky, 2007), combining the dysfunctional cyp82e5v2 mutation with the cyp82e4v2 knockout mutation had remarkably little impact on leaf nornicotine levels. As illustrated in Table 1, plants homozygous for a double mutation (e4e4/e5e5) had an average nicotine conversion of 2.3%, compared with an average conversion of 2.6% in those plants harboring only the cyp82e4v2 (e4e4) mutation. A modest difference in average conversion between the two genotypes was not statistically significant (P = 0.118). However, one of the RNAi-silenced CYP82E4v2 transgenic lines included in this study had an average conversion of 0.7%, a significantly lower (P < 0.001) than that obtained in either the e4e4 or e4e4/e5e5 genotypes. Therefore, another gene with high homology to CYP82E4v2 must exist within the tobacco genome that contributes to nornicotine production in the plant.

Example 1: Isolation and characterization of a nicotine demethylase gene cyp82e10

[0126] To identify other genes in the tobacco genome that have the potential to encode nicotine demethylase enzymes, homology searches using the BLASTN and BLASTX algorithms (Altschul et al., 1990, 1997) were directed against the N. tabacum expressed sequence tag (EST) databases in GenBank, using the DNA and protein sequences of CYP82E4v2 as the respective query sequences. In addition to identifying sequences corresponding to previously characterized members of the CYP82E superfamily (such as CYP82E2, CYP82E3, and CYP82E5v2), seven ESTs were discovered that did not align perfectly with any previously characterized member of this family. Interestingly, all seven ESTs originated from both the cDNA libraries and root-specific cDNA libraries made from mixed tissues that included roots. This observation suggested that the novel CYP82E gene is specifically expressed in root tissue, a property that could explain why this particular member of the CYP82E P450 superfamily has previously eluded detection, given that previous efforts focused on characterizing CYP82E genes expressed in leaf tissue. Because no single EST sequence was long enough to cover the entire coding region of this novel gene, PCR primers were designed to allow amplification of the entire cDNA sequence from a first-strand cDNA generated from RNA isolated from tobacco root tissue. Furthermore, the primers were used to amplify the corresponding genomic region of the gene, which includes a large, central intron. This novel CYP82E cDNA shares 92.4% nucleotide identity with tobacco cDNA CYP82E4v2 and 91.1% predicted identity at the amino acid level. Following guidelines for P450 gene nomenclature, this novel gene was named CYP82E10. Of all characterized members of the CYP82E superfamily, CYP82E10 shows the highest sequence similarity to CYP82E5v2, sharing 96.5% nucleotide identity at the cDNA level and 95.7% predicted amino acid sequence identity. The CYP82E10 DNA sequence and its predicted protein sequence are illustrated in Figure 1.

[0127] Although the cDNAs of the various CYP82E family members tend to be highly conserved, the genomic versions of these genes show much greater sequence diversity. This is primarily due to the considerable sequence divergence observed within the large, central intron. An alignment of the CYP82E4v2, CYP82E5v2, and CYP82E10 genomic sequences is illustrated in Figure 2. As calculated using the EMBOSS Pairwise Alignment algorithm (www.ebi.ac.uk/Tools/emboss/align/index.html), the CYP82E4v2 and CYP82E10 genes share 78.3% nucleotide identity, with the CYP82E10 gene being 84.9% identical to the CYP82E5v2 gene as they both exist within the tobacco genome (the CYP82E4v2 and CYP82E5v2 genomic sequences share 75% identity).

[0128] As explained in several publications, most of the CYP82E superfamily genes that are found in the tobacco genome do not encode functional nicotine demethylase enzymes (Siminszky et al., 2005; Chakrabarti et al., 2007; Dewey et al., 2007; Gavilano et al., 2007; Xu et al., 2007a). Therefore, sequence homology alone is not a sufficiently accurate indicator of gene function for the CYP82E family. Instead, expression analysis in both transgenic plants (Siminszky et al., 2005) and yeast (Gavilano and Siminszky, 2007; Xu et al., 2007a) has become the conventional way to determine whether individual members of this gene family encode nicotine demethylase activity.

[0129] To determine whether CYP82E10 functions as a nicotine demethylase gene, its cDNA was cloned into the yeast expression vector pYeDP60 and transformed into yeast strain W(R). Strain W(R) is the yeast cell line engineered to overexpress yeast NADPH-dependent P450 reductase, an enzyme that functions as the direct electron donor to P450s; this system greatly enhances the detection of off-target P450 enzyme activities expressed in yeast (Pompon et al., 1995). Nicotine demethylase assays were performed by incubating yeast microsomal membrane preparations with [14C]-nicotine and resolving the products by thin layer chromatography as described in Siminszky et al. (2005).

[0130] As illustrated in Figure 3, no nicotine demethylase activity could be detected using yeast microsomes from the W(R) strain that only expressed the pYeDP60 vector. However, very robust nicotine demethylase activity could be measured from yeast-derived microsomal cells expressing the CYP82E10 cDNA. By measuring CYP82E10 enzyme activity over a wide range of [14C]- nicotine concentrations, a substrate saturation curve was established and an apparent Km of 3.9 μM nicotine was calculated using the microsomal assay. This kinetic parameter for CYP82E10 is very similar to the Kms reported for CYP82E4v2 and CYP82E5v2 enzymes when the similarity was expressed in yeast (Gavilano et al., 2007; Gavilano and Siminszky, 2007; Xuet al., 2007a).

Example 2: Identification of plants that have mutant alleles of CYP82E10

[0131] To accurately assess the specific contribution of CYP82E10 to the total nornicotine content of the tobacco plant, it was necessary to: (1) identify a tobacco plant with a knock-out mutation within this gene; and (2) combine this mutation with the cyp82e4v2 and cyp82e5v2 mutations originating from plants 775 and 1013, respectively. To identify potentially debilitating mutations in CYP82E10, the EMS-mutagenized population DH98-325-6 was identified by high-throughput DNA sequence analysis using primers that specifically amplify portions of CYP82E10 (without simultaneously amplifying other members of the CYP82E superfamily). To specifically amplify exon 1 of CYP82E10, the following PCR primers were used: 5′-GTGATAGTTTGATTCCCAAGTGC-3′ (forward) and 5′-CTCCCAAAGTTAGATTAGTCCG-3′ (reverse); specific amplification of exon 2 was achieved using primers 5′-AGGTCGCGCTGATTCTTG-3′ (forward) and 5′-AGATGAATACCCATCTATCTAGGAGT-3′ (reverse). To ensure maximum specificity, the reverse primer for exon 1 and the forward primer for exon 2 correspond to sequences within the CYP82E10 intron (Fig. 1). PCR amplification and sequence analysis of the mutagenized plants was performed using a 96-well format as described in Dewey et al. (2007).

[0132] High-throughput sequence analysis of over 1,200 individuals from the mutagenized tobacco population resulted in the identification of 15 individuals with mutations in CYP82E10. The most notable of these mutations are indicated in Table 2. The nucleotide and amino acid residues mutated in these plants are also illustrated in Figure 1. Although no truncating mutations were observed among these individuals, in several cases mutations were identified that changed an amino acid residue within a highly conserved region of the enzyme. To determine the effects of a particular mutation on CYP82E10 enzyme activity, site-directed mutagenesis was used to introduce the specific mutations corresponding to seven of the nine mutations indicated in Table 2 into the CYP82E10 cDNA within the yeast expression vector pYeDP60. Microsomal preparations from yeast strains expressing each of the seven CYP82E10 variants were tested in vitro for nicotine demethylase activity using both unsaturated (2.45 μM) and saturated (50 μM) concentrations of [14C]-nicotine. The results of the yeast expression assays revealed that the mutations found in plants 693, 817, and 1035 did not alter enzyme activity, whereas the mutations found in plants 1041, 1512, and 2476 caused complete enzyme inactivation. The mutation observed in plant 1442 resulted in a 75% reduction in activity compared to the wild-type CYP82E10 enzyme.

[0133] Thin layer chromatography data for the in vitro yeast expression assays corresponding to the plant 1041 mutation are shown in Figure 3. This specific mutation was selected for further investigation. To provide further confirmation that a Pro to Ser substitution at amino acid position 381 that defines the plant 1041 mutation is incompatible with nicotine demethylation function, this same mutation was introduced into a CYP82E4v2 cDNA that had been similarly cloned into the pYeDP60 vector. The results of these yeast assays are shown in Table 3. Whether introduced into the CYP82E10 or CYP82E4v2 enzyme, the Ser to Pro substitution at position 381 causes complete ablation of nicotine demethylase activity in this assay. Interestingly, although the activities of wild-type CYP82E10 and CYP82E4v2 enzymes were comparable at the non-saturating concentration of [14C]-nicotine (2.45 μM), at a substrate level of 25 μM, the rate of [14C]-nornicotine synthesis was almost threefold higher in microsomal preparations harboring the CYP82E10 enzyme than in preparations containing CYP82E4v2. Table 2. EMS-treated DH98-325-6 lines with mutations in the CYP82E10 gene aReferring to the start codon of the CYP82E10 cDNA sequence.bRelative to the wild-type enzyme expressed in yeast.Table 3. Nicotine demethylase activity of CYP82E4v2 and CYP82E10 enzymes harboring the 1041 (Pro381Ser) mutation. aCounts per minute of microsomal [14C]-nornicotine protein/mg.bStandard deviation of two technical replicates.

[0134] The nicotine demethylase activities of wild-type and 1041 mutant yeast cells expressing CYP82E10 were also tested in vivo. Yeast cultures were shaken overnight in the presence of 55 μM [14C]-nicotine, extracted with methanol, and analyzed by thin layer chromatography. [14C]-nornicotine could be detected in the extracts of yeast expressing wild-type CYP82E10, but not the gene in its 1041 mutant version (data not shown). Cumulatively, the yeast expression assays strongly suggest that CYP82E10 function is completely abolished by the introduction of the 1041 mutation.

Example 3: Combination of mutant alleles of cyp82e10, cyp82e4v2, and cyp82e5v2

[0135] Since the original 1041 mutation is in a genetic background (DH98-325-6) that contains both a strong CYP82E4v2 converter allele and the wild-type CYP82E5v2 gene, the only way to accurately assess the specific contribution of CYP82E10 to the total nornicotine content of the plant is to introduce the 1041 mutation into tobacco plants that also carry CYP82E4v2 and CYP82E5v2 knock-out mutations. To achieve this goal, plants heterozygous for the 1041 mutation (e10E10) were crossed with plants heterozygous for the above-described 775 and 1013 mutations (e4E4/e5E5). The latter plants represent progeny of the cross 775/1013//TN90/3/TN90/4/TN90. F1 plants heterozygous for the three nicotine demethylase mutations (e4E4/e5E5/e10E10) were identified by molecular genotyping and were allowed to self-pollinate. Molecular genotyping was also used to identify over 400 F2 progeny and subsequently group them into the following genotypic classes: E4E4/E5E5/e10e10 (3 plants total); e4e4/E5E5/e10e10 (4 plants total); E4E4/e5e5/e10e10 (5 plants total) and ee4e4/e5e5/e10e10 (5 plants total).

[0136] All of the above-described plants were transplanted and grown in the field at the Upper Coastal Plains Research Station in Rocky Mount, North Carolina, USA, in the summer of 2009. Also included in this research were two of the genotypes tested in the 2008 field trial shown in Table 1. Specifically, ten DH98-325-6 plants homozygous only for the cyp82e4v2 mutation (e4e4/E5E5/E10E10) and eleven DH98-325-6 plants possessing the double homozygous genotype e4e4/e5e5/E10E10 were included as comparisons. As controls, randomly selected individual plants from a commercial "low-conversion" seed lot (TN90LC), wild-type DH98-325-6 individuals, and plants from one of the best transgenic lines with CYP82E4v2 RNAi suppression were also included in the research. Once the plants reached an average height of approximately 30 cm (35 days after transplanting), leaves with similar stem positions were harvested, treated with ethephon, and cured outdoors according to the protocol established by Jack et al. (2007). The alkaloid content of the cured leaf materials was determined by gas chromatography as described in the same protocol.

[0137] Table 4 and Figure 4 indicate the results of the alkaloid analyses for the 2009 field trial. Consistent with previous observations, the cyp82e4v2 knock-out mutation alone negates the strong converter phenotype of the DH98-325-6 line and also confers a substantially lower nornicotine accumulation phenotype than plants from commercial seed TN90LC (2.2% versus 7.1% conversion, respectively). As observed in the 2008 field trial (Table 1), combining the cyp82e5v2 mutation with cyp82e4v2 did not produce further reductions in nornicotine content. Indeed, the average nicotine conversion for e4e4/E5E5/E10E10 plants was actually lower than that observed for e4e4/e5e5/E10E10 individuals (2.2% versus 2.3%), although this subtle difference was not statistically significant. As expected, the cyp82e10 mutation did not impact the high nornicotine levels conferred by an active CYP82E4v2 gene, either alone (E4E4/E5E5/e10e10 genotypes), or in combination with a cyp82e5v2 mutant allele (E4E4/e5e5/e10e10 genotypes) (Fig. 4A). Similar to the cyp82e4v2 and cyp82e5v2 double mutant results (Tables 1 and 4), introducing cyp82e10 into a cyp82e4v2 background was not effective in reducing nornicotine levels below those that could be achieved by the cyp82e4v2 mutation alone (Fig. 4B). The e4e4/E5E5/e10e10 genotypes had an average conversion of 1.85%, which was not significantly different from the average conversion levels of 2.2% observed for e4e4/E5E5/E10E10 individuals (P = 0.235). Table 4. Alkaloid profiles for experimental materials evaluated in a 2009 field trial. Measurements collected from leaves harvested 35 days after transplanting. Percentage values represent an average. aThe number in parentheses indicates the total number of plants analyzed. bNumbering relative to the start codon of the cDNA sequence. cThe percentages were calculated based on the dry weight of the tobacco. dThe percentage of nicotine conversion is equal to [% nornicotine/(% nornicotine + % nicotine)] X 100.

[0138] Although the cyp82e5v2 and cyp82e10 mutations did not serve to significantly reduce the nornicotine content of cyp82e4v2 plants when combined individually, pyramiding the three nicotine demethylase mutations produced a very notable effect. The conversion of nicotine to nornicotine in triple mutant plants (e4e4/e5e5/e10e10) averaged only 0.55%, a percentage virtually identical to the 0.54% observed in the RNAi-suppressed transgenic lines (P = 0.893; Fig. 4B). This represents a more than threefold reduction in nicotine conversion beyond that mediated by the cyp82e4v2 mutation alone. Statistically, the differences in percent nicotine conversion (and nornicotine accumulation as a percent of total dry weight) between e4e4/E5E5/E10E10 and e4e4/e5e5/e10e10 genotypes were highly significant (P < 0.0001). Similar to the investigation of RNAi-mediated suppression of nicotine conversion (Lewis et al., 2008), the present non-transgenic alteration of nicotine demethylase activities in tobacco plants did not appear to significantly alter the content of the minor alkaloid species anatabine and anabasine.

[0139] The effects of pyramiding the three independent nicotine demethylase genetic mutations were also tested in a field trial conducted during the 2010 growing season. For this trial, crosses were performed entirely within a DH98-325-6 genetic background (unlike the 2009 trial, in which a TN90 mother was also used). Molecular genotyping was again used to create every possible combination necessary to determine the respective contributions of each CYP82E locus to the nornicotine phenotype. Alkaloid data were collected on tobacco plants grown to maturity and cured according to standard industry practices. As illustrated in Table 5, a high level of nicotine conversion (from 52.4 to 65.59%) was observed in all genotypes homozygous for a wild-type CYP82E4v2 gene (genotypes E4E4/E5E5/E10E10, E4E4/e5e5/E10E10, E4E4/E5E5/e10e10, and E4E4/e5e5/e10e10). Plants homozygous for only one cyp82e4v2 mutation (e4e4/E5E5/E10E10) had an average nicotine-to-nornicotine conversion of 2.91%. Similar to the 2009 results, the effects of the cyp82E5v2 and cyp82E10 mutations were not additive and were only manifested when the three mutant loci were pyramided together. DH98-325-6(e4e4/E5E5/e10e10) plants averaged 2.89% conversion, while DH98-325-6(e4e4/e5e5/E10E10) individuals averaged 2.52%, values that were not statistically different from those observed with the cyp82e4v2 mutation alone. However, the reduction in nicotine observed in the triple mutant genotype DH98-325-6(e4e4/e5e5/e10e10) (1.11% nicotine conversion) was 2.6-fold lower than that obtained via the cyp82e4v2 mutation alone. The reduction in nicotine conversion attributable to the triple mutant combination was highly significant (P<0.001) compared with cyp82e4v2 or either double mutant combination. Table 5. Alkaloid profiles for genotypes DH98-325-6 harboring different mutation combinations at the CYP82E4v2 (E4), CYP82Ev25 (E5), and CYP82E10 (E10) loci. Data represent the average of five replicates and are generated from soil samples collected from the fourth and fifth leaves from the top of the plant. Alkaloid percentages were calculated on a dry weight basis. The percent nicotine conversion is equal to [% nornicotine/(%nornicotine + % nicotine)] X 100.

Conclusions

[0140] Through the present discovery and characterization of a novel nicotine demethylase gene, CYP82E10, it has been possible to develop a strategy to reduce nicotine conversion rates (and thus nornicotine levels) in outdoor-cured commercial-type tobacco plants to levels previously only possible using transgenic approaches. This non-GMO (genetically modified organism)-based technology can reduce nornicotine levels to a similar degree to that achieved using transgenic strategies, but offers the tremendous advantage of serving as a means to develop ultra-low nornicotine tobacco varieties while avoiding the considerable complications associated with commercializing transgenic crops, such as: (1) negotiating and paying license fees for the various enabling technologies required to generate transgenic plants; (2) avoiding delays and high costs associated with disrupting a transgenic event; and (3) address the possibility that the product will be rejected by end users who are philosophically opposed to GMOs. The discovery presented here represents a major advance in our ability to reduce levels of one of the best-documented strong carcinogens found in tobacco products, compared with previously described anti-GMO strategies that only targeted mutations in the nicotine demethylase gene CYP82E4v2 (Julio et al., 2008; Xu et al., 2007b) or combined CYP82E4v2 and CYP82E5v2 mutations (Dewey et al., 2007). Using transgenic technologies, it was previously demonstrated that reducing nicotine conversion levels from ~2.6% to ~0.5% in cured leaf also caused a commensurate reduction in leaf NNN content (Lewis et al., 2008). Similar reductions in NNN content would be expected in tobacco leaves containing the triple mutant combination (e4e4/e5e5/e10e10) described in this paper. Although originally developed for outdoor-cured tobaccos, this technology will be beneficial for oven-cured varieties as well. As heat exchangers age, their ability to remove NOx gases during oven curing may decrease. Furthermore, recent research has shown that a considerable amount of TSNA formation can occur during storage of cured leaf. Minimizing nornicotine levels by introducing a triple mutant combination into oven-cured varieties may act as a safeguard mechanism against NNN formation, either during storage or the consequences of inefficient heat exchange during the curing process.

REFERENCES

[0141] Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410.

[0142] Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.

[0143] Brogan, A.P., Dickerson, T.J., Boldt, G.E. and Janda, K.D. 2005. Altered retinoid homeostasis catalyzed by nicotine metabolite: implications in macular degeneration and normal development. Proc. Natl. Academic. Sci USA 102: 10433-10438.

[0144] Boyette, M.D. and Hamm, L.A. 2001. Results of year 2000 TSNA sampling program in flue-cured tobacco. Rec. Adv. Tob. Sci. 27: 17-22.

[0145] Bush, L.P., Cui, M., Shi, H., Burton, H.R., Fannin, F.F., Lei, L. and Dye, N. 2001. Formation of tobacco-specific nitrosamines in air-cured tobacco. Rec. Adv. Tob. Sci. 27: 23-46.

[0146] Chakrabarti, M., Meekins, K.M., Gavilano, L.B. and Siminszky, B. 2007. Inactivation of the cytochrome P450 gene CYP82E2 by degenerative mutations was a key event in the evolution of the alkaloid profile of modern tobacco. New Phytol. 175: 565-574.

[0147] Dewey, R.E., Siminszky, B., Bowen, S.W. and Gavilano, L. 2007. Alteration of tobacco alkaloid content through modification of specific cytochrome P450 genes. U.S. Patent Application 60/987,243.

[0148] Dickerson, T.J. and Janda, K.D. 2002. A previously undescribed chemical link between smoking and metabolic disease. Proc. Natl. Academic. Sci. USA 99: 15084-15088.

[0149] Gavilano, L.B., Coleman, N.P., Bowen, S.W. and Siminszky, B. 2007. Functional analysis of nicotine demethylase genes reveals insights into the evolution of modern tobacco. J. Biol. Chem. 282: 249-256.

[0150] Gavilano, L. B. and Siminszky, B. 2007. Isolation and characterization of the cytochrome P450 gene CYP82E5v2 that mediates nicotine to nornicotine conversion in the green leaves of tobacco. Plant Cell Physiol. 48: 1567-1574.

[0151] Hecht, S.S. 1998. Biochemistry, biology and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 11: 559-603.

[0152] Hecht, S.S. 2003. Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nature Rev. 3: 733-744.

[0153] Hecht, S.S. and Hoffmann, D. 1990. The relevance of tobacco specific nitrosamines to human cancer. Cancer Surveys 8: 273-294.

[0154] Hoffmann, D., Brunnemann, K. D., Prokopczyk, B. and Djordjevic, M. V. 1994. Tobacco-specific N-nitrosamines and Areca-derived N-nitrosamine chemistry, biochemistry, carcinogenicity and relevance to humans. J. Toxicol. Environ. Health 41: 1-52.

[0155] Jack, A., Fannin, N. and Bush, L.P. 2007. Implications of reducing nornicotine accumulation in burley tobacco: Appendix A - The LC Protocol. Rec. Adv. Tob. Sci. 33: 58-79.

[0156] Julio, E., Laporte, F., Reis, S., Rothan, C. and Dorlhac de Borne, F. 2008. Reducing the content of nornicotine in tobacco via targeted mutation breeding. Mol. Breed. 21:369381.

[0157] Katz, J., Caudle, R.M., Bhattacharyya, I., Stewart, C.M. and Cohen, D.M. 2005. Receptor for advanced glycation end product (RAGE) upregulation in human gingival fibroblasts incubated with nornicotine. J. Periodontol. 76: 1171-1174.

[0158] Lewis, R.S., Jack, A.M., Morris, J.W., Robert, V.J.M., Gavilano, L., Siminszky, B., Bush, L.P., Hayes, A.J. and Dewey, R.E. 2008. RNAi-induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves. Plant Biotech. J. 6: 346-354.

[0159] Peele, D.M. and Gentry, J. S. 1999. Formation of tobacco-specific nitrosamines in flue-cured tobacco. CORESTA Meeting, Agro-Phyto Groups, Suzhou, China.

[0160] Pompon, D., Louerat, B., Bronne, A. and Urban, P. 1995.Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol. 272: 51-64.

[0161] Siminszky, B., Gavilano, L., Bowen, S. W. and Dewey, R. E. 2005. Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase. Proc. Natl. Academic. Sci USA 102: 14919-14924.

[0162] Wernsman, E. A. and Matzinger, D. F. 1968. Time and site of nicotine conversion in tobacco. Tob. Sci. 12: 226-228.

[0163] Xu, D., Shen, Y., Chappell, J., Cui, M. and Nielsen, M. 2007a. Biochemical and molecular characterization of nicotine demethylase in tobacco. Physiol. Plantarum 129: 307-319.

[0164] Xu, D., Nielsen, M.T. and Shen, Y. 2007b. Tobacco plants having a mutation in a nicotine demethylase gene. U.S. Patent Application 20070199097.

[0165] Various modifications and other embodiments of the inventions disclosed herein will come to the mind of those skilled in the art to which the inventions pertain, with the benefit of the teachings presented in the preceding descriptions and their corresponding figures. Therefore, it is important to understand that the inventions are not to be limited to the specific embodiments described and that modifications and other embodiments are intended to be included within the scope of the list of embodiments and appended claims. Although specific terms are used herein, they are used for general and descriptive purposes only and are not intended to be limiting.

[0166] All publications and patent applications mentioned in the specification are indicative of the level of skill in the art to which this invention belongs. All publications and patent applications are incorporated herein by reference in the same manner as if each of the publications or patent applications had been specifically and individually indicated to be incorporated by reference.

Claims (20)

1. Tobacco product prepared from a tobacco plant or part of a tobacco plant, CHARACTERIZED by the fact that said tobacco plant or part thereof contains a CYP82E10 nicotine demethylase comprising an amino acid sequence having the sequence selected from the group consisting of the sequences defined by SEQ ID NO: 2, 5, 6, 7, 8 and 9, wherein said CYP82E10 nicotine demethylase has a mutation selected from the group consisting of: a) a serine substitution at the glycine residue of position 79; b) a serine substitution at the proline residue of position 107; c) a serine substitution at the proline residue of position 381; d) a serine substitution at the proline residue of position 419; and e) any of their combinations, wherein said mutation causes reduced expression or function of said CYP82E10 nicotine demethylase; and wherein said tobacco product is selected from the group consisting of filter cigarettes, thin cigars, cigars, gums, lozenges and dissolving strips. 2. The tobacco product of claim 1, further comprising a CYP82E4 nicotine demethylase having a sequence selected from the group consisting of the sequences defined by SEQ ID NO: 14, 15, 16, 17, 18, 19, and 20, wherein said CYP82E4 nicotine demethylase has a mutation selected from the group consisting of: a) a stop codon substitution at the tryptophan residue at position 329; b) an asparagine substitution at the lysine residue at position 364; c) a methionine substitution at the valine residue at position 376; d) a serine substitution at the proline residue at position 381; e) a serine substitution at the proline residue at position 458; and f) any combination thereof; wherein said mutation causes reduced expression or function of said CYP82E4 nicotine demethylase. 3. The tobacco product of claim 1 or 2 further comprising a CYP82E5 nicotine demethylase comprising an amino acid sequence having a sequence selected from the group consisting of the sequences defined by SEQ ID NO: 26, 27, 28, 29, 30, 31, and 32, wherein said CYP82E5 nicotine demethylase has a mutation selected from the group consisting of: a) a stop codon substituted at the tryptophan residue at position 422; b) a leucine substituted at the proline residue at position 449; and c) any combination thereof; wherein said mutation causes reduced expression or function of said CYP82E5 nicotine demethylase. 4. The tobacco product of claim 3 comprising a CYP82E4 nicotine demethylase having a sequence selected from the group consisting of the sequences defined by SEQ ID NO: 14, 15, 16, 17, 18, 19, and 20, wherein said CYP82E4 nicotine demethylase has a mutation selected from the group consisting of a) a stop codon substituted at the tryptophan residue of position 329; b) an asparagine substitution at the lysine residue of position 364; c) a methionine substitution at the valine residue of position 376; d) a serine substitution at the proline residue of position 381; e) a serine substitution at the proline residue of position 458; and f) any combination thereof. 5. The tobacco product of any one of claims 1 to 4, wherein said CYP82E10 nicotine demethylase comprises a serine substitution at the proline residue of position 381, said CYP82E4 nicotine demethylase comprises a substituted stop codon at the tryptophan residue of position 329, and said CYP82E5 nicotine demethylase comprises a substituted stop codon at the tryptophan residue at position 422, wherein such numbering is in accordance with SEQ ID NO:2, 14, and 26, respectively. 6. The tobacco product of claim 4 or 5 wherein the plant or plant part has less than 1.5% conversion of nicotine to nornicotine. 7. Tobacco product according to claim 6, CHARACTERIZED by the fact that said plant or part of plant has at most 0.5% conversion of nicotine to nornicotine. 8. A method for reducing the carcinogenic potential of a tobacco product, comprising preparing said tobacco product from a tobacco plant, or a part or progeny of the plant, as defined in any one of claims 1 to 7. 9. A method for reducing the level of nornicotine, or reducing the rate of conversion of nicotine to nornicotine, in a tobacco plant or part of a plant, the method comprising introducing into the genome of said plant a mutation within at least one allele of each of at least three nicotine demethylase genes, wherein said mutation reduces the expression of said nicotine demethylase gene, and wherein a first of said nicotine demethylase genes encodes a root-specific nicotine demethylase involved in the metabolic conversion of nicotine to nornicotine in a tobacco plant or part of a plant; wherein said root-specific nicotine demethylase gene is a CYP82E10 nicotine demethylase comprising a nucleic acid sequence defined by SEQ ID NO: 1; wherein said nucleic acid sequence has a substitution selected from the group consisting of G235A, C319T, C1141T, and C1255T. 10. The method of claim 9, wherein a second of the nicotine demethylase genes comprises a nucleic acid sequence defined by SEQ ID NO: 38; wherein said nucleic acid sequence has a G986A substitution. 11. The method of claim 9 or 10, wherein a third of the nicotine demethylase genes comprises a nucleic acid sequence defined by SEQ ID NO: 39; wherein the nucleic acid sequence has a G1266A substitution. 12. The method of any one of claims 9 to 11, wherein the introduction includes a cultivation protocol. 13. The method of any one of claims 9 to 12, wherein the plant is a Burley, Virginia, kiln-cured, air-cured, fire-cured, Oriental, or dark tobacco plant. 14. The tobacco product of any one of claims 1 to 7 wherein the plant is a Burley, Virginia, kiln-cured, air-cured, fire-cured, Oriental, or dark tobacco plant. 15. Method for identifying a tobacco plant with low levels of nornicotine, said method being CHARACTERIZED by the fact that it comprises examining a DNA sample from a tobacco plant of interest in search of the presence of a mutation in SEQ ID NO: 1 or 3. 16. The method of claim 15, wherein said tobacco plant is a non-converting plant. 17. Method according to claim 15 or 16, CHARACTERIZED by the fact that the examination is performed using a sequence selected from the group consisting of SEQ ID NO: 1, 3, 35, 36, 37 and 38. 18. Isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, and 13; wherein said amino acid sequence has a modification at an amino acid residue corresponding to position 79, 107, 381, or 419 of SEQ ID NO:2, or any combination thereof; wherein said mutation is selected from the group consisting of:a) a serine substitution at the glycine residue at position 79;b) a serine substitution at the proline residue at position 107;c) a serine substitution at the proline residue at position 381;d) a serine substitution at the proline residue at position 419. 19. Tobacco product prepared from a tobacco plant or part thereof, CHARACTERIZED by the fact that said tobacco plant or part thereof is homozygous for a mutation in a gene encoding a CYP82E10 nicotine demethylase, a gene encoding a CYP82E4 nicotine demethylase and a gene encoding a CYP82E5 nicotine demethylase, wherein said mutation causes reduced expression or function of said CYP82E10, CYP82E4 and CYP82E5 nicotine demethylase, wherein said CYP82E10 nicotine demethylase gene comprises a nucleic acid sequence consisting of SEQ ID NO: 1, and comprises a C1141T mutation, said CYP82E4 nicotine demethylase gene comprises a nucleotide sequence consisting of SEQ ID NO: 38, and comprises a G986A substitution, said CYP82E5 nicotine demethylase gene demethylase comprises a nucleic acid sequence consisting of SEQ ID NO: 39, and comprises a G1266A substitution; and wherein said tobacco product is selected from the group consisting of filter cigarettes, slim cigars, cigars, gums, lozenges, and dissolving strips. 20. Tobacco product prepared from a tobacco plant or part thereof, CHARACTERIZED by the fact that said tobacco plant or part thereof has a CYP82E10 nicotine demethylase comprising an amino acid sequence selected from the group consisting of the sequences defined by SEQ ID NO: 2, 5, 6, 7, 8 and 9, wherein said CYP82E10 nicotine demethylase has a mutation selected from the group consisting of: a) a serine substitution at the glycine residue at position 79; b) a serine substitution at the proline residue at position 107; c) a serine substitution at the proline residue at position 381; d) a serine substitution at the proline residue at position 419; ee) any combination thereof; a CYP82E4v2 nicotine demethylase that inhibits the activity in green leaves of a nicotine demethylase comprising an amino acid sequence selected from the group consisting of the sequences defined in SEQ ID NO: 14, 15, 16, 17, 18, 19 and 20; wherein such CYP82E4v2 nicotine demethylase has a mutation selected from the group consisting of: a) a stop codon substitution at the tryptophan residue of position 329; b) an asparagine substitution at the lysine residue of position 364; c) a methionine substitution at the valine residue of position 376; d) a serine substitution at the proline residue of position 381; e) a serine substitution at the proline residue of position 458; and f) any combination thereof; and a CYP82E5v2 nicotine demethylase that inhibits the activity in green leaves of a nicotine demethylase comprising an amino acid sequence selected from the group consisting of the sequences defined in SEQ ID NO: 26, 27, 28, 29, 30, 31 and 32; wherein such CYP82E5v2 nicotine demethylase has a mutation selected from the group consisting of a) a stop codon substituted at the tryptophan residue of position 422; b) a leucine substituted at the proline residue of position 449; and c) any combination thereof; and wherein said tobacco product is selected from the group consisting of filter cigarettes, slim cigars, cigars, gums, lozenges and dissolving strips.