Patent Description:
The predominant alkaloid found in commercial tobacco varieties is nicotine, typically accounting for <NUM> - <NUM>% of the total alkaloid pool. The remaining alkaloid fraction is comprised 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 usually represents less than <NUM>% of the total pyridine alkaloid pool, but through a process termed "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 that have genetically converted (termed "converters"), the great majority of nornicotine production occurs during the senescence and curing of the mature leaf (<NPL>). Burley tobaccos are particularly prone to genetic conversion, with rates as high as <NUM>% per generation observed in some cultivars.

During the curing and processing of the tobacco leaf, a portion of the nornicotine is metabolized to the compound N-nitrosonornicotine (NNN), a tobacco-specific nitrosamine (TSNA) that has been asserted to be carcinogenic in laboratory animals (<NPL>; Hoffmann et al.

<NPL>; <NPL>). In flue-cured tobaccos, TSNAs are found to be predominantly formed through the reaction of alkaloids with the minute amounts of nitrogen oxides present in combustion gases formed by the direct-fired heating systems found in traditional curing barns (<NPL>.

Meeting, Agro-Phyto Groups, Suzhou, China). Retrofitting these curing barns with heat-exchangers virtually eliminated the mixing of combustion gases with the curing air and dramatically reduced the formation of TSNAs in tobaccos cured in this manner (<NPL>. In contrast, in the air-cured Burley tobaccos, TSNA formation proceeds primarily through reaction of tobacco alkaloids with nitrite, a process catalyzed by leaf-borne microbes (<NPL>). Thus far, attempts to reduce TSNAs through modification of curing conditions while maintaining acceptable quality standards have not proven to be successful for the air-cured tobaccos.

Aside from serving as a precursor for NNN, recent studies suggest that the nornicotine found in tobacco products may have additional undesirable health consequences. <NPL> demonstrated that nornicotine causes aberrant protein glycation within the cell. Concentrations of nornicotine-modified proteins were found to be much higher in the plasma of smokers compared to nonsmokers. This same study also showed that nornicotine can covalently modify commonly prescribed steroid drugs such as prednisone. Such modifications have the potential of altering both the efficacy and toxicity of these drugs. Furthermore, studies have been reported linking the nornicotine found in tobacco products with age-related macular degeneration, birth defects, and periodontal disease (<NPL>; <NPL>).

In Burley tobaccos, a positive correlation has been found between the nornicotine content of the leaf and the amount of NNN that accumulates in the cured product (<NPL>; <NPL>). Therefore, strategies that could effectively reduce the nornicotine content of the leaf would not only help ameliorate the potential negative health consequences of the nornicotine per se as described above, but should also concomitantly reduce NNN levels. This correlation was further solidified in the recent study by <NPL> who demonstrated that lowering nornicotine levels using an RNAi transgene construct directed against the CYP82E4v2 gene, which encodes a senescence-induced nicotine demethylase, lead to concomitant reductions in the NNN content of the cured leaf. Although this study demonstrated that transgenic technologies.

can be used to greatly reduce the nornicotine and NNN content of tobacco, a combination of public perception and intellectual property issues make it very difficult for commercialization of products derived from transgenic plants.

Therefore a great need exists for a means to effectively minimize nornicotine accumulation in tobacco that does not rely on the use of transgenics.

<CIT> discloses compositions and methods for reducing the level of nornicotine and N'-nitrosonomicotine in Nicotiana plants and plant parts thereof.

The following listing sets forth the sequence information for the Sequence Listing. Standard notation for amino acid substitutions is used. Thus, for example, CYP82E10 P419S indicates the variant protein has a serine substitution for the proline residue at position <NUM>, where the numbering is with respect to the wild-type sequence, in this case, the CYP82E10 sequence set forth in SEQ ID NO:<NUM>. As another example, CYP82E4 P38L indicates the variant protein has a leucine substitution for the proline residue at position <NUM>, where the numbering is with respect to the wild-type sequence, in this case, the CYP82E4 sequence set forth in SEQ ID NO: <NUM>. As yet another example, CYP82E5 P72L indicates the variant protein has a leucine substitution for the proline residue at position <NUM>, where the numbering is with respect to the wild-type sequence, in this case, the CYP82E5 sequence set forth in SEQ ID NO:<NUM>.

The present invention includes methods for inhibiting expression or function of root-specific nicotine demethylase polypeptides that are involved in the metabolic conversion of nicotine to nornicotine in the roots of a plant, particularly plants of the Nicotiana genus, including tobacco plants of various commercial varieties.

As used herein, "inhibit," "inhibition" and "inhibiting" are defined as any method known in the art or described herein, which decreases the expression or function of a gene product of interest (i.e., the target gene product), in this case a nicotine demethylase, such as a root-specific nicotine demethylase of the disclosure. 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, knock out approaches such as mutagenesis, and the like. In the invention methods are used that knock out, or knock down, expression and/or function of the CYP82E10 nicotine demethylase.

By "favorable mutation" is intended a mutation that results in a substitution, insertion, deletion, or truncation of the CYP82E10 polypeptide such that its nicotine demethylase activity is inhibited. In some embodiments, the nicotine demethylase activity is inhibited by at least <NUM>%, <NUM>%, <NUM>, <NUM>%, <NUM>, <NUM>%, <NUM>%, or <NUM>% when compared to the activity of the wild-type CYP82E10 polypeptide under the same test conditions. In other embodiments, the nicotine demethylase activity is inhibited by at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%. In preferred embodiments, the favorable mutation provides for complete inhibition (i.e., <NUM>% inhibition), and the nicotine demethylase activity is knocked out (i.e., its activity cannot be measured).

"Inhibiting" can be in the context of a comparison between two plants, for example, a genetically altered plant versus a wild-type plant. The comparison can be between plants, for example, a wild-type plant and one of which lacks a DNA sequence capable of producing a root-specific nicotine demethylase that converts nicotine to nornicotine. Inhibition of expression or function of a target gene product also can be in the context of a comparison between plant cells, organelles, organs, tissues or plant parts within the same plant or between different plants, and includes comparisons between developmental or temporal stages within the same plant or plant part or between plants or plant parts.

"Inhibiting" can include any relative decrement of function or production of a gene product of interest, in this case, a root-specific nicotine demethylase, up to and including complete elimination of function or production of that gene product. When levels of a gene product are compared, such a comparison is preferably carried out between organisms with a similar genetic background. Preferably, a similar genetic background is a background where the organisms being compared share <NUM>% or greater, more preferably <NUM>% or greater, and, even more preferably <NUM>% or greater sequence identity of nuclear genetic material. A similar genetic background is a background where the organisms being compared are plants, and the plants are isogenic except for any genetic material originally introduced using plant transformation techniques or a mutation generated by human intervention. Measurement of the level or amount of a gene product may be carried out by any suitable method, non-limiting examples of which include, but are not limited to, comparison of mRNA transcript levels, protein or peptide levels, and/or phenotype, especially the conversion of nicotine to nornicotine. As used herein, mRNA transcripts can include processed and non-processed mRNA transcripts, and polypeptides or peptides can include polypeptides or peptides with or without any post-translational modification.

As used herein, "variant" means a substantially similar sequence. A variant can have different function or a substantially similar function as a wild-type polypeptide of interest. For a nicotine demethylase, a substantially similar function is at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% of wild-type enzyme function of converting nicotine to nornicotine under the same conditions or in a near-isogenic line. A wild-type CYP82E10 is set forth in SEQ ID NO:<NUM>. A wild-type CYP82E4 is set forth in SEQ ID NO: <NUM>. A wild-type CYP82E5 is set forth in SEQ ID NO:<NUM>. Exemplary variants of the wild-type CYP82E10 include polypeptides comprising the sequence set forth in SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. The variant set forth in SEQ ID NO:<NUM> (CYP82E10 P419S) advantageously has a favorable mutation that results in the enzyme having only about <NUM>% of the nicotine demethylase activity of the wild-type CYP82E10 polypeptide. The variants set forth in SEQ ID NOs: <NUM> (CYP82E10 G79S), <NUM> (CYP82E10 with P107S), and <NUM> (CYP82E10 with P382S) advantageously have favorable mutations that result in their nicotine demethylase activity being knocked out (i.e., <NUM>% inhibition, and thus a nonfunctional polypeptide). In like manner, exemplary variants of the wild-type CYP82E4 include polypeptides comprising the sequence set forth in SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. The variant set forth in SEQ ID NO:<NUM> (CYP82E4 V376M) advantageously has a favorable mutation that results in the enzyme having only about <NUM>% of the nicotine demethylase activity of the wild-type CYP82E4 polypeptide. The variants set forth in SEQ ID NOs: <NUM> (CYP82E4 W329Stop), <NUM> (CYP82E4 K364N), <NUM> (CYP82E4 P382S), and <NUM> (CYP82E4 P458S) advantageously have favorable mutations that result in their nicotine demethylase activity being knocked out (i.e., <NUM>% inhibition). Similarly, exemplary variants of the wild-type CYP82E4 include polypeptides comprising the sequence set forth in SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. The variant set forth in SEQ ID NO:<NUM> (CYP82E5 P449L) advantageously has a favorable mutation that results in inhibition of its nicotine demethylase activity, and the variant set forth in SEQ ID NO:<NUM> advantageously has a favorable mutation that results in its nicotine demethylase activity being knocked out (i.e., <NUM>% inhibition).

As used herein, a "variant polynucleotide" or "variant polypeptide" means a nucleic acid or amino acid sequence that is not wild-type.

A variant can have one addition, deletion or substitution; two or less additions, deletions or substitutions; three or less additions, deletions or substitutions; four or less additions, deletions or substitutions; or five or less additions, deletions or substitutions. A mutation includes additions, deletions, and substitutions. Such deletions or additions can be at the C-terminus, N-terminus or both the C- and N-termini. Fusion polypeptides or epitope-tagged polypeptides are also disclosed. "Silent" nucleotide mutations do not change the encoded amino acid at a given position. Amino acid substitutions can be conservative. A conservative substitution is a change in the amino acid where the change is to an amino acid within the same family of amino acids as the original amino acid. The family is defined by the side chain of the individual amino acids. A family of amino acids can have basic, acidic, uncharged polar or nonpolar side chains. See, <NPL>). A deletion, substitution or addition can be to the amino acid of another CYP82E family member in that same position. As used herein, a "fragment" means a portion of a polynucleotide or a portion of a polypeptide and hence protein encoded thereby.

As used herein, "plant part" means plant cells, plant protoplasts, plant cell tissue cultures from which a whole plant can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants such as embryos, pollen, anthers, ovules, seeds, leaves, flowers, stems, branches, fruit, roots, root tips and the like. Progeny, variants and mutants of regenerated plants are also included, provided that they comprise the introduced polynucleotides of the invention. As used herein, "tobacco plant material" means any portion of a plant part or any combination of plant parts.

The present invention relates to a novel nicotine demethylase gene, CYP82E10 (genomic sequence set forth in SEQ ID NO:<NUM>), and its encoded CYP82E10 nicotine demethylase (SEQ ID NO:<NUM>), that is involved in root-specific conversion of nicotine to nornicotine in roots of tobacco plants and its use in reducing or minimizing nicotine to nornicotine conversion and thus reducing levels of nornicotine in tobacco plants and plant parts thereof. By "root-specific" is intended it is preferentially expressed within the roots of tobacco plants, as opposed to other plant organs such as leaves or seeds. By introducing selected favorable mutations into this root-specific nicotine demethylase or variants thereof having nicotine demethylase activity, in combination with one or more selected favorable mutations within a gene encoding a green-leaf nicotine demethylase (for example, CYP82E5 set forth in SEQ ID NO:<NUM>) or variant thereof having nicotine demethylase activity, and further in combination with one or more selected favorable mutations within a gene encoding a senescence-induced nicotine demethylase (for example, CYP82E4 set forth in SEQ ID NO:<NUM>) or variant thereof having nicotine demethylase activity, it is possible to produce nontransgenic tobacco plants having minimal nicotine to nornicotine conversion, where the conversion rate is less than about <NUM>%, preferably less than about <NUM>%.

Lowering nornicotine levels in tobacco is highly desirable because this alkaloid serves as a precursor to the well-documented carcinogen N'-nitrosonomicotine (NNN). Two genes encoding proteins having nicotine demethylase activity in tobacco have been previously identified and designated as CYP82E4v2 and CYP82E5v2. The CYP82E4 polypeptide (SEQ ID NO:<NUM>) 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 <CIT>, which published as <CIT>. The CYP82E5 polypeptide (SEQ ID NO:<NUM>) 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 <CIT>, which published as <CIT>.

Plants homozygous for favorable mutant cyp82e4v2 and cyp82e5v2 alleles (i.e., mutant alleles that knock down, or knock out, expression of these respective nicotine demethylase genes), however, can still metabolize more than <NUM>% of their nicotine to nornicotine, which represent nornicotine levels that can still lead to substantial NNN formation. The discovery of the CYP82E10 nicotine demethylase gene provides a further avenue for minimizing the nicotine to nornicotine conversion rate in tobacco plants, and thus further reducing the levels of nornicotine and thus NNN in tobacco plants and plant materials derived therefrom. Combining favorable mutant cyp82e10 alleles with favorable mutant cyp82e4v2 and cyp82e5v2 alleles provides for tobacco plants possessing more than a <NUM>-fold reduction in nornicotine when compared to that observed for tobacco plants having the cyp82e4v2 mutation alone, or the cyp82e5v2 mutations together. In one embodiment, the present invention provides a homozygous triple mutant combination of nicotine demethylase genes cyp82e4v2, cyp82e5v2, and cyp82e10) as defined in the claims that results in nontransgenic tobacco plants that produce very low levels of nornicotine comparable to that only previously achieved via transgenic gene suppression approaches, such as those described in <CIT> and <CIT>.

Disclosed herein is the CYP82E10 polypeptide and variants and fragments thereof. Such nicotine demethylase polynucleotides and polypeptides are involved in the metabolic conversion of nicotine to nornicotine in plants, including commercial varieties of tobacco plants. In particular, disclosed herein are isolated polypeptides comprising the amino acid sequences as shown in SEQ ID NOs:<NUM>, and <NUM>-<NUM>, isolated polynucleotides comprising the nucleotide sequences as shown in SEQ ID NOs:<NUM>, <NUM>, and <NUM>, and isolated polynucleotides encoding the amino acid sequences of SEQ ID NOs:<NUM> and <NUM>-<NUM>. The polynucleotides can find use in inhibiting expression of nicotine demethylase polypeptides or variants thereof that are involved in the metabolic conversion of nicotine to nornicotine in plants, particularly tobacco plants. The inhibition of polypeptides is effective in lowering nornicotine levels in tobacco lines where genetic conversion occurs in less than <NUM>%, <NUM>%, <NUM>%, <NUM>% of the population, such as flue-cured tobaccos. The inhibition of polypeptides is effective in lowering nornicotine levels in tobacco populations where genetic conversion occurs in at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% of a plant population. A population preferably contains greater than about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM>, <NUM>,<NUM>, or <NUM>,<NUM> plants where, more preferably at least about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% of the plants comprise a polypeptide disclosed herein.

The nicotine demethylase polynucleotides and encoded polypeptides include a novel cytochrome P450 gene, designated the CYP82E10 nicotine demethylase gene, that is newly identified as having a role in the metabolic conversion of nicotine to nornicotine in roots of tobacco plants. Transgenic approaches such as sense, antisense, and RNAi suppression (not part of the invention) may be used to knock down expression of this nicotine demethylase, in a manner similar to that described for the CYP82E4 and CYP82E5 nicotine demethylases, as described in <CIT> and <CIT>. In the invention, one or more favorable mutations as defined in the claims are introduced into this gene, as this approach advantageously provides nontransgenic tobacco plants having reduced nicotine to nornicotine conversion rates, and thus reduced levels of nornicotine and NNN. Such approaches include, but are not limited to, mutagenesis, and the like, as described elsewhere herein below.

The invention encompasses isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the <NUM>' and <NUM>' 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 can contain less than about <NUM> kb, <NUM> kb, <NUM> kb, <NUM> kb, <NUM> kb, <NUM> kb, or <NUM> kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% (by dry weight) of contaminating protein. When the protein or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about <NUM>% <NUM>%, <NUM>%, <NUM>%, or <NUM>% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

Naturally occurring variants include those variants that share substantial sequence identity to the CYP82E10 polynucleotides and polypeptides as defined herein below. Naturally occurring variants may also share substantial functional identity to the CYP82E10 polynucleotides disclosed herein. Such CYP82E10 polypeptides can possess the relevant nicotine demethylase activity, i.e., involvement in the metabolic conversion of nicotine to nornicotine in plants, or not. Such variants may result from, for example, genetic polymorphism or from human manipulation as occurs with breeding and selection, including mutagenesis approaches. Biologically active variants of a CYP82E10 protein, for example, variants of the polypeptide set forth in SEQ ID NO:<NUM> and <NUM>-<NUM>, will have at least about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or more sequence identity to the amino acid sequence for the wild-type protein as determined by sequence alignment programs and parameters described elsewhere herein, and can be characterized by their functional involvement in the metabolic conversion of nicotine to nornicotine in plants, or lack thereof. In the invention, a variant has at least <NUM>% sequence identity to SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. A biologically active variant of a protein may differ from that protein by as few as <NUM>-<NUM> amino acid residues, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, or as few as <NUM> amino acid residue. A biologically inactive variant of a protein may differ from that protein by as few as <NUM>-<NUM> amino acid residues, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, as few as <NUM>, or as few as <NUM> amino acid residue.

Variants of a particular polynucleotide include those naturally occurring polynucleotides that encode a CYP82E10 polypeptide that is involved in the metabolic conversion of nicotine to nornicotine in the roots of plants. Such polynucleotide variants can comprise a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide disclosed herein and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. Because of 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 disclosure. Naturally occurring variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as are known in the art and disclosed herein. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still share substantial sequence identity to the naturally occurring sequences disclosed herein, and thus can be used in the methods of the invention to inhibit the expression or function of a nicotine demethylase that is involved in the metabolic conversion of nicotine to nornicotine, including the nicotine demethylase polypeptides set forth in SEQ ID NOS:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>. Generally, variants of a particular polynucleotide, for example, the polynucleotide sequence of SEQ ID NO:<NUM> or the polynucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:<NUM>, and <NUM>-<NUM>, will have at least about <NUM>%,<NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.

Variants of a particular polynucleotide (also referred to as the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by the reference polynucleotide and the polypeptide encoded by a variant polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or more sequence identity.

Furthermore, the polynucleotides can be used to isolate corresponding root-specific nicotine demethylase sequences, particularly CYP82E10 sequences, from other members of the Nicotiana genus. PCR, hybridization, and other like methods can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Such sequences include sequences that are orthologs of the disclosed sequences.

According to the present invention, "orthologs" are genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode for a nicotine demethylase polypeptide that is involved in the nicotine-to-nornicotine metabolic conversion and which hybridize under stringent conditions to the CYP82E10 sequence disclosed herein, or to variants or fragments thereof, are contemplated. Such sequences can be used to inhibit expression of nicotine demethylase polypeptides that are involved in the metabolic conversion of nicotine to nornicotine in plants. Using PCR, oligonucleotide primers can be designed for use in PCR reactions to amplify 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 disclosed in<NPL>); <NPL>); <NPL>); and <NPL>). Known methods of PCR 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, and the like.

Hybridization techniques involve 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 DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.

Hybridization may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least <NUM>-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are <NUM>% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about <NUM> nucleotides in length, optimally less than <NUM> nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about <NUM> Na ion, typically about <NUM> to <NUM> Na ion concentration (or other salts) at pH <NUM> to <NUM> and the temperature is at least about <NUM>. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of <NUM> to <NUM>% formamide, <NUM> NaCl, <NUM>% SDS (sodium dodecyl sulphate) at <NUM>, and a wash in 1X to 2X SSC (20X SSC = <NUM> NaCl/<NUM> trisodium citrate) at <NUM> to <NUM>. Exemplary moderate stringency conditions include hybridization in <NUM> to <NUM>% formamide, <NUM> NaCl, <NUM>% SDS at <NUM>, and a wash in <NUM>. 5X to 1X SSC at <NUM> to <NUM>. Exemplary high stringency conditions include hybridization in <NUM>% formamide, <NUM> NaCl, <NUM>% SDS at <NUM>, and a wash in <NUM>. 1X SSC at <NUM> to <NUM>. Optionally, wash buffers may comprise about <NUM>% to about <NUM>% SDS. Duration of hybridization is generally less than about <NUM> hours, usually about <NUM> to about <NUM> hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

Stringency conditions include hybridization in a solution containing 5X SSC, <NUM>% SDS, 5X Denhardt's, <NUM> ug/ul Poly A RNA, <NUM> ug/ul calf thymus DNA and <NUM>% formamide at <NUM>, and at least one post-hybridization wash in a solution comprising from about <NUM>. 01X SSC to about l X SSC. The duration of hybridization is from about <NUM> to about <NUM> hours.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of <NPL>: Tm = <NUM> + <NUM> (log M) + <NUM> (%GC) - <NUM> (% form) - <NUM>/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. The Tm is the temperature (under defined ionic strength and pH) at which <NUM>% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about <NUM> for each <NUM>% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≥<NUM>% identity are sought, the Tm can be decreased <NUM>. Generally, stringent conditions are selected to be about <NUM> lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However; severely stringent conditions can utilize a hybridization and/or wash at <NUM>, <NUM>, <NUM>, or <NUM> lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm, of less than <NUM> (aqueous solution) or <NUM> (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in <NPL>); and <NPL>). See <NPL>).

Hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as <NUM>P, or any other delectable marker. For example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the CYP82E10 polynucleotides sequences of the present invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in <NPL>).

For example, the CYP82E10 polynucleotide sequences disclosed herein, or one or more portions thereof, may be used as probes capable of specifically hybridizing to corresponding root-specific nicotine demethylase polynucleotides and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among the CYP82E10 polynucleotide sequences or unique to one of the CYP82E10 polynucleotide sequences, including upstream regions <NUM>' to the coding sequence and downstream regions <NUM>' to the coding sequence and an intron region (for example, SEQ ID NO:<NUM>), and are optimally at least about <NUM> contiguous nucleotides in length, more optimally at least about <NUM> contiguous nucleotides in length, more optimally at least about <NUM> contiguous nucleotides in length, more optimally at least about <NUM> contiguous nucleotides in length, and more optimally at least about <NUM> contiguous nucleotides in length. Such probes may be used to amplify corresponding CYP82E10 polynucleotides. This technique may 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 hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, <NPL>).

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

As used herein, the term "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, where the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the deference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least <NUM> contiguous nucleotides in length, and optionally can be <NUM>, <NUM>, <NUM>, <NUM>, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of <NPL>; the local alignment algorithm of<NPL>; the global alignment algorithm of <NPL>; the search-for-local alignment method of <NPL>; the algorithm of <NPL>, modified as in <NPL>.

The BLAST programs of <NPL>are based on the algorithm of Karlin and Altschul (<NUM>) supra. BLAST nucleotide searches can be performed with the BLASTN program, score = <NUM>, wordlength = <NUM>, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score = <NUM>. wordlength = <NUM>, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST <NUM>) can be utilized as described in<NPL>. Alternatively, PSI-BLAST (in BLAST <NUM>) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (<NUM>) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used (See www. Alignment may also be performed manually by inspection.

The sequence identity/similarity values provided herein may be calculated using the BLASTX (Altschul et al. (<NUM>) supra), Clustal W (<NPL>), and GAP (University of Wisconsin Genetic Computing Group software package) algorithms using default parameters. The present disclosure also encompasses the use of any equivalent program thereof for the analysis and comparison of nucleic acid and protein sequences. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by BLASTX. Clustal W, or GAP. For purposes of the foregoing discussion of variant nucleotide and polypeptide sequences, the term "sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity. " Means for malting this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of <NUM> and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and <NUM>. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

The term "percentage of sequence identity" as used herein means the value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise 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 yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by <NUM> to yield the percentage of sequence identity.

Thus, CYP82E10 polynucleotide and polypeptide sequences can be identified using the sequences provided herein. Such methods include obtaining a polynucleotide or polypeptide sequence at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence identity with the polynucleotide sequence of SEQ ID NO: <NUM>, <NUM>, or <NUM> or a complement or fragment thereof, or a polypeptide sequence of SEQ ID NO: <NUM>, or <NUM>-<NUM>. A preferred aspect includes a polypeptide corresponding to SEQ ID NO:<NUM> that has a serine at position <NUM>, <NUM>, or <NUM> of the CYP82E10 polypeptide, where the numbering corresponds to SEQ ID NO:<NUM>.

Methods of reducing the concentration, content, and/or activity of a CYP82E10 polypeptide in a Nicotiana plant or plant part, particularly the root tissue, are provided. Many methods may be used, alone or in combination, to reduce or eliminate the activity of the CYP82E10 polypeptide (SEQ ID NO:<NUM>), and variants thereof that retain nicotine demethylases activity (for example, SEQ ID NOs:<NUM>, <NUM>, <NUM>, and <NUM>). In addition, combinations of methods may be employed to reduce or eliminate the activity of two or more different nicotine demethylases, more particularly the root-specific CYP82E10 nicotine demethylase and one or both of the green-leaf CYP82E5 and senescence-induced CYP82E4 nicotine demethylases. In a particular embodiment, the CYP82E5 is a polypeptide with at least one amino acid mutation in the sequence of SEQ ID NO: <NUM> that negatively affects conversion in green leaves and the CYP82E4 has the sequence set forth in SEQ ID NO: <NUM> with at least one amino acid mutation that negatively affects conversion in senescent leaves.

In accordance with the present invention, the expression of a CYP82E10 nicotine demethylase 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 where these plants have been cultured and harvested using the same protocols. In particular embodiments of the invention, the protein level of the CYP82E10 polypeptide in a modified plant according to the invention is less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>% of the protein level of the same CYP82E10 polypeptide in a plant that is not a mutant or that has not been genetically modified to inhibit the expression of that CYP82E10 polypeptide and which has been cultured and harvested using the same protocols. The expression level of the CYP82E10 polypeptide may be measured directly, for example, by assaying for 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, and include, but are not limited to, Northern blot analysis and RNA differentiation assays. Methods for determining the activity of a targeted CYP82E10 polypeptide in converting nicotine to nornicotine are known in the art and described elsewhere herein below, and include, but are not limited to, alkaloid analysis using gas chromatography.

The present invention provides methods for reducing the level of nornicotine, or reducing the rate of conversion of nicotine to nornicotine, in a tobacco plant, or plant part thereof. The methods of the invention comprise 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 reduces expression of the nicotine demethylase gene, and wherein a first of these nicotine demethylase genes encodes a root-specific nicotine demethylase involved in the metabolic conversion of nicotine to nornicotine in a tobacco plant or a plant part thereof. The root-specific nicotine demethylase is CYP82E10 or variant thereof as defined in the claims. In other emodiments, these methods comprise introducing into the genome of a tobacco plant a mutation within at least one allele of a nicotine demethylase gene encoding CYP82E10 or variant thereof as defined in the claims, and a mutation within at least one allele of a nicotine demethylase encoding CYP82E4 or variant thereof as defined in the claims, and/or a nicotine demetyylase encoding CYP82E5 or variant thereof as defined in the claims.

A number of approaches have been used to combine mutations in one plant including sexual crossing. A plant having a favorable mutation in a CYP82E10 gene that inhibits the nicotine demethylases activity in roots can be crossed with a plant having a favorable mutation in a CYP82E4v2 gene that inhibits the nicotine demethylase activity in senescent leaves, or be crossed with a plant having a favorable mutation in a CYP83E5v2 gene that inhibits nicotine demethylase activity in green leaves to produce a plant having reduced nicotine to nornicotine conversion. Crosses may be made in order to introduce a favorable mutation within a CYP82E10, CYP82E4v2, and CYP82E5v2 gene within the same plant. In this manner, a plant having a favorable mutation in a CYP82E10 gene that inhibits the nicotine demethylases activity in roots is crossed with a plant having a favorable mutation in a CYP82E4v2 gene that inhibits the nicotine demethylase activity in senescent leaves and a favorable mutation in a CYP83E5v2 gene that inhibits nicotine demethylase activity in green leaves. Alternatively, a plant having a favorable mutation in a CYP82E4v2 gene that inhibits the nicotine demethylase activity in senescent leaves is crossed with a plant having a favorable mutation in a CYP82E10 gene that inhibits the nicotine demethylase activity in roots and a favorable mutation in a CYP83E5v2 gene that inhibits nicotine demethylase activity in green leaves. In yet another aspect, a plant having a favorable mutation in a CYP82E5v2 gene that inhibits the nicotine demethylase activity in green leaves is crossed with a plant having a favorable mutation in a CYP82E10 gene that inhibits the nicotine demethylase activity in roots and a favorable mutation in a CYP83E4v2 gene that inhibits nicotine demethylase activity in senescent leaves. By introducing a favorable mutation into each of these nicotine demethylases genes it is possible to produce a plant having reduced nicotine to nornicotine conversion rates with conversion levels lower than about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%.

A plant having one or more favorable mutations that results in a modification of the CYP82E10 polypeptide at position <NUM>, <NUM>, or <NUM> (where the numbering is according to SEQ ID NO:<NUM>) can be crossed with a plant having one or more favorable mutations that results in a modification of the CYP82E polypeptide at position <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> and/or having one or more favorable mutations that results in a modification of the CYP82E5 polypeptide at position <NUM> or <NUM> to produce a plant with conversion levels lower than <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%. A particularly preferred conversion level of nicotine to nornicotine can be between <NUM>% - <NUM>%, between <NUM> - <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, or between <NUM>% - <NUM>%.

The domains of cytochrome P450 proteins are known in the art. See, for example, <NPL>. By crossing plants having a nonfunctional or inhibited CYP82E10 gene with plants having a nonfunctional or inhibited CYP82E4v2 gene, a nonfunctional or inhibited CYP82E5v2 gene, or nonfunctional or inhibited CYP82E4v2 and CYP82E5v2 genes, nornicotine levels can be reduced in a tobacco plant.

The activity of a CYP82E10, CYP82E4, or CYP82E5 nicotine demethylase polypeptide 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 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 that nicotine demethylase polypeptide and which has been cultured and harvested using the same protocols. In particular embodiments, activity of a nicotine demethylase polypeptide in converting nicotine to nornicotine in a modified tobacco plant or plant part according to the invention is inhibited if the activity is less than <NUM>%, less than <NUM>%, less than <NUM>% less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>% less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>% 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 has been cultured and harvested using the same protocols. The activity of a nicotine demethylase polypeptide in converting nicotine to nornicotine in a tobacco plant or plant part is eliminated according to the invention when it is not detectable by the assay methods described elsewhere herein. Methods of determining the activity of a nicotine demethylase polypetide in converting nicotine to nornicotine in a tobacco plant using gas chromatography are disclosed in the examples here in below.

In some embodiments, the favorable mutation is introduced into a tobacco plant or plant part using a mutagenesis approach, and the introduced mutation is selected using methods known to those of skill in the art such as, but not limited to, Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. A plant or plant part altered or modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to modulate the concentration and/or activity of polypeptides of the present invention in the plant. Plant forming conditions are well known in the art and discussed briefly elsewhere herein.

A modified tobacco plant containing a favorable mutation in a nicotine demethylase described herein has a reduced level of conversion of nicotine to nornicotine. In particular embodiments, conversion of nicotine to nornicotine in a modified tobacco plant or plant part according to the invention is less than <NUM>%, less than <NUM>%, less than <NUM>% less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>% less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>% of the conversion in a tobacco plant that that has not been genetically modified to inhibit the expression of that nicotine demethylase polypeptide and which has been cultured and harvested using the same protocols. In some embodiments, the modified tobacco plant is a converter tobacco plant. In other embodiments, the modified tobacco plant is a nonconverter tobacco plant. In some embodiments, the modified tobacco plant has a conversion rate lower than the rate observed in commercial non-converter tobacco plants.

According to the present invention, changes in levels, ratios, activity, or distribution of CYP82E10 polypeptides, or changes in tobacco plant or plant part phenotype, particularly reduced accumulation of nornicotine and its carcinogenic metabolite, NNN, could be measured by comparing a subject plant or plant part to a control plant or plant part, where the subject plant or plant part and the control plant or plant part have been cultured and/or harvested using the same protocols. As used herein, a subject plant or plant part is one in which genetic alteration, for example, by mutagenesis, has been affected as to the nicotine demethylase polypeptide of interest, or is a tobacco plant or plant part that is descended from a tobacco plant or plant part so altered and which comprises the alteration. A control plant or plant part provides a reference point for measuring changes in phenotype of the subject plant or plant part. The measurement of changes in phenotype can be measured at any time in a plant or plant part, including during plant development, senescence, or after curing. In other embodiments, the measurement of changes in phenotype can be measured in plants grown under any conditions, including from plants grown in growth chamber, greenhouse, or in a field. In one embodiment, changes in phenotype can be measured by determining the nicotine to nornicotine conversion rate. In a preferred embodiment, conversion can be measured by dividing the percentage of nornicotine (as a percentage of the total tissue weight) by the sum of the percentage nicotine and nornicotine (as percentages of the total tissue weight) and multiplying by <NUM>.

According to the present invention, a control plant or plant part may comprise a wild-type tobacco plant or plant part, i.e., of the same genotype as the starting material for the genetic alteration that resulted in the subject plant or plant part. A control plant or plant part may also comprise a tobacco plant or plant part of the same genotype as the starting material but that has been transformed with a null construct (i.e., with a construct that has no known effect on the trait of interest, such as a construct comprising a selectable marker gene). In all such cases, the subject plant or plant part and the control plant or plant part are cultured and harvested using the same protocols.

The activity of a nicotine demethylase polypeptide may be reduced or eliminated by disrupting the gene encoding the nicotine demethylase polypeptide. The invention provides mutagenized plants that carry mutations in nicotine demethylase genes, where the mutations reduce expression of the nicotine demethylase gene or inhibit the activity of an encoded nicotine demethylase polypeptide.

The gene encoding the nicotine demethylase polypeptide may be disrupted by any method known in the art, for example, by transposon tagging or by mutagenizing plants using random or targeted mutagenesis and selecting for plants that have reduced nicotine demethylase activity or mutations in CYP82E10, alone or in combination with mutations in CYP82E4 or CYP82E5.

Transposon tagging may be used to reduce or eliminate the activity of one or more CYP82E10 nicotine demethylase polypeptides. Transposon tagging comprises inserting a transposon within an endogenous nicotine demethylase gene to reduce or eliminate expression of the nicotine demethylase polypeptide.

Methods for the transposon tagging of specific genes in plants are well known in the art. See, for example, <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>).

Additional methods for decreasing or eliminating the expression of endogenous genes in plants are also known in the art and can be similarly applied to the instant invention. These methods include other forms of mutagenesis, using mutagenic or carcinogenic compounds including ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted. For examples of these methods see<NPL>;<NPL>; and<NPL>. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention.

Mutations that impact gene expression or that interfere with the function of the encoded nicotine demethylase protein can be determined using methods that are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues can be particularly effective in inhibiting the metabolic function of the encoded protein. Conserved residues of plant nicotine demethylase polypeptides suitable for mutagenesis with the goal to eliminate activity of a nicotine demethylase polypeptide in converting nicotine to nornicotine in a tobacco plant or plant part have been described. See Figure 1A-C of <CIT>, where the residues that differ from the other P450 polypeptides are shaded in grey. The conserved residue is that which is not shaded in grey at each position. Such mutants can be isolated according to well-known procedures.

Dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of a duplicated gene locus. See, for example, <NPL>.

The CYP82E10 polynucleotides, and variants and fragments thereof, can be used in the methods to inhibit expression or function of CYP82E10 nicotine demethylases that are involved in the metabolic conversion of nicotine to nornicotine in a plant. In this manner, favorable mutations as defined in the claims can be introduced into the CYP82E10 gene of interest. The methods do not depend on a particular method for introducing the favorable mutation into the CYP82E10 nicotine demethylase gene.

The methods of the invention can be used to reduce the nornicotine content, particularly 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 can also be practiced using any varieties 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 flue or bright varieties, Burley varieties, dark varieties and oriental/Turkish varieties. In some embodiments, the tobacco plant of interest is a Burley, Virginia, flue-cured, air-cured, fire-cured, Oriental, or a dark tobacco plant.

The tobacco plants and varieties described herein are suitable for conventional growing and harvesting techniques, such as cultivation in manure rich soil or without manure, bagging the flowers or no bagging, or topping or no topping. The harvested leaves and stems may be used in any traditional tobacco product including, but not limited to, pipe, cigar and cigarette tobacco, and chewing tobacco in any form including leaf tobacco, shredded tobacco, or cut tobacco.

Thus the present invention provides a tobacco plant, or plant part thereof, comprising a mutation in a gene encoding a CYP82E10 nicotine demethylase (as defined in the claims), wherein said mutation results in reduced expression or function of said.

CYP82E10 nicotine demethylases, and a reduced amount of nornicotine and N'-nitrosonomicotine. As used herein, the term "a reduced amount" or "a reduced level" is intended to refer to an amount of nornicotine and/or N'-nitrosonomicotine in a plant of the present invention or a plant part or tobacco product thereof that is less than what would be found in a plant of the genus Nicotiana or a plant part or tobacco product from the same variety of tobacco, processed (i.e., cultured and harvested) in the same manner, that has not been genetically modified for reduced nornicotine and/or N'-nitrosonornicotine. The amount of nornicotine may be reduced by about <NUM>% to greater than about <NUM>%, including greater than about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, and about <NUM>%. The conversion of nicotine to nornicotine can be less than <NUM>%, less than <NUM>%, less than <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, between <NUM>% - <NUM>%, or between <NUM>% - <NUM>% in plants, plant parts, and products, and more specifically in plants, plant parts having mutations in CYP82E10, CYP82E4v2, and CYP825v2.

The term "tobacco products" as used herein include, but are not limited to, smoking materials (e.g., any cigarette, including a cigarillo, a non-ventilated or vented recess filter cigarette, a cigar, pipe tobacco), smokeless products (e.g., snuff, chewing tobacco, biodegradable inserts (e.g., gum, lozenges, dissolving strips)). See, for example, <CIT>. The present disclosure also encompasses a range of tobacco product blends that can be made by combining conventional tobacco with differing amounts of the low nornicotine and/or N'-nitrosonornicotine tobacco described herein. The plant or plant part of the genus Nicotiana as described above may be cured tobacco.

The tobacco product reduces the carcinogenic potential of tobacco smoke that is inhaled directly with consumption of a tobacco product such as cigars, cigarettes, or pipe tobacco, or inhaled as secondary smoke (i.e., by an individual that inhales the tobacco smoke generated by an individual consuming a tobacco product such as cigars, cigarettes, or pipe tobacco). The cured tobacco described herein can be used to prepare a tobacco product, particularly one that undergoes chemical changes due to heat, comprising a reduced amount of nornicotine and/or N'-nitrosonornicotine in the smoke stream that is inhaled directly or inhaled as secondary smoke. In the same manner, the tobacco products may be useful in the preparation of smokeless tobacco products such as chewing tobacco, snuff and the like.

The tobacco products derived from the tobacco plants of the present invention thus find use in methods for reducing the carcinogenic potential of these tobacco products, and reducing the exposure of humans to the carcinogenic nitrosamine NNN, particularly for individuals that are users of these tobacco products. The following examples are offered by way of illustration and not by way of limitation.

The citations mentioned in the following discussion are provided at the close of the Experimental section.

The knowledge that CYP82E4v2 represents the nicotine demethylase locus responsible for the high nornicotine accumulation observed in Converter plants (Siminszky et al. , <NUM>), opened the door for nontransgenic, as well as transgenic, approaches toward overcoming the conversion problem and lowering the nornicotine content of the senescent, cured leaf. Specifically, it became possible for researchers to generate tobacco populations that had been exposed to a chemical mutagen, and select for individuals possessing nonfunctional alleles at the CYP82E4v2 locus. In fact, three independent groups have already generated nonconverting tobacco lines based on this strategy (Dewey et al. , <NUM>; Xu et al. , 2007b; Julio et al.

As previously reported, a tobacco plant designated <NUM> was identified from an EMS-mutagenized population of Burley line DH98-<NUM>-<NUM> and shown to possess a knockout mutation within the CYP82E4v2 gene (Dewey et al. In the summer of <NUM>, plants homozygous for the <NUM> mutation were grown at the Upper Coastal Plains research station in Rocky Mount, NC, and air-cured according to standard industry practice. Alkaloid analysis of these materials was conducted using the "LC Protocol" described by Jack et al. As shown in Table <NUM>, plants possessing the <NUM> mutation averaged <NUM>% nicotine to nornicotine conversion, In contrast, ><NUM>% conversion was observed in the parental line DH98-<NUM>-<NUM>, a strong converter genotype. Nearly identical results were reported by Julio et al. (<NUM>), who recorded conversion percentages ranging from <NUM> to <NUM> for plants homozygous for a cyp82e4v2 knockout mutant within the strong converter burley genotype BB16NN (parental conversion rates ranged between <NUM> - <NUM>%). Thus, debilitating mutations in CYP82E4v2 alone appear to be effective in eliminating the problems arising from the unstable genetic phenomenon associated with the generation of Converter plants.

Although the utilization of tobacco plants possessing the <NUM>, or comparable, mutations in CYP82E4v2 can be an effective means of eliminating the introduction of Converter plants within tobacco populations, a low, but significant amount of nornicotine remains in these plants. Given that nicotine to nornicotine conversion rates as low as <NUM>% were observed in transgenic plants expressing an RNAi-based construct directed against CYP82E4v2 (Lewis et al. , <NUM>), it was apparent that at least one other gene with high DNA sequence homology to CYP82E4v2 must be responsible for the majority of the nornicotine synthesis that is observed within both Nonconverter plants and Converter plants possessing an inactivated CYP82E4v2 gene. This possibility was further supported by the discovery of CYP82E5v2, a gene that shares <NUM>% DNA sequence identity with CYP82E4v2 that was also shown to encode a functional nicotine demethylase enzyme (Dewey et al. , <NUM>; Gavilano and Siminszky, <NUM>). The CYP82E5v2 nicotine demethylase gene is expressed at low levels in green tobacco leaves of Converter and Nonconverter plants alike, in contrast to CYP82E4v2 which is expressed at very high levels, but only in the leaves of Converter plants during senescence and air-curing.

As outlined in Dewey et al. (<NUM>), screening of an EMS-mutagenized DH98-<NUM>-<NUM> tobacco population lead to the identification of an individual (plant <NUM>) possessing a knockout 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 <NUM> and <NUM>. Molecular genotyping of numerous F<NUM> individuals derived from the F<NUM> 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 <NUM> field trial. Despite the fact the CYP82E5v2 has been shown to encode a functional nicotine demethylase enzyme (Dewey et al. , <NUM>; Gavilano and Siminszky, <NUM>), combining the dysfunctional cyp82e5v2 mutation with the knockout cyp82e4v2 mutation had remarkably little impact on leaf nornicotine levels. As shown in Table <NUM>, plants homozygous for the double mutation (e4e4/e5e5) averaged <NUM>% nicotine conversion, compared with an average of <NUM>% conversion for plants possessing only the cyp82e4v2 mutation (e4e4). The modest difference in mean conversion between the two genotypes was not statistically significant (P = <NUM>). In contrast, one of the CYP82E4v2 RNAi-silenced transgenic lines that was included in this study averaged <NUM>% conversion, an amount significantly lower (P < <NUM>) than that obtained from either the e4e4 or e4e4/e5e5 genotypes. Thus, another gene with high homology to CYP82E4v2 must exist within the tobacco genome that contributes toward nornicotine production in the plant.

To identify other genes in the tobacco genome that have the potential of encoding nicotine demethylase enzymes, homology searches using the BLASTN and BLASTX algorithms (Altschul et al. , <NUM>, <NUM>) were directed against the N. tabacum expressed sequenced tagged (EST) databases in GenBank, using the DNA and protein sequences of CYP82E4v2 as the respective query sequences. In addition to identifying cDNA 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 gene family. Interestingly, all seven of the ESTs originated from either root-specific cDNA libraries, or cDNA libraries made up of mixed tissues that included roots. This observation suggested that the new CYP82E gene is expressed specifically in root tissue, a property that could explain why this particular member of the CYP82E P450 superfamily has eluded detection previously, as prior efforts have focused on the characterization of CYP82E genes expressed in leaf tissue. Because no individual EST sequence was long enough to cover the entire coding region of this novel gene, PCR primers were designed that enabled amplification of the entire cDNA sequence from first-strand cDNA that had been generated from RNA isolated from tobacco root tissue. In addition, primers were used to amplify the corresponding genomic region of the gene that includes a central, large intron. This novel CYP82E cDNA shares <NUM>% nucleotide identity with the tobacco CYP82E4v2 cDNA, and a <NUM>% predicted identity at the amino acid level. In keeping with the guidelines for P450 gene nomenclature, this new gene was designated CYP82E10. Of all the characterized members of the CYP82E superfamily, CYP82E10 displays that highest sequence similarity with CYP82E5v2, sharing <NUM>% nucleotide identity at the cDNA level and <NUM>% predicted amino acid sequence identity. The DNA sequence of CYP82E10 and its predicted protein sequence are shown in <FIG>.

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 due primarily to the substantial sequence divergence observed within the large, central intron. An alignment of CYP82E4v2, CYP82E5v2, and CYP82E10 genomic sequences is shown in <FIG>. As calculated using the EMBOSS Pairwise Alignment algorithm (www. uk/Tools/emboss/align/index. html), the CYP82E4v2 and
CYP82E10 genes share <NUM>% nucleotide identity, and CYP82E10 is <NUM>% identical to the CYP82E5v2 gene as they exist within the tobacco genome (CYP82E4v2 and CYP82E5v2 genomic sequences share <NUM>% identity).

As detailed in several publications, most of the genes of the CYP82E superfamily that are found in the tobacco genome do not encode functional nicotine demethylase enzymes (Siminszky et al. , <NUM>; Chakrabarti et al. , <NUM>; Dewey et al. , <NUM>; Gavilano et al. , <NUM>; Xu et al. Therefore, sequence homology alone is not a very accurate indicator of gene function for the CYP82E family. Instead, expression analysis in either transgenic plants (Siminszky et al. , <NUM>) or in yeast (Gavilano and Siminszky, <NUM>; Xu et al. , 2007a) has become the established means for determining whether individual members of this gene family encode nicotine demethylase activity.

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 a yeast cell line that was engineered to overexpress the yeast NADPH-dependent P450 reductase, an enzyme that serves as the direct electron donor to P450s; this system greatly enhances the detection of foreign P450 enzyme activities that are expressed in yeast (Pompon et al. Nicotine demethylase assays were conducted by incubating yeast microsomal membrane preparations with [<NUM>C]-nicotine, and resolving the products by thin layer chromatography as described in Siminszky et al.

As shown in <FIG>, no nicotine demethylase activity could be detected using yeast microsomes from the W(R) strain expressing only the pYeDP60 vector. In contrast, a very robust nicotine demethylase activity could be measures from microsomes derived from yeast cells expressing the CYP82E10 cDNA. By measuring CYP82E10 enzyme activity across a wide range of [<NUM>C]-nicotine concentrations, a substrate saturation curve was established and an apparent Km of <NUM> nicotine was calculated using the microsomal assay. This kinetic parameter for CYP82E10 is very similar to the Kms reported for the CYP82E4v2 and CYP82E5v2 enzymes when similarly expressed in yeast (Gavilano et al. , <NUM>; Gavilano and Siminszky, <NUM>; Xu et al.

In order to accurately assess the specific contribution of CYP82E10 toward the total nornicotine content of the tobacco plant, it was necessary to: (<NUM>) identify a tobacco plant with a knockout mutation within this gene; and (<NUM>) combine this mutation with the cyp82e4v2 and cyp82e5v2 mutations originating from plants <NUM> and <NUM>, respectively. To identify potentially debilitating mutations in CYP82E10, the EMS-mutagenized DH98-<NUM>-<NUM> population was screened 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 <NUM> of CYP82E10, the following PCR primers were used: <NUM>'-GTGATAGTTTGATTCCCAAGTGC-<NUM>' (forward) and <NUM>'-CTCCCAAAGTTAGATTAGTCCG-<NUM>' (reverse); specific amplification of exon <NUM> was achieved using the primers <NUM>'-AGGTCGCGCTGATTCTTG-<NUM>' (forward) and <NUM>'-AGATGAATACCCATCTATCTAGGAGT-<NUM>' (reverse). To ensure maximal specificity, the reverse primer for exon <NUM> and the forward primer for exon <NUM> correspond to sequences within the CYP82E10 intron (<FIG>). PCR amplification and sequence analysis of the mutagenized plants was conducted using a <NUM>-well format as described in Dewey et al.

High-throughput sequence analysis of over <NUM>,<NUM> individuals from the mutagenized tobacco population resulted in the identification of <NUM> individuals with mutations in CYP82E10. The most notable of these are shown in Table <NUM>. The nucleotide and amino acid residues mutated in these plants are also highlighted in <FIG>. Although no truncation mutations were observed among these individuals, in several cases, mutations were identified that altered 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 shown in Table <NUM> into the CYP82E10 cDNA within the pYeDP60 yeast expression vector. Microsomal preparations from yeast strains expressing each of the seven CYP82E10 variants were assayed in vitro for nicotine demethylase activity using both non-saturating (<NUM>) and saturating (<NUM>) concentrations of [<NUM>C]-nicotine. The results from the yeast expression assays showed that mutations found plants <NUM>, <NUM> and <NUM> did not alter enzyme activity, whereas the mutations found in plants <NUM>, <NUM> and <NUM> lead to complete enzyme inactivation. The mutation observed in plant <NUM> resulted in a <NUM>% reduction in activity compared to the wild type CYP82E10 enzyme.

The thin layer chromatographic data for the in vitro yeast expression assays corresponding to the plant <NUM> mutation are shown in <FIG>. This particular mutation was selected for more extensive investigation. To provide additional confirmation that the Pro to Ser substitution at amino acid position <NUM> that defines the plant <NUM> 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 displayed in Table <NUM>. Whether introduced into the CYP82E10 or CYP82E4v2 enzymes, a Ser substitution for Pro at position <NUM> leads to the complete ablation of nicotine demethylase activity in this assay. Interestingly, although the activities of the wild type CYP82E10 and CYP82E4v2 enzymes were comparable at the non-saturating [<NUM>C]-nicotine concentration (<NUM>), at the <NUM> substrate level, the rate of [<NUM>C]-nornicotine synthesis was nearly three times greater in microsomal preparations possessing the CYP82E10 enzyme than preparations containing CYP82E4v2.

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

Given that the original <NUM> mutation is in a genetic background (DH98-<NUM>-<NUM>) that contains both a strong converter CYP82E4v2 allele as well as a wild type CYP82E5v2 gene, the only way to accurately assess the specific contribution of CYP82E10 toward total plant nornicotine content is to introduce the <NUM> mutation into tobacco plants possessing knockout CYP82E4v2 and CYP82E5v2 mutations as well. To accomplish this, plants heterozygous for the <NUM> mutation (e10E10) were crossed with plants heterozygous for both the <NUM> and <NUM> mutations described above (e4E4/e5E5). The latter plants represent progeny of from the cross <NUM>/<NUM>//TN90/<NUM>/TN90/<NUM>/TN90. F<NUM> plants heterozygous for all three nicotine demethylase mutations (e4E4/e5E5/e10E10) were identified by molecular genotyping, and allowed to self-pollinate. Molecular genotyping was also used to screen over <NUM> F<NUM> progeny and subsequently group them into the following genotypic classes: E4E4/E5E5/e10e10 (<NUM> plants total); e4e4/E5E5/e10e10 (<NUM> plants total); E4E4/e5e5/e10e10 (<NUM> plants total); and e4e4/e5e5/e10e10 (<NUM> plants total).

All of the plants described above were transplanted and grown in the field at the Upper Coastal Plains research station in Rocky Mount, NC in the summer of <NUM>. Also included in this study were two of the genotypes tested in the <NUM> field trial shown in Table <NUM>. Specifically, ten DH98-<NUM>-<NUM> plants homozygous for only the cyp82e4v2 mutation (e4e4/E5E5/E10E10) and eleven DH98-<NUM>-<NUM> plants possessing the double homozygous e4e4/e5e5/E10E10 genotype were included for comparison. As controls, individual plants randomly selected from a commercial "low converter" seedlot (TN90LC), wild type DH98-<NUM>-<NUM> individuals, and plants from one of the best CYP82E4v2 RNAi-suppressed transgenic lines was also included in the study. After the plants were about an average of <NUM> tall (<NUM> days after transplanting) leaves from similar stalk positions where collected, treated with ethephon and air-cured according to the protocol established by Jack et al. Alkaloid content of the cured leaf materials was determined by gas chromatography as described in the same protocol.

Table <NUM> and <FIG> shows the results of the alkaloid analyses for the <NUM> field trial. Consistent with previous observations, the cyp82e4v2 knockout mutation alone negates the strong converter phenotype of line DH98-<NUM>-<NUM>, and also confers a substantially lower nornicotine accumulation phenotype than plants from the commercial TN90LC seed (<NUM>% conversion versus <NUM>%, respectively). As observed in the <NUM> field trial (Table <NUM>), combining the cyp82e5v2 mutation with cyp82e4v2 did not lead to further reductions in nornicotine content. In fact, the mean nicotine conversion for the e4e4/E5E5/E10E10 plants was actually lower than that observed for e4e4/e5e5/E10E10 individuals (<NUM>% versus <NUM>%), though this slight difference was not statistically significant. As expected, the cyp82e10 mutation had no impact on the high nornicotine levels conferred by an active CYP82E4v2 gene, either alone (E4E4/E5E5/e10e10 genotypes), or when combined with a mutant cyp82e5v2 allele (E4E4/e5e5/e10e10 genotypes) (<FIG>). Similar to the cyp82e4v2 and cyp82e5v2 double mutant results (Tables <NUM> and <NUM>), introducing cyp82e10 into a cyp82e4v2 background was not effective in reducing nornicotine levels below than that which could be achieved by the cyp82e4v2 mutation alone (<FIG>). The e4e4/E5E5/e10e10 genotypes averaged <NUM> % conversion which was not significantly different than the <NUM>% mean conversion levels observed for e4e4/E5E5/E10E10 individuals (P = <NUM>).

Although the cyp82e5v2 and cyp82e10 mutations did not serve to significantly decrease the nornicotine content of cyp82e4v2 plants when combined individually, pyramiding all three nicotine demethylase mutations had a very notable effect. Nicotine to nornicotine conversion in triple mutant plants (e4e4/e5e5/e10e10) averaged only <NUM> %, a percentage virtually identical to the <NUM>% observed in the RNAi-suppressed transgenic line (P = <NUM>; <FIG>). This represents over a <NUM>-fold reduction in nicotine conversion beyond that which was mediated by the cyp82e4v2 mutation alone. Statistically, the differences in percent nicotine conversion (and nornicotine accumulation as a percentage of total dry weight) between e4e4/E5E5/E10E10 and e4e4/e5e5/e10e10 genotypes was highly significant (P < <NUM>). Similar to the investigation of RNAi-mediated suppression of nicotine conversion (Lewis et al. , <NUM>), the present nontransgenic alteration of nicotine demethylase activities in the tobacco plant did not appear to significantly alter the content of the minor alkaloid species anatabine and anabasine.

The effects of pyramiding the three independent nicotine demethylase gene mutations were also tested in a field trial conducted during the <NUM> growing season. For this study, the crosses were conducted entirely within the DH98-<NUM>-<NUM> genetic background (in contrast to the <NUM> study where a TN90 parent was also used). Molecular genotyping was again used to create every possible combination needed to determine the respective contributions of each CYP82E locus on the nornicotine phenotype. Alkaloid data were collected on tobacco plants that were grown to maturity and cured according to standard industry practice. As shown in Table <NUM>, a high level of nicotine conversion (ranging from <NUM> - <NUM>%) 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 just the cyp82e4v2 mutation (e4e4/E5E5/E10E10) averaged <NUM>% nicotine to nornicotine conversion. Similar to the <NUM> results, the effects of the cyp82E5v2 and cyp82E10 mutations were not additive, and were only manifest when all three mutant loci were pyramided together. DH98-<NUM>-<NUM> (e4e4/E5E5/e10e10) plants averaged <NUM>% conversion and DH98-<NUM>-<NUM> (e4e4/e5e5/E10E10) individuals averaged <NUM>%, values that were not statistically different than that observed with the cyp82e4v2 mutation alone. In contrast, the reduction in nornicotine observed in the triple mutant DH98-<NUM>-<NUM> (e4e4/e5e5/e10e10) genotype (<NUM>% nicotine conversion) was <NUM>-fold lower than that attained via the cyp82e4v2 mutation alone. The reduction in nicotine conversion attributable to the triple mutant combination was highly significant (P<<NUM>) compared with either cyp82e4v2 alone or any double mutant combination.

Claim 1:
A tobacco plant, or plant part thereof, comprising a mutation in a gene encoding a CYP82E10 nicotine demethylase, wherein said mutation is in a gene encoding a CYP82E10 nicotine demethylase which comprises an amino acid sequence having at least <NUM>% sequence identity to SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, said mutation results in a modification of the amino acid sequence of said CYP82E10 nicotine demethylase at an amino acid residue corresponding to position <NUM>, <NUM> or <NUM> of SEQ ID NO: <NUM>, or any combination thereof, and wherein said mutation results in reduced expression or function of said CYP82E10 nicotine demethylase.