Method for reducing nitrosamine content in tobacco using constitutive high affinity cationic efflux proteins

Abstract

The present invention relates to a method for reducing the content of at least one tobacco-specific nitrosamine (TSNA) or TSNA precursor in tobacco, which comprises expressing a deregulated cationic efflux protein in a tobacco plant or plant part or plant cell thereof.

Description

Technical Field

[0001] The present invention relates to reducing the specific nitrosamines or their precursors in tobacco and products derived therefrom (e.g., propagation material, harvested leaves, processed tobacco and tobacco industry products). In particular, the present invention relates to cationic efflux proteins and their use in regulating (e.g., reducing) the specific nitrosamines or their precursors in tobacco. Background Art

[0002] Tobacco pyridine alkaloids are precursors to tobacco-specific nitrosamines (TSNAs), which are formed during the curing of leaves after harvest. The four main TSNAs found in flue-cured tobacco leaves are N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanatabine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). TSNAs ( Figure 1 ). NAT and NAB are formed via the nitrosation of the secondary alkaloids anatabine and anabasine, respectively. Although early studies claimed that NNN was derived from both nicotine and nornicotine, recent reports have confirmed that the appearance of NNN in flue-cured tobacco leaves is associated with nornicotine content rather than nicotine (Bush et al., Rec. Adv. Tob. Sci. 27; 23-46 (2001); Lewis et al., Plant Biotech J. 6: 346-354 (2008)). Nornicotine is a demethylated derivative of nicotine, which is the main alkaloid in tobacco, accounting for 90% of the total alkaloid content (Saitoh et al., 1985 Phytochemistry, 24, pp. 477-480). The precursor / product relationship of NNK formation is less clear. Some studies indicate that NNK is a nitrosation product of nicotine, but since the reaction rate of nicotine nitrosation is slow, it is likely that oxidative derivatives of nicotine rather than nicotine itself serve as the direct precursor of NNK (Caldwell et al. Ann. NY Acad. Sci. 686, 213-228 (1993)). It is of great importance to identify the genes responsible for the production and regulation of TSNA precursors.

[0003] Although nornicotine usually accounts for only 2-4% of the total pyridine alkaloid content in tobacco plants, genetic instability leading to the spontaneous emergence of plants with high nornicotine content conversion is a long-standing problem in tobacco production. Maintaining low nornicotine levels can prevent the unpleasant flavor and aroma associated with this alkaloid, as well as reduce the formation of N-nitrosonornicotine (NNN), for which nornicotine is the direct precursor, in tobacco industry products.

[0004] The gene responsible for most of the nicotine to nornicotine conversion is the nicotine demethylase gene CYP82E4, which encodes a cytochrome P450 monooxygenase (Siminszky et al., Proc. Natl. Acad. Sci. USA, 102 (2005), pp. 14919-14924; Xu et al., Physiol. Plantarum, 129 (2007), pp. 307-319). The nicotine demethylase gene family in tobacco has been extensively characterized, but little is known about other cellular processes that can affect nornicotine levels.

[0005] There remains a great need to devise methods that can further reduce TSNA levels in tobacco plants and products produced from tobacco plants.

[0006] Cationic efflux proteins are pumps that remove metal ions such as cadmium (Cd), zinc (Zn) and cobalt (Co) from cells. These metals are not only essential micronutrients for plant metabolism, they also act as second messengers, affecting several enzymatic reactions and signal transduction cascades. Co, Cd and Zn increase the activity of N-demethylases, cytochrome P-450 and microsomal heme content in mice (Kadiiska et al. E. Arch Toxicol (1985) 56: 167). It is proposed that these cations exert enzyme induction effects on liver monooxygenases.

[0007] Cationic efflux proteins typically include an N-terminal cytoplasmic domain that often contains a metal binding site, 4-6 transmembrane domains, followed by a C-terminal cation efflux domain. It is proposed that by interacting with key metal binding residues in the central cytoplasmic domain, the N-terminal cytoplasmic domain shows negative regulation of the protein, preventing metal transport. When the metal concentration in the cell increases to a certain threshold, the combination of the metal and the N-terminal domain induces a conformational change that relaxes the inhibition of the cytoplasmic binding site. Cationic efflux becomes a high affinity state and allows metal transport (Futai et al., Handbook of ATPases: Biochemistry, Cell Biology, Pathophysiology 2004). Summary of the invention

[0008] According to one aspect, the present invention provides a method for reducing the level of at least one tobacco-specific nitrosamine (TSNA) or TSNA precursor in tobacco, comprising expressing a deregulated cationic efflux protein in a tobacco plant or plant part or plant cell thereof.

[0009] The cation efflux protein of deregulation can be a constitutive high affinity cation efflux transporter. Compared with the wild-type cation efflux transporter, the cation efflux protein of deregulation can show increased metal ion transport. The cation efflux protein of deregulation can lack a functional regulatory domain, preferably, the cation efflux protein of deregulation lacks at least a portion of a regulatory domain. The cation efflux protein of deregulation can lack a functional cytoplasmic domain, preferably, the cation efflux protein of deregulation lacks at least a portion of a cytoplasmic domain. When compared with a wild-type cation efflux protein comprising an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto, the cation efflux transporter of deregulation can be deregulated. The deregulated cation efflux transporter may comprise one or more mutations compared to an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto. The method may comprise introducing mutations within a polynucleotide encoding a cation efflux protein into the genome of the plant or plant cell such that the polynucleotide encodes a deregulated cation efflux protein. The mutations introduced into the plant genome may generate deletions, splice mutants or codons encoding non-tolerant amino acid substitutions in the polynucleotide encoding the protein.

[0010] The deregulated cation efflux protein may comprise an amino acid sequence lacking at least a portion of the N-terminus when compared to an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto. The method may comprise introducing an exogenous polynucleotide sequence into the genome of the plant or plant cell, the exogenous polynucleotide sequence encoding the deregulated cation efflux protein.

[0011] Deregulated cation efflux proteins can be:

[0012] a) a truncated cationic efflux protein lacking at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein lacking at least the amino acids corresponding to amino acids 2-25, or 2-50, or 2-75, or 2-100, 2-114 or 2-117 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0013] b) a truncated cationic efflux protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30.

[0014] The TSNA is N'-nitrosonornicotine (NNN) and / or the precursor may be nornicotine.

[0015] In another aspect, a method of producing tobacco having a reduced content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors is provided, comprising:

[0016] a. crossing a donor tobacco plant that produces reduced levels of tobacco-specific nitrosamines (TSNAs) or TSNA precursors and comprises a deregulated cationic efflux protein with a recipient tobacco plant that does not produce reduced levels of tobacco-specific nitrosamines (TSNAs) or TSNA precursors and has a commercially desirable trait;

[0017] b. separating genetic material from offspring of the donor plant hybridized with the recipient plant; and

[0018] c. performing molecular marker-assisted selection using molecular markers, comprising:

[0019] i. Identifying introgression regions comprising mutations in the polynucleotide sequence encoding the protein defined in a.

[0020] The deregulated cation efflux protein may comprise an amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto. The deregulated cation efflux protein may be encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 34, or a sequence having at least 70% sequence identity thereto, preferably a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0021] In another aspect, an isolated polynucleotide is provided, comprising a nucleotide sequence selected from the group consisting of:

[0022] a. comprising the nucleotide sequence of SEQ ID No.34;

[0023] b. a nucleotide sequence having at least 70% sequence identity to the entirety of the sequence shown in SEQ ID No. 34, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants;

[0024] c. a nucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No. 33 herein, or a fragment thereof comprising at least 200, or at least 250, or at least 300, or at least 350, or at least 400 consecutive residues of SEQ ID No. 33;

[0025] d. A nucleotide sequence encoding a truncated polypeptide or protein, wherein the truncated polypeptide lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and the protein has at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, wherein the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and the protein has at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, and the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130;

[0026] e. A nucleotide sequence encoding a truncated polypeptide or protein, the truncated polypeptide consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, the protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein, the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0027] f. a nucleotide sequence encoding a polypeptide comprising an amino acid sequence corresponding to amino acids 125-324 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and / or comprising an amino acid sequence corresponding to amino acids 323-413 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants;

[0028] g. A nucleotide sequence according to any one of the preceding items (a) to (f), further comprising a mutation in the nucleotide sequence, for example wherein the mutation produces a deletion, a splice mutant or a codon encoding a non-tolerant amino acid substitution in the polynucleotide encoding the protein; or

[0029] h. A nucleotide sequence complementary to the sequence according to any one of the aforementioned items (a) to (g).

[0030] In a further aspect, an isolated polypeptide is provided, comprising an amino acid sequence selected from the group consisting of:

[0031] a. comprising an amino acid sequence of SEQ ID No.33;

[0032] b. an amino acid sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID No.33;

[0033] c. an amino acid sequence comprising amino acids corresponding to amino acids 125-324 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and / or comprising amino acids corresponding to amino acids 323-413 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, wherein the polypeptide is involved in cation efflux in plants;

[0034] d. It is an amino acid sequence of a fragment of SEQ ID No.3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, said fragment comprising at least 200, or at least 250, or at least 300, or at least 350, or at least 400 consecutive residues of SEQ ID No.3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0035] e. an amino acid or protein which is a truncated amino acid sequence, said truncated amino acid sequence lacking at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, said protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, said truncated protein lacking at least the amino acids corresponding to SEQ ID No. amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 of ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30;

[0036] f. an amino acid or protein which is a truncated amino acid sequence, the truncated amino acid sequence consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, the protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein, the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0037] or

[0038] g. An amino acid sequence having at least 70% sequence identity to the entirety of the sequence shown in SEQ ID No. 33.

[0039] In a further aspect, a construct or vector is provided, comprising a polynucleotide according to the invention or a polynucleotide encoding a polypeptide according to the invention.

[0040] In another aspect, provided is a tobacco plant or part thereof, which:

[0041] a. modified to achieve a reduction in TSNA or TSNA precursors compared to an unmodified plant, wherein the modified plant or part thereof comprises a deregulated cationic efflux protein;

[0042] b. having a mutation in a plant gene encoding a cationic efflux protein, wherein the mutation deregulates the cationic efflux protein, wherein the gene before the mutation comprises a sequence as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7, SEQ ID No.8, SEQ ID No.10, SEQ ID No.11, SEQ ID No.13, SEQ ID No.14, SEQ ID No.16, SEQ ID No.17, SEQ ID No.19, SEQ ID No.20, SEQ ID No.22, SEQ ID No.23, SEQ ID No.25, SEQ ID No.26, SEQ ID No.28 or SEQ ID No.29, or a sequence having at least 70% sequence identity thereto;

[0043] c. obtained or obtainable by the method according to the present invention;

[0044] d. comprising an exogenous gene encoding a deregulated cation efflux protein, wherein the protein comprises a truncated amino acid sequence which lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, wherein the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130;

[0045] e. comprising a deregulated cationic efflux protein comprising the amino acid sequence shown in SEQ ID No.33 or a sequence having at least 70% identity thereto, or the polynucleotide sequence shown in SEQ ID No.34 or a sequence having at least 70% identity thereto; or

[0046] f. comprising a construct or a vector according to the invention.

[0047] In another aspect, provided is a cell, which:

[0048] a. comprising a deregulated cation efflux protein;

[0049] b. having a mutation in a gene encoding a cationic efflux protein, wherein the mutation deregulates the cationic efflux protein, wherein the gene before the mutation comprises a sequence as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7, SEQ ID No.8, SEQ ID No.10, SEQ ID No.11, SEQ ID No.13, SEQ ID No.14, SEQ ID No.16, SEQ ID No.17, SEQ ID No.19, SEQ ID No.20, SEQ ID No.22, SEQ ID No.23, SEQ ID No.25, SEQ ID No.26, SEQ ID No.28 or SEQ ID No.29, or a sequence having at least 70% sequence identity thereto;

[0050] c. obtained or obtainable by the method according to the invention;

[0051] d. comprising an exogenous gene encoding a deregulated cation efflux protein, wherein the protein comprises a truncated amino acid sequence which lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, wherein the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130;

[0052] e. comprising a deregulated cationic efflux protein comprising the amino acid sequence shown in SEQ ID No.33 or a sequence having at least 70% identity thereto, or the polynucleotide sequence shown in SEQ ID No.34 or a sequence having at least 70% identity thereto; or

[0053] f. comprising a construct or a vector according to the invention.

[0054] The cell can be a plant cell (eg, a tobacco plant cell) or a yeast cell.

[0055] In a further aspect, provided is a cell culture comprising a cell or cell population according to the invention.

[0056] In another embodiment, provided is plant propagation material (eg, plant seeds) obtainable from a plant according to the invention.

[0057] Plants for use in the present invention may be from the species Nicotiana tabacum or Nicotiana rustica.

[0058] In another aspect, provided is the use of a cell according to the invention, or a tobacco plant or tobacco plant part thereof according to the invention, or a cell culture according to the invention, or a deregulated cationic efflux protein as defined herein for producing a tobacco industry product.

[0059] In a further aspect, provided is a tobacco plant according to the invention, or the use of a plant propagation material according to the invention to grow a tobacco plant.

[0060] In a further aspect, provided is the use of a tobacco plant according to the invention, or a plant propagation material according to the invention, for growing crops.

[0061] In another aspect, provided is the use of a tobacco plant according to the invention, or a plant propagation material according to the invention, for producing processed (preferably cured) tobacco leaves.

[0062] In another aspect, provided are harvested leaves of a tobacco plant according to the invention, or harvested leaves of a tobacco plant obtainable from propagation material according to the invention, or harvested leaves of a tobacco plant obtainable by a method according to the invention, or harvested leaves of a tobacco plant obtainable by use according to the invention.

[0063] In one aspect, provided are harvested leaves of a tobacco plant according to the present invention, wherein the harvested leaves are shredded harvested leaves.

[0064] In a further aspect, provided is processed tobacco leaf (preferably non-viable processed tobacco leaf) which:

[0065] a. comprising a plant cell according to the present invention;

[0066] b. obtainable from processing tobacco plants according to the present invention;

[0067] c. a tobacco plant obtainable from the plant propagation material according to the invention propagated; or

[0068] d. can be obtained by processing the harvested leaves according to the present invention;

[0069] Processed tobacco leaves may be processed by curing, fermentation, pasteurization or a combination thereof. Processed tobacco leaves may be cut processed tobacco leaves.

[0070] In a further aspect, the present invention provides a flue-cured tobacco material made from a plant or part thereof according to the present invention, or obtained (or obtainable) from a plant propagation material according to the present invention, or obtained (or obtainable) by a method according to the present invention.

[0071] In one aspect, there is provided a tobacco blend comprising a flue-cured tobacco material according to the present invention.

[0072] In another aspect, provided is a tobacco industry product which:

[0073] a. prepared from a tobacco plant or a part thereof according to the present invention;

[0074] b. prepared from a tobacco plant or part thereof (preferably leaves harvested from a plant) obtained or obtainable by the method according to the present invention;

[0075] c. prepared from plants (preferably leaves) propagated from plant propagation material according to the present invention;

[0076] d. prepared from harvested leaves according to the present invention;

[0077] e. Prepared by processing blades according to the present invention;

[0078] f. prepared by cells according to the present invention;

[0079] g. or prepared by a cell culture according to the present invention;

[0080] h. prepared from a flue-cured tobacco material according to the present invention; or

[0081] i. Prepared from a tobacco blend according to the present invention.

[0082] The tobacco industry product may be a combustible smoking article or a smokeless tobacco industry product or a non-combustible aerosol delivery system, such as a tobacco heating device (eg, an aerosol generating device).

[0083] In another aspect, provided is a combustible smoking article, a non-combustible gas mist supply system, a smokeless tobacco industrial product or a tobacco heating device comprising a plant or part thereof from the species Nicotiana tabacum or Nicotiana rustica according to the invention, or obtainable (e.g. obtained) from a plant propagation product according to the invention, or obtainable (e.g. obtained) from a method according to the invention.

[0084] In another aspect, provided is a nucleotide sequence encoding a deregulated cation efflux protein comprising an amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70% identity thereto, or the use of a nucleotide sequence encoding the following for selecting plants having a modified content of TSNA or TSNA precursors: a deregulated cation efflux protein, wherein the protein comprises a truncated amino acid sequence which lacks at least the amino acid sequence corresponding to the amino acid sequence from SEQ ID 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 amino acids on the N-terminal side of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with a truncated protein lacking at least the amino acid sequence corresponding to SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 amino acids.

[0085] In another aspect, provided is a plant mutant carrying a heritable mutation in a nucleotide sequence of at least one gene encoding a deregulated cation efflux protein comprising an amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70% identity thereto, or a nucleotide sequence encoding the following: a deregulated cation efflux protein, wherein the protein comprises a truncated amino acid sequence lacking at least the amino acid sequence corresponding to the amino acid sequence from SEQ ID 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 amino acids on the N-terminal side of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with a truncated protein lacking at least the amino acid sequence corresponding to SEQ ID or 2-130 of amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 of amino acids 2-3, 6, 9, 12, 15, 18, 21, 24, 27, or 30; wherein the heritable mutation reduces the content of at least one TSNA or TSNA precursor relative to a comparable plant not carrying the heritable mutation.

[0086] In a further aspect, provided are progeny or seeds of a mutant plant carrying a heritable mutation according to the invention.

[0087] In another aspect, provided is a harvested leaf, processed leaf or cured tobacco material produced by a plant comprising a nucleotide sequence encoding a deregulated cation efflux protein comprising an amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70% identity thereto, or a nucleotide sequence encoding: a deregulated cation efflux protein, wherein the protein comprises a truncated amino acid sequence lacking at least the amino acid sequence corresponding to the amino acid sequence from SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 amino acids on the N-terminal side of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with a truncated protein lacking at least the amino acid sequence corresponding to SEQ ID or 2-130 of amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, ...

[0088] In another aspect, provided is a method, tobacco leaf, tobacco plant, tobacco plant propagation material, harvested leaf, processed tobacco, tobacco industry product, cell, cell culture, use or combination thereof substantially as described herein with reference to the specification and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0090] Figure 1 It was shown that tobacco-specific nitrosamines (TSNAs) are formed from precursors such as nicotine and nornicotine via nitrosation reactions in tobacco smoke.

[0091] Figure 2 Shown are the nornicotine contents of 5-week-old tobacco leaves that have been modified by virus-induced gene silencing of a cationic efflux protein.

[0092] Figure 3Shown are the nornicotine contents of 5-week-old tobacco leaves modified to express a luciferase control (SEQ ID No. 44), a wild-type sequence (SEQ ID No. 2), an antisense (SEQ ID No. 39), a cation efflux_Δ117C variant (SEQ ID No. 32), or a cation efflux_Δ117N variant (SEQ ID No. 34).

[0093] Figure 4a Schematic representation of the proposed mechanism for low- and high-affinity confirmation of cation efflux transporters is shown.

[0094] Figure 4b Schematic diagram of the proposed mechanism for the expression of cation efflux_Δ117C and cation efflux_Δ117N is shown.

[0095] Figure 5 The genomic sequence encoding the protein according to the invention is shown - SEQ ID No. 1.

[0096] Figure 6 The cDNA sequence encoding the protein according to the present invention is shown - SEQ ID No. 2.

[0097] Figure 7 The polypeptide sequence of the protein according to the present invention is shown as SEQ ID No. 3 - the polypeptide sequence comprises a cation efflux transmembrane domain (amino acids 125-324) and a cation efflux cytoplasmic domain (amino acids 323-413).

[0098] Figure 8 The genomic sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 4.

[0099] Fig. 9 The cDNA sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 5.

[0100] Fig.10 The polypeptide sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 6.

[0101] Fig.11 The genomic sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 7.

[0102] Fig.12 The cDNA sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 8.

[0103] Fig.13The polypeptide sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 9.

[0104] Fig.14 The genomic sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 10.

[0105] Fig.15 The cDNA sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 11.

[0106] Fig.16 The polypeptide sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 12.

[0107] Fig.17 The genomic sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 13.

[0108] Fig.18 The cDNA sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 14.

[0109] Fig.19 The polypeptide sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 15.

[0110] Fig. 20 The genomic sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 16.

[0111] Fig.21 The cDNA sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 17.

[0112] Fig. 22 The polypeptide sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 18.

[0113] Fig.23 The genomic sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 19.

[0114] Fig.24 The cDNA sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 20.

[0115] Fig.25 The polypeptide sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 21.

[0116] Fig.26The genomic sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 22.

[0117] Fig. 27 The cDNA sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 23.

[0118] Fig.28 The polypeptide sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 24.

[0119] Fig.29 The genomic sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 25.

[0120] Fig.30 The cDNA sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 26.

[0121] Fig.31 The polypeptide sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 27.

[0122] Fig.32 The genomic sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 28.

[0123] Fig.33 The cDNA sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 29.

[0124] Fig.34 The polypeptide sequence encoding the homologous protein from Nicotiana tabacum is shown - SEQ ID No. 30.

[0125] Fig.35 An alignment of SEQ ID No. 3 and the homologous protein sequence from Nicotiana tabacum is shown.

[0126] Fig.36 The transmembrane domain and cytoplasmic domain of SEQ ID No. 3 and the homologous protein sequence from Nicotiana tabacum are shown. The protein sequence of SEQ ID No. 3 indicates the presence of a cation efflux transmembrane domain (amino acid positions 125-324) and a cation efflux cytoplasmic domain (amino acid positions 323-413).

[0127] Fig.37 An alignment of SEQ ID No. 3 and the homologous protein sequence from Nicotiana tabacum is shown.

[0128] Fig.38The polypeptide sequence of a mutant cationic efflux protein based on SEQ ID No. 3 is shown, wherein the last 117 amino acids of the C-terminal domain have been deleted (SEQ ID No. 31).

[0129] Fig.39 The cDNA sequence of a mutant cationic efflux protein based on SEQ ID No. 3 is shown, wherein the last 117 amino acids of the C-terminal domain have been deleted (SEQ ID No. 32).

[0130] Fig.40 The polypeptide sequence of a mutant cationic efflux protein based on SEQ ID No. 3 is shown, wherein the first 117 amino acids (ie from the N-terminal domain) have been deleted (SEQ ID No. 33).

[0131] Fig.41 The cDNA sequence of a mutant cationic efflux protein based on SEQ ID No. 3 is shown, wherein the first 117 amino acids (ie from the N-terminal domain) have been deleted (SEQ ID No. 34).

[0132] Fig.42 The sequence of primer M13.fw is shown (SEQ ID No. 36).

[0133] Fig.43 The sequence of primer M13.rv is shown (SEQ ID No. 37).

[0134] Fig.44 The sequence of the Gateway Destination Binary (GDB) expression vector is shown (SEQ ID No. 38).

[0135] Fig.45 The sequence of the cationic efflux antisense (AS) is shown (SEQ ID No. 39).

[0136] Fig.46 The sequence of primer AS.fw is shown (SEQ ID No. 40).

[0137] Fig.47 The sequence of primer AS.rv is shown (SEQ ID No. 41).

[0138] Fig.48 The sequence of primer attB1 is shown (SEQ ID No. 42).

[0139] Fig.49 The sequence of primer attB2 is shown (SEQ ID No. 43).

[0140] Fig.50 The sequence of the luciferase control sequence is shown (SEQ ID No. 44).

[0141] Fig.51 The sequence of primer Δ117C fw is shown (SEQ ID No. 45).

[0142] Fig.52 The sequence of primer Δ117C rv is shown (SEQ ID No. 46).

[0143] Fig.53 The sequence of primer Δ117N fw is shown (SEQ ID No. 47).

[0144] Fig.54 The sequence of primer Δ117N rv is shown (SEQ ID No. 48).

[0145] Fig.55 The sequence of Tobacco Rattle Virus (TRV) RNA1 is shown (SEQ ID No. 49).

[0146] Fig.56 The sequence of TRV RNA2 is shown (SEQ ID No. 50).

[0147] Fig.57 The sequence of the virus-induced gene silencing (VIGS) target sequence is shown (SEQ ID No. 51).

[0148] Fig.58 The sequence of the VIGS control sequence is shown (SEQ ID No. 52).

[0149] Fig.59 The sequence of the yeast Fw primer used in Example 5 is shown (SEQ ID No. 53).

[0150] Fig.60 The sequence of the yeast Rv primer used in Example 5 is shown (SEQ ID No. 54).

[0151] Fig.61 The sequence of the modified pTES3 vector pYES3 / UT.1 used in Example 5 is shown (SEQ ID No. 55).

[0152] Fig.62 The sequence of the Δ117C Fw primer used in Example 5 is shown (SEQ ID No. 56).

[0153] Fig.63 The sequence of the Δ117C Rv primer (SEQ ID No. 57) used in Example 5 is shown.

[0154] Fig.64 The sequence of the pTES3 vector pYES3 / UT.2 used in Example 5 is shown (SEQ ID No. 58).

[0155] Fig.65 The sequence of the Δ117N Fw primer used in Example 5 is shown (SEQ ID No. 59).

[0156] Fig.66 The sequence of the Δ117N Rv primer used in Example 5 is shown (SEQ ID No. 60).

[0157] Fig.67 Shown is the predicted 3D structure of SEQ ID No. 3. The homodimer is predicted to contain two zinc docking sites.

[0158] Fig.68 Shown are the nornicotine levels of tobacco plants treated with the indicated concentrations of zinc, cadmium or nickel.

[0159] Fig.69a Shown are the nornicotine levels of tobacco plants expressing the full-length zinc transporter, the Δ117C and Δ117N variants after treatment with the indicated concentrations of zinc.

[0160] Fig.69b Shown are the phenotypes of tobacco leaves expressing the indicated constructs after treatment with 10 mM zinc.

[0161] Fig.69c Shows Fig.69b Chlorophyll intensity of treated leaf areas in . Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with P values ​​≤ 0.001 when compared to metal-free samples and analyzed with one-way ANOVA and Tukey's multiple comparison post hoc test.

[0162] Fig.70 It was shown that expression of the Δ117N metal efflux variant induces growth retardation in yeast. DETAILED DESCRIPTION

[0163] The groundbreaking discovery of the present invention is that a deregulated cationic efflux protein can be used to modulate at least one tobacco-specific nitrosamine (TSNA) or a precursor thereof in tobacco.

[0164] Based on this discovery, deregulated (eg, constitutive high affinity cation efflux transporters) can be used to reduce TSNA or TSNA precursor levels in tobacco. In one embodiment, the TSNA is N'nitrosonornicotine (NNN) and / or the precursor is nornicotine.

[0165] Cationic efflux proteins are pumps that remove metal ions such as cadmium, zinc and cobalt from cells. These metals are not only essential micronutrients for plant metabolism, they also act as second messengers that initiate intracellular signal transduction cascades.

[0166] Based on these surprising findings, there is provided a method of reducing or lowering the content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in tobacco, comprising expressing a deregulated cationic efflux protein in a tobacco plant or plant part or plant cell thereof. Suitably, the reduction in TSNA content or TSNA precursors is compared to a tobacco plant or part thereof that does not contain a deregulated cationic efflux protein. In other words, the reduction in TSNA content or TSNA precursors is compared to a tobacco plant or part thereof that has not been modified to express a deregulated cationic efflux protein.

[0167] In one embodiment, the nicotine concentration and / or total content in tobacco plants or parts thereof, such as leaves (e.g., cured leaves), is not reduced by the method for reducing the content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in tobacco plants or plant parts thereof according to the present invention.

[0168] In one embodiment, the nicotine concentration and / or total content in tobacco plants or parts thereof, such as leaves (e.g., cured leaves), is not altered (e.g., is not significantly altered, e.g., is altered by less than 10%, less than 5%, less than 3%, less than 2%, less than 1%) by the method for reducing the content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in tobacco plants or plant parts thereof according to the present invention. In one embodiment, the nicotine concentration and / or total content in tobacco plants or parts thereof, such as leaves (e.g., cured leaves), can be increased by the method for reducing the content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in tobacco plants or plant parts thereof according to the present invention.

[0169] In a preferred embodiment, the content of TSNAs or precursors thereof is reduced in tobacco (eg, tobacco leaves) by expressing a deregulated cationic efflux protein in tobacco plants or plant parts or plant cells thereof.

[0170] In one embodiment, the method of the invention comprises introducing a mutation in a polynucleotide encoding a cation efflux protein into the genome of said plant or part thereof or plant cell. Suitably, said mutation results in a mutant polynucleotide encoding a deregulated cation efflux protein.

[0171] As used herein, a "mutation" may be a modification selected from deletion, insertion, substitution.

[0172] In a preferred embodiment, the mutation is a deletion. In another embodiment, the mutation may be a substitution with an unnatural amino acid.

[0173] In one embodiment, the method of the present invention comprises introducing a mutation into the genome of the plant in a polynucleotide encoding a protein comprising a sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto.

[0174] In one embodiment, the method of the present invention comprises introducing a mutation into the genome of the plant in a polynucleotide encoding a protein comprising a sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0175] In one embodiment, the method comprises introducing a mutation in a polynucleotide into the genome of a tobacco plant, wherein the polynucleotide comprises a sequence as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7, SEQ ID No.8, SEQ ID No.10, SEQ ID No.11, SEQ ID No.13, SEQ ID No.14, SEQ ID No.16, SEQ ID No.17, SEQ ID No.19, SEQ ID No.20, SEQ ID No.22, SEQ ID No.23, SEQ ID No.25, SEQ ID No.26, SEQ ID No.28 or SEQ ID No.29, or a sequence having at least 70% sequence identity thereto.

[0176] In one embodiment, the method comprises introducing a mutation in a polynucleotide into the genome of a tobacco plant, wherein the polynucleotide comprises a sequence as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7, SEQ ID No.8, SEQ ID No.10, SEQ ID No.11, SEQ ID No.13, SEQ ID No.14, SEQ ID No.16, SEQ ID No.17, SEQ ID No.19, SEQ ID No.20, SEQ ID No.22, SEQ ID No.23, SEQ ID No.25, SEQ ID No.26, SEQ ID No.28 or SEQ ID No.29, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0177] In one embodiment, a protein comprising a sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto, is encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 28 or SEQ ID No. 29, or a sequence having at least 70% sequence identity thereto.

[0178] In one embodiment, a protein comprising a sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto, is encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 28 or SEQ ID No. 29, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0179] The mutation may be any mutation that results in deregulated expression of the cation efflux protein.

[0180] In one embodiment, the mutation may produce a deletion, a splice mutant, or a codon encoding a non-tolerant amino acid substitution in the polynucleotide encoding the protein. Suitably, the mutant may comprise a deletion, such as a deletion of at least a portion of a domain or a deletion of an entire domain. Suitably, the mutant may comprise a deletion of at least a portion of a cytoplasmic domain.

[0181] In one embodiment, the mutation (e.g., deletion) may be in the N-terminal cytoplasmic domain of the protein. Suitably, the mutation may comprise a deletion of at least a portion of the N-terminus when compared to an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto.

[0182] Suitably, the mutation may comprise a deletion of at least part of the N-terminus when compared to the amino acid sequence shown as SEQ ID No. 3, or a sequence having at least 70% identity thereto.

[0183] Suitably, the mutation may be present in the amino acids corresponding to at least amino acids 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 of SEQ ID No. 3. Preferably, the mutation is in the amino acids corresponding to amino acids 2-117 of SEQ ID No. 3. Preferably, the mutation is a deletion.

[0184] In one embodiment, the method of the invention comprises expressing in a tobacco plant, or a plant part or plant cell thereof:

[0185] a) a truncated protein which lacks at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%) sequence identity with a truncated protein which lacks at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114 or 2-117 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0186] b) a truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30.

[0187] Suitably, the method may comprise expressing in a tobacco plant or plant part or plant cell thereof a truncated protein lacking at least the amino acids corresponding to amino acids 2-117 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%) sequence identity to the truncated protein lacking at least the amino acids corresponding to amino acids 2-117 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30.

[0188] Suitably, the method may comprise expressing in a tobacco plant or plant part or plant cell thereof a truncated protein lacking at least the amino acids corresponding to amino acids 2-117 from the N-terminal side of SEQ ID No. 3, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%) sequence identity with the truncated protein lacking at least the amino acids corresponding to amino acids 2-117 of SEQ ID No. 3.

[0189] In one embodiment, the method according to the invention comprises expressing a protein in a tobacco plant or a plant part or plant cell thereof, said protein comprising (or having or consisting of) an amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70% or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98% or at least 99% sequence identity thereto.

[0190] In another embodiment, the method according to the present invention comprises expressing in a tobacco plant or a plant part or plant cell thereof a protein encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 34, or a sequence having at least 70% sequence identity thereto, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0191] The tobacco plant, part thereof, or plant cell may express the protein using any method known in the art.

[0192] Suitably, the method according to the invention may comprise introducing a nucleotide sequence encoding the protein into a plant or part thereof or a plant cell. This may be achieved, for example, by introducing a construct or vector encoding the protein. Suitably, the method may comprise introducing a construct or vector according to the invention into a plant or part thereof or a plant cell.

[0193] Alternatively, the method may comprise gene editing an endogenous nucleotide sequence to encode a variant protein.

[0194] In one embodiment, the TSNA may be one or more selected from the group consisting of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanatabine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

[0195] In a preferred embodiment, the TSNA is N'-nitrosonornicotine (NNN).

[0196] TSNAs can be measured in processed tobacco, such as flue-cured tobacco or reconstituted tobacco. In one embodiment, TSNA content is measured and / or modified (eg, reduced) in a flue-cured tobacco plant or part thereof (eg, flue-cured tobacco leaves).

[0197] As used herein, the term "tobacco-specific nitrosamines" or "TSNAs" has its ordinary meaning in the art, i.e., nitrosamines found only in tobacco products or other nicotine-containing products. Suitably, at least one tobacco-specific nitrosamine may be 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT) or N'-nitrosoanatabine (NAB).

[0198] More suitably, the at least one tobacco-specific nitrosamine may be NNK or NNN.

[0199] In one embodiment, the tobacco-specific nitrosamine is NNN.

[0200] When used with respect to at least one tobacco-specific nitrosamine, the term "precursor thereof" refers to one or more chemicals or compounds of the tobacco plant that cause the formation of tobacco-specific nitrosamines or involve a nitrosative reaction that causes tobacco-specific nitrosamines to be produced. Suitably, the term "precursor thereof" may refer to nitrates, nitrites or nitrogen oxides.

[0201] In one embodiment, the precursor of TSNA is one or more selected from nornicotine, ananicotine, anatabine and an oxidized derivative of nicotine such as pseudo-oxidized nicotine (PON).

[0202] In one embodiment, the TSNA precursor is an immediate precursor to TSNA.

[0203] In one embodiment, the immediate precursor of TSNA is one or more selected from nornicotine, anabasine, anatabine and an oxidized derivative of nicotine such as pseudo-oxidized nicotine (PON).

[0204] In a preferred embodiment, the precursor of TSNA is nornicotine.

[0205] In one embodiment, the precursor of TSNA is PON.

[0206] In one embodiment, the TSNA precursor is not nicotine.

[0207] Precursors of TSNA (e.g., NNN, NNK, NAB and / or NAT) can be measured in green tobacco leaves, e.g., before processing, e.g., before curing. In one embodiment, precursors of TSNA (e.g., NNN, NNK, NAB and / or NAT) are measured and / or modified (e.g., reduced) in green tobacco leaves, e.g., before processing, e.g., before curing.

[0208] In one embodiment, performance of the methods and or uses of the invention results in a reduction in at least one TSNA or precursor thereof in the modified tobacco plant (or part thereof) when compared to a tobacco plant (or part thereof) not modified according to the invention.

[0209] The term "reduction of at least one TSNA or a precursor thereof" or "reduction of at least one TSNA or a precursor thereof" is used herein to mean that the concentration and / or total content of at least one TSNA or a precursor thereof is lower in the product, method or use of the invention relative to a comparable product, method or use. For example, a comparable tobacco industry product is derived from a tobacco plant that has not been modified according to the invention, but in which all other relevant characteristics are the same (e.g., plant species, growing conditions, method of processing the tobacco, etc.).

[0210] As defined herein, the term "comparable product" can mean a tobacco plant or part thereof, such as tobacco leaves, harvested leaves, cut harvested leaves, processed tobacco leaves or tobacco plant propagation material, or a tobacco industry product or a combination thereof, which can be obtained or is obtained from a tobacco plant that has not been altered to express a deregulated cationic efflux protein.

[0211] In one embodiment, a comparable product may be obtained or is obtained from a tobacco plant that does not express a truncated protein that lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the ... No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, 2-114 or 2-117 amino acids.

[0212] In another embodiment, a comparable product may be obtained or is obtained from a tobacco plant that does not express a protein comprising the amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0213] Suitably, the comparable product may be obtainable or obtained from a tobacco plant which does not express a protein encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 34, or having at least 70% sequence identity thereto, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0214] As defined herein, the term "unmodified plant" is a tobacco plant that has not been modified according to the present invention to express a deregulated cationic efflux protein. In another embodiment, the unmodified plant is a tobacco plant that has not been modified according to the present invention to express a truncated protein that lacks at least 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 amino acids from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, the truncated protein lacking at least 2-30 amino acids from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, 2-114 or 2-117, and wherein all other relevant characteristics are the same (e.g., plant species, growing conditions, method of processing tobacco, etc.).

[0215] Any method known in the art for determining the concentration and / or level of at least one TSNA or its precursor may be used. In particular, a method may include the addition of a deuterium-labeled internal standard, aqueous extraction and filtration, followed by analysis using reverse phase high performance liquid chromatography with tandem mass spectrometry (LC-MS / MS). Other examples for determining the concentration and / or level of tobacco-specific nitrosamine precursors include methods such as the one detailed in CORESTA Recommended Method CRM-72: Determination of Tobacco-Specific Nitrosamines in Tobacco and Tobacco Products by LC-MS / MS; CRM is being developed as ISO / DIS 21766 or Wagner et al. Analytical Chemistry (2005), 77(4), 1001-1006, which are incorporated herein by reference in their entirety.

[0216] Suitably, the concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor can be reduced by implementing the method and / or purposes of the present invention. Suitably, the concentration and / or level of at least one tobacco-specific nitrosamine or its precursor can be reduced in the tobacco plant of the present invention (e.g., obtainable or obtained by the method and / or purposes of the present invention) when compared with the concentration and / or level of at least one tobacco-specific nitrosamine or its precursor in the tobacco plant not modified according to the present invention.

[0217] The concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor may be reduced in tobacco leaves, harvested leaves, processed leaves, tobacco industry products, or a combination thereof, obtainable or obtained from a tobacco plant (or part of a tobacco plant) of the present invention (or a part of a tobacco plant) when compared to tobacco leaves, harvested leaves, processed leaves, tobacco industry products, or a combination thereof, obtainable or obtained from a tobacco plant (or part of a tobacco plant) that has not been modified according to the present invention.

[0218] Suitably, the concentration and / or total content of at least one tobacco-specific nitrosamine or a precursor thereof may be reduced in the processed tobacco leaves.

[0219] Suitably, the concentration and / or level of at least one tobacco-specific nitrosamine or a precursor thereof may be reduced in the tobacco industry product.

[0220] In one embodiment, at least one tobacco-specific nitrosamine or its precursor can be reduced by at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some embodiments, at least one tobacco-specific nitrosamine or its precursor can be reduced by about 5% to about 95%, about 10% to about 90%, 20% to about 80%, 30% to about 70%, or about 40% to 60%.

[0221] With regard to processed tobacco leaves (e.g., cured or reconstituted), at least one tobacco-specific nitrosamine or its precursor can be reduced by about 5000 ng / g to about 50 ng / g, about 4000 ng / g to about 100 ng / g, about 3000 ng / g to 500 ng / g, or 2000 ng / g to 1000 ng / g. In some embodiments, at least one tobacco-specific nitrosamine or its precursor can be reduced by at least about 5000 ng / g, at least about 4000 ng / g, at least about 3000 ng / g, at least about 2000 ng / g, at least about 1000 ng / g, at least about 500 ng / g, at least about 100 ng / g, or at least about 50 ng / g.

[0222] Cationic efflux proteins

[0223] Cationic efflux proteins are pumps that remove metal ions such as cadmium, zinc and cobalt from cells. These metals are not only essential micronutrients for plant metabolism, they also act as second messengers that initiate intracellular signal transduction cascades.

[0224] In one embodiment, the wild-type cationic efflux protein comprises an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence that is at least 90% identical thereto (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical thereto).

[0225] Suitably, the wild-type cationic efflux protein according to the present invention may comprise the amino acid sequence shown in SEQ ID No. 3, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0226] Suitably, the wild-type cationic efflux protein may comprise the amino acid sequence shown in SEQ ID No. 6, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0227] Suitably, the wild-type cationic efflux protein may comprise the amino acid sequence shown in SEQ ID No. 9, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0228] Suitably, the wild-type cationic efflux protein may comprise the amino acid sequence shown in SEQ ID No. 12, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0229] Suitably, the wild-type cationic efflux protein may comprise the amino acid sequence shown in SEQ ID No. 15, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0230] Suitably, the wild-type cationic efflux protein may comprise the amino acid sequence shown in SEQ ID No. 18, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0231] Suitably, the wild-type cationic efflux protein may comprise the amino acid sequence shown in SEQ ID No. 21, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0232] Suitably, the wild-type cationic efflux protein according to the present invention may comprise the amino acid sequence shown in SEQ ID No. 24, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0233] Suitably, the wild-type cationic efflux protein according to the present invention may comprise the amino acid sequence shown in SEQ ID No. 27, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0234] Suitably, the wild-type cationic efflux protein according to the present invention may comprise the amino acid sequence shown in SEQ ID No. 30, or a sequence having at least 90% identity thereto (preferably at least 95%, at least 97% or at least 99% identity).

[0235] In one embodiment, the wild-type cationic efflux protein comprises an amino acid sequence selected from the group consisting of: SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30.

[0236] Suitably, the cationic efflux protein may be derived from Nicotiana tabacum.

[0237] In one embodiment, the wild-type cationic efflux protein is encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7, SEQ ID No.8, SEQ ID No.10, SEQ ID No.11, SEQ ID No.13, SEQ ID No.14, SEQ ID No.16, SEQ ID No.17, SEQ ID No.19, SEQ ID No.20, SEQ ID No.22, SEQ ID No.23, SEQ ID No.25, SEQ ID No.26, SEQ ID No.28 or SEQ ID No.29, or a sequence having at least 70% sequence identity thereto.

[0238] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 1, or a sequence having at least 90% sequence identity thereto.

[0239] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 2, or a sequence having at least 90% sequence identity thereto.

[0240] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 4, or a sequence having at least 90% sequence identity thereto.

[0241] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 5, or a sequence having at least 90% sequence identity thereto.

[0242] Suitably, the protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence as shown in SEQ ID No. 7, or a sequence having at least 90% sequence identity thereto.

[0243] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 8, or a sequence having at least 90% sequence identity thereto.

[0244] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 10, or a sequence having at least 90% sequence identity thereto.

[0245] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 11, or a sequence having at least 90% sequence identity thereto.

[0246] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 13, or a sequence having at least 90% sequence identity thereto.

[0247] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 14, or a sequence having at least 90% sequence identity thereto.

[0248] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 16, or a sequence having at least 90% sequence identity thereto.

[0249] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 17, or a sequence having at least 90% sequence identity thereto.

[0250] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 19, or a sequence having at least 90% sequence identity thereto.

[0251] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 20, or a sequence having at least 90% sequence identity thereto.

[0252] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 22, or a sequence having at least 90% sequence identity thereto.

[0253] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 23, or a sequence having at least 90% sequence identity thereto.

[0254] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 25, or a sequence having at least 90% sequence identity thereto.

[0255] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 26, or a sequence having at least 90% sequence identity thereto.

[0256] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 28, or a sequence having at least 90% sequence identity thereto.

[0257] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence shown as SEQ ID No. 29, or a sequence having at least 90% sequence identity thereto.

[0258] In one embodiment, the wild-type cationic efflux protein is encoded by a polynucleotide sequence, wherein the gene (before mutation) is selected from: SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7, SEQ ID No.8, SEQ ID No.10, SEQ ID No.11, SEQ ID No.13, SEQ ID No.14, SEQ ID No.16, SEQ ID No.17, SEQ ID No.19, SEQ ID No.20, SEQ ID No.22, SEQ ID No.23, SEQ ID No.25, SEQ ID No.26, SEQ ID No.28 or SEQ ID No.29.

[0259] Suitably, the wild-type protein for use according to the present invention may be encoded by a polynucleotide sequence from Nicotiana tabacum.

[0260] Deregulated cation efflux protein

[0261] In one embodiment, the protein for use according to the invention is a deregulated cation efflux protein.

[0262] As used herein, "cation efflux protein" has its ordinary meaning in the art and refers to a protein that transports cations, eg, transports cations out of a cell.

[0263] Cation efflux proteins are typically integral membrane proteins that increase tolerance to divalent metal ions such as zinc, cadmium, and cobalt. Cation efflux proteins can act as efflux pumps to remove these ions from the cell.

[0264] Cation efflux proteins may be able to transport zinc, cobalt, cadmium, or nickel.

[0265] In one embodiment, the cation efflux protein is capable of transporting zinc. In one embodiment, the cation efflux protein is a zinc transporter.

[0266] As used herein, "deregulated cation efflux protein" means that the cation efflux protein for use in accordance with the present invention does not respond to metal ion concentrations in cells, or shows a reduced response to metal ion concentrations, when compared to the wild-type cation efflux protein found in tobacco plants, i.e., a cation efflux protein that is not deregulated.

[0267] In other words, such cation efflux proteins have reduced sensitivity to, or are no longer responsive to, changes in metal ion concentrations within the cell.

[0268] For example, upon modulation of metal ion concentration within a cell (ie, from low metal ion concentration to high metal ion concentration), a deregulated cation efflux protein cannot undergo conformational changes.

[0269] A cation efflux protein that is no longer able to change conformation in response to intracellular metal ion concentration may be referred to herein as a deregulated cation efflux protein.

[0270] In one embodiment, the deregulated cation efflux protein for use according to the present invention is a constitutive high affinity cation efflux transporter.

[0271] As used herein, the term "high affinity cation efflux protein" or "high affinity cation efflux transporter" refers to a protein that is in an open conformation that allows metal ion transport. For example, the protein can be in a constitutively open conformation.

[0272] In one embodiment, the high affinity cation efflux protein is in a conformation that allows metal binding to the cytoplasmic binding site between the transmembrane domains. Figure 4a In other words, in high-affinity cation efflux proteins, the cytoplasmic binding site between the transmembrane domains is not shielded by the cytoplasmic regulatory domain.

[0273] In one embodiment, the deregulated cationic efflux protein is a protein that transports cations (e.g., zinc ions) out of the cell at a higher rate than a comparable wild-type cationic efflux protein. Suitably, the wild-type cationic protein may have an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30.

[0274] In one embodiment, compared with wild-type cation efflux transporter, the cation efflux protein of deregulation shows increased metal ion transport.Compared with wild-type cation efflux transporter, the cation efflux protein of deregulation can show more than at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% metal ion transport.Suitably, metal ion transport can be measured under comparable conditions.

[0275] The activity of a cation efflux transporter (eg, metal ion transport) can be measured using any method known in the art.

[0276] For example, fluorescent cation sensors such as fluorescent Zn 2+ The sensor, fluorescence microscopy (described by Carpenter et al., Arch Biochem Biophys. 2016 Dec 1;611:20–29, incorporated herein by reference) was used to measure metal ion transport.

[0277] In one embodiment, cells expressing a deregulated cation efflux protein exhibit lower intracellular metal ion concentrations (e.g., lower intracellular zinc ion concentrations) compared to cells that do not express the deregulated cation efflux protein, e.g., compared to cells that express only a wild-type cation efflux transporter.

[0278] Suitably, the wild-type cationic protein may have an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30.

[0279] Cells expressing a deregulated cation efflux protein can exhibit a decrease in ion transport by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in intracellular cation concentration (e.g., intracellular zinc concentration) compared to a wild-type cation efflux transporter.

[0280] The concentration of cations (e.g., zinc) in cells can be measured by first washing the cells, removing the extracellular matrix and lysing the cells, and then measuring the concentration of cations (e.g., zinc). Cells expressing a deregulated cation efflux protein according to the present invention can have a lower intracellular cation (e.g., zinc) concentration than cells that do not express the deregulated cation efflux protein, i.e., cells that only express wild-type cation efflux proteins.

[0281] In one embodiment, the deregulated cation efflux protein increases the tolerance of the cell to divalent metal ions (eg, zinc).

[0282] Without wishing to be bound by theory, a deregulated cation efflux protein may reduce the intracellular concentration of cations (e.g., zinc), thereby increasing tolerance to divalent metal ions. Suitably, a deregulated cation efflux protein may allow cells to tolerate higher concentrations of divalent metal ions (e.g., zinc) compared to cells that do not express the deregulated cation efflux protein.

[0283] The deregulated cation efflux protein can increase tolerance to divalent metal ions (e.g., zinc) by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to a wild-type cation efflux transporter.

[0284] Increased tolerance to metal ions (e.g., zinc) can be measured using any method in the art. For example, leaf mass can be determined, e.g., Fig.69b Tolerance was measured by measuring the chlorophyll intensity of leaves treated with metal ions as shown in Without wishing to be bound by theory, leaves of plants expressing a deregulated cation efflux protein show tolerance to treatment with metal ions because the cation efflux protein reduces the intracellular concentration of said metal ions.

[0285] In one embodiment, upon treatment with a metal ion (e.g., zinc), cells expressing a deregulated cation efflux protein exhibit a leaf phenotype (e.g., chlorophyll intensity) similar to leaves not treated with the metal ion (e.g., zinc) - see e.g. Fig.69c Suitably, cells expressing a deregulated cation efflux protein may show a reduction in chlorophyll intensity which is not statistically significant when compared to leaves not treated with the metal ion, for example using a one-way ANOVA and Tukey's multiple comparison post hoc test.

[0286] Cells expressing a deregulated cation efflux protein may show less than 15%, less than 10%, less than 5% reduction in chlorophyll intensity when treated with metal ions compared to leaves not treated with the metal ions.

[0287] In one embodiment, a deregulated cation efflux protein may lack a functional regulatory domain; preferably, a deregulated cation efflux protein lacks at least a portion of a regulatory domain.

[0288] As used herein, the term "regulatory domain" has its usual meaning in the art, i.e., a conserved portion of a protein sequence that controls protein activity. Regulatory domains include activation domains, repression domains, and epigenetic effector domains.

[0289] In one aspect, the regulatory domain according to the invention is a cytoplasmic domain.

[0290] In one aspect, the regulatory domain is a sensor of metal ion levels in the cell.

[0291] When a protein lacks a functional regulatory domain according to the invention, the activity of said protein can no longer be regulated by this regulatory domain.

[0292] In one aspect, a cation efflux protein lacking a functional regulatory domain is unable to switch between a low-affinity conformation and a high-affinity conformation (e.g., Figure 4a ).

[0293] Suitably, a deregulated cation efflux protein may lack a functional cytoplasmic domain.

[0294] In one embodiment, the cytoplasmic domain can be rendered non-functional by mutation. In another embodiment, the cytoplasmic domain can be rendered non-functional by deletion. Suitably, when compared to the wild-type protein, the cation efflux protein to be deregulated may lack at least a portion of the cytoplasmic domain. Suitably, when compared to the wild-type protein, the cation efflux protein to be deregulated may lack at least a portion of the N-terminal.

[0295] As used herein, the term "cytoplasmic domain" has its ordinary meaning in the art and describes the topology of a protein domain as an intracellular domain that interacts with the interior of the cell.

[0296] The topological domains of proteins, including cytoplasmic domains, can be annotated based on predictions provided by algorithms, such as the program TMHMM provided by the Technical University of Denmark (DTU) Department of Bio and Health Informatics; http: / / www.cbs.dtu.dk / services / TMHMM / .

[0297] Alternatively, the cytoplasmic domain can be annotated or predicted by comparing the amino acid sequence with known protein structures. For example, the cytoplasmic domain of the cation efflux protein can be identified by alignment against SEQ ID No. 3, wherein the amino acids 1-128 of SEQ ID No. 3 indicate the cytoplasmic domain.

[0298] In one embodiment, the cytoplasmic domain is the protein region corresponding to amino acids 1 to 128 of the N-terminus of SEQ ID No. 3. In one embodiment, the cytoplasmic domain is the protein region corresponding to amino acids 2 to 128 of the N-terminus of SEQ ID No.3.

[0299] In one embodiment, the first amino acid (amino acid number 1) is not counted as part of the N-terminus.The first amino acid (amino acid number 1) is always required for protein translation.

[0300] In the context of the present invention, the cytoplasmic domain regulates the activity of the cation efflux protein. The cytoplasmic domain contains a metal binding site. When the metal concentration in the cell increases to a threshold, the binding of the metal to the N-terminal domain induces a conformational change in the cation efflux protein (see Figure 4).

[0301] In one embodiment, the deregulated cation efflux transporter is deregulated when compared to the wild-type cation efflux protein.

[0302] In one embodiment, the wild-type cationic efflux protein comprises an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto).

[0303] In one embodiment, the wild-type cationic efflux protein is encoded by a polynucleotide sequence as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7, SEQ ID No.8, SEQ ID No.10, SEQ ID No.11, SEQ ID No.13, SEQ ID No.14, SEQ ID No.16, SEQ ID No.17, SEQ ID No.19, SEQ ID No.20, SEQ ID No.22, SEQ ID No.23, SEQ ID No.25, SEQ ID No.26, SEQ ID No.28 or SEQ ID No.29, or a nucleic acid sequence having at least 70% identity thereto (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto).

[0304] Suitably, the method according to the invention may comprise modifying an endogenous wild-type sequence to encode a deregulated cation efflux protein.The endogenous sequence may be modified, for example, by gene editing.

[0305] In one embodiment, the deregulated cation efflux protein comprises one or more mutations compared to the amino acid sequence shown as SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto.

[0306] The mutation can be a deletion, a splice mutant, or a codon encoding a non-tolerant amino acid substitution. The mutation can be in the cytoplasmic domain. The mutation can be in the N-terminus.

[0307] In one embodiment, the deregulated cationic efflux protein comprises an amino acid sequence lacking at least a portion of the N-terminus when compared to the amino acid sequence shown as SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% identity thereto.

[0308] In another embodiment, the deregulated cation efflux protein comprises:

[0309] a) a truncated protein which lacks at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein which lacks at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114 or 2-117 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30; or

[0310] b) a truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30.

[0311] In one embodiment, the deregulated cationic efflux protein comprises the amino acid sequence shown in SEQ ID No. 33, or a sequence having at least 70% or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0312] In another embodiment, the deregulated cationic efflux protein is encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 34, or a sequence having at least 70% sequence identity thereto, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0313] In one embodiment, the deregulated cationic efflux protein comprises an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 90% identity thereto (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto), wherein after alignment with the amino acid sequence SEQ ID No. 3, the deregulated cationic efflux protein comprises:

[0314] methionine at amino acid position number 3;

[0315] Aspartic acid residue at amino acid position number 7: and / or

[0316] The threonine residue at amino acid position number 68,

[0317] The amino acid numbers refer to SEQ ID No. 3.

[0318] In some embodiments, the invention provides the cation efflux protein of deregulation.Relative to wild-type cation efflux protein, the cation efflux protein of deregulation can be modified to promote the formation of dimer (e.g., homodimer) or oligomer or to stabilize it.In one embodiment, relative to wild-type cation efflux protein, the cation efflux protein of deregulation according to the present invention forms a more stable dimer (e.g., homodimer) or other oligomer.Suitably, the sudden change in the amino acid sequence can promote dimerization (e.g., homodimerization) or oligomerization of the cation efflux protein of deregulation.

[0319] In some embodiments, a deregulated cation efflux protein can comprise one or more modified transmembrane domains (eg, relative to a wild-type cation efflux protein).

[0320] Suitably, the deregulated cation efflux protein may comprise an additional transmembrane domain or may comprise a stabilized transmembrane domain.

[0321] Sequence identity

[0322] Sequence identity comparisons can be performed visually, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the % identity between two or more sequences.

[0323] % identity can be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called a "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0324] Although this is a very simple and consistent approach, it fails to take into account that, for example, in an otherwise identical pair of sequences, an insertion or deletion renders the following amino acid residues unalignable, thus potentially leading to a substantial reduction in % identity when an overall alignment is performed. Thus, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without overly penalizing the overall identity score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local identity.

[0325] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting a higher relatedness between the two compared sequences - achieves a higher score than an alignment with many gaps. An "affine gap cost" is often used, which imposes a relatively high cost for the presence of a gap, and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Of course, a high gap penalty will produce an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, when using such software for sequence comparison, it is preferred to use the default values.

[0326] Therefore, calculation of maximum % identity first requires producing an optimal alignment taking into account gap penalties. A suitable computer program for performing such an alignment is Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, for example, the BLAST package (see Ausubel et al. 1999 Short Protocols in Molecular Biology, 4th edition - Chapter 18), BLAST 2 (see FEMS Microbiol Lett 1999 174 (2): 247-50; FEMS Microbiol Lett 1999 177 (1): 187-8 and tatiana@ncbi.nlm.nih.gov), FASTA (Altschul et al. 1990 J. Mol. Biol. 403-410) and AlignX. At least BLAST, BLAST 2 and FASTA can be used for offline and online searching (see Ausubel et al. 1999, pages 7-58 to 7-60).

[0327] Although final % identity can be measured according to identity, the alignment process itself is usually not based on all-or-no paired comparisons. On the contrary, a scaled similarity score matrix is ​​generally used, which scores each paired comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the default matrix of the BLOSUM62 matrix-BLAST program suite. The Vector NTI program generally uses disclosed default values ​​or a custom symbol comparison table (if provided) (for further details, see the user manual). For some applications, the default values ​​of the Vector NTI software package are preferably used.

[0328] Alternatively, percent identity can be calculated based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244) using the multiple alignment feature in Vector NTI (Invitrogen Corp.).

[0329] Once the software has generated an optimal alignment, it can calculate % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0330] Gap penalties should be used when determining sequence identity and the following parameters can be used for pairwise alignments:

[0331]

[0332]

[0333] In one embodiment, BLAST may be used with gap penalty and gap extension sets as defined above.

[0334] In one embodiment, CLUSTAL may be used with gap penalty and gap extension sets as defined above.

[0335] In some embodiments, the gap penalty for BLAST or CLUSTAL alignments may be different from those detailed above. The skilled person will appreciate that the standard parameters for performing BLAST and CLUSTAL alignments may change periodically, and can select appropriate parameters based on the standard parameters detailed at the time for the BLAST or CLUSTAL alignment algorithm.

[0336] Suitably, the degree of identity with respect to a nucleotide sequence or amino acid sequence is determined over at least 20 consecutive nucleotides / amino acids, preferably over at least 30 consecutive nucleotides / amino acids, preferably over at least 40 consecutive nucleotides / amino acids, preferably over at least 50 consecutive nucleotides / amino acids, preferably over at least 60 consecutive nucleotides / amino acids, preferably over at least 100 consecutive nucleotides / amino acids.

[0337] Suitably, the degree of identity with respect to nucleotide sequences or amino acid sequences may be determined over the entire sequence.

[0338] The sequences may also have deletions, insertions or substitutions of amino acid residues which produce silent changes and result in functionally equivalent substances. Deliberate amino acid substitutions may be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and uncharged polar head groups of amino acids with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0339] Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second column, and preferably in the same row in the third column, can be substituted for each other:

[0340]

[0341] The present invention also encompasses homologous substitutions that may occur (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue with an alternative residue), i.e., like-for-like, such as basic substitution for basic, acidic substitution for acidic, polar substitution for polar, etc. Non-homologous substitutions may also occur, i.e., from one class of residues to another, or alternatively involve the inclusion of unnatural amino acids, such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine and phenylglycine.

[0342] Substitutions can also be made with unnatural amino acids, including; α* and α-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-C1-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-aminobutyric acid*, L-γ-aminobutyric acid*, L-α-aminoisobutyric acid*, L-ε-aminocaproic acid# , 7-aminoheptanoic acid*, L-methionine sulfone # *, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline # , L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino) # , L-Tyr(methyl)*, L-Phe(4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)*, L-diaminopropionic acid # and L-Phe(4-benzyl)*. The notation * has been used for the purposes discussed above (in relation to homologous or nonhomologous substitution) to indicate the hydrophobic nature of the derivative, while # has been used to indicate the hydrophilic nature of the derivative, with #* indicating an amphiphilic character.

[0343] The variant amino acid sequence may include a suitable spacer, which may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl, plus amino acid spacers such as glycine or β-alanine residues. Further variant forms involve the presence of one or more amino acid residues in a peptoid form, which is well understood by those skilled in the art. For the avoidance of doubt, "peptoid form" is used to refer to variant amino acid residues in which the α-carbon substituent is located on the nitrogen atom of the residue rather than on the α-carbon. Methods for preparing peptides in a peptoid form are known in the art, such as Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134.

[0344] The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention, or sequences that are capable of hybridizing to the sequences of the present invention or to sequences that are complementary thereto.

[0345] As used herein, the term "hybridization" shall include "the process by which a strand of nucleic acid binds to a complementary strand through base pairing," as well as the process of amplification as performed in polymerase chain reaction (PCR) techniques.

[0346] The present invention also relates to nucleotide sequences that can hybridize to the nucleotide sequences of the present invention, including the complementary sequences of those presented herein.

[0347] Preferably, hybridization is measured under stringent conditions (eg, 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015 M Na 3 citrate, pH 7.0}).

[0348] More preferably, hybridization is measured under high stringency conditions (eg, 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na 3 citrate, pH 7.0}).

[0349] Reduce or prevent expression and / or function

[0350] In one aspect, the present invention provides a method for reducing the concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor in tobacco, comprising expressing a deregulated cationic efflux protein in a tobacco plant or a plant part or plant cell thereof.

[0351] In another aspect, the present invention provides a method for reducing the concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor in tobacco, comprising expressing in a tobacco plant or a plant part or plant cell thereof:

[0352] a) a truncated cationic efflux protein lacking at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein lacking at least the amino acids corresponding to amino acids 2-25, or 2-50, or 2-75, or 2-100, 2-114 or 2-117 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0353] b) a truncated cationic efflux protein in a tobacco plant or plant part or plant cell thereof consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0354] c) a protein comprising the amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto; or

[0355] d) a protein encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 34, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto. Any method known in the art for expressing proteins can be used in the present method.

[0356] For example, the method may include:

[0357] ● providing a mutation (e.g., a deletion) in a nucleic acid sequence encoding a protein comprising an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or an amino acid sequence having at least 70% sequence identity thereto;

[0358] ● providing a mutation in a regulatory region (e.g., a cytoplasmic domain) of a protein comprising an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or an amino acid sequence having at least 70% sequence identity thereto;

[0359] ● providing a mutation (e.g., a deletion) in a regulatory region (e.g., a cytoplasmic domain, preferably an N-terminal cytoplasmic domain) of a protein comprising an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or an amino acid sequence having at least 70% sequence identity thereto;

[0360] ● Providing a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto;

[0361] ●Provide a polynucleotide comprising a sequence as shown in SEQ ID No.34, or a sequence having at least 70% sequence identity thereto, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0362] Each of the above methods results in a reduction in the content of at least one TSNA or TSNA precursor in tobacco. Suitably, each of the above methods may result in the expression of a deregulated cationic efflux protein.

[0363] As used herein, the term "mutation" encompasses both natural genetic variants and engineered variants. In particular, the term "mutation" refers to a variation in a nucleotide sequence encoding an amino acid sequence or an amino acid sequence compared to a sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or an amino acid sequence having at least 70% sequence identity thereto. Suitably, the mutation may reduce the content of at least one TSNA or TSNA precursor in tobacco.

[0364] In one embodiment, the method according to the present invention may include providing a nucleic acid sequence according to the present invention to a plant or its part or plant cell. Suitably, the nucleic acid sequence may be introduced into a plant or its part or cell. Suitably, an endogenous nucleic acid sequence in a plant or its part or cell may be modified to encode a polypeptide according to the present invention (e.g., by gene editing).

[0365] In a preferred embodiment, each copy of the nucleic acid sequence encoding a protein present in the plant is modified, e.g., mutated as defined herein (e.g., each genomic copy of the gene encoding the protein in the plant is mutated), the protein comprising a sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% sequence identity thereto. For example, each copy of a gene in the allotetraploid genome of Nicotiana tabacum may be mutated.

[0366] In a preferred embodiment, all homologues of the cationic efflux protein are modified, e.g. mutated. Suitably, SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 or corresponding sequences having at least 70% sequence identity thereto are all modified, e.g. mutated.

[0367] In a preferred embodiment, the plant or plant cell according to the invention is homozygous. Suitably, the plant or plant cell may be homozygous for a modification, such as a mutation.

[0368] In one embodiment, preferably, the plant or plant cell according to the invention expresses only modified, e.g. mutated nucleic acids. In other words, in some embodiments, no endogenous (or endogenous and functional proteins) are present in the plant according to the invention. In other words, if any endogenous protein is present, it is preferably in an inactive form.

[0369] In one embodiment, the method may comprise providing a mutation in a sequence as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 28 or SEQ ID No. 29, or a nucleic acid sequence having at least 70% identity thereto.

[0370] The mutation can change the plant genome so that the nucleic acid sequence encoding the protein is completely or partially deleted or otherwise rendered non-functional, wherein the protein comprises an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or an amino acid sequence having at least 70% sequence identity thereto.

[0371] The nucleotide sequence may comprise one or more nucleotide changes that reduce or prevent protein expression or influence protein transport. For example, the expression of the protein may be reduced or prevented by introducing one or more of a frameshift, a splice mutant or a non-tolerant amino acid substitution in an open reading frame.

[0372] Frameshift mutations (also called framing errors or reading frame shifts) are mutations caused by insertion-deletions (insertions or deletions) of multiple nucleotides that are not divisible by three in a nucleic acid sequence. Due to the triplet nature of gene expression through codons, insertions or deletions can change the reading frame, resulting in a translation that is completely different from the original. Frameshift mutations often cause the codons after mutation to be read to encode different amino acids. Frameshift mutations usually result in the introduction of premature stop codons.

[0373] Splicing mutants insert, delete or change many nucleotides at specific sites, where splicing occurs during the processing of precursor messenger RNA into mature messenger RNA. The loss of splicing sites causes one or more introns to remain in mature mRNA and may lead to the production of abnormal proteins.

[0374] Non-tolerant amino acid substitutions refer to mutations that cause non-synonymous amino acid substitutions in proteins, which result in reduced or abolished function of the protein.

[0375] Any method known in the art for providing a mutation in a nucleotide sequence can be used in the present method. For example, homologous recombination can be used to produce a vector in which the nucleotide sequence in question is mutated and used to transform a plant or plant cell. The recombinant plant or plant cell expressing the mutant sequence can then be selected.

[0376] The nucleic acid sequence may be deleted in whole or in part. The deletion may be continuous or may comprise multiple segments of the sequence. The deletion preferably removes a sufficient amount of nucleotide sequence such that the nucleic acid sequence no longer encodes a functional protein.

[0377] Deletion can remove at least a portion of one or more domains of a cationic efflux protein. Deletion can for example remove at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the coding portion of the nucleic acid sequence of at least one domain.

[0378] The deletion may, for example, remove at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the coding portion of the nucleic acid sequence.

[0379] The N-terminal regulatory cytoplasmic domain is shown in Fig.37 The N-terminal regulatory cytoplasmic domain can be identified by alignment with the sequence of SEQ ID NO. 1. In one embodiment, the amino acids encoding the N-terminal regulatory cytoplasmic domain correspond to amino acids 1-128 when aligned with SEQ ID No. 3.

[0380] Suitably, at least a portion of the N-terminal regulatory cytoplasmic domain may be deleted. The deletion may, for example, remove at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the coding portion of the cytoplasmic domain. Suitably, the deletion may remove the entire N-terminal regulatory cytoplasmic domain.

[0381] The cation efflux transmembrane domain and the cation efflux cytoplasmic domain are shown in Fig.36 middle.

[0382] Suitably, at least a portion of the cation efflux transmembrane domain may be deleted. The deletion may for example remove at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the encoding portion of the cation efflux transmembrane domain.

[0383] Suitably, at least a portion of the cation efflux cytoplasmic domain may be deleted. The deletion may for example remove at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the encoding portion of the cation efflux cytoplasmic domain.

[0384] Methods for deleting nucleotide sequences in plants are known in the art. For example, homologous recombination can be used to produce a vector in which the relevant nucleotide sequence lacks, and to transform plants or plant cells. Recombinant plants or plant cells expressing the new sequence portion can then be selected.

[0385] Plant cells transformed with a vector as described above can be grown and maintained according to well-known tissue culture methods, for example, by culturing the cells in an appropriate medium supplied with necessary growth factors such as amino acids, plant hormones, vitamins and the like.

[0386] Modification of nucleic acid sequences can be performed using targeted mutagenesis methods (also known as targeted nucleotide exchange (TNE) or oligonucleotide-directed mutagenesis (ODM)). Targeted mutagenesis methods include, but are not limited to, methods using zinc finger nucleases, TALENs (see WO2011 / 072246 and WO2010 / 079430), Cas9-like, Cas9 / crRNA / tracrRNA, Cas9 / gRNA or other CRISPR systems (see WO 2014 / 071006 and WO2014 / 093622), meganucleases (see WO2007 / 047859 and WO2009 / 059195), or targeted mutagenesis methods using mutagenic oligonucleotides, which may contain chemically modified nucleotides having sequence complementarity with the gene, for enhancing mutagenesis in plant protoplasts (e.g. or TALEN).

[0387] Alternatively, mutagenesis systems such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al., 2000, Nat Biotech 18:455, and McCallum et al. 2000, Plant Physiol. 123, 439-442, both of which are incorporated herein by reference) can be used to generate plant lines containing protein-encoding genes with mutations. TILLING uses traditional chemical mutagenesis (e.g., ethyl methanesulfonate (EMS) mutagenesis to generate random mutations), followed by high-throughput screening of mutations. Thus, plants, seeds, and tissues containing genes with desired mutations can be obtained.

[0388] The method may further comprise the steps of: making plant seed mutagenesis (for example EMS mutagenesis), the merging of plant individual or DNA, the pcr amplification of target region, heteroduplex formation and high throughput detection, the identification of mutant plant, the sequencing of mutant pcr product.Should be understood that other mutagenesis and selection methods can be used equally for generating such modified plants.Seed can for example be irradiated or chemically treated, and can be with regard to the phenotype screening plant of modification.

[0389] Fast neutron deletion mutagenesis can be used in a reverse genetics sense (i.e., using PCR) to identify plant lines carrying deletions in endogenous genes. See, e.g., 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, which are incorporated herein by reference.

[0390] In another approach, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of duplicated loci. See, e.g., Kusaba et al. (2003) Plant Cell 15: 1455-1467 (incorporated herein by reference).

[0391] The modified plants can be distinguished from unmodified plants, ie wild-type plants, by molecular means, such as the presence of a mutation in the DNA, and by the phenotypic characteristics of the modification. The modified plants can be homozygous or heterozygous for the mutation.

[0392] In one embodiment, the method of reducing the level of at least one tobacco-specific nitrosamine (TSNA) or TSNA precursor in tobacco does not include treating the plant with a chemical (eg, an agrochemical).

[0393] Other ways to reduce or prevent expression or activity will be apparent to those skilled in the art and include the use of microRNA silencing, RNAi, antisense, tDNA insertion, virus-induced gene silencing (VIG), or dominant negative constructs (or antimorphic mutations).

[0394] In one embodiment, a nucleic acid encoding a protein comprising an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or an amino acid sequence having at least 70% sequence identity thereto, can be modified or mutated by a targeted mutagenesis-based system.

[0395] In one embodiment, the nucleic acid encoding the protein can be modified (e.g., mutated or at least partially deleted) by gene editing, such as a CRISPR-based system, and the protein comprises an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or an amino acid sequence having at least 70% sequence identity thereto.

[0396] In one embodiment, the nucleic acid encoding the protein can be modified (e.g., mutated or at least partially deleted) by zinc finger nucleases, TALENs, meganucleases, mutagenic oligonucleotides or TILLING, and the protein comprises an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or an amino acid sequence having at least 70% sequence identity thereto.

[0397] Measuring the level or amount of a gene product can be performed by any suitable method, including, for example, comparing mRNA transcript levels, protein or peptide levels and / or the phenotype of a plant between a modified plant and a comparable plant not modified according to the invention.

[0398] The ability of a protein to act as a cation efflux protein can be determined using any method known in the art, including, for example, plant assays (such as those described by Lang et al., 2011. Journal of Experimental Botany, Vol. 62, No. 13, pp. 4467-4480 (incorporated herein by reference)), or yeast complementation assays (such as those described by Pervans et al. 2001, PNAS, Vol. 98, No. 17 (incorporated herein by reference)).

[0399] In one embodiment, a plant assay can be used to determine the ability of a protein to function as a cation efflux protein.

[0400] Suitably, the shoot biomass production of the plant comprising the target polynucleotide can be measured under metal stress and compared with the shoot biomass production of wild-type plants. Relative to the shoot biomass production of wild-type plants, the increase in shoot biomass production under metal stress indicates that the target polynucleotide has cation efflux transporter functionality.

[0401] Suitably, the transcriptional expression of the target polynucleotide can be measured under metal stress (e.g., Zn, Cd, Co, Ni) and compared with the basal level of wild-type plants. Compared with the shoot biomass production of wild-type plants, the increase in the transcriptional level of the target polynucleotide under metal stress indicates that the target polynucleotide has cation efflux transporter functionality.

[0402] In one embodiment, the ability of a protein to function as a cation efflux protein can be measured using a yeast complementation assay, such as the assay described herein.

[0403] Suitably, the cDNA of the polynucleotide of interest may be cloned into a yeast expression vector and transformed into a cot1zrc1 double mutant yeast strain (sensitive to Co, Ni, Cd and Zn). Suitably, the yeast is grown on a plate surrounding a filter disc impregnated with cations,

[0404] The determination can be performed by comparing the growth inhibition zone of yeast strains containing the target polynucleotide with yeast containing a control vector. When the inhibition zone area of ​​the yeast containing the target polynucleotide is less than the inhibition zone area of ​​the yeast containing the control vector, this indicates that the target polynucleotide has cation efflux transporter functionality.

[0405] In one aspect, the present invention provides methods for reducing the concentration and / or total content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in tobacco plants.

[0406] The method may comprise expressing in the plant a polynucleotide (eg, an exogenous polynucleotide) comprising:

[0407] a) a truncated cationic efflux protein lacking at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein lacking at least the amino acids corresponding to amino acids 2-25, or 2-50, or 2-75, or 2-100, 2-114 or 2-117 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0408] b) a truncated cationic efflux protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0409] c) a protein comprising the amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto; or

[0410] d) a protein encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 34, or a sequence having at least 70% sequence identity thereto, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0411] In one aspect, the invention provides a method for increasing the nicotine concentration and / or total content in a tobacco plant. Suitably, the method can be used to produce nicotine for use in a non-combustible system.

[0412] The method may comprise expressing in the plant a polynucleotide (eg, an exogenous polynucleotide) comprising:

[0413] a) a truncated cationic efflux protein lacking at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein lacking at least the amino acids corresponding to amino acids 2-25, or 2-50, or 2-75, or 2-100, 2-114 or 2-117 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0414] b) a truncated cationic efflux protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0415] c) a protein comprising the amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto; or

[0416] d) a protein encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 34, or a sequence having at least 70% sequence identity thereto, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0417] Promoter

[0418] The nucleic acid sequence may be operably linked to a heterologous promoter for directing transcription of the nucleic acid sequence in the plant.

[0419] For example, in some embodiments, a promoter can be operably linked to a nucleotide sequence in a construct or vector for reducing the concentration and / or total content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in a tobacco plant or part thereof.

[0420] In one embodiment, the promoter is operably linked to a nucleic acid sequence encoding a deregulated cation efflux protein according to the present invention.

[0421] Suitably, the promoter may be operably linked to:

[0422] a. comprising the nucleotide sequence of SEQ ID No.34;

[0423] b. a nucleotide sequence having at least 70% sequence identity to the entirety of the sequence shown in SEQ ID No. 34, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants;

[0424] c. a nucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No. 33 herein, or a fragment thereof comprising at least 200, or at least 250, or at least 300, or at least 350, or at least 400 consecutive residues of SEQ ID No. 33;

[0425] d. A nucleotide sequence encoding a truncated polypeptide or protein, wherein the truncated polypeptide lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and the protein has at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, wherein the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and the protein has at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, and the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130;

[0426] e. A nucleotide sequence encoding a truncated polypeptide or protein, the truncated polypeptide consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, the protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein, the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0427] f. A nucleotide sequence encoding a polypeptide comprising an amino acid sequence corresponding to amino acids 125-324 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and / or comprising an amino acid sequence corresponding to amino acids 323-413 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants.

[0428] In some embodiments, the promoter can be operably linked to a nucleotide sequence in a construct or vector for increasing the concentration and / or total content of nicotine in a cell or cell culture or a tobacco plant or part thereof.

[0429] In some embodiments, the promoter can be selected from the group consisting of: a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, and an inducible promoter.

[0430] In one embodiment, the promoter may be a constitutive promoter.

[0431] Constitutive promoters direct the expression of a gene continuously throughout various parts of the plant during plant development, although the gene may not be expressed at the same level in all cell types. Examples of known constitutive promoters include those associated with the cauliflower mosaic virus 35S transcript (Odell JT, Nagy F, Chua NH. (1985). Identification of DNA sequences required for activity of the cauliflowermosaic virus 35S promoter. Nature. 313810-2), the rice actin 1 gene (Zhang W, McElroy D, Wu R. (1991). Analysis of rice Act 1 5' region activity in transgenic rice plants. Plant Cell 3 1155-65), and the maize ubiquitin 1 gene (Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE. (1993). Activity of a maize ubiquitin promoter in transgenic rice. Plant Molec. Biol. 23 567-81). A constitutive promoter, for example, the carnation etched ring virus (CERV) promoter (Hull R, Sadler J, Longstaff M (1986) (CaMV / 35S), figwortmosaic virus 35S promoter. The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses. EMBO Journal, 5(2): 3083-3090).

[0432] The constitutive promoter may be selected from: the Carnation Etch Ring Virus (CERV) promoter, the Cauliflower Mosaic Virus (CaMV35S promoter), the promoter from the rice actin 1 gene or the maize ubiquitin 1 gene.

[0433] The promoter can be a tissue-specific promoter. A tissue-specific promoter is a promoter that directs the expression of a gene in one (or several) parts of a plant, usually throughout the life cycle of those plant parts. The category of tissue-specific promoters also generally includes promoters whose specificity is not absolute, i.e., they can also direct expression at lower levels in tissues other than preferred tissues. Tissue-specific promoters include the phaseolin promoter, the legumin b4 promoter, the usp promoter, the sbp promoter, the ST-LS1 promoter, and the B33 (patatin class I promoter).

[0434] In another embodiment, the promoter can be a developmentally regulated promoter.

[0435] Developmentally regulated promoters direct changes in the expression of a gene in one or more parts of a plant at specific times during plant development. The gene may be expressed in that plant part at other times at different (usually lower) levels, and may also be expressed in other plant parts.

[0436] In one embodiment, the promoter may be an inducible promoter.

[0437] Inducible promoter can respond to inducer to guide the expression of gene. In the absence of inducer, gene is not expressed. Inducer can act directly on promoter sequence, or can work by offsetting the effect of repressor molecule. Inducer can be chemical reagent such as metabolite, protein, growth regulator (for example auxin and salicylic acid that activates OCS promoter) or toxic element, physiological stress such as heat, light (for example soybean SSU promoter), wound (for example nos, nopaline synthase promoter) or osmotic pressure, or the indirect consequence of pathogen or pest action. Developmental regulated promoter can be described as the inducible promoter of specific type, and its response is the environmental stimulation of the specific moment of endogenous inducer produced by plant or in the life cycle of plant. Examples of known inducible promoters include, for example, promoters associated with wound response as described by Warner SA, Scott R, Draper J. ((1993) Plant J. 3 191-201), promoters associated with temperature response as disclosed by Benfey & Chua (1989) (Benfey, PN and Chua, NH. ((1989) Science 244 174-181), and promoters associated with chemical induction as described by Gatz ((1995) Methods in Cell Biol. 50 411-424).

[0438] The present invention also provides a construct or vector comprising a nucleic acid sequence encoding a protein according to the present invention.

[0439] In one embodiment, a construct or vector according to the present invention may comprise:

[0440] a. comprising the nucleotide sequence of SEQ ID No.34;

[0441] b. a nucleotide sequence having at least 70% sequence identity to the entirety of the sequence shown in SEQ ID No. 34, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants;

[0442] c. a nucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No. 33 herein, or a fragment thereof comprising at least 200, or at least 250, or at least 300, or at least 350, or at least 400 consecutive residues of SEQ ID No. 33;

[0443] d. A nucleotide sequence encoding a truncated polypeptide or protein, wherein the truncated polypeptide lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and the protein has at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, wherein the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and the protein has at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, and the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130;

[0444] e. A nucleotide sequence encoding a truncated polypeptide or protein, the truncated polypeptide consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, the protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein, the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0445] f. A nucleotide sequence encoding a polypeptide comprising an amino acid sequence corresponding to amino acids 125-324 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and / or comprising an amino acid sequence corresponding to amino acids 323-413 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants.

[0446] The present invention further provides the use of a nucleic acid sequence according to the present invention for reducing the content of tobacco-specific nitrosamines (TSNA) or TSNA precursors in tobacco.

[0447] The present invention also provides a chimeric construct comprising a promoter operably linked to a nucleic acid sequence according to the present invention.

[0448] Suitable promoter sequences may be constitutive, non-constitutive, tissue-specific, developmentally regulated, or inducible / repressible.

[0449] In one embodiment, a suitable promoter may be a promoter selected from the group consisting of a cauliflower mosaic virus 35S promoter, a dianthus etch ring virus (CERV) promoter, a pea plastocyanin promoter, a rubisco promoter, a nopaline synthase promoter, a chlorophyll a / b binding promoter, a high molecular weight glutenin promoter, an α,β-gliadin promoter, a hordein promoter, a patatin promoter, or a wilt-specific promoter.

[0450] The construct may be contained in a vector. Suitably, the vector may be a plasmid.

[0451] Exogenous polynucleotides can be introduced into plants according to the method of the present invention with the aid of a suitable vector, such as a plant transformation vector. The plant transformation vector may comprise an expression cassette comprising a promoter sequence in the transcription direction 5'-3', a target gene (e.g., a nucleic acid sequence encoding a protein according to the present invention) coding sequence, optionally including introns, and optionally a 3' untranslated terminator sequence, including a termination signal for RNA polymerase and a polyadenylation signal for polyadenylate enzyme. The promoter sequence may be present in one or more copies, and such copies may be identical or variants of the promoter sequence as described above. The terminator sequence may be derived from a plant, bacterial or viral gene. Suitable terminator sequences are, for example, the pea rbcS E9 terminator sequence, the nos terminator sequence derived from the nopaline synthase gene of Agrobacterium tumefaciens, and the 35S terminator sequence from cauliflower mosaic virus. Other suitable terminator sequences will be readily known to those skilled in the art.

[0452] The expression cassette can also include a gene expression enhancement mechanism to increase the strength of the promoter. The example of this type of enhancer element is an enhancer element derived from a part of the promoter of the pea plastocyanin gene, and is the subject of International Patent Application No. WO 97 / 20056. Suitable enhancer elements can be, for example, the nos enhancer element derived from the nopaline synthase gene of Agrobacterium tumefaciens, and the 35S enhancer element from cauliflower mosaic virus. These regulatory regions can be derived from the gene identical to the promoter DNA sequence, or can be derived from different genes, for example derived from a plant of the Solanaceae family. All regulatory regions should be able to work in the cell of tissue to be transformed.

[0453] The promoter DNA sequence may be derived from a gene identical to the coding sequence of the target gene used in the present invention (e.g., a gene to which the promoter is intended to direct, such as a gene encoding a modification of a plant to increase the activity or expression of a protein comprising a sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or an amino acid sequence having at least 70% sequence identity thereto), or may be derived from a different gene, such as a plant of the Solanaceae family.

[0454] The expression cassette can be incorporated into a basic plant transformation vector, for example pBIN 19Plus, pBI 101 or other suitable plant transformation vectors known in the art. In addition to the expression cassette, the plant transformation vector also contains such sequences as are necessary for the transformation process. These can include Agrobacterium vir genes, one or more T-DNA border sequences, and selectable markers or other means for identifying transgenic plant cells.

[0455] The term "plant transformation vector" means a construct capable of in vivo or in vitro expression. Preferably, the expression vector is incorporated into the genome of the organism. The term "incorporated" preferably encompasses stable incorporation into the genome.

[0456] The technology for transforming plants is well known in the art and includes, for example, Agrobacterium-mediated transformation. The basic principle in the construction of genetically modified plants is to insert genetic information into the plant genome in order to obtain stable maintenance of the inserted genetic material. A review of general techniques can be found in the papers by Potrykus (Annu Rev Plant Physiol Plant Mol Biol

[1991] 42: 205-225) and Christon (AgroFood-Industry Hi-Tech March / April 1994 17-27).

[0457] Usually, in the transformation mediated by Agrobacterium, by the co-cultivation of Agrobacterium and the explant from the target plant, the binary vector carrying the target exogenous DNA is transferred to the target plant from the appropriate Agrobacterium strain.Then the plant tissue of the transformation is regenerated on the selection medium, and the selection medium comprises a selectable marker and a plant growth hormone.The alternative method is the flower immersion method (Clough & Bent, 1998), which contacts the flower bud of the complete plant with the suspension of the Agrobacterium strain containing the mosaic gene, and after fruiting, by the growth on the selection medium, the individual of the transformation is germinated and identified.Direct infection of plant tissue by Agrobacterium is a simple technique, which has been widely adopted, and is described in Butcher DN et al., (1980), Tissue Culture Methods for Plant Pathologists, editors: DS Ingrams and JPHelgeson, 203-208.

[0458] Further suitable transformation methods include direct gene transfer into protoplasts, for example using polyethylene glycol or electroporation techniques, particle bombardment, microinjection and the use of silicon carbide fibers.

[0459] Use ballistic transformation to comprise that silicon carbide whisker technology is transformed plant in Frame BR, Drayton PR, Bagnaall SV, Lewnau CJ, Bullock WP, Wilson HM, Dunwell JM, Thompson JA & Wang K (1994). Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation is taught in The Plant Journal 6:941-948) and viral transformation technology is taught in for example Meyer P, Heidmmm I & Niedenhof I (1992). The purposes of cassava mosaic virus as the carrier system for plant is taught in Gene 110:213-217. Further instruction about plant transformation can be found in EP-A-0449375.

[0460] In a further aspect, the present invention relates to a vector system, which carries a nucleotide sequence encoding a target gene (e.g., a nucleic acid sequence according to the present invention) and introduces it into the genome of an organism such as a plant. The vector system can comprise a vector, but it can comprise two vectors. In the case of two vectors, the vector system is generally referred to as a binary vector system. The binary vector system is further described in detail in Gynheung Anetal, (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.

[0461] A widely used system for transforming plant cells uses the Ti plasmid from Agrobacterium tumefaciens, or the Ri plasmid from Agrobacterium rhizogenes (Anetal., (1986), Plant Physiol.81, 301-305, and Butcher DN et al., (1980), Tissue Culture Methods for Plant Pathologists, eds.: DS Ingrams and JP Helgeson, 203-208). After each method of introducing the desired foreign gene in the plant according to the present invention, the presence and / or insertion of further DNA sequences may be necessary. The use of T-DNA for plant cell transformation has been extensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters BB, Amsterdam, 1985, Chapter V; Fraley et al., Crit. Rev. Plant Sci., 4: 1-46; and Anetal., EMBO J (1985) 4: 277-284.

[0462] Plant cells transformed with a foreign gene encoding a target protein (e.g., a protein according to the present invention) can be grown and maintained according to well-known tissue culture methods, for example, by culturing the cells in a suitable medium supplied with necessary growth factors such as amino acids, plant hormones, vitamins, etc.

[0463] As described herein, the term "transgenic plant" in relation to the present invention includes any plant comprising a foreign gene encoding a target gene, such as a protein according to the present invention. Preferably, the foreign gene is incorporated into the genome of the plant.

[0464] When the terms "transgenic plants" and "foreign genes" are under the control of their native promoters (which are also in their natural environment), they do not encompass the native nucleotide coding sequences in their natural environment.

[0465] Therefore, in one embodiment, the present invention relates to a method for producing a transgenic plant, which comprises introducing an exogenous gene (chimeric construct or vector) encoding a protein according to the present invention into an unmodified plant.

[0466] In one embodiment, the present invention relates to a method for producing a transgenic plant, which comprises transforming a plant cell with a construct or vector (e.g., a chimeric construct) comprising a nucleic acid encoding a protein according to the present invention; and regenerating a plant from the transformed plant cell.

[0467] In another aspect, provided is the use of an exogenous nucleic acid sequence (construct or vector or chimeric construct) according to the invention for reducing the content of tobacco-specific nitrosamines (TSNA) or TSNA precursors in tobacco plants, for example by transforming the plants with the exogenous nucleic acid sequence (construct or vector or chimeric construct).

[0468] In one embodiment, the present invention further relates to a host cell comprising an exogenous nucleic acid sequence (construct or vector or chimeric construct) according to the present invention.

[0469] In one embodiment, a mutation in an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% sequence identity thereto, can deregulate the protein, relative to the protein as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% sequence identity thereto.

[0470] In some embodiments, relative to a protein as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% sequence identity thereto, a mutation in an amino acid sequence as shown in SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a sequence having at least 70% sequence identity thereto may not alter the level or expression of the protein, but may reduce the activity of the protein.

[0471] Biomass production

[0472] In one aspect, the invention provides a method for producing biomass, comprising:

[0473] Cells that have been engineered to express a deregulated cation efflux protein are grown under conditions that produce biomass.

[0474] In one embodiment, the invention provides a method of producing biomass having a modified (e.g., increased) nicotine concentration and / or total content, comprising growing cells that have been engineered to:

[0475] a) expression of a deregulated cationic efflux protein;

[0476] b) expressing a truncated protein which lacks at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein which lacks at least the amino acids corresponding to 2-25, or 2-50, or 2-75, or 2-100, 2-114 or 2-117 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0477] c) expressing a truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, or a protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0478] d) expressing a protein comprising the amino acid sequence as shown in SEQ ID No. 33, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto;

[0479] e) expressing a protein encoded by a polynucleotide comprising a sequence as shown in SEQ ID No. 34, or a sequence having at least 70% sequence identity thereto, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto.

[0480] Suitably, the biomass may contain a higher concentration and / or total content of nicotine than biomass produced from comparable cells which have not been modified according to the invention.

[0481] Suitably, the cells used in biomass production may be plant cells, such as tobacco cells.

[0482] Suitably, the cells used in biomass production may be yeast cells.

[0483] In one embodiment, the cell (e.g., yeast cell) can be further modified to include one or more sequences that increase nicotine alkaloid biosynthesis. Suitably, the one or more sequences can be incorporated into a nucleic acid construct suitable for cell (e.g., yeast cell) transformation. One or more sequences can be overexpressed in a cell (e.g., yeast cell). The sequence can be selected from one or more of the following genes: MPO (or methylputrescine oxidase or MPO1 or MPO2); A622 (or isoflavone reductase-like protein or isoflavone reductase homolog or isoflavone reductase-like protein); BBL (or berberine bridge enzyme or berberine bridge enzyme-like or BBE or NBB1); PMT (or putrescine N-methyltransferase or putrescine methyltransferase or S-adenosyl-L-methionine: putrescine N-methyltransferase or PMT or PMT1 or PMT2 or PMT3 or PMT4) and QPT (or quinolinate phosphoribosyltransferase). In one embodiment, the sequence may be selected from one or more of the following genes: BBL, A622, PMT and MPO (MPO1 or MPO2). For example, genes suitable for modification in this manner may be taught in US2016032299, which is incorporated herein by reference.

[0484] Commercially desirable traits

[0485] The term "commercially desirable trait" includes traits such as yield, quality (e.g., leaf quality, appropriately cured leaf quality), abiotic (e.g., drought) stress tolerance, herbicide tolerance and / or biotic (e.g., insect, bacterial or fungal) stress tolerance and / or disease tolerance.

[0486] For example, according to the United States Department of Agriculture (USDA) grades and standards, leaf quality can be measured based on the color, texture, and aroma of the roasted leaves.

[0487] Tobacco grades are assessed based on factors including, but not limited to, petiole position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, gloss (related to the intensity and depth of coloration and brightness of the leaf), hygroscopicity (the ability of the leaf to absorb and retain moisture from the environment), and green nuances or fall-off.

[0488] Leaf grade can be determined using standard methods known in the art, such as using the official standard grades (7 U.S.C. § 511) published by the Agricultural Marketing Service of the USD department of Agriculture. See, for example, the official standard grades for burley tobacco (U.S. type 31 and foreign type 93), effective November 5, 1990 (55 F.R. 40645); the official standard grades for flue-cured tobacco (U.S. types 11, 12, 13, 14 and foreign type 92), effective March 27, 1989 (54 F.R. 7925); the official standard grades for Pennsylvania Seedle af Tobacco (U.S. type 41), effective January 8, 1965 (29 F.R. 16854); the official standard grades for Ohio Cigar-Leaf tobacco, effective December 8, 1963 (29 F.R. 16854); the official standard grades for cigar-leaf tobacco (29 F.R. 16854), ...); the official standard grades for cigar-leaf tobacco (29 F.R. 16854); the official standard grades for cigar- Tobacco) (U.S. Types 42, 43 and 44) ​​(28 F.R. 11719 and 28 F.R. 11926); Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061), effective November 20, 1969; Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061), effective November 20, 1969; Georgia and Florida Shade Grown Cigar-Wrapper Tobacco (U.S. Type 62) (34 F.R. 17061), effective April 1971. USDA grade index values ​​may be determined based on industry recognized grade indices. See, e.g., Bowman et al., Tobacco Science, 32:39-40 (1988); Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al., 1990, Tobacco Intern., 192:55-57 (all of the foregoing references are incorporated herein in their entirety).

[0489] In one aspect, the USDA grade index is a 0-100 numerical representation of the received federal grade and is a weighted average of all shank positions. A higher grade index indicates higher quality. Alternatively, the blade grade can be determined via hyperspectral imaging. See, for example, WO 2011 / 027315 (which is incorporated herein by reference).

[0490] In one embodiment, the tobacco plants of the present invention provide commercially acceptable grades of tobacco.

[0491] Suitably, the tobacco plants of the present invention provide commercially acceptable grades of flue-cured tobacco.

[0492] In one embodiment, when growing under similar growth conditions, tobacco plant of the present invention can produce its USDA grade index value is the leaf blade at least about 70% of the USDA grade index value of comparable plant leaf.Suitably, when growing under similar growth conditions, tobacco plant disclosed herein may be able to produce its USDA grade index value is the leaf blade at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% of the USDA grade index value of control plant. Suitably, the tobacco plants disclosed herein may be capable of producing leaves having a USDA grade index value that is 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the USDA grade index value of a comparable plant.

[0493] In one aspect, the tobacco plants of the present invention are capable of producing leaves having a USDA Grade Index value of at least 50. Suitably, the tobacco plants disclosed herein may be capable of producing leaves having a USDA Grade Index value of 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more.

[0494] Unless otherwise indicated, tobacco yield as used herein refers to cured leaf yield, which is calculated based on the weight of cured leaf per acre under standard field conditions following standard agronomic and curing practices.

[0495] In one aspect, tobacco plants of the invention have a yield that is 50% to 150%, 55% to 145%, 60% to 140%, 65% to 135%, 70% to 130%, 75% to 125%, 80% to 120%, 85% to 115%, 90% to 110%, 95% to 105%, 50% to 100%, 55% to 100%, 60% to 100%, 6 ... From 1% to 100%, from 70% to 100%, from 75% to 100%, from 80% to 100%, from 85% to 100%, from 90% to 100%, from 95% to 100%, from 100% to 150%, from 105% to 150%, from 110% to 150%, from 115% to 150%, from 120% to 150%, from 125% to 150%, from 130% to 150%, from 135% to 150%, from 140% to 150%, or from 145% to 150%.

[0496] In another aspect, the tobacco plants of the invention have a yield that is about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 times greater than the yield of comparable plants when grown under similar field conditions.

[0497] In another aspect, the yield of tobacco plants of the present invention is comparable to the yield of comparable plants that are cured when grown under similar field conditions.

[0498] In one aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of about 1200 to 3500, 1300 to 3400, 1400 to 3300, 1500 to 3200, 1600 to 3100, 1700 to 3000, 1800 to 2900, 1900 to 2800, 2000 to 2700, 2100 to 2600, 2200 to 2500, and 2300 to 2400 pounds per acre.

[0499] In another aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of about 1200 to 3500, 1300 to 3500, 1400 to 3500, 1500 to 3500, 1600 to 3500, 1700 to 3500, 1800 to 3500, 1900 to 3500, 2000 to 3500, 2100 to 3500, 2200 to 3500, 2300 to 3500, 2400 to 3500, 2500 to 3500, 2600 to 3500, 2700 to 3500, 2800 to 3500, 2900 to 3500, 3000 to 3500, and 3100 to 3500 pounds per acre.

[0500] In a further aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of about 1200 to 3500, 1200 to 3400, 1200 to 3300, 1200 to 3200, 1200 to 3100, 1200 to 3000, 1200 to 2900, 1200 to 2800, 1200 to 2700, 1200 to 2600, 1200 to 2500, 1200 to 2400, 1200 to 2300, 1200 to 2200, 1200 to 2100, 1200 to 2000, 1200 to 1900, 1200 to 1800, 1200 to 1700, 1200 to 1600, 1200 to 1500, and 1200 to 1400 pounds per acre.

[0501] Plant cultivation

[0502] In one embodiment, the present invention provides a method for producing a tobacco plant having a reduced content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors, comprising:

[0503] a. crossing a donor tobacco plant that produces reduced levels of tobacco-specific nitrosamines (TSNAs) or TSNA precursors and comprises a deregulated cationic efflux protein with a recipient tobacco plant that does not produce reduced levels of tobacco-specific nitrosamines (TSNAs) or TSNA precursors and has a commercially desirable trait;

[0504] b. separating genetic material from offspring of the donor plant hybridized with the recipient plant; and

[0505] c. performing molecular marker-assisted selection using molecular markers, comprising:

[0506] d. Identifying the introgression region comprising a mutation in the polynucleotide sequence encoding the protein defined in a.

[0507] Molecular marker-assisted selection may include performing PCR to identify introgressed nucleic acid sequences comprising:

[0508] a. comprising the nucleotide sequence of SEQ ID No.34;

[0509] b. a nucleotide sequence having at least 70% sequence identity to the entirety of the sequence shown in SEQ ID No. 34, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants;

[0510] c. a nucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID No. 33 herein, or a fragment thereof comprising at least 200, or at least 250, or at least 300, or at least 350, or at least 400 consecutive residues of SEQ ID No. 33;

[0511] d. A nucleotide sequence encoding a truncated polypeptide or protein, wherein the truncated polypeptide lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and the protein has at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, wherein the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and the protein has at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, and the truncated protein lacks at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130;

[0512] e. A nucleotide sequence encoding a truncated polypeptide or protein, the truncated polypeptide consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, the protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein, the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30;

[0513] f. a nucleotide sequence encoding a polypeptide comprising an amino acid sequence corresponding to amino acids 125-324 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and / or comprising an amino acid sequence corresponding to amino acids 323-413 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants;

[0514] Tobacco plants and parts thereof

[0515] As used herein, term " tobacco plant " refers to the plant in the Nicotiana (Nicotiana) used in the production of tobacco industry products.The non-limiting examples of suitable tobacco plants include common tobacco and yellow flower tobacco (for example, LAB21, LN KY171, T1 1406, Basma, Galpao, Perique, Beinhart 1000-1 and Petico).

[0516] Thus, in one embodiment, the tobacco plant does include Nicotiana tabacum (Nicotiana tabacum baginifolia).

[0517] Tobacco material can be derived from the mutation of common tobacco species, is commonly referred to as burley tobacco mutation, flue-cured tobacco or light mutation, dark mutation and oriental / Turkey mutation.In some embodiments, tobacco material is derived from burley tobacco, Virginia, baking, air-drying, fire-cured, oriental or dark tobacco plant.Tobacco plant can be selected from Maryland tobacco, rare tobacco, special tobacco, expansion tobacco etc.

[0518] The present invention also considers the use of tobacco cultivars and superior tobacco cultivars.Therefore, the tobacco plant used herein can be a tobacco cultivar or an superior tobacco cultivar.

[0519] Particularly useful common tobacco varieties include Burley, dark, flue-cured, and Oriental tobaccos.

[0520] In some embodiments, the tobacco plant can be, for example, selected from one or more of the following varieties: Nicotiana tabacum AA 37-1, Nicotiana tabacum B13P, Nicotiana tabacum Xanthi (Mitchell-Mor), Nicotiana tabacum KT D#3 Hybrid 107, Nicotiana tabacum Bel-W3, Nicotiana tabacum 79-615, Nicotiana tabacum Samsun Holmes NN, F4 from a cross of Nicotiana tabacum BU21 x Nicotiana tabacum Hoja Parado, Line 97, Nicotiana tabacum KTRDC#2 Hybrid 49, Nicotiana tabacum KTRDC#4 Hybrid 110, Nicotiana tabacum Burley 21, Nicotiana tabacum PM016, Nicotiana tabacum KTRDC#5KY 160SI, Nicotiana tabacum KTRDC#7FCA, Nicotiana tabacum KTRDC#6TN 86SI, Nicotiana tabacum PM021, Nicotiana tabacum K 149, Nicotiana tabacum K 326, Nicotiana tabacum K 346, Nicotiana tabacum K 358, Nicotiana tabacum K 394, Nicotiana tabacum K 399, Tobacco K 730, Tobacco KY10, Tobacco KY 14, Tobacco KY 160, Tobacco KY 17, Tobacco KY 8959, Tobacco KY 9, Tobacco KY 907, Tobacco MD 609, Tobacco McNair 373, Tobacco NC 2000, Tobacco PG 01, Tobacco PG 04, Tobacco P01, Tobacco P02, Tobacco P03, Tobacco RG 1 1, Tobacco RG17, Tobacco RG 8, Tobacco Speight G-28, Tobacco TN 86, Tobacco TN 90, Tobacco VA509, Tobacco AS44, Tobacco Banket A1, Tobacco Basma Drama B84 / 31, Tobacco BasmaI Zichna ZP4 / B, Tobacco Basma Xanthi BX 2A, Tobacco Batek, Tobacco Besuki Jember, Tobacco C104, Tobacco Coker 319, Tobacco Coker 347, Tobacco Criollo Misionero, Tobacco PM092, Tobacco Delcrest, Tobacco Djebel 81, Tobacco DVH 405, Tobacco Galpao Comum, Tobacco HB04P, Tobacco Hicks Broadleaf, Tobacco Kabakulak Elassona, Tobacco PM102, Tobacco Kutsage E1, Tobacco KY 14xL8, Tobacco KY 171, Tobacco LA BU 21, Tobacco McNair 944, Tobacco NC2326, NC 71, NC297, NC 3, PVH 03, PVH 09, PVH 19, PVH21 10, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, PM132, Wislica, Yayaldag, NC 4, TR Madole, Prilep HC-72, Prilep P23, Prilep PB156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, tobacco T′I-1068, tobacco KDH-960, tobacco TI-1070, tobacco TW136, tobacco PM204, tobacco PM205, tobacco Basma, tobacco TKF 4028, tobacco L8, tobacco TKF 2002, tobacco TN90, tobacco GR141, tobacco Basma xanthi, tobacco GR149, tobacco GR153, and tobacco Petit Havana.

[0521] Non-limiting examples of varieties or cultivars are: BD 64, CC 101, CC 200, CC 27, CC 301, CC400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF91 1, DT 538LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501LC, K149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY160, KY 17, KY 171, KY 907, KY907LC, KTY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129.NC 2002.Neal Smith Madole, OXFORD207, PD 7302LC, PD 7309LC, PD 7312LC 'Periq'e' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-1 1, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51. RS1410, Speight168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, Tl1406, Tl 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TND950, TR (Tom Rosson) Madole, VA 309, VA359, AA 37-1, B 13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, SamsunHolmes NN, KTRDC No. 2 Hybrid 49, Burley 21, KY 8959, KY 9, MD 609, PG 01, PG 04, P01, P02, P03, RG 1 1, RG 8, VA509, AS44, Banket A1, Basma Drama B84 / 31, Basma I ZichnaZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, CriolloMisionero, Delcrest, Djebel 81.DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LABU 21, NC2326, NC 297, PVH 21 10, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, TI-1068, KDH-960, Tl-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi, GR149, GR153, PetitHavana. Even though not specifically indicated herein, the above-mentioned low converter subvarieties are contemplated.

[0522] In one embodiment, the tobacco plant is a Burley-type tobacco plant, suitably Burley Nicotiana tabacum PH2517.

[0523] In one embodiment, the plant propagation material may be obtainable from a tobacco plant of the present invention.

[0524] As used herein, "plant propagation material" refers to any plant matter taken from a plant from which further plants can be produced. Suitably, the plant propagation material may be selected from seeds, plant callus and plant pieces.

[0525] Suitably, the plant propagation material may be seeds. Suitably, the plant propagation material may be plant callus. Suitably, the plant propagation material may be plant pieces.

[0526] In one embodiment, the cell (e.g., tobacco cell), tobacco plant and / or plant propagation material may be obtainable (e.g., obtained) by the method according to the invention. In one embodiment, the cell (e.g., tobacco cell), tobacco plant and / or plant propagation material of the present invention may comprise:

[0527] a nucleic acid sequence encoding a deregulated cation efflux protein;

[0528] A nucleotide sequence comprising SEQ ID No.34;

[0529] A nucleotide sequence having at least 70% sequence identity to the entirety of the sequence shown in SEQ ID No. 34, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants;

[0530] A nucleotide sequence encoding a polypeptide comprising the amino acid sequence shown herein as SEQ ID No. 33, or a fragment thereof comprising at least 200, or at least 250, or at least 300, or at least 350, or at least 400 consecutive residues of SEQ ID No. 33;

[0531] A nucleotide sequence encoding a truncated polypeptide or protein, the truncated polypeptide lacking at least the amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130 from the N-terminal side of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, the protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity with the truncated protein, the truncated protein ... No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-117, or 2-120, or 2-125, or 2-128, or 2-130;

[0532] A nucleotide sequence encoding a truncated polypeptide or protein, the truncated polypeptide consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, the protein having at least 70% (preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%) sequence identity to the truncated protein, the truncated protein consisting essentially of or consisting of at least 375, at least 350, or at least 300, or at least 250, or at least 200, or at least 150 amino acids from the C-terminus of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30; or

[0533] A nucleotide sequence encoding a polypeptide comprising an amino acid sequence corresponding to amino acids 125-324 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, and / or comprising an amino acid sequence corresponding to amino acids 323-413 of SEQ ID No. 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants.

[0534] Suitably, the tobacco plant according to the invention may have, for example, a reduced content of tobacco-specific nitrosamines (TSNAs) or TSNA precursors when compared to an unmodified tobacco plant, wherein the modification is the expression of a deregulated cationic efflux protein according to the invention, or the expression of a nucleic acid sequence according to the invention.

[0535] In one embodiment, a tobacco plant according to the invention comprises a tobacco cell of the invention.

[0536] In another embodiment, the plant propagation material may be obtainable (eg, obtained) from a tobacco plant of the present invention.

[0537] In one embodiment, provided is the use of a tobacco plant as described herein to grow tobacco plants.

[0538] In another embodiment, the present invention also provides use of the tobacco plant of the preceding embodiment for producing tobacco industry products.

[0539] In another embodiment, provided is the use of the tobacco plant of the present invention for growing crops.

[0540] In one embodiment, provided is the use of a cell as provided in the preceding embodiments for producing a tobacco industry product.

[0541] In one embodiment, the invention provides cell cultures (eg, in vitro cultures).

[0542] Tobacco cell culture can be a cell suspension culture. These in vitro cultured cells can be incorporated into the tobacco industry product, for example as a substitute for conventional tobacco particles, tobacco shreds, fine cutting or long cutting tobacco flakes, as an additive component, or both as a substitute and as an additive. Suitably, cell culture can produce nicotine.

[0543] In one embodiment, provided is the use of a cell culture, eg, a harvested and / or processed cell culture according to the invention, for producing a tobacco industry product.

[0544] Cells harvested from in vitro culture may be dried, such as freeze-dried, for example to produce a powder.

[0545] In one embodiment, the cell culture is a tobacco cell culture.

[0546] The technician will know the known methods for setting up the in vitro culture of tobacco cell.Only as an example, following method can be used: collect the seed from the target tobacco plant, and sterilize its outside to eliminate undesirable organism, plant said seed to grow the target tobacco plant, from tobacco plant (for example from tobacco stem), take out tissue for use as explant, set up callus culture from tobacco explant, set up cell suspension culture from callus culture, and gather in the crops culture material (for example comprising tobacco cell) to produce tobacco cell culture.

[0547] Tobacco cells can be harvested by various methods including filtration such as vacuum filtration. The sample can be washed in a filter by adding water and the remaining liquid removed by filtration such as vacuum filtration.

[0548] The harvested tobacco cell culture may be further processed, such as dried, such as air-dried and / or freeze-dried.The harvested tobacco cell culture or the dried harvested tobacco cell culture may be incorporated into a tobacco industry product according to the present invention.

[0549] In one embodiment, the cell culture is a yeast cell culture. In one embodiment, the cell (e.g., yeast cell) may be further modified to include one or more sequences that increase nicotine alkaloid biosynthesis. Suitably, the one or more sequences may be incorporated into a nucleic acid construct suitable for cell (e.g., yeast cell) transformation. One or more sequences may be overexpressed in a cell (e.g., yeast cell). The sequence may be selected from one or more of the following genes: MPO (or methylputrescine oxidase or MPO1 or MPO2); A622 (or isoflavone reductase-like protein or isoflavone reductase homolog or isoflavone reductase-like protein); BBL (or berberine bridge enzyme or berberine bridge enzyme-like or BBE or NBB1); PMT (or putrescine N-methyltransferase or putrescine methyltransferase or S-adenosyl-L-methionine: putrescine N-methyltransferase or PMT or PMT1 or PMT2 or PMT3 or PMT4) and QPT (or quinolinate phosphoribosyltransferase). In one embodiment, the sequence may be selected from one or more of the following genes: BBL, A622, PMT and MPO (MPO1 or MPO2). For example, genes suitable for modification in this manner may be taught in US2016032299, which is incorporated herein by reference.

[0550] product

[0551] The invention also provides products obtainable or obtained from tobacco according to the invention.

[0552] In one embodiment, provided is the use of the tobacco plant of the present invention to produce tobacco leaves.

[0553] Suitably, the tobacco leaves may be subjected to downstream applications such as processing.

[0554] Therefore, in one embodiment, the use of the foregoing embodiments can provide processed tobacco leaves. Suitably, the tobacco leaves can be subjected to curing, fermentation, pasteurization or a combination thereof. In another embodiment, the tobacco leaves can be shredded. In some embodiments, the tobacco leaves can be shredded before or after being subjected to curing, fermentation, pasteurization or a combination thereof.

[0555] In one embodiment, the invention provides harvested leaves of the tobacco plant of the invention.

[0556] In a further embodiment, the harvested leaves may be obtainable (eg, obtained) from a tobacco plant propagated from the propagation material of the invention.

[0557] In another embodiment, provided are harvested leaves obtainable from the methods or uses of the invention.

[0558] Suitably, the harvested leaves may be shredded harvested leaves.

[0559] In some embodiments, the harvested leaves may contain living tobacco cells. In other embodiments, the harvested leaves may be subjected to further processing.

[0560] Also available is processed tobacco leaves.

[0561] Processed tobacco leaves may be obtainable from a tobacco plant of the invention. Suitably, processed tobacco leaves may be obtainable from a tobacco plant obtained according to any of the methods and / or uses of the invention.

[0562] In another embodiment, processed tobacco leaves may be obtainable from tobacco plants propagated from tobacco plant propagation material according to the present invention.

[0563] The processed tobacco leaf of the present invention may be obtainable by processing the harvested leaf of the present invention.

[0564] As used herein, the term "processed tobacco leaf" refers to tobacco leaf that has undergone one or more processing steps to which tobacco is subjected in the art."Processed tobacco leaf" contains no or substantially no living cells.

[0565] The term "living cell" refers to a cell that is capable of growth and / or metabolic activity. Thus, if a cell is said to be not alive, also known as "non-viable", the cell does not exhibit the characteristics of a living cell.

[0566] The term "substantially free of viable cells" means that less than about 5% of all cells are viable. Preferably, less than about 3%, more preferably less than about 1%, even more preferably less than about 0.1% of all cells are viable.

[0567] In one embodiment, the processed tobacco leaves may be processed by one or more of: curing, fermenting, and / or pasteurizing.

[0568] Suitably, the processed tobacco leaves may be processed by curing.

[0569] The tobacco leaves can be cured by any method known in the art. In one embodiment, the tobacco leaves can be cured by one or more curing methods selected from the following: air curing, fire curing, baking and sun curing.

[0570] Suitably, the tobacco leaves may be air-cured.

[0571] Typically, air-curing is achieved by hanging the leaves in a well-ventilated barn and allowing them to dry. This is usually done over a period of four to eight weeks. Air-curing is particularly suitable for Burley tobacco.

[0572] Suitably, the tobacco leaves may be fire-cured. Fire-curing is usually achieved by hanging the leaves in large barns where hardwood fires are kept smoldering continuously or intermittently and typically takes from three days to ten weeks depending on the process and the tobacco.

[0573] In another embodiment, the tobacco leaves can be cured. Curing can include stringing the tobacco leaves onto tobacco stems and hanging them from tier-poles in a curing barn. The barn usually has a flue that enters from an externally supplied firebox. Typically, this results in tobacco that has been heat-cured without being exposed to smoke. Typically, the temperature rises slowly during the curing process, with the entire process taking about 1 week.

[0574] Suitably, the tobacco leaves may be sun-cured. This process generally involves exposing uncovered tobacco to the sun.

[0575] Suitably, the process tobacco may be processed by fermentation.

[0576] Fermentation can be carried out in any manner known in the art. Usually, during fermentation, tobacco leaves are piled into a flue-cured tobacco pile (pile) covered in, for example, a sack, to retain moisture. The combination of the residual water inside the leaf and the tobacco weight generates the natural heat that makes tobacco mature. The temperature in the center of the pile is monitored every day. In some methods, the whole pile is opened every week. The leaves are then moved to shake and moisten, and the pile is turned over so that the inner leaves are outside and the bottom leaves are placed on the top of the pile. This ensures the uniform fermentation of the whole pile. The other moisture on the leaf adds the actual turning over of the leaf itself to generate heat, releases the natural ammonia of tobacco and reduces nicotine, while also deepening the color and improving the aroma of tobacco. Usually, the fermentation process lasts up to 6 months, which depends on the variety of tobacco, the handle position on the leaf, the thickness of the leaf and the intended use.

[0577] Suitably, the processed tobacco leaves may be processed by pasteurisation.Pasteurisation may be particularly preferred when the tobacco leaves are to be used in the preparation of smokeless tobacco industrial products, most preferably snus.

[0578] Tobacco pasteurization can be carried out by any method known in the art. For example, pasteurization can be carried out as described in J Foulds, L Ramstrom, M Burke, K Fagerstrom. Effect of smokeless tobacco (snus) on smoking and public health in Sweden. Tobacco Control (2003) 12 :349–359, the teachings of which are incorporated herein by reference.

[0579] During snus production, pasteurization is usually carried out by a process in which the tobacco is heat treated with steam for 24-36 hours, reaching a temperature of about 100° C. This results in a nearly sterile product and, without wishing to be bound by theory, one of the consequences of this is believed to be limiting further TSNA formation.

[0580] In one embodiment, the pasteurization may be steam pasteurization.

[0581] In some embodiments, the processed tobacco leaves may be shredded. The processed tobacco leaves may be shredded before or after processing. Suitably, the processed tobacco leaves may be shredded after processing.

[0582] In one embodiment, use of the foregoing embodiments can provide reconstituted tobacco.

[0583] As used herein, "reconstituted" may also be referred to as reconstituted, recycled or homogenized sheet tobacco, and refers to tobacco material generated from processed tobacco leaf residues. Reconstituted tobacco allows the production of consistent, high quality blends, and allows the ratios of the various components to be adjusted.

[0584] Reconstituted tobacco can be nanofiber reconstituted (nanofibers can be extracted in solid or liquid form), paper reconstituted (which uses stems, chips, midribs, etc. as raw materials), or pulp-type reconstituted (which uses a mixture of fine powder and tobacco stems ground into powder, mixed with water and a vegetable binder. The soluble residue is formed into flakes by extracting water.)

[0585] Any method known in the art can be used to prepare reconstituted tobacco, for example, see CORESTA Congress, Sapporo, 2012, Smoke Science / Product Technology Groups, SSPT 12 (incorporated herein by reference).

[0586] In some embodiments, tobacco plant, the harvest leaf of tobacco plant and / or processing tobacco leaf can be used to extract nicotine.The extraction of nicotine can be realized using any method known in the art.For example, the method for extracting nicotine from tobacco is taught in US2,162,738, which is incorporated herein by reference.

[0587] In one aspect, the present invention provides a cured tobacco material made from a tobacco plant or part thereof according to the present invention.

[0588] In another aspect, the present invention provides a tobacco blend comprising a tobacco material made from a tobacco plant or part thereof according to the present invention, or a tobacco cell culture according to the present invention. In one aspect, the present invention provides a tobacco blend comprising a flue-cured tobacco material according to the present invention.

[0589] Suitably, tobacco blends according to the present invention may include about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% tobacco from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may include about 10% tobacco from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may include about 20% tobacco from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may include about 30% tobacco from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may include about 40% tobacco from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may include about 50% tobacco from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends may include about 60% tobacco from tobacco plants according to the present invention or parts thereof, or from tobacco cell cultures according to the present invention. Suitably, tobacco blends can comprise approximately 70% coming from tobacco plant according to the present invention or its part or from tobacco of tobacco cell culture according to the present invention. Suitably, tobacco blends can comprise approximately 80% coming from tobacco plant according to the present invention or its part or from tobacco of tobacco cell culture according to the present invention. Suitably, tobacco blends can comprise approximately 90% coming from tobacco plant according to the present invention or its part or from tobacco of tobacco cell culture according to the present invention.

[0590] In one aspect, the tobacco blend products of the present invention comprise at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 percent by dry weight of tobacco cured from a tobacco plant or part thereof according to the present invention, or a tobacco cell culture according to the present invention.

[0591] Suitably, the cured tobacco material may be air-cured. Suitably, the cured tobacco material may be baked. Suitably, the cured tobacco material may be sun-cured. Suitably, the cured tobacco material may be fire-cured.

[0592] Tobacco industry products or smoking articles according to the invention may comprise a tobacco material according to the invention (eg a cured tobacco material or a reconstituted tobacco material).

[0593] In another aspect, the invention provides tobacco industry products.

[0594] In one embodiment, the tobacco industry product according to the invention may be a blended tobacco industry product. Suitably, the tobacco blend may comprise a flue-cured tobacco material according to the invention.

[0595] In one embodiment, tobacco industry products can be prepared from the tobacco plants or parts thereof of the present invention.

[0596] Suitably, tobacco plants or parts thereof may be propagated from tobacco plant propagation material according to the invention.

[0597] As used herein in the context of tobacco plant, term " its part " refers to a part of tobacco plant. Suitably, " its part " can be leaf, root or stem or flower of tobacco plant. Suitably, " its part " can be leaf, root or stem of tobacco plant.

[0598] Tobacco industry products

[0599] As used herein, the term "tobacco industry product" is intended to include combustible smoking articles, such as cigarettes, cigarillos, cigars, tobacco for pipes or roll-your-own cigarettes (whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, tobacco substitutes or other smokeable materials), non-combustible gas aerosol delivery systems (e.g., heating products that release compounds from a matrix material without combustion, such as electronic cigarettes, tobacco heating products, and hybrid systems that generate aerosols from a combination of matrix materials, such as hybrid systems containing liquid or gel or solid matrices), and aerosolizable matrix materials used in these aerosol delivery systems; as well as aerosol-free delivery products, such as lozenges, chewing gum, patches, products containing breathable powders, and smokeless tobacco industry products such as snus and snus, which aerosol-free delivery products may or may not deliver nicotine.

[0600] In one embodiment, a tobacco industry product can be prepared from (eg, can comprise) a tobacco plant or part thereof of the invention.

[0601] Suitably, tobacco plants or parts thereof may be propagated from tobacco plant propagation material according to the invention.

[0602] As used herein in the context of tobacco plants, the term "parts thereof" refers to a part of the tobacco plant. Preferably, the "parts thereof" are leaves of the tobacco plant.

[0603] In another embodiment, tobacco industry products can be prepared from the harvested leaves of the present invention.

[0604] In a further embodiment, tobacco industry products can be prepared from the processed tobacco leaves of the present invention.

[0605] Suitably, tobacco industry products may be prepared from tobacco leaves processed by one or more of the following: curing, fermentation and / or pasteurization.

[0606] Suitably, the tobacco industry product may comprise cut tobacco leaves, optionally processed according to the preceding embodiments.

[0607] In another embodiment, tobacco industry products can be prepared from tobacco cell cultures according to the present invention.

[0608] In another embodiment, a tobacco industry product can be prepared from (eg can comprise) a flue-cured tobacco material according to the invention.

[0609] In another embodiment, a tobacco industry product can be prepared from (eg, can include) a tobacco blend according to the invention.

[0610] In one embodiment, the tobacco industry product may be a smoking article.

[0611] As used herein, the term "smoking article" may include smokeable products such as cigarettes, cigarettes, cigars and cigarillos, whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco or tobacco substitutes.

[0612] In another embodiment, the tobacco industry product can be a smokeless tobacco industry product.

[0613] As used herein, the term "smokeless tobacco industry product" refers to a tobacco industry product that is not intended to be smoked and / or subjected to combustion.

[0614] Smokeless tobacco industry products (including heat-not-burn materials) can contain tobacco in any form, including dry particles, shreds, granules, powders, or slurries deposited on, mixed in, surrounded by, or combined with other ingredients, which take any form, such as sheets, films, tabs, foams, or beads.

[0615] In one embodiment, smokeless tobacco industry products can include snus, snuff, chewing tobacco, and the like.

[0616] In one embodiment, the tobacco industry product is a combustible smoking article selected from the group consisting of cigarettes, cigarillos, and cigars.

[0617] In one embodiment, the tobacco industry product comprises one or more components of a combustible smoking article, such as filters, filter rods, filter rod segments, tobacco, tobacco rods, tobacco rod segments, spills, additive release components such as capsules, threads, beads, paper (e.g., plug wrap, tipping paper, or cigarette paper).

[0618] In one embodiment, the tobacco industry product is a non-combustible aerosol delivery system.

[0619] In one embodiment, the tobacco industry product comprises one or more components of a non-combustible gas aerosol delivery system, such as a heater and an aerosolizable substrate.

[0620] In one embodiment, the aerosol supply system is an electronic cigarette, also known as a vaping device.

[0621] In one embodiment, the electronic cigarette includes a heater, a power source capable of powering the heater, an aerosolizable substrate such as a liquid or gel, a housing, and optionally a mouthpiece.

[0622] In one embodiment, the aerosolizable substrate is contained in a substrate container. In one embodiment, the substrate container is associated with or includes a heater.

[0623] In one embodiment, the tobacco industry product is a heating product that releases one or more compounds by heating rather than burning a matrix material. The matrix material is an atomizable material, which may be, for example, tobacco or other non-tobacco products, which may or may not contain nicotine. In one embodiment, the heating product is a tobacco heating product.

[0624] In one embodiment, the heating product is an electronic device.

[0625] In one embodiment, the tobacco heating product comprises a heater, a power source capable of powering the heater, and an atomizable substrate such as a solid or gel material.

[0626] In one embodiment, the heating product is a non-electronic article.

[0627] In one embodiment, the heating product comprises an atomizable substrate, such as a solid or gel material, and a heat source capable of supplying thermal energy to the atomizable substrate without any electronic means, for example by burning a combustion material such as charcoal.

[0628] In one embodiment, the heated product further comprises a filter capable of filtering the aerosol generated by heating the aerosolizable substrate.

[0629] In some embodiments, the aerosolizable base material may comprise a vapor or aerosol generating agent or a humectant, such as glycerin, propylene glycol, triacetin, or diethylene glycol.

[0630] In one embodiment, the tobacco industry product is a hybrid system, generates aerosol with the combination of the non-combustion host material by heating.The host material can comprise for example solid, liquid or gel, which may or may not contain nicotine.In one embodiment, the hybrid system comprises liquid or gel matrix and solid matrix.Solid matrix can be for example tobacco or other non-tobacco product, which may or may not contain nicotine.In one embodiment, the hybrid system comprises liquid or gel matrix and tobacco.

[0631] In a further embodiment, the tobacco industry product may be a tobacco heating device or a mixing device or an electronic cigarette or the like.

[0632] Typically in a tobacco heating device or mixing device, aerosol is generated by transferring heat from a heat source to a physically separate aerosol-forming substrate or material, which may be located within, around or downstream of the heat source. During smoking, volatile compounds are released from the aerosol-forming substrate by heat transfer from the heat source and are entrained in the air drawn in by the smoking article. As the released compounds cool, they condense to form an aerosol that is inhaled by the user.

[0633] Aerosol-generating articles and devices for consuming or smoking tobacco heating devices are known in the art. They may include, for example, electrically heated aerosol-generating devices, wherein aerosol is generated by transferring heat from one or more electrical heating elements of the aerosol-generating device to an aerosol-forming substrate of the tobacco heating device.

[0634] Suitably, the tobacco heating device may be an aerosol generating device.

[0635] Preferably, the tobacco heating device may be a heat-not-burn device. Heat-not-burn devices are known in the art and release compounds by heating rather than burning tobacco.

[0636] Examples of suitable heat-but-no-burn devices may be those taught in WO2013 / 034459 or GB2515502, which are incorporated herein by reference.

[0637] In one embodiment, the aerosol-forming substrate of a tobacco heating device may be a tobacco industry product according to the present invention.

[0638] In one embodiment, the tobacco heating device may be a hybrid device.

[0639] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY, Harper Perennial, NY (1991), provide a general dictionary of many of the terms used in the present disclosure for those of skill in the art.

[0640] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure. Numerical ranges include the numerical values ​​of the limited ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction, respectively.

[0641] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0642] Amino acids are referred to herein using either the amino acid name, three letter abbreviation, or the single letter abbreviation.

[0643] As used herein, the term "protein" includes proteins, polypeptides, and peptides.

[0644] As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide."

[0645] The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids is as defined by the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It should also be understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0646] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, as these may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be restrictive, as the scope of the present disclosure is limited only by the appended claims.

[0647] In the case of providing a range of values, it should be understood that, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of the scope to one-tenth of the lower limit unit is also specifically disclosed. Each smaller range between any specified value or intermediate value in a given range and any other specified value or intermediate value in the given range is included in the present disclosure. The upper and lower limits of these smaller ranges can be independently included in the scope or excluded from the scope, and each range wherein includes any limit, does not include a limit or includes two limits in a smaller range is also included in the present disclosure, subject to any specifically excluded limit in the given range. When a given range includes one or two limits, excluding these includes any or both of the scopes in the limit and is also included in the present disclosure.

[0648] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" or a "nucleic acid sequence" includes a plurality of such candidate agents and equivalents thereof known to those skilled in the art, and so forth.

[0649] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art for the claims appended hereto.

[0650] The invention will now be described, by way of example only, with reference to the following figures and examples.

[0651] Example

[0652] Example 1

[0653] Methods and Materials

[0654] Plant materials

[0655] Plant material from the genus Nicotiana includes common tobacco, for example, Burley and Virginia varieties, and other species, such as Nicotiana rustica.

[0656] Bacterial strains

[0657] Escherichia coli (E. coli) strain TOP10 F-[F-mcrAΔ(mrr-hsdRMS-mcrBC)Φ80lacZΔM15ΔlacX74 recA1 araD139Δ(araleu)7697galU galKrpsL(StrR)endA1 nupG] was used for cloning and plasmid DNA production. E. coli strain TOP10 was transformed by chemical methods.

[0658] Agrobacterium tumefaciens strain GV3101::pMP90 was used for binary vector transformation in planta assays.

[0659] Alkaloid measurement

[0660] The relative content of pyridine alkaloids was determined by reverse phase high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS).Chromatographic separation was achieved using a Gemini-NX column (100 mm × 3.0 mm, particle size 3 μm, Phenomenex) and a gradient chromatographic separation using 6.5 mM ammonium acetate buffer (aq) (pH 10) and methanol.

[0661] The mass spectrometer was operated in electrospray (ESI) positive mode using scheduled MRM data acquisition. Two MRM transitions were monitored for each analyte and one for an isotopically labeled internal standard.

[0662]

[0663] Statistical analysis

[0664] Statistical significance based on one-way ANOVA analysis was performed using Prism 5.01 software (GraphPad Software).

[0665] Virus-induced gene silencing (VIGS)

[0666] TRV vectors containing both SEQ ID No.49 (TRV RNA1) and SEQ ID No.50 (TRV RNA2) containing SEQ ID No.51 (targeting nucleotide sequence) were propagated separately in Agrobacterium tumefaciens. These cultures were mixed (1:1) and infiltrated into 2-week-old tobacco plants with a syringe. The silencing effect of the virus two weeks after infection was evaluated by evaluating the expression level of the target gene.

[0667] result

[0668] The nornicotine content of 5-week-old tobacco leaves expressing the VIGS target sequence (SEQ ID No.51) and the VIGS control sequence (SEQ ID No.52) is shown in Figure 2 middle.

[0669] Nornicotine content is expressed relative to the control (SEQ ID No. 52) and contains three biological replicates analyzed by one-way ANOVA and Tukey's multiple comparison post hoc test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.001.

[0670] Conclusion Silencing of the cationic efflux protein encoded by SEQ ID No. 2 leads to an increase in nornicotine content.

[0671] Example 2

[0672] Methods and Materials

[0673] clone

[0674] GDB-cation efflux expression vector

[0675] Using primers located outside the attB1 and atB2 sites flanking the gene sequence (M13.fw [SEQ ID No.36] and M13.rv [SEQ ID No.37]), the TM The gene sequence (SEQ ID No. 2) was amplified from a compatible cDNA library and then transferred to a GDB expression vector (SEQ ID No. 38).

[0676] GDB-cation efflux_AS expression vector

[0677] Using the GDB-cation efflux expression vector as a template, a first set of gene-specific primers (cation efflux_AS.fw [SEQ ID No.40]] and cation efflux_AS.rv [SEQ ID No.41]), and a second set of Gateway TM Compatible primers (attB1 [SEQ ID No. 42] and attB2 [SEQ ID No. 43]), through two-step amplification and Gateway TM The AS construct (SEQ ID No. 39) was generated by cloning. The amplified product was inserted into Gateway TM pDON TM / Zeo vector (ThermoFisherScientific). The sequence was then transferred to the GDB expression vector (SEQ ID No. 38).

[0678] GDB-cation efflux_Δ117C expression vector

[0679] Using the GDB-cation efflux expression vector as a template, the first set of gene-specific primers (cation efflux_Δ117C.fw [SEQ ID No.45] and cation efflux_Δ117C.rv [SEQ ID No.46]), and the second set of Gateway TM Compatible primers (attB1 [SEQ ID No. 42] and attB2 [SEQ ID No. 43]), through two-step amplification and Gateway TM The Δ117C variant (SEQ ID No. 32) was generated by cloning. The amplified product was inserted into Gateway TM pDON TM / Zeo vector (ThermoFisher Scientific). The sequence was then transferred to the GDB expression vector (SEQ ID No. 38).

[0680] GDB-cation efflux_Δ117N expression vector

[0681] Using the GDB-cation efflux expression vector as a template, the first set of gene-specific primers (cation efflux_Δ117N.fw [SEQ ID No.47] and cation efflux_Δ117N.rv [SEQ ID No.48]), and the second set of Gateway TM Compatible primers (attB1 [SEQ ID No. 42] and attB2 [SEQ ID No. 43]), through two-step amplification and Gateway TM The Δ117N variant (SEQ ID No. 34) was generated by cloning. The amplified product was inserted into Gateway TM pDON TM / Zeo vector (ThermoFisher Scientific). The sequence was then transferred to the GDB expression vector (SEQ ID No. 38).

[0682] The resulting plasmid was sequenced and transformed into Agrobacterium tumefaciens GV3101pMP90 by heat shock and transient expression in TN90 leaves.

[0683] Transient gene expression

[0684] Agrobacterium tumefaciens GV3101 strains carrying the target construct were grown overnight in Luria-Bertani (LB) medium supplemented with appropriate antibiotics. The culture was spun down and resuspended to OD600=0.6 in a buffer containing 10 mM MgCl2, 10 mM MES pH 5.6 and 100 μM acetosyringone and incubated at room temperature for 1 hour. Infiltration into TN90 leaves was performed with a needleless syringe. Samples were obtained 5 days after infiltration.

[0685] The test was performed with two biological replicates, each containing twelve technical replicates.

[0686] Statistical analysis

[0687] Statistical significance based on one-way ANOVA analysis was performed using Prism 5.01 software (GraphPad Software).

[0688] result

[0689] Nornicotine content of 5-week-old TN90 leaves expressing the indicated constructs is shown in Figure 3 . Nornicotine content is expressed relative to luciferase control (SEQ ID No. 44), and the results include three biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.05.

[0690] in conclusion

[0691] Deletion of the N-terminal domain results in a decrease in nornicotine content.

[0692] The proposed mechanism for the expression of cation efflux_Δ117C and cation efflux_Δ117N is shown in FIG4 .

[0693] Example 3

[0694] To determine the ligand transported by SEQ ID No. 3, Phyre2 (Kelley LA et al. Nature Protocols 10, 845-858 (2015), incorporated herein by reference) and Swiss-Model were used to perform 3D modeling of the protein structure. Phyre2 uses an alignment of a hidden Markov model (HMM) via Hhsearch ( J. Bioinformatics 21, 951-960 (2005), incorporated herein by reference), to significantly determine the accuracy and detection rate of the alignment. The materials and methods used by Swiss-Model are described at swissmodel.expasy.org.

[0695] result

[0696] Using Phyre 2 Protein modeling indicated zinc transport with 100% confidence.

[0697] Protein modeling using an evolutionarily related structure matching SED ID No. 3 indicated two Zn ion binding sites upon homodimer formation (Swiss-Model, see e.g. FIG. 4 c ).

[0698] in conclusion

[0699] These results indicated that SEQ ID No. 3 was likely involved in zinc efflux.

[0700] Example 4

[0701] To evaluate the correlation between the substrates of the identified metal transporters and the nornicotine content in plants, transgenic tobacco plants overexpressing the wild-type cation efflux (encoded by SEQ ID No. 2), the cation efflux Δ117C variant (SEQ ID No. 32), and the cation efflux Δ117N variant (SEQ ID No. 34) were treated with various concentrations of substrate metals: Zn (3 mM, 5 mM, 10 mM), and Cd (70 μM, 100 μM, 150 μM) or Ni (50 μM, 300 μM, 1 mM), and nornicotine levels were measured as described in Example 2.

[0702] result

[0703] Nornicotine content of tobacco plants treated with the indicated concentrations of zinc, cadmium or nickel is shown in Fig.68 middle.

[0704] Zinc treatment induced an increase in nornicotine levels. Fig.69a As shown in Figure -c, this effect was restored upon expression of the constitutively high-affinity Δ117N zinc transporter.

[0705] in conclusion

[0706] Zinc levels correlate with nornicotine levels, indicating that zinc acts as a regulator in nicotine turnover. This was further demonstrated by insensitivity to zinc and reduced symptoms to zinc treatment when a constitutive high affinity Δ117N zinc transporter was expressed, which detoxifies cytoplasmic zinc ions via exocytosis.

[0707] Example 5

[0708] Since zinc acts as a catalytic or structural cofactor for many proteins, depletion of intracellular zinc content leading to zinc deficiency induces growth arrest and apoptosis in severe cases (Siklar et al., 2003 J Trop Pediatr. 49(3):187-8; Eide 2009 Journal of Biological Chemistry 10; 284(28):18565–18569). Zinc starvation has been reported to severely impair yeast growth and induce autophagy (Kawamata et al. 2017 J Biol Chem. 2017 May 19; 292(20):8520-8530).

[0709] To confirm that Δ117N acts as a constitutive high-affinity zinc transporter, the cation efflux proteins according to the present invention, Δ117C and Δ117N variants were engineered into yeast, and colony growth was monitored.

[0710] yeast

[0711] For yeast expression, BY4742 strain was transformed by chemical methods.

[0712] Cloning of cationic efflux yeast expression vector

[0713] Using In-Fusion PCR primers (SEQ ID No.50 and SEQ ID No.51), TM The gene sequence (SEQ ID No. 2) was amplified from a compatible cDNA library with primers that allowed In-Fusion cloning into the pYES2 vector (pYES3 / UT.1 [SEQ ID No. 52]) (Thermo Fisher Scientific). Prior to In-Fusion cloning, the vector was digested with HindIII and BamHI restriction enzymes (Promega).

[0714] Cation efflux_Δ117C yeast expression vector

[0715] The cationic efflux_Δ117C yeast expression vector was generated by In-Fusion cloning using the GDB-cationic efflux expression vector as a template and In-Fusion PCR primers (SEQ ID No. 53 and SEQ ID No. 54) that allow In-Fusion cloning into the pYES2 vector (SEQ ID No. 55). Prior to In-Fusion cloning, the vector was digested with HindIII and BamHI restriction enzymes (Promega).

[0716] Cationic efflux_Δ117N yeast expression vector

[0717] The cationic efflux_Δ117N yeast expression vector was generated by In-Fusion cloning using the GDB-cationic efflux expression vector as a template and In-Fusion PCR primers (SEQ ID No. 56 SEQ ID No. 57) that allow In-Fusion cloning into the pYES2 vector (SEQ ID No. 55). Prior to In-Fusion cloning, the vector was digested with HindIII and BamHI restriction enzymes (Promega).

[0718] The activity of cation efflux proteins was measured using the yeast metal tolerance assay.

[0719] Yeast metal tolerance assay

[0720] Transform yeast wild-type strains with target constructs using ScEasyComp TM Yeast transformation was performed using the Transformation Kit (Thermo Fisher).

[0721] Positive colonies were selected on synthetic dropout (SD) plates containing appropriate selectable markers. Yeast strains expressing empty vector, wild-type cation transporter (Seq ID No. 2), or mutant variants (Δ117C and Δ117N) were pre-cultured for 16 hours at 28°C in SD liquid medium containing appropriate selectable markers. The optical density was normalized to 0.5, and the strains were grown for 4 hours with selection medium + galactose 2% to induce gene expression. Three 10-fold dilutions of 5 μL of the culture (optical density at 600 nm was 0.005, 0.0005, and 0.00005) were spotted onto SD agar plates containing appropriate selectable markers, galactose, and ZnCl2 (3 mM and 5 mM).

[0722] result

[0723] Growth of wild-type yeast expressing the indicated constructs is shown in Fig.70 middle.

[0724] in conclusion

[0725] Expression of the Δ117N cation efflux variant in yeast resulted in reduced growth. Thus, the Δ117N cation efflux variant was identified as a deregulated constitutive high affinity transporter.

[0726] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the present invention should not be overly limited to such specific embodiments. In fact, it will be apparent to those skilled in the art of biochemistry and biotechnology or related fields that various modifications of the modes for implementing the present invention are contemplated within the scope of the following claims.

Claims

1. A method for reducing the content of N'-nitrosonicotine or nornicotine in tobacco, comprising expressing a deregulated cationic efflux protein in a tobacco plant or plant part or plant cell thereof, The deregulated cationic efflux protein is a truncated cationic efflux protein lacking amino acids 2-25, or 2-50, or 2-75, or 2-100, 2-114, 2-117, 2-120, 2-125, or 2-128 from the N-terminal side of SEQ ID No.

3.

2. The method according to claim 1, wherein the deregulated cation efflux protein is a constitutive high affinity cation efflux transporter.

3. The method according to any preceding claim, wherein the deregulated cation efflux protein shows increased metal ion transport compared to a wild-type cation efflux transporter.

4. A method according to claim 1 or claim 2, wherein the deregulated cation efflux transporter is deregulated when compared to a wild-type cation efflux protein comprising the amino acid sequence shown in SEQ ID No.

3.

5. A method according to claim 1 or claim 2, comprising introducing a mutation into the genome of the plant or plant cell within a polynucleotide encoding a cation efflux protein such that the polynucleotide encodes a deregulated cation efflux protein as defined in claim 1.

6. The method according to claim 5, wherein the mutation generates a deletion in the polynucleotide encoding the deregulated cation efflux protein.

7. The method according to any one of claims 1-2, comprising introducing an exogenous polynucleotide sequence into the genome of the plant or plant cell, the exogenous polynucleotide sequence encoding the deregulated cation efflux protein.

8. The method according to claim 1 or claim 2, wherein the deregulated cationic efflux protein is a truncated cationic efflux protein consisting of: At least 375, at least 350, or at least 300 amino acids from the C-terminus of SEQ ID No.

3.

9. A method for producing tobacco with reduced N'-nitrosonicotine or nicotine content, comprising: a. hybridizing a donor tobacco plant having a reduced N'-nitrosonicotine or nornicotine content and comprising a deregulated cationic efflux protein as defined in any one of claims 1 to 8 with a recipient tobacco plant having an N'-nitrosonicotine or nornicotine content and having a commercially desirable trait; b. separating genetic material from offspring of the donor tobacco plant hybridized with the recipient tobacco plant; and c. performing molecular marker-assisted selection using molecular markers, comprising: i. Identifying introgression regions comprising mutations in the polynucleotide sequence encoding the protein defined in a.

10. The method according to any one of claims 1, 2, 6 or 9, wherein the deregulated cationic efflux protein consists of the amino acid sequence shown in SEQ ID No.

33.

11. The method according to any one of claims 1, 2, 6 or 9, wherein the deregulated cationic efflux protein is encoded by a polynucleotide consisting of the sequence shown in SEQ ID No.

34.

12. An isolated polynucleotide encoding a deregulated cation efflux protein, wherein the nucleotide sequence of the isolated polynucleotide is selected from the group consisting of: a. a nucleotide sequence consisting of SEQ ID No.34; b. a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID No.33; c. A nucleotide sequence encoding a truncated polypeptide lacking amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128 from the N-terminal side of SEQ ID No. 3; d. a nucleotide sequence encoding a polypeptide, the polypeptide consisting of the amino acid sequence of positions 125-413 as shown in SEQ ID No.3, wherein the nucleotide sequence encodes a polypeptide involved in cation efflux in plants; e. A nucleotide sequence complementary to the sequence according to any one of the above (a) to (d).

13. An isolated polypeptide which is a deregulated cation efflux protein, wherein the amino acid sequence of the isolated polypeptide is selected from the group consisting of: a. an amino acid sequence consisting of SEQ ID No.33; b. an amino acid sequence consisting of amino acids 125-413 as shown in SEQ ID No. 3, wherein the polypeptide is involved in cation efflux in plants; or c. A truncated amino acid sequence lacking amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128 from the N-terminal side of SEQ ID No.

3.

14. A construct or vector comprising a polynucleotide according to claim 12 or a polynucleotide encoding a polypeptide according to claim 13.

15. A method of producing a tobacco plant or part thereof, said tobacco plant or part thereof: a. A plant modified to achieve a reduction in N'-nitrosonicotine or nicotine compared to an unmodified plant, wherein the modified plant or part thereof comprises a deregulated cationic efflux protein as defined in any one of claims 1 to 8; b. obtained by a method according to any one of claims 1-11; c. comprising an exogenous gene encoding a deregulated cationic efflux protein, wherein the deregulated cationic efflux protein consists of a truncated amino acid sequence lacking amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128 from the N-terminal side of SEQ ID No.3; d. a deregulated cationic efflux protein comprising the amino acid sequence shown in SEQ ID No.33, or a polynucleotide sequence shown in SEQ ID No.34; or e. comprising a construct or vector according to claim 14.

16. A method for producing a tobacco plant cell or a yeast cell, wherein the tobacco plant cell or the yeast cell: a. comprising a deregulated cationic efflux protein as defined in claim 1; b. obtained by a method according to any one of claims 1-11; c. comprising an exogenous gene encoding a deregulated cationic efflux protein, wherein the deregulated cationic efflux protein consists of a truncated amino acid sequence lacking amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128 from the N-terminal side of SEQ ID No.3; d. a deregulated cationic efflux protein comprising the amino acid sequence shown in SEQ ID No.33, or a polynucleotide sequence shown in SEQ ID No.34; or e. comprising a construct or vector according to claim 14.

17. A method for producing a cell culture comprising the cell or cell population according to claim 16, wherein a tobacco plant cell culture or a yeast cell culture is obtained.

18. A method of producing plant propagation material obtained from a plant as defined in claim 15.

19. The method of claim 18, wherein the plant propagation material is a plant seed.

20. The method according to any one of claims 1, 2, 9, 15, 16, 17 or 18, wherein the plant is from the species Nicotiana tabacum or Nicotiana rustica.

21. Use of a cell as defined in claim 16, or a tobacco plant or tobacco plant part thereof as defined in claim 15, or a cell culture as defined in claim 17, or a deregulated cationic efflux protein as defined in any one of claims 1 to 13 for producing a tobacco industry product.

22. Use of a tobacco plant as defined in claim 15 or a plant propagation material as defined in claim 18 for growing a tobacco plant.

23. Use of a tobacco plant as defined in claim 15 or a plant propagation material as defined in claim 18 for growing crops.

24. Use of a tobacco plant as defined in claim 15 or a plant propagation material as defined in claim 18 for producing processed tobacco leaves.

25. The use of claim 24, wherein the processed tobacco leaf is a flue-cured tobacco leaf.

26. The method of claim 15, further comprising harvesting leaves from the tobacco plant, thereby obtaining harvested leaves.

27. The method of claim 26, wherein the harvested leaves are shredded harvested leaves.

28. A processed tobacco leaf, which: a. comprising a plant cell as defined in claim 16; b. obtained from processing tobacco plants as defined in claim 15; c. a tobacco plant derived from a plant propagation material as defined in claim 18; or d. obtained by processing harvested leaves of a plant as defined in claim 15.

29. The processed tobacco leaf according to claim 28, wherein the plant or leaf is processed by curing, fermentation, pasteurization or a combination thereof.

30. The processed tobacco leaf according to claim 28 or 29, wherein the processed tobacco leaf is cut processed tobacco leaf.

31. The processed tobacco leaf of claim 28 or 29, wherein the processed tobacco leaf is a non-viable processed tobacco leaf.

32. A cured tobacco material made from a plant or part thereof as defined in claim 15, or obtained from plant propagation material as defined in claim 18, or obtained by a method according to any one of claims 1 to 11.

33. A tobacco blend comprising the flue-cured tobacco material of claim 32.

34. A tobacco industry product, which: a. prepared from a tobacco plant or a part thereof as defined in claim 15; b. prepared by a tobacco plant or part thereof obtained by a method according to any one of claims 1-11; c. Prepared from a plant propagated from a plant propagation material as defined in claim 18; d. Prepared by processing the blade according to any one of claims 28-31; e. prepared from tobacco plant cells as defined in claim 16; f. or prepared by a tobacco plant cell culture as defined in claim 17; g. prepared from a flue-cured tobacco material according to claim 32; or h. Prepared from a tobacco blend according to claim 33.

35. The tobacco industry product of claim 34, wherein in (b) the tobacco plant part is leaves harvested from the plant.

36. The tobacco industry product of claim 34, wherein the tobacco industry product is prepared from leaves of a tobacco plant propagated from a plant propagation material as defined in claim 18.

37. The tobacco industry product according to any one of claims 34 to 36, wherein the tobacco industry product is a combustible smoking article or a smokeless tobacco industry product or a non-combustible gas aerosol supply system.

38. The tobacco industry product of claim 37, wherein the tobacco industry product is a tobacco heating device.

39. The tobacco industry product of claim 38, wherein the tobacco heating device is an aerosol generating device.

40. A combustible smoking article, a non-combustible gas mist delivery system or a smokeless tobacco industrial product comprising a plant or a part thereof as defined in claim 15, wherein the plant is from the species Nicotiana tabacum or Nicotiana rustica, or is obtained from a plant propagation product as defined in claim 18, or is obtained from a method according to any one of claims 1 to 11.

41. The combustible smoking article, non-combustible gas aerosol delivery system or smokeless tobacco industry product of claim 40, wherein the non-combustible gas aerosol delivery system is a tobacco heating device.

42. Use of a nucleotide sequence encoding a deregulated cation efflux protein consisting of the amino acid sequence shown in SEQ ID No.33, or a nucleotide sequence encoding the following for selecting plants with reduced content of N'-nitrosonicotine or nornicotine: a deregulated cation efflux protein, wherein the deregulated cation efflux protein consists of a truncated amino acid sequence lacking amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128 from the N-terminal side of SEQ ID No.

3.

43. A method for producing a mutant plant, the mutant plant carrying a heritable mutation in a nucleotide sequence encoding a deregulated cation efflux protein, wherein the deregulated cation efflux protein consists of a truncated amino acid sequence, the truncated amino acid sequence lacking amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128 from the N-terminal side of SEQ ID No. 3; wherein the heritable mutation reduces the content of N'-nitrosonicotine or nornicotine relative to a tobacco plant that does not carry the heritable mutation.

44. A method of producing progeny of a mutant plant carrying a heritable mutation according to claim 43.

45. A method of producing mutant plant seeds carrying the heritable mutation according to claim 43.

46. ​​A processed tobacco leaf or cured tobacco material produced by a tobacco plant having a nucleotide sequence encoding a deregulated cationic efflux protein, wherein the deregulated cationic efflux protein consists of a truncated amino acid sequence lacking amino acids 2-25, or 2-50, or 2-75, or 2-100, or 2-114, or 2-117, or 2-120, or 2-125, or 2-128 from the N-terminal side of SEQ ID No. 3; wherein the processed tobacco leaf or cured tobacco material has a reduced content of N'-nitrosonicotine or nornicotine relative to a tobacco plant not carrying the modification.

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