Modulation of nitrate levels in plants via mutation of nitrate reductase

Abstract

To provide the art for reducing nitrate levels in plants.SOLUTION: The present invention provides a plant cell comprising: (a) a polynucleotide sequence encoding a nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO: 5 or SEQ ID NO: 7, wherein methionine is substituted for an amino acid that reduces nitrate reductase activity in the plant cell as compared to a control plant cell; (b) a polypeptide sequence encoded by the polynucleotide sequence set forth in (a); or (c) a construct, vector or expression vector comprising the polynucleotide sequence set forth in (b).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to plants having reduced nitrate levels, plant cells derived from the plants, products produced from the plants, and methods for regulating nitrate levels in plants by mutating the nitrate reductase enzyme. [Background technology]

[0002] Certain plants, such as tobacco plants, accumulate high levels of free nitrates in their leaves. This is undesirable because high levels of nitrates are associated with the formation of carcinogenic compounds called tobacco-specific nitrosamines (TSNAs). TSNAs are a class of compounds that are primarily formed during the curing of tobacco leaves, but can also arise during the subsequent processing and storage of the leaves, and can also be formed via thermosynthesis during combustion. Two TSNAs found in cured leaves, N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), are classified as Group I carcinogens (highest indication) by the International Agency for Research on Cancer. Due to evidence implicating these compounds in various tobacco-related cancers, the World Health Organization has recommended mandatory implementation to ensure that future tobacco products have reduced levels of these toxicants. TSNAs are the nitrosation products of representative tobacco alkaloids. There is general agreement that in air-cured tobacco, nitrite is the substance directly involved in the formation of TSNAs. However, due to its cytotoxicity, endogenous nitrite levels in plant tissues are generally very low. Instead, most of the nitrite involved in TSNA formation is thought to be derived from nitrate reductase activity of microorganisms living on the leaf surface during the 6-10 week desiccation process, during which a portion of the leaf nitrate pool is converted to nitrite as cell membranes and organelles are degraded.

[0003] TSNAs are primarily formed during leaf curing and involve the nitrosation of tobacco alkaloids. Genetic strategies to reduce TSNA content and levels in cured leaves have focused on targeting either (1) the alkaloid precursors or (2) the nitrosating agents involved. Most efforts to reduce TSNAs at the level of altering tobacco genetics have targeted the alkaloid precursors to TSNAs. Such strategies provide a significant reduction in NNN by downregulating the gene family involved in the synthesis of the alkaloid precursor nornicotine.

[0004] Modified tobacco plants with reduced nitrate levels have been described by the applicant in WO2016 / 046288. As described herein, the expression or activity of the nitrate reductase enzyme is attenuated. The attenuated nitrate reductase enzyme has an amino acid substitution at a position corresponding to position 523 of the polypeptide encoding nitrate reductase. The mutation is called S523D. Overexpression of the mutant nitrate reductase enzyme results in a dramatic increase in nitrate assimilation, resulting in a reduced nitrate / TSNA content and increased amino acid levels. The plants showed 90% less nitrate compared to the control.

[0005] The development of non-genetically modified organism (non-GMO) methods to reduce nitrate accumulation may be desirable. As it becomes more difficult to grow and market genetically modified crops in countries, including Europe, it may be advantageous to work with mutants characterized by single nucleotide polymorphisms rather than mutants obtained through the use of genetic engineering techniques. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a continuing need in the art to reduce nitrate levels in plants, particularly through non-genetic engineering techniques. [Means for solving the problem]

[0007] The present invention is based, at least in part, on the discovery that by mutating Nicotiana tabacum nitrate reductase (NIA2) via non-genetic techniques, it is possible to obtain plants or plant materials whose desiccated leaves contain modulated (e.g., reduced) levels of nitrate compared to desiccated leaves derived from control plants. One such mutation described herein is located in the nitrate reductase kinase recognition site within the hinge 1 domain of Nicotiana tabacum nitrate reductase.

[0008] Hardin et al. (2009) Biochem. J. 422:305-312 teach that oxidation of methionine at position 538 in the Arabidopsis NIA2 protein is a mechanism that inhibits phosphorylation of the NIA protein. The oxidation prevents 14-3-3 from binding to nitrate reductase and prevents nitrate reductase from being inactivated when exposed to reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals. Since nitrate reductase converts nitrate to nitrite, resulting in the production of ammonia and amino acids, high nitrate reductase activation would be expected to reduce nitrate levels, as demonstrated in WO2016 / 046288. Thus, oxidation of M538 to methionine sulfoxide in the Arabidopsis NIA2 protein would be expected to lead to reduced nitrate levels. M527 in Nicotiana tabacum NIA2 protein corresponds to M538 in Arabidopsis thaliana NIA2. The properties of mutant Nicotiana tabacum plants described herein are unexpected. Nicotiana tabacum NIA2 mutant M527I (as well understood in the art, M527I means that methionine (M) at position 527 is replaced with isoleucine (I)) contains a replacement of methionine at position 537 with isoleucine, which cannot be oxidized. Methionine is known to be oxidized on sulfur residues to become methionine sulfoxide, but isoleucine does not have any sulfur residues in its structure. Based on this difference, Nicotiana tabacum NIA2 mutant M527I is not expected to stimulate nitrate reductase activation upon exposure to ROS, and as a result, the mechanism of reducing plant nitrate levels is eliminated. However, it has surprisingly been found that despite the substitution of methionine, the mutant plants of the present disclosure exhibit reduced nitrate levels.

[0009] There are many other advantages to producing the plants described in the present disclosure. For example, the plants described herein can be non-genetically modified plants, which overcomes the problems associated with growing and marketing genetically modified crops. As a further example, the reduction of nitrate levels can be achieved without affecting the total harvested biomass of the plant, and nicotine, total alkaloids, ammonia, or reducing the sugar content of the leaves. This can improve the commercial acceptability of the plant, plant material, or plant product.

[0010] In one aspect, disclosed is: (a) a polynucleotide sequence encoding a nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO:5 or SEQ ID NO:7, wherein a methionine is replaced with an amino acid that reduces nitrate reductase activity in the plant cell compared to a control plant cell; (b) a polypeptide sequence encoded by the polynucleotide sequence described in (a); or (c) a plant cell comprising a construct, vector, or expression vector comprising the polynucleotide sequence described in (b).

[0011] Preferably, the methionine is replaced with a different amino acid, more preferably, the replacement amino acid is isoleucine, and more preferably, the polypeptide comprises a contiguous polypeptide sequence of SEQ ID NO:6 or SEQ ID NO:8.

[0012] Suitably, the nitrate reductase polypeptide comprises an amino acid substitution at a position corresponding to position 527 of a sequence having at least 80% sequence identity to SEQ ID NO:1.

[0013] Suitably, the nitrate reductase polypeptide comprises, consists of, or consists essentially of the polypeptide sequence set forth in SEQ ID NO:3.

[0014] Suitably, the polynucleotide sequence comprises, consists or consists essentially of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO:4.

[0015] Suitably, the polynucleotide sequence comprises, consists or consists essentially of the polynucleotide sequence set forth in SEQ ID NO:4.

[0016] Suitably, the plant cell is a Nicotiana tabacum plant cell.

[0017] Suitably, the plant cell is a plant cell of the Nicotiana tabacum cultivar AA37.

[0018] In another aspect, a plant or part thereof comprising a plant cell according to any of the preceding claims is disclosed, wherein preferably the desiccated leaves of the plant or part thereof contain lower levels of nitrate compared to a control plant or part thereof, preferably the level of nitrate is reduced by about 37% or more compared to a control plant or part thereof.

[0019] In another aspect, there is a disclosed plant material, dried plant material, or homogenized plant material derived from a plant or part thereof as described herein, preferably the dried plant material is air-dried or sun-dried or flue-cured plant material, preferably the plant material, dried plant material, or homogenized plant material comprises biomass, seeds, stems, flowers, or leaves of a plant or part thereof.

[0020] In another aspect, a tobacco product is disclosed that includes a plant cell described herein, a part of a plant described herein, or a plant material described herein.

[0021] In another aspect, a method for producing a plant as described herein is disclosed, comprising: (a) providing a plant cell as described herein; and (b) propagating the plant cell into a plant.

[0022] In another aspect, a method for producing a desiccated plant material having an altered amount of nitrate compared to a control plant material is disclosed, comprising the steps of: (a) providing a plant or part thereof as described herein or a plant material as described herein; (b) optionally harvesting plant material from the plant or part thereof; and (c) desiccating the plant material.

[0023] Suitably the plant material comprises dried leaves.

[0024] Preferably, the drying process is selected from the group consisting of air drying, flame drying, smoke drying, and hot air tube drying. [Brief description of the drawings]

[0025] [Figure 1] Figure 1 shows the domains and consensus sequences of the NtNIA2 protein sequence. (A) Major domains of the Nicotiana tabacum nitrate reductase NIA2 protein (GenBank: X14059). (B) The nitrate reductase kinase recognition site (LKKSISTPFM) and the 14-3-3 recognition site in hinge 1 of NtNIA2. The positions of the S523D mutation from WO2016 / 046288 and the M527I mutation described herein are indicated. [Figure 2-1]Figure 2 is a series of bar graphs showing chemical profiling of dried leaves from the second harvest and total biomass of undried leaves. (A) Total harvested leaf wet mass expressed as grams per plant (FW), (B) Nicotine content of dried leaves expressed as micrograms per gram of dried material, (C) Total alkaloid content expressed as percentage of dry mass of dried material, (D) Ammonia content expressed as percentage of dry mass of dried material, (E) Reducing sugar content expressed as percentage of dry mass of dried material, and (F) Nitrate content expressed as percentage of dry mass of dried material. AA37 control indicates lines that did not receive EMS treatment (n=6 plots), NIA2_M527I_WT indicates AA37 out-segregant wild type lines (n=15 plots), and NIA2_M527I_HOMO indicates NtNIA2 M527I homozygous lines (n=11 plots). Error bars indicate 95% confidence intervals calculated using the Student's t test. [Figure 2-2] Same as above.

[0026] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document, including definitions, governs. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting.

[0027] As used herein, the terms "comprise," "include," "having," "having," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or configurations.

[0028] The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise.

[0029] The term "and / or" means either (a) or (b), or both (a) and (b).

[0030] The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0031] As recitations of numerical ranges herein, each intermediate number therebetween is specifically contemplated with the same degree of precision. For example, in the range 6-9, the numbers 7 and 8 are specifically contemplated in addition to the numbers 6 and 9, and in the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are specifically contemplated.

[0032] As used throughout the specification and claims, the following terms have the following meanings.

[0033] "Coding sequence" or "encoding a polynucleotide" refers to a nucleotide (RNA or DNA molecule) that comprises a polynucleotide that codes for a polypeptide. The coding sequence can further comprise initiation and termination signals operably linked to control elements, including a promoter and polyadenylation signal, capable of directing expression in cells of an individual or mammal to which the polynucleotide is administered. The coding sequence can be codon optimized.

[0034] "Complement" or "complementary" can mean Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs. "Complementarity" refers to the property shared between two polynucleotides, e.g., when aligned antiparallel to each other, that the nucleotide bases at every position are complementary.

[0035] "Construct" refers to a double-stranded recombinant polynucleotide fragment that contains one or more polynucleotides. A construct contains a "template strand" bases that are base-paired with a complementary "sense or coding strand." A given construct can be inserted into a vector in either of two possible orientations: the same (or sense) orientation or the opposite (or antisense) orientation relative to the orientation of a promoter placed in the vector (e.g., an expression vector).

[0036] The term "control" in the context of a control plant or control plant cell refers to a plant or plant cell in which the expression, function, or activity of one or more genes or polypeptides is not altered (e.g., increased or decreased), and thus can provide a comparison with a plant in which the expression, function, or activity of one or more genes or polypeptides is altered. As used herein, a "control plant" is a plant that is substantially equivalent to a test plant or modified plant in all parameters except the test parameter. For example, when referring to a plant in which a mutation has been introduced, the control plant is an equivalent plant in which the mutation has not been introduced. A control plant can be an out-segregant control plant.

[0037] "Expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment can refer to transcription of the polynucleotide fragment (e.g., transcription resulting in an mRNA or functional RNA) and / or translation of an mRNA into a precursor or mature polypeptide.

[0038] "Functional" and "fully functional" describe a polypeptide that has a biological function or activity. A "functional gene" refers to a gene that is transcribed into mRNA that is translated into a functional or active polypeptide.

[0039] "Genetic construct" refers to a DNA or RNA molecule that comprises a polynucleotide that encodes a polypeptide. The coding sequence can include initiation and termination signals that are operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression.

[0040] The term "homology" or "similarity" refers to the degree of sequence similarity between two polypeptide molecules or two polynucleotide molecules compared by sequence alignment. The degree of homology between two discontinuous polynucleotides being compared is a function of the number of identical or matching nucleotides at comparable positions.

[0041] "Identical" or "identity" in the context of two or more polynucleotides or polypeptides means that the sequences have a certain ratio of residues that are the same over a particular region. The ratio is calculated by optimally aligning the two sequences, comparing the two sequences over a particular region, determining the number of positions where identical residues exist in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the particular region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences have different lengths, or if the alignment results in one or more staggered ends and the particular region being compared contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or by using computer sequence algorithms such as ClustalW, ClustalX, BLAST, FASTA, or Smith-Waterman. The general multiple alignment program ClustalW (Nucleic Acids Research (1994) 22, 4673-4680; Nucleic Acids Research (1997), 24, 4876-4882) is a suitable method for creating multiple alignments of polypeptides or polynucleotides. Suitable parameters for ClustalW may be as follows: for polynucleotide alignments: gap opening penalty = 15.0, gap extension penalty = 6.66, and matrix = Identity. for polypeptide alignments: gap opening penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet. for DNA and protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that these and other parameters may need to be varied for optimal sequence alignment. A percentage identity calculation is then suitably calculated from such an alignment as (N / T), where N is the number of positions where the sequences share identical residues and T is the total number of positions being compared, including gaps but not including overhangs.

[0042] The terms "isolated" or "purified" refer to a material that is substantially or essentially free from components that normally accompany it when found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. In particular, an isolated polynucleotide is separated from open reading frames that flank the desired gene and encode polypeptides other than the desired polypeptide. As used herein, the term "purified" means that the polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel. Specifically, this means that the polynucleotide or polypeptide is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. An isolated polynucleotide can be purified from a naturally occurring host cell. Conventional polynucleotide purification methods known to those of skill in the art can be used to obtain an isolated polynucleotide. The term also encompasses recombinant and chemically synthesized polynucleotides.

[0043] "Modulate" or "modulation" refers to causing or promoting a qualitative or quantitative change, alteration, or modification in a process, pathway, function, or activity of a subject. Without being limited thereto, such a change, alteration, or modification can be an increase or decrease in the relative process, pathway, function, or activity of a subject. For example, gene expression or polypeptide expression, or polypeptide function or activity can be modulated. Typically, the relative change, alteration, or modification will be determined by comparing with a control.

[0044] The term "non-natural" describes entities such as polynucleotides, genetic mutations, polypeptides, plants, plant cells, and plant materials that are not formed or exist in nature. Such non-natural or artificial entities can be created, synthesized, initiated, modified, intervened, or manipulated by methods described herein or known in the art. Such non-natural or artificial entities can be created, synthesized, initiated, modified, intervened, or manipulated by humans. By way of example, non-natural entities can be entities that have been mutated by methods known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically induced mutagenesis, radiation-induced mutagenesis, mutagenesis using modified bases, mutagenesis using gapped duplex DNA, double-strand break mutagenesis, mutagenesis using repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling, and other equivalent methods. For example, chemical mutagenesis can be used, which involves the use of exogenously added chemicals, such as mutagenic, teratogenic, or carcinogenic organic compounds, to induce mutations. Mutants with advantageous properties can then be selected and identified.

[0045] "Oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, polynucleotide also encompasses the complementary strand of the depicted single strand. Many variants of a polynucleotide can be used for the same purpose as a given polynucleotide. Thus, polynucleotide also encompasses substantially identical polynucleotides and their complements. A single strand provides a probe that can hybridize to a given sequence under stringent hybridization conditions. Thus, polynucleotide also encompasses probes that hybridize under stringent hybridization conditions. A polynucleotide can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Polynucleotides can be DNA, both genomic and cDNA, RNA, or hybrids, and can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.

[0046] The specificity of single-stranded DNA for hybridizing complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)). Hybridization stringency increases as the tendency to form DNA duplexes decreases. In polynucleotide hybridization reactions, stringency can be selected to favor specific hybridization (high stringency), which can be used, for example, to identify full-length clones from a library. Less specific hybridization (low stringency) can be used to identify related, but not exactly the same (homologous but not identical) DNA molecules or segments. DNA duplexes are stabilized by (1) the number of complementary base pairs, (2) the type of base pair, (3) the salt concentration (ionic strength) of the reaction mixture, (4) the reaction temperature, and (5) the presence of certain organic solvents, such as formamide, which reduce the stability of DNA duplexes. In general, the longer the probe, the higher the temperature required for proper annealing. A common approach is to vary the temperature; the higher the relative temperature, the more stringent the reaction conditions. Hybridization under "stringent conditions" describes hybridization protocols in which polynucleotide sequences that are at least 60% homologous to each other remain hybridized. Typically, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the particular sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to a given sequence hybridize to the given sequence at equilibrium. A given sequence is generally present in excess at the Tm, so that 50% of the probes are occupied at equilibrium.

[0047] "Stringent hybridization conditions" are conditions that allow a probe, primer, or oligonucleotide to hybridize only to its specific sequence. Stringent conditions are sequence-dependent and vary. Stringent conditions typically include: (1) low ionic strength and high temperature washing, e.g., 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50°C; (2) denaturing agents during hybridization, e.g., 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate, pH 6.5) at 42°C; or (3) 50% formamide. Also, the wash solution typically comprises 5×SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate at 42° C., with a high stringency wash consisting of 0.2×SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a wash in 0.1×SSC with EDTA at 55° C. Suitably, the conditions are such that sequences that are at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.

[0048] "Moderately stringent conditions" use wash solutions and hybridization conditions that are less stringent such that polynucleotides hybridize to the entire polynucleotide, fragments, derivatives, or analogs. One example includes hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS, and 100 μg / mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1xSSC, 0.1% SDS at 37°C. Temperature, ionic strength, etc. can be adjusted to suit experimental factors such as probe length. Other moderate stringency conditions have been described (see Ausubel et al., Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., Hoboken, NJ (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, NY (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, NY (1988)).

[0049] "Low stringency conditions" use wash solutions and hybridization conditions that are less stringent than moderate stringency such that polynucleotides hybridize to the entire polynucleotide, fragments, derivatives, or analogs. Non-limiting examples of low stringency hybridization conditions include hybridization in 35% formamide, 5xSSC, 50mM Tris HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg / mL denatured salmon sperm DNA, 10% (weight / volume) dextran sulfate at 40°C, followed by one or more washes in 2xSSC, 25mM Tris HCl (pH 7.4), 5mM EDTA, and 0.1% SDS at 50°C.

[0050] The term "plant" refers to any plant at any stage of its life cycle or development, and its progeny. In one embodiment, the plant is a tobacco plant, which refers to a plant belonging to the genus Nicotiana. The term includes reference to whole plants, plant organs, plant tissues, plant propagules, plant seeds, plant cells, and their progeny. Plant cells include, but are not limited to, seeds, suspension cultures, embryos, meristematic tissue sites, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and pollen grain cells. Suitable species, cultivars, hybrids, and varieties of tobacco plants are described herein.

[0051] "Polynucleotide", "polynucleotide sequence", or "polynucleotide fragment" are used interchangeably herein and refer to a polymer of RNA or DNA that is single-stranded or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are designated by the following single-letter designations: "A" for adenylic acid or deoxyadenylic acid (in RNA or DNA, respectively), "C" for cytidylic acid or deoxycytidylic acid, "G" for guanylic acid or deoxyguanylic acid, "U" for uridylic acid, "T" for deoxythymidylic acid, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide. A polynucleotide may be, but is not limited to, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragments thereof. Moreover, a polynucleotide may be single-stranded or double-stranded, a mixture of single-stranded and double-stranded regions, a hybrid molecule comprising DNA and RNA, or a hybrid molecule having a mixture of single-stranded and double-stranded regions or fragments thereof. Furthermore, a polynucleotide may be composed of triplex regions comprising DNA, RNA, or both, or fragments thereof. A polynucleotide may contain one or more modified bases, such as phosphothioates, and may be a peptide nucleic acid (PNA). In general, a polynucleotide may be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or combinations of the foregoing. Although the polynucleotides described herein are shown as DNA sequences, the polynucleotides include their corresponding RNA sequences, and their complementary (e.g., fully complementary) DNA or RNA sequences, including their reverse complements. The polynucleotides of the present disclosure are set forth in the attached sequence listing.

[0052] "Polypeptide" or "polypeptide sequence" refers to a polymer of amino acids in which one or more amino acid residues are artificial chemical analogues of a corresponding naturally occurring amino acid, as well as naturally occurring polymers of amino acids. These terms also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The polypeptides of the present disclosure are set forth in the accompanying sequence listing.

[0053] As used herein, "recombinant" refers to the artificial combination of two otherwise separate segments of sequence, for example, by chemical synthesis or by the manipulation of isolated segments of polynucleotides by recombinant genetic techniques. The term also includes reference to a cell or vector that has been modified by the introduction of a heterologous polynucleotide, or a cell derived from a cell so modified, but does not encompass the alteration of a cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation or transduction or transposition), such as those that occur without deliberate human intervention.

[0054] The term "tobacco" is used in a collective sense to refer to tobacco crops (e.g., tobacco plants that are grown in a field and are not hydroponically grown), tobacco plants, and parts thereof, including but not limited to roots, stems, leaves, flowers, and seeds, prepared and / or obtained as described herein. "Tobacco" is understood to refer to the Nicotiana tabacum plant and its products.

[0055] The term "tobacco products" refers to consumer tobacco products, including, but not limited to, smoking materials (e.g., cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials, and ingredients for the manufacture of consumer tobacco products. Suitably, these tobacco products are made from tobacco leaves and stems harvested from tobacco plants and cut, dried, cured, and / or fermented according to conventional techniques in tobacco preparation.

[0056] "Variant" with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in sequence by amino acid insertions, deletions, or conservative substitutions, but that retains at least one biological function or activity. Variant can also refer to a polypeptide that retains at least one biological function or activity. Conservative substitutions of amino acids, i.e., replacing one amino acid with a different amino acid that has similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are recognized in the art as typically involving minor changes.

[0057] The term "variety" refers to a group of plants that share certain characteristics that distinguish them from other plants of the same species. While possessing one or more distinctive traits, varieties are further characterized by very minor overall variations among individuals within the variety. Varieties are often commercially available.

[0058] "Vector" refers to a polynucleotide vehicle that includes a combination of polynucleotide components to allow for transport, such as polynucleotides, polynucleotide constructs, and polynucleotide conjugates. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. Suitable vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and vectors of any other origin.

[0059] As used herein, an "expression vector" is a polynucleotide vehicle that contains a combination of polynucleotide components to allow expression, such as polynucleotides, polynucleotide constructs, and polynucleotide conjugates. Suitable expression vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and functionally equivalent expression vectors of any other origin. An expression vector includes at least a promoter, as defined below, located upstream and operably linked to a polynucleotide, polynucleotide construct, or polynucleotide conjugate.

[0060] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and polypeptide and polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. The meaning and scope of the terms must be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise specified by context, singular terms shall include the plural and plural terms shall include the singular.

[0061] "Genome editing" refers to the alteration of an endogenous gene encoding an endogenous polypeptide, resulting in the expression of a truncated endogenous polypeptide or an endogenous polypeptide with an amino acid modification, such as a substitution. Genome editing can include replacing a targeted endogenous gene region or replacing the entire endogenous gene with a copy gene with a truncation or amino acid substitution by a repair mechanism, such as homologous recombination repair. Genome editing can also include the generation of an amino acid substitution in an endogenous gene by generating a double-stranded break in the endogenous gene, which is then repaired using NHEJ. NHEJ can add or delete at least one base pair during repair, during which an amino acid substitution can occur. Genome editing can also include deleting a gene segment by the simultaneous action of two nucleases on the same DNA strand to create a break between two nuclease target sites and repair of the DNA break by NHEJ.

[0062] "Homologous recombination repair" or "HDR" refers to the cell's mechanism for repairing double-stranded DNA damage when a piece of homologous DNA is present in the nucleus, mainly in the G2 and S phases of the cell cycle.HDR can be used to guide repair using donor DNA or donor template and to create specific sequence changes to genomes, including targeted addition of whole genes.If donor template is provided with site-specific nuclease, then the cell's mechanism repairs the break by homologous recombination, which is multiplied by orders of magnitude in the presence of DNA break.If no piece of homologous DNA is present, NHEJ can be performed instead.

[0063] As used herein, "non-homologous end joining (NHEJ) pathway" refers to a pathway that repairs double-strand breaks in DNA by directly ligating the broken ends without the need for a homologous template. Template-independent religation of DNA ends by NHEJ is a stochastic, error-prone repair process that introduces random microinsertions and microdeletions (indels) at the DNA breakpoint. This method can be used to intentionally disrupt, delete, or change the reading frame of a target gene sequence. NHEJ typically uses short homologous DNA sequences, called microhomologies, to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of the double-strand break. NHEJ usually repairs the break accurately when the overhangs are perfectly compatible, and imprecise repairs leading to nucleotide losses can also occur, but are even more common when the overhangs are incompatible.

[0064] "Site-specific nuclease" refers to an enzyme that can specifically recognize and cleave a DNA sequence. Site-specific nucleases can be engineered. Examples of engineered site-specific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), CRISPR / Cas9-based systems, and meganucleases.

[0065] "Transcription Activator-Like Effector" or "TALE" refers to a polypeptide structure that recognizes and binds to specific DNA sequences. "TALE DNA-binding domain", also known as RVD modules, refers to a DNA-binding domain that contains a tandem 33-35 amino acid repeat array, each of which specifically recognizes a single base pair of DNA. The RVD modules can be arranged in any order to assemble an array that recognizes a defined sequence. The binding specificity of a TALE DNA-binding domain is determined by the RVD array followed by a single truncated repeat of 20 amino acids. A TALE DNA-binding domain can have 12-27 RVD modules, each of which contains an RVD and recognizes a single base pair of DNA. Specific RVDs that recognize each of the four possible DNA nucleotides (A, T, C, and G) have been identified. Because TALE DNA-binding domains are modular, four different DNA nucleotide-recognizing repeats can be linked together to recognize any specific DNA sequence. These targeted DNA binding domains can then be combined with catalytic domains to create functional enzymes, including artificial transcription factors, methyltransferases, integrases, nucleases, and recombinases.

[0066] As used interchangeably herein, "transcription activator-like effector nuclease" or "TALEN" refers to an engineered fusion polypeptide of the catalytic domain of a nuclease, such as the endonuclease FokI, and an engineered TALE DNA binding domain capable of targeting a common DNA sequence.

[0067] "TALEN monomer" refers to an engineered fusion polypeptide having a catalytic nuclease domain and a designed TALE DNA binding domain. Two TALEN monomers can be designed to target and cleave the TALEN target region.

[0068] "Zinc finger" refers to a polypeptide structure that recognizes and binds to DNA sequences. Zinc finger domains are the most common DNA-binding motif in the human proteome. A single zinc finger contains about 30 amino acids, and the domain typically functions by binding to three consecutive DNA base pairs through interactions of one amino acid side chain per base pair.

[0069] "Zinc finger nuclease" or "ZFN" refers to a chimeric polypeptide molecule that contains at least one zinc finger DNA binding domain operatively linked to at least one nuclease or portion of a nuclease that, when fully assembled, is capable of cleaving DNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] In one aspect, an isolated polynucleotide sequence is provided that encodes a nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO:5, wherein a methionine is replaced with an amino acid that reduces nitrate reductase activity compared to a control plant cell.

[0071] Suitably, the nitrate reductase polypeptide has a contiguous polypeptide sequence of SEQ ID NO:7, in which a methionine has been replaced with an amino acid that reduces nitrate reductase activity compared to a control plant cell.

[0072] Suitably, the isolated polynucleotide sequence encodes a nitrate reductase polypeptide in which methionine is replaced with a different amino acid, preferably an aliphatic non-polar amino acid, more preferably isoleucine.

[0073] Preferably, the aliphatic non-polar amino acid is selected from the group of amino acids consisting of glycine, alanine, proline, isoleucine, leucine, or valine.

[0074] Suitably, the aliphatic non-polar amino acid is isoleucine. According to this embodiment, the nitrate reductase has a substituted contiguous polypeptide sequence of SEQ ID NO:6 or SEQ ID NO:8.

[0075] Preferably, the isolated polynucleotide sequence encodes a nitrate reductase polypeptide comprising, consisting of, or consisting essentially of an amino acid substitution at a position corresponding to position 527 of a sequence having at least 80% sequence identity to SEQ ID NO:1.

[0076] Suitably, the isolated polynucleotide sequence encodes a nitrate reductase polypeptide comprising, consisting of, or consisting essentially of the sequence set forth in SEQ ID NO:3.

[0077] Suitably, the isolated polynucleotide sequence comprises, consists or consists essentially of a sequence having at least 80% sequence identity to SEQ ID NO:2.

[0078] Suitably, the polynucleotide sequence comprises, consists or consists essentially of the polynucleotide sequence set forth in SEQ ID NO:4.

[0079] Exemplary substitutional mutations according to the present disclosure are set forth in Table 1.

[0080] In certain embodiments, the polynucleotide sequence comprises, consists of, or consists essentially of a sequence having at least 80% sequence identity to any sequence described herein, including any polynucleotide shown in the sequence listing. Suitably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0081] Preferably, the polynucleotides described herein encode active nitrate reductase polypeptides having at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the function or activity of the polypeptides shown in the sequence listing.

[0082] In another embodiment, there is provided a polynucleotide fragment of SEQ ID NO:4 that encodes a polypeptide sequence comprising an M527I mutation having substantial homology (i.e., sequence similarity) or substantial identity to, having at least about 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to the corresponding fragment of SEQ ID NO:4.

[0083] Also disclosed are fragments of polynucleotides incorporating the mutations described herein. Polynucleotide fragments typically contain at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, or at least 200 contiguous nucleotides.

[0084] The polynucleotides described herein can include polymers of nucleotides that can be native or denatured deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Thus, the polynucleotides can be, but are not limited to, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragments or truncations thereof. Moreover, the polynucleotides can be single-stranded or double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules that include DNA and RNA, or hybrid molecules that include a mixture of single-stranded and double-stranded regions or fragments thereof. Furthermore, the polynucleotides can be composed of triple-stranded regions that include DNA, RNA, or both, or fragments thereof. In general, polynucleotides can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or combinations of the foregoing. Although the polynucleotides described herein are shown as DNA sequences, they include their corresponding RNA sequences, and their complementary (e.g., fully complementary) DNA or RNA sequences, including their reverse complements.

[0085] The polynucleotides described herein generally contain phosphodiester bonds. Other analog polynucleotides include those with cationic backbones, nonionic backbones, and non-ribose backbones. Modification of the ribose-phosphate backbone can be performed for various reasons, such as to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of natural polynucleotides and analogs, or mixtures of different polynucleotide analogs, and mixtures of natural polynucleotides and analogs can be made.

[0086] Analog polynucleotides can include those with cationic, non-ionic, and non-ribose backbones. Also included are polynucleotides containing one or more carbocyclic sugars.

[0087] Other analogs include peptide polynucleotides, which are peptide polynucleotide analogs. These backbones are substantially non-ionic under neutral conditions, in contrast to the multivalent phosphodiester backbones of natural polynucleotides. This can result in advantages. First, peptide polynucleotide backbones can exhibit improved hybridization kinetics. Peptide polynucleotides have a larger change in melting temperature of mismatched bases compared to perfectly matched base pairs. DNA and RNA typically exhibit a 2-4°C decrease in melting temperature for internal mismatches. With non-ionic peptide polynucleotide backbones, this decrease approaches 7-9°C. Similarly, due to their non-ionic nature, hybridization of bases attached to these backbones is relatively insensitive to salt concentration. Additionally, peptide polynucleotides may not be degraded, or may not be degraded to a lesser extent, by cellular enzymes, and therefore may be more stable.

[0088] Among the uses of the disclosed polynucleotides and fragments thereof are the use of the fragments as probes in hybridization assays or primers for use in amplification assays.Such fragments generally comprise at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides of DNA sequence.In other embodiments, the DNA fragments comprise at least about 10, 15, 20, 30, 40, 50 or 60 or more consecutive nucleotides of DNA sequence.Therefore, in one aspect, methods for detecting polynucleotides are also provided, comprising the use of probes or primers or both.Exemplary primers are described herein.

[0089] The basic parameters that influence the selection of hybridization conditions, and the guidance for devising suitable conditions, are described by Sambrook, J., EF Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Using knowledge of the genetic code combined with the polypeptide sequences described herein, a set of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful, for example, as primers in polymerase chain reactions (PCR), in which DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for genetic libraries. Such libraries include cDNA libraries, genomic libraries, and even electronic expressed sequence tags or DNA libraries. The homologous sequences identified by this method can then be used as probes to identify homologs of the sequences identified herein.

[0090] Also of potential use is the use of polynucleotides and oligonucleotides (e.g., primers or probes) that hybridize to polynucleotides under low stringency conditions, typically under moderate stringency conditions, and usually under high stringency conditions, as described herein.The basic parameters that influence the selection of hybridization conditions, and the guidance for devising suitable conditions, are described in Sambrook, J., EF Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), and can be easily determined by those skilled in the art, for example, based on the length or base composition of the polynucleotide.

[0091] One way to achieve moderate and high stringency conditions is defined herein. Of course, the washing temperature and washing salt concentration can be adjusted as necessary to achieve the desired degree of stringency by applying the basic principles governing hybridization reactions and duplex stability, known to those skilled in the art and described further below (see, for example, Sambrook, J., EF Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)). When hybridizing a polynucleotide to a polynucleotide of unknown sequence, the length of the hybrid is assumed to be the length of the hybridizing polynucleotide. When hybridizing a polynucleotide of known sequence, the length of the hybrid can be determined by aligning the sequences of the polynucleotides and identifying the region of optimal sequence complementarity. The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5-10°C lower than the melting temperature of the hybrid, where the melting temperature is determined by the following equation: For hybrids less than 18 base pairs in length, the melting temperature (°C) = 2 (number of A+T bases) + 4 (number of G+C bases). For hybrids greater than 18 base pairs in length, the melting temperature (°C) = 81.5 + 16.6 (log10 [Na+]) + 0.41 (% G+C) - (600 / N), where N is the number of bases in the hybrid and [Na+] is the sodium ion concentration in the hybridization buffer ([Na+] = 0.165M in 1 x standard sodium citrate). Typically, each such hybridizing polynucleotide has a length that is at least 25% (generally at least 50%, 60%, or 70%, most commonly at least 80%) of the length of the polynucleotide to which it hybridizes and has at least 60% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the polynucleotide to which it hybridizes.

[0092] As can be understood by those skilled in the art, linear DNA has two possible orientations: 5'-3' and 3'-5'. For example, if a first sequence is arranged in a 5'-3' direction and a second sequence is arranged in a 5'-3' direction in the same polynucleotide molecule / strand, the first and second sequences are oriented in the same direction, i.e., have the same orientation. Typically, the promoter sequence and the gene of interest under the control of a given promoter are arranged in the same orientation. However, if the second sequence is arranged in a 3'-5' direction in the same polynucleotide molecule / strand with respect to the first sequence arranged in a 5'-3' direction, the first and second sequences are oriented in the antisense direction, i.e., have an antisense orientation. Alternatively, two sequences that have an antisense orientation relative to each other can be described as having the same orientation when the first sequence (5'-3' direction) and the reverse complementary sequence of the first sequence (the first sequence arranged in a 5'-3' direction) are arranged in the same polynucleotide molecule / strand. The sequences set forth herein are presented in the 5'-3' orientation.

[0093] Also provided is a vector that contains recombinant polynucleotide constructs, such as those described herein.Suitable vector backbones include those that are routinely used in the art, such as plasmid, virus, artificial chromosome, bacterial artificial chromosome, yeast artificial chromosome, or bacteriophage artificial chromosome.Suitable expression vectors include, but are not limited to, plasmid and viral vectors derived from bacteriophage, baculovirus, and retrovirus.Many vectors and expression systems are commercially available.

[0094] In one aspect, an isolated polypeptide is provided that comprises, consists of, or consists essentially of a polypeptide having at least 80% sequence identity to any polypeptide described herein, including any polypeptide shown in the sequence listing. Suitably, the isolated polypeptide comprises, consists of, or consists essentially of a sequence having at least 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity thereto.

[0095] In another aspect, disclosed are polypeptide sequences encoded by the polynucleotide sequences presented herein.

[0096] In another aspect, a nitrate reductase polypeptide is disclosed that comprises a contiguous polypeptide sequence of SEQ ID NO:5, in which a methionine is replaced with an amino acid that reduces nitrate reductase activity compared to a control plant cell. The sequence of SEQ ID NO:5 serves as a recognition site for binding of nitrate reductase kinase, which is involved in phosphorylation of NtNIA2 amino acid S523.

[0097] Suitably, the nitrate reductase comprises the contiguous polypeptide sequence of SEQ ID NO: 7, wherein a methionine has been replaced with an amino acid that reduces nitrate reductase activity compared to a control plant cell.

[0098] Suitably, the methionine is replaced with a different amino acid, such as an aliphatic non-polar amino acid, which may be selected from the group of amino acids consisting of, for example, glycine, alanine, proline, isoleucine, leucine, or valine.

[0099] Suitably, the aliphatic non-polar amino acid is I. According to this embodiment, the nitrate reductase comprises a contiguous polypeptide sequence of SEQ ID NO:6 or SEQ ID NO:8.

[0100] Suitably, the nitrate reductase polypeptide comprises, consists of, or consists essentially of an amino acid substitution at a position corresponding to position 527 of a sequence having at least 80% sequence identity to SEQ ID NO:1.

[0101] Suitably, the nitrate reductase polypeptide comprises, consists or consists essentially of the amino acid substitution M527I in a sequence having at least 80% sequence identity to SEQ ID NO:1.

[0102] Suitably, the nitrate reductase polypeptide comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO:3.

[0103] Also disclosed are fragments of polypeptides incorporating the mutations described herein. A fragment of a polypeptide typically retains some or all of the function or activity of the full-length sequence. A polypeptide fragment typically comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, or at least 200 consecutive amino acids. The polypeptides disclosed herein comprise at least one mutation that is generated by introducing one or more modifications of any type that may be naturally isolated. Preferably, the function or activity of the nitrate reductase polypeptide in which the mutation is introduced is modulated (e.g., reduced) by the introduction of the mutation. Preferably, the mutation is a substitution. Substitution refers to the replacement of at least one amino acid of the nitrate reductase polypeptide with another amino acid that has similar properties (e.g., similar hydrophobicity, hydrophilicity, antigenicity, tendency to form or break an alpha-helical or beta-sheet structure, etc.).

[0104] Preferably, the mutation is a substitution of methionine in the contiguous polypeptide sequence of SEQ ID NO:5 or SEQ ID NO:7, and the methionine is substituted with an amino acid that causes the plant having the substitution to have reduced nitrate reductase activity compared to control plants.In one embodiment, the methionine is substituted with an aliphatic non-polar amino acid, such as glycine, alanine, proline, isoleucine, leucine, or valine.In one embodiment, the aliphatic non-polar amino acid is isoleucine, and preferably, the contiguous polypeptide sequence is SEQ ID NO:6 or SEQ ID NO:8.In one embodiment, the substitution is M527I.An exemplary nitrate reductase polypeptide sequence having an M527I mutation is SEQ ID NO:3.

[0105] Disclosed are plants or plant cells that contain or have a mutation in one or more polynucleotides or polypeptides described herein, which mutation results in a regulated function or activity of nitrate reductase. The mutations described herein may include artificial or synthetic mutations. The mutations in the polynucleotides and polypeptides described herein may be mutations that are obtained or obtainable by a process that includes an in vitro or in vivo manipulation step. The mutations in the polynucleotides and polypeptides described herein may be mutations that are obtained or obtainable by a process that includes human intervention. A method is provided for modulating the level of a polypeptide in a (dried) plant or in a (dried) plant material, the method comprising introducing one or more mutations into the genome of the plant that modulate the expression of at least one gene, the at least one mutation being selected from a sequence according to the present disclosure, e.g., M527I. Advantageously, the gene encodes a Nicotiana tabacum nitrate reductase polypeptide described herein.

[0106] Also disclosed are plants or plant cells that are heterozygous or homozygous for one or more mutations according to the present disclosure, which mutations result in the modulation of expression of a gene or the function or activity of a polypeptide encoded thereby.

[0107] The function or activity of one or more of the polypeptides of the present disclosure in a plant is increased or decreased when the function or activity is lower or higher than the function or activity of the same polypeptide in a plant that has not been modified to inhibit the function or activity of the polypeptide and that has been grown, harvested, and dried using the same protocols.

[0108] Also disclosed is the method for obtaining mutant polynucleotides and polypeptides described herein.The plant of interest, such as tobacco, including plant cell or plant material, can be genetically modified by various methods known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically induced mutagenesis, radiation-induced mutagenesis, mutagenesis using modified bases, mutagenesis using gapped duplex DNA, double-strand break mutagenesis, mutagenesis using repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling, and other methods discussed below.

[0109] Methods for randomly introducing mutations into polynucleotides can include chemical mutagenesis and radiation mutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals, such as mutagenic, teratogenic, or carcinogenic organic compounds, to induce mutations. Mutagens that primarily cause point mutations, as well as short deletions, insertions, missense mutations, simple sequence repeats, transversions, and / or transitions, can be used to induce mutations, including chemical mutagens or radiation. Mutagens include ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfane, diepoxyalkanes (diepoxyoctane, diepoxybutane, etc.), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride, and formaldehyde.

[0110] Methods for introducing one or more targeted mutations into a polynucleotide sequence include, but are not limited to, genome editing techniques, in particular zinc finger nuclease-mediated mutagenesis, TILLING (targeting induced local lesions in genomes), homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis.

[0111] Zinc finger polypeptides can be used to introduce one or more targeted mutations into a polynucleotide sequence. In various embodiments, a genomic DNA sequence, including part or all of the coding sequence of a polynucleotide, is modified by zinc finger nuclease-mediated mutagenesis. The genomic DNA sequence is searched for a unique site for zinc finger polypeptide binding. Alternatively, the genomic DNA sequence is searched for two unique sites for zinc finger polypeptide binding, where both sites are on opposite strands and are close to each other, e.g., 1, 2, 3, 4, 5, 6 or more base pairs apart. Thus, zinc finger polypeptides that bind to polynucleotides are provided. The zinc finger polypeptides can be engineered to recognize selected target sites within a gene. The zinc finger polypeptides can include any combination of motifs derived from natural and non-natural zinc finger DNA binding domains by a process of truncation or extension or site-directed mutagenesis coupled with a selection method, such as, but not limited to, phage display selection, bacterial two-hybrid selection, or bacterial one-hybrid selection. The term "non-natural zinc finger DNA binding domain" refers to a zinc finger DNA binding domain that binds a 3-base pair sequence that is in a polynucleotide target and does not occur in the cell or organism that contains the polynucleotide to be modified.Methods for designing zinc finger polypeptides that bind specific polynucleotides that are unique to target genes are known in the art.Methods for delivering zinc finger polypeptides and zinc finger nucleases to plants are similar to the methods described below for delivering meganucleases.

[0112] Meganucleases such as I-CreI can be used to introduce one or more targeted mutations into a polynucleotide sequence. Natural meganucleases as well as recombinant meganucleases can be used to specifically cause double-strand breaks at a single site or at relatively few sites in the genomic DNA of a plant, allowing the cleavage of one or more of the polynucleotides described herein. The meganuclease can be an engineered meganuclease with altered DNA recognition properties. Meganuclease polypeptides can be delivered to plant cells by a variety of different mechanisms known in the art. Meganucleases can be used to cleave meganuclease recognition sites in the coding region of a polynucleotide. Such cleavage frequently results in the deletion of DNA at the meganuclease recognition site after mutagenic DNA repair by non-homologous end joining. Such mutations in the gene coding sequence are typically sufficient to inactivate the gene. This method for modifying plant cells first involves the delivery of a meganuclease expression cassette into the plant cell using a suitable transformation method. For highest efficiency, it is desirable to link the meganuclease expression cassette to a selection marker and select successfully transformed cells in the presence of a selection agent. This approach results in integration of the meganuclease expression cassette into the genome, which may not be desirable if the plant is likely to require regulatory approval. In such cases, the meganuclease expression cassette (and the linked selection marker gene) may be segregated in subsequent plant generations using conventional breeding techniques. After delivery of the meganuclease expression cassette, the plant cells are initially cultured under conditions that are typical for the particular transformation procedure used. This may mean culturing the transformed cells in a medium at a temperature below 26° C., often in the dark. Such standard conditions can be used for a period of time, preferably 1 to 4 days, to allow the plant cells to recover from the transformation process.Any time after this initial recovery period, the culture temperature can be increased to stimulate activity of the engineered meganuclease, cleaving the meganuclease recognition site and causing mutations.

[0113] One method of genome editing involves the use of transcription activator-like effector nucleases (TALENs) that induce double-stranded breaks to which the cell can respond with repair mechanisms. NHEJ rejoins DNA from either side of a double-stranded break with very little or no sequence overlap for annealing. This repair mechanism induces errors in the genome due to insertions or deletions, or chromosomal rearrangements. Such errors may result in a gene product being coded non-functional at that location. In certain applications, it may be desirable to precisely remove a polynucleotide from the genome of a plant. Such applications are possible using a pair of engineered meganucleases, each of which cuts a meganuclease recognition site on either side of the intended deletion. TALENs can also be used that can recognize and bind to a gene and introduce a double-stranded break into the genome.

[0114] Another method of genome editing involves the use of bacterial CRISPR / Cas systems. Bacteria and archaea exhibit chromosomal elements called clustered regularly interspaced short palindromic repeats (CRISPR), which are part of the adaptive immune system that protects against viral and plasmid DNA invasion. In type II CRISPR systems, CRISPR RNA (crRNA) functions with transactivating crRNA (tracrRNA) and CRISPR-associated (Cas) polypeptides to introduce double-stranded breaks in the target DNA. Target cleavage by Cas9 requires base pairing between the crRNA and tracrRNA, and between the crRNA and the target DNA. Target recognition is facilitated by the presence of a short motif called a protospacer adjacent motif (PAM), which matches the sequence NGG. This system can be exploited for genome editing. Cas9 is usually programmed by a duplex RNA composed of crRNA and tracrRNA. However, the core components of these RNAs can be combined into a single hybrid "guide RNA" for Cas9 targeting. The use of non-coding RNA guides to target DNA for site-specific cleavage promises to be much simpler than existing technologies such as TALEN. Using the CRISPR / Cas strategy, retargeting the nuclease complex only requires the introduction of new RNA sequences, without the need to redesign the specificity of polypeptide transcription factors. CRISPR / Cas technology was implemented in plants in the manner of International Application No. WO2015 / 189693, which discloses a virus-mediated genome editing platform that is broadly applicable across plant species. The RNA2 genome of Tobacco Rattles Virus (TRV) was engineered to carry and deliver guide RNA to Nicotiana benthamiana plants overexpressing Cas9 endonuclease. In the context of the present disclosure, the guide RNA can be derived from any of the sequences disclosed herein, and the teachings of WO2015 / 189693 can be applied to edit the genome of plant cells to obtain desired mutant plants.Fast-paced technological development has given rise to a wide variety of protocols with broad applicability in the plant kingdom, which are well catalogued in many recent scientific review articles (e.g., Plant Methods (2016) 12:8, and Front Plant Sci. (2016) 7:506). A review of the CRISPR / Cas system with a particular focus on its applications can be found in Biotechnology Advances (2015) 33, 1, 41-52). More recent developments in the use of CRISPR / Cas to manipulate plant genomes are reviewed in Acta Pharmaceutica Sinica B (2017) 7, 3, 292-302, and Curr. Op. in Plant Biol. (2017) 36, 1-8. CRISPR / Cas9 plasmids for use in plants are listed in "addgene", a non-profit plasmid repository (addgene.org), and CRISPR / Cas plasmids are commercially available.

[0115] One or more introduced mutations, such as those described herein, can be identified or selected using methods known to those of skill in the art, such as Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that affect gene expression or interfere with the function of the encoded polypeptide can be determined using methods well known in the art.

[0116] A plant or plant cell according to the present disclosure comprises any combination of the mutations described and optionally one or more additional mutations in one or more genes. For example, a plant or plant cell may have a single mutation in a single gene, multiple mutations in a single gene, a single mutation in two or more, three or more, or four or more genes, or multiple mutations in two or more, three or more, or four or more genes. An example of such a mutation is described herein.

[0117] In one embodiment, seeds from the plant are mutagenized and then grown into first generation mutant plants. The first generation plants are then self-pollinated, and seeds from the first generation plants are grown into second generation plants, which are then screened for mutations such as those described herein at the locus. Mutagenized plant material can be screened for mutations, but the advantage of screening second generation plants is that all somatic mutations correspond to germline mutations. Those skilled in the art will understand that a variety of plant materials, including but not limited to seeds, pollen, plant tissues, or plant cells, can be mutagenized to create mutant plants. However, the type of mutagenized plant material may affect when the plant's polynucleotides are screened for mutations. For example, if pollen is subject to mutagenesis before pollination of a non-mutagenized plant, the seeds resulting from that pollination will grow into first generation plants. All cells of the first generation plants will contain the mutations created in the pollen, and therefore these first generation plants can then be screened for mutations without waiting until the second generation.

[0118] Polynucleotides prepared from individual plants, plant cells, or plant materials can be optionally pooled to facilitate screening for at least the mutations described herein in a population of plants originating from the mutagenized plant tissues, cells, or materials. One or more subsequent generations of plants, plant cells, or plant materials can be screened. The size of the optionally pooled population depends on the sensitivity of the screening method used.

[0119] After the samples are optionally pooled, they can be subjected to polynucleotide-specific amplification techniques such as PCR. Any one or more primers or probes specific to genes or sequences immediately adjacent to the gene can be utilized to amplify sequences in the optionally pooled samples. Preferably, one or more primers or probes are designed to amplify the region of the locus where useful mutations are most likely to occur. Most preferably, primers are designed to detect mutations within the region of the polynucleotide. Furthermore, primers and probes preferably avoid known polymorphic sites to facilitate screening for point mutations. To facilitate detection of the amplification product, one or more primers or probes can be labeled using any conventional labeling method. Primers or probes can be designed based on the sequences described herein using methods well understood in the art.

[0120] To facilitate detection of the amplification products, primers or probes may be labeled using any conventional labeling method, which can be designed based on the sequences described herein using methods well understood in the art.

[0121] Polymorphisms can be identified by means known in the art, several of which have been described in the literature.

[0122] Thus, in a further aspect, there is provided a method of preparing a plant comprising a mutation as described herein. The method comprises providing at least one cell of the plant comprising a gene encoding a functional nitrate reductase polynucleotide. The at least one cell of the plant is then treated under conditions effective to modulate the function of the nitrate reductase polynucleotide. The at least one mutant plant cell is then grown in a mutant plant, where the mutant plant has a modulated level of the nitrate reductase polypeptide compared to a control plant. In one embodiment, the treating step involves subjecting at least one cell to a chemical mutagen as described herein and under conditions effective to obtain at least one mutant plant cell. In another embodiment of the method, the treating step involves subjecting at least one cell to a radiation source under conditions effective to obtain at least one mutant plant cell. The term "mutant plant" includes mutant plants whose genotype has been altered compared to a control plant, preferably by means other than genetic engineering or genetic modification.

[0123] In certain embodiments, the mutant plant, mutant plant cell, or mutant plant material may contain one or more mutations that naturally occur in another plant, plant cell, or plant material and that confer a desired characteristic. The mutation can be incorporated (e.g., introgressed) into another plant, plant cell, or plant material (e.g., a plant, plant cell, or plant material having a different genetic background than the plant in which the mutation was induced) to confer the characteristic thereto. Thus, as an example, a naturally occurring mutation in a first plant can be introduced into a second plant (e.g., a second plant having a different genetic background than the first plant). Thus, one skilled in the art can search for and identify plants that naturally carry in their genome one or more mutant alleles of the genes described herein that confer the desired characteristic. The naturally occurring mutant alleles can be transferred to a second plant by various methods, including breeding, backcrossing, and introgression, to generate a line, variety, or hybrid having one or more mutations in the genes described herein. The same technique can be applied to the introgression of one or more non-natural mutations from a first plant to a second plant. Plants exhibiting desired characteristics can be screened out from a pool of mutant plants. Advantageously, the selection is performed using knowledge of the polynucleotide sequence as described herein. As a result, screening for genetic characteristics compared to a control is possible. Such screening techniques may involve the application of traditional amplification and / or hybridization techniques as discussed herein. Thus, a further aspect of the present disclosure relates to a method for identifying a mutant plant, the method comprising the steps of (a) providing a sample comprising a polynucleotide from a plant, and (b) determining the sequence of the polynucleotide, wherein a difference in the sequence of the polynucleotide compared to the polynucleotide of a control plant indicates that the plant is a mutant plant.In another aspect, a method is provided for identifying mutant plants that accumulate reduced levels of nitrate compared to a control plant, the method comprising: (a) providing a sample from a plant to be screened; (b) determining whether the sample contains one or more mutations described herein in a nitrate reductase polynucleotide; and (c) determining the level of nitrate in said plant. Preferably, the level of nitrate is determined in desiccated leaves. In another aspect, a method is provided for preparing a mutant plant having reduced levels of nitrate compared to a control plant, the method comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations described herein in a nitrate reductase polynucleotide that results in reduced levels of nitrate; and (c) transferring the one or more mutations to a second plant. Preferably, the level of nitrate is determined in desiccated leaves. The mutation can be transferred to the second plant using various methods known in the art, such as genetic recombination, genetic engineering, introgression, plant breeding, backcrossing, etc. In one embodiment, the first plant is a wild-type plant. In one embodiment, the second plant has a different genetic background than the first plant. In another aspect, a method is provided for preparing a mutant plant having a reduced level of nitrate compared to a control plant, the method comprising: (a) providing a sample from the first plant; (b) determining whether the sample contains one or more mutations described herein in a nitrate reductase polynucleotide that results in a reduced level of nitrate; and (c) introgressing the one or more mutations from the first plant to the second plant. Preferably, the level of nitrate is determined in desiccated leaves. In one embodiment, the introgression step comprises plant breeding, optionally including backcrossing, etc. In one embodiment, the first plant is a wild-type plant. In one embodiment, the second plant has a different genetic background than the first plant. In one embodiment, the first plant is not a cultivar or elite cultivar. In one embodiment, the second plant is a cultivar or elite cultivar.Further aspects relate to mutant plants (including mutant plants of cultivars or elite cultivars) obtained or obtainable by the methods described herein. In certain embodiments, a "mutant plant" may have one or more mutations localized only to a particular region of the plant, such as within the sequence of one or more polynucleotides described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be identical or substantially identical to the plant prior to mutagenesis.

[0124] In a further aspect, a method is provided for identifying a plant, plant cell, or plant material comprising a mutation in a gene encoding a nitrate reductase polynucleotide, the method comprising: (a) subjecting the plant, plant cell, or plant material to mutagenesis; (b) obtaining a sample from the plant, plant cell, or plant material, or a progeny thereof; and (c) determining the presence of a mutated polynucleotide sequence having a mutation as described herein.

[0125] The disclosed compositions and methods can be applied to any species belonging to the genus Nicotiana, including N. rustica and N. tabacum (e.g., LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico). Other species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, N. benavidesii, N. spp. ... navidesii, N. benthamiana, N. bigelovii, N. borariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N. .N.forgetiana, N.fragrans, N.glauca, N.glutinosa, N.goodspeedii, N.gossei, N.hybrid, N.ingulba, N.kawakamii, N.knightiana, N.langus N. langsdorffii, N. linearis, N. longiflora, N. maritima, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N.occidentalis, N. occidentalis subsp. hesperis, N. otophora, N. paniculata, N. pauciflora, N. petunioides, N. plumbaginifolia, N. quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata subsp. ingulva subsp.ingulba), N.rotundifolia, N.setchellii, N.simulans, N.solanifolia, N.spegazzinii, N.stocktonii, N.suaveolens, N.sylvestris , N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides and Nx sanderae.

[0126] In one embodiment, the plant is N. tabacum.

[0127] The use of tobacco cultivars and elite tobacco cultivars is also contemplated herein. Thus, the plant may be a tobacco cultivar or an elite tobacco cultivar that contains one or more genetic mutations. The genetic mutation (e.g., one or more polymorphisms) may be a mutation that does not naturally occur in the particular tobacco cultivar or tobacco cultivar (e.g., elite tobacco cultivar), or may be a naturally occurring genetic mutation, where the mutation does not naturally occur in the particular tobacco cultivar or tobacco cultivar (e.g., elite tobacco cultivar).

[0128] Nicotiana tabacum varieties include burley, dark, flue-cured, and orient tobacco. Non-limiting examples of varieties or cultivars include AA37, BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KY14xL8 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, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, PD 7302 LC, PD 7309 LC, PD 7312 LC, 'Perique' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, SpeightG-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR(Tom Rosson)Madole, VA 309, VA359, AA 37-1, B13P, Xanthi(Mitchell-Mor), Bel-W3, 79-615, Samsung Holmes NN, KTRDC number 2 Hybrid 49, Burley 21, KY8959, KY9, MD 609, PG01, PG04, PO1, PO2, PO3, RG11, RG 8, VA509, AS44, Banket A1, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81, DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, 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, TI-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi, GR149, GR153, Petit Havana. Low converter variants of the above are also contemplated, even if not specifically identified herein.

[0129] In one embodiment, the cultivar is AA37, which is generally understood to be a cross between South American dark tobacco and American burley germplasm.

[0130] The embodiments are also directed to compositions and methods for generating plants that are modified to regulate the expression or function of the polynucleotides described herein (or any combination thereof described herein). Advantageously, the resulting plants can be similar or substantially identical in overall appearance to control plants. Various phenotypic characteristics can be evaluated by field observation, such as maturity, number of leaves per plant, stem height, leaf insertion angle, leaf size (width and length), internode distance, and leaf blade to midvein ratio.

[0131] One aspect relates to the seed of the plant described herein.Preferably, the seed is a tobacco seed.A further aspect relates to the pollen or ovule of the plant described herein.Furthermore, the plant described herein is provided, further comprising a polynucleotide that confers male sterility.

[0132] Also provided are tissue cultures of regenerable cells of the plants described herein, which culture will regenerate plants capable of expressing all the morphological and physiological characteristics of the parent. Regenerable cells include, but are not limited to, cells derived from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and parts thereof, ovules, shoots, stems, pith, and bolls, or callus or protoplasts derived therefrom.

[0133] One objective is to provide a plant or part thereof that exhibits a regulated level of nitrate in the plant material, e.g., in desiccated leaves. Suitably, the plant or part thereof exhibits a regulated level of nitrate compared to a control plant.

[0134] Suitably, the plants or parts thereof have substantially the same total harvested biomass (expressed as wet leaf biomass per plant) as the control plants.

[0135] Suitably, the plant or part thereof has substantially the same level of nicotine leaf content as a control plant.

[0136] Suitably, the plant or part thereof has substantially the same level of total alkaloid leaf content as a control plant.

[0137] Suitably, the plant or part thereof has substantially the same level of ammonia leaf content as a control plant.

[0138] Suitably, the plant or part thereof has substantially the same level of reducing sugar content as a control plant.

[0139] Thus, the present specification describes a plant or part thereof or a plant cell that has a regulated level of nitrate compared to a control cell or a control plant.The plant or plant cell is modified to regulate the synthesis or function of one or more of the polypeptides described herein by regulating the expression of the corresponding one or more of the polynucleotides described herein.Preferably, the regulated level of nitrate is observed in cured leaves.In certain embodiments, the level of nitrate in a plant, such as cured leaves or cured tobacco, can be reduced.

[0140] Further aspects relate to plants or plant cells, in which the expression or function of one or more of the polypeptides described herein is modulated, and a part of the plant (e.g., cured leaves, or cured tobacco) has a nitrate level therein that is reduced by at least about 37% compared to a control plant in which the expression or function of the polypeptide is not modulated. In certain embodiments, the nitrate level in a plant, such as cured leaves or cured tobacco, may be reduced by at least about 30% or more, or by about 35% or more, or by about 37% or more, or by about 40% or more.

[0141] Still further embodiments relate to cured plant material, such as cured leaves or cured tobacco, derived or derivable from a plant or plant cell described herein, wherein expression of one or more polynucleotides described herein or the function of the polypeptides encoded thereby is modulated and nitrate levels are modulated by at least about 37% compared to a control plant, such as at least about 30% or more, or about 35% or more, or about 40% or more.

[0142] Embodiments are also directed to compositions and methods for producing plants or plant cells that have been modified to modulate the expression or function of one or more polynucleotides or polypeptides described herein, which can result in plants or plant components (e.g., leaves, such as cured leaves, or tobacco) or plant cells having modulated nitrate content.

[0143] The increase in function or activity compared to a control can be from about 5% to about 100%, or can be an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or more than 100%, e.g., an increase of 200%, 300%, 500%, 1000% or more.

[0144] The decrease in function or activity compared to a control can be from about 5% to about 100%, or can be at least a 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% decrease.

[0145] Plants carrying the mutations described herein in the nitrate reductase polynucleotide can be used in plant breeding programs to generate useful lines, varieties, and hybrids. In particular, the mutations can be introgressed into the commercially important varieties described above. Thus, a method for breeding plants is provided, comprising crossing a plant described herein with a plant of a different genetic identity. The method may further comprise crossing the progeny plant with another plant, and optionally repeating the crossing until progeny with the genetic trait or genetic background are obtained. One purpose served by such breeding methods is to introduce desirable genetic characteristics into other varieties, breeding lines, hybrids, or cultivars, especially those of commercial interest. Another purpose is to facilitate stacking of genetic modifications of different genes in a single plant variety, line, hybrid, or cultivar. Intra- and inter-species crosses are contemplated. The progeny plants resulting from such crosses are also referred to as breeding lines, and are examples of plants of the present disclosure.

[0146] In one embodiment, a method for producing a plant is provided, comprising: (a) crossing a plant of the present disclosure with a second plant to obtain a progeny tobacco seed; (b) cultivating the progeny tobacco seed under plant cultivation conditions to obtain a non-natural plant. The method may further comprise: (c) crossing the non-natural plant of the previous generation with itself or another plant to obtain a progeny tobacco seed; (d) cultivating the progeny tobacco seed of step (c) under plant cultivation conditions to obtain an additional non-natural plant; and (e) repeating the crossing and cultivating steps of (c) and (d) multiple times to generate additional generations of the non-natural plant. Optionally, the method may comprise, prior to step (a), providing a parent plant with a characterized genetic identity that is not identical to the plant of the present disclosure. In some embodiments, depending on the breeding program, the crossing and cultivating steps are repeated 0-2 times, 0-3 times, 0-4 times, 0-5 times, 0-6 times, 0-7 times, 0-8 times, 0-9 times, or 0-10 times to generate a generation of non-native plants. Backcrossing is an example of such a method in which the progeny is crossed with one of its parents or another plant genetically similar to the parent to obtain a next generation progeny plant with a genetic identity close to that of one of the parents. Plant breeding, particularly plant breeding techniques, are well known and can be used in the methods of the present disclosure. In certain embodiments, the step of selecting the plants is excluded.

[0147] According to the present disclosure, in a breeding program, successful crosses produce F1 plants that are fertile. The selected F1 plants can be crossed with one of the parents, and the first generation plants of the backcross are self-pollinated to generate a population that is screened again for mutant gene expression (e.g., a null version of the gene). The backcross, self-pollinated, and screening process is repeated, for example, at least four times until a final screen produces a plant that is fertile and reasonably similar to the recurrent parent. If desired, the plant is self-pollinated, and then the progeny is screened again to confirm that the plant shows mutant gene expression. In some embodiments, the F2 generation plant population is screened for mutant gene expression, for example, by using PCR methods with primers based on the polynucleotide sequence information of the polynucleotides described herein (or any combination thereof described herein) according to standard methods to identify plants that do not express the polypeptide due to a defect in the gene.

[0148] Apart from the mutations described herein, a plant or plant cell described herein can have one or more further mutations, either in the same polynucleotides or polypeptides as described herein, or in one or more other polynucleotides or polypeptides within the genome.

[0149] Without being limited thereto, the plants and parts thereof described herein can be modified either before or after the expression, function, or activity of one or more polynucleotides and / or polypeptides according to the present disclosure is modulated.

[0150] One or more of the following additional genetic modifications (eg, mutations) may be present in the plant and parts thereof:

[0151] One or more genes involved in the conversion of nitrogen metabolism intermediates can be modified (e.g., mutated) to result in reduced levels of at least one tobacco-specific nitrosamine (TSNA). Non-limiting examples of such genes include those encoding nicotine demethylases, such as CYP82E4, CYP82E5, and CYP82E10, as described in WO2006 / 091194, WO2008 / 070274, WO2009 / 064771, and WO2011 / 088180, and nitrate reductases, as described in WO2016046288.

[0152] One or more genes involved in heavy metal uptake or heavy metal transport can be modified (e.g., mutated) to reduce heavy metal content.Non-limiting examples include genes belonging to the multidrug resistance-related polypeptide family, the cation diffusion facilitator (CDF) family, the Zrt-Irt-like polypeptide (ZIP) family, the cation exchanger (CAX) family, the copper transporter (COPT) family, the heavy metal ATPases family (e.g., HMA as described in WO2009 / 074325 and WO2017 / 129739), the macrophage polypeptide (e.g., NRAMP) homolog family associated with natural resistance, and other members of the ATP-binding cassette (ABC) transporter family (e.g., MRP) as described in WO2012 / 028309, which are involved in the transport of heavy metals such as cadmium.

[0153] Other exemplary modifications (e.g., mutations) can result in plants with regulated expression or function of isopropylmalate synthase, resulting in changes in sucrose ester composition that can be used to alter the flavor profile (see WO2013029799).

[0154] Other exemplary modifications (e.g., mutations) can result in plants with regulated expression or function of threonine synthase, which can modulate levels of methional (see WO2013029800).

[0155] Other exemplary modifications (e.g., mutations) can result in plants with regulated expression or function of one or more of neoxanthin synthase, lycopene beta cyclase, and 9-cis-epoxycarotenoid dioxygenase to modulate beta-damascenone content and alter flavor profile (see WO2013064499).

[0156] Other exemplary modifications (e.g., mutations) can result in plants having regulated expression or function of members of the CLC family of chloride channels to regulate nitrate levels therein (see WO2014096283 and WO2015197727).

[0157] Other exemplary modifications (e.g., mutations) can result in plants in which the expression or function of one or more asparagine synthetases are modulated to regulate the levels of asparagine in leaves and the levels of acrylamide in the aerosol produced upon heating or burning of leaves (see WO2017042162).

[0158] Other examples of modifications (eg, mutations) include herbicide resistance; for example, glyphosate is the active ingredient in many broad-spectrum herbicides.

[0159] Another exemplary modification (e.g., mutation) results in plants that are resistant to insects. Bacillus thuringiensis (Bt) toxins can provide an effective way to delay the development of Bt-resistant pests, as recently demonstrated in broccoli, where the cry1Ac and cry1CBt genes in cones have eliminated diamondback moths that are resistant to either single polypeptide and significantly delayed the evolution of resistant insects.

[0160] Another exemplary modification (e.g., mutation) results in a plant that is resistant to a disease caused by a pathogen (e.g., virus, bacteria, fungus). Plants expressing the Xa21 gene (resistant to bacterial spot) have been engineered along with plants expressing both a Bt fusion gene and a chitinase gene (resistant to the yellow borer and resistant to coleoptera).

[0161] Another exemplary modification (eg, mutation) results in altered fertility, such as male sterility.

[0162] Another exemplary modification (eg, mutation) results in a plant that is tolerant to abiotic stress (eg, drought, temperature, salinity).

[0163] Another exemplary modification (e.g., mutation) results in a plant in which the activity of one or more glycosyltransferases (e.g., N-acetylglucosaminyltransferase, β(1,2)-xylosyltransferase, and a(1,3)-fucosyltransferase) is modulated (see WO / 2011 / 117249).

[0164] Another exemplary modification (e.g., mutation) results in a plant in which the activity of one or more nicotine N-demethylases is modulated such that the levels of nornicotine and nornicotine metabolites formed during the desiccation process can be regulated (see WO2015169927).

[0165] Other exemplary modifications (e.g., mutations) can result in plants with improved polypeptide and oil storage capacity, plants with increased photosynthetic efficiency, plants with extended shelf life, plants with increased carbohydrate content, and plants that are resistant to fungi.

[0166] One or more genes involved in the nicotine synthesis pathway can be modified (e.g., mutated) to result in a plant or part of a plant that has a regulated level of nicotine when subjected to a dry treatment. The nicotine synthesis gene can be selected from the group consisting of A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MPO1, MPO2, MYC2a, MYC2b, NBB1, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4, and QPT, or one or more combinations thereof.

[0167] One or more genes involved in controlling the amount of one or more alkaloids can be modified (e.g., mutated) to result in a plant or part of a plant producing regulated levels of alkaloids. The alkaloid level control genes can be selected from the group consisting of BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MYC2a, MYC2b, nic1, nic2, NUP1, and NUP2, or a combination of two or more thereof.

[0168] The plant parts described herein, particularly the blades and midribs of such plants, can be incorporated into or used to manufacture various consumable products, including, but not limited to, aerosol-forming materials, aerosol-forming devices, smoking articles, smokable articles, smokeless products, medical or cosmetic products, intravenous preparations, tablets, powders, and tobacco products. Examples of aerosol-forming materials include tobacco compositions, tobacco, tobacco extracts, cut tobacco, cut fillers, flue-cured tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobacco. Smoking articles and smokable articles are types of aerosol-forming devices. Examples of smoking articles or smokable articles include cigarettes, cigarillos, and cigars. Examples of smokeless products include chewing tobacco and snuff. In certain aerosol-forming devices, rather than combustion, the tobacco composition or another aerosol-forming material is heated by one or more electric heating elements to generate an aerosol. In another type of heated aerosol-forming device, the aerosol is generated by transferring heat from a combustible fuel element or heat source to a physically separated aerosol-forming material that may be located within, around, or downstream of the heat source. Smokeless tobacco products and various tobacco-containing aerosol-forming materials may contain tobacco in any form, including dry particles, pieces, granules, powders, or slurries, as well as deposited on, mixed with, surrounded by, or combined with other components in any form, such as flakes, films, tabs, foams, or beads. As used herein, the term "smoke" is used to describe the type of aerosol generated by a smoking article, such as a cigarette, or by burning an aerosol-forming material.

[0169] In one embodiment, dried plant material derived from the plants described herein is also provided. Processes for drying fresh tobacco leaves are known to those skilled in the art and include, but are not limited to, air drying, flame drying, hot air tube drying, and sun drying, as described herein.

[0170] In another embodiment, a tobacco product is described that includes a tobacco-containing aerosol-forming material, including plant material such as leaves, preferably cured leaves, derived from a tobacco plant as described herein. The tobacco product described herein may be a blended tobacco product, which may further include unmodified tobacco.

[0171] Products and methods for crop management and agriculture

[0172] The plants may have other uses, for example in agriculture, for example, the plants described herein can be used to produce animal feed and food for humans.

[0173] The present disclosure also provides a method for producing seeds, comprising cultivating the plants described herein and collecting seeds from the cultivated plants.Seeds from the plants described herein can be prepared by means known in the art and packaged in packaging material to form an article of manufacture.Packaging materials such as paper and cloth are well known in the art.The seed package can have a label, such as a tag or label fixed to the packaging material, a label printed on the package that describes the nature of the seeds contained therein, and the like.

[0174] Compositions, methods, and kits for genotyping plants for identification, selection, or breeding can include a means for detecting the presence of a polynucleotide (or any combination thereof as described herein) in a sample of polynucleotides. Thus, compositions are described as comprising one or more primers for specifically amplifying at least a portion of one or more polynucleotides, and optionally one or more probes, and optionally one or more reagents for amplification or detection.

[0175] Thus, disclosed are gene-specific oligonucleotide primers or probes that comprise about 10 or more contiguous polynucleotides corresponding to the polynucleotides described herein, which may comprise or consist of more than about 15, 20, 25, 30, 40, 45, or 50 contiguous polynucleotides that hybridize (e.g., specifically hybridize) to the polynucleotides described herein.

[0176] In some embodiments, a primer or probe may comprise or consist of about 10-50 contiguous nucleotides, about 10-40 contiguous nucleotides, about 10-30 contiguous nucleotides, or about 15-30 contiguous nucleotides that may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) or isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques), or gene detection (e.g., as one or more amplification primers in amplification or detection). One or more specific primers or probes can be designed and used to amplify or detect some or all of the polynucleotides. As a specific example, two primers may be used in a PCR protocol to amplify a polynucleotide fragment. PCR may also be performed using one primer derived from the polynucleotide sequence and a second primer that hybridizes to a sequence upstream or downstream of the polynucleotide sequence (e.g., a promoter sequence, the 3' end of a pre-mRNA, or a sequence derived from a vector). Examples of temperature and isothermal techniques useful for in vitro amplification of polynucleotides are well known in the art. The sample may be or may be derived from a plant, plant cell, or plant material, or a tobacco product produced from or derived from a plant, plant cell, or plant material as described herein.

[0177] In a further aspect, a method of detecting a polynucleotide as described herein (or any combination thereof as described herein) in a sample is also provided, the method comprising the steps of (a) providing a sample containing or suspected to contain a polynucleotide, (b) contacting the sample with one or more primers or one or more probes to specifically detect at least a portion of the polynucleotide, and (c) detecting the presence of an amplification product, where the presence of the amplification product is indicative of the presence of the polynucleotide in the sample. In a further aspect, the use of one or more primers or probes to specifically detect at least a portion of the polynucleotide is also provided. Kits for detecting at least a portion of the polynucleotide are also provided, which include one or more primers or probes to specifically detect at least a portion of the polynucleotide. The kits may include reagents for polynucleotide amplification (e.g., PCR) or for probe hybridization detection techniques (e.g., Southern blot, Northern blot, in situ hybridization, or microarray). The kits may include reagents for antibody binding detection techniques such as Western blot, ELISA, SELDI mass spectrometry, or test strips. The kit may include reagents for DNA sequencing. The kit may include the reagents and instructions for using the kit.

[0178] In some embodiments, the kits may include instructions for one or more of the described methods. The described kits may be useful for determining genetic identity, phylogenetic studies, genotyping, haplotypes, pedigree analysis, or plant breeding, particularly in co-dominance assessments.

[0179] The present disclosure also provides a method of genotyping a plant, plant cell, or plant material comprising a polynucleotide described herein. Genotyping provides a means of distinguishing between chromosomal pair homologs and can be used to identify segregants in a population of plants. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships between crop varieties, identifying hybrids or somatic hybrids, localizing segments of chromosomes that affect single genetic traits, map-based cloning, and quantitative inheritance studies. Specific methods of genotyping may employ any number of molecular marker analysis techniques, including amplified fragment length polymorphism (AFLP). AFLP is the product of allelic differences between amplified fragments due to polynucleotide variability. Thus, the present disclosure further provides a means of tracking the segregation of one or more genes or polynucleotides, as well as chromosomal sequences genetically associated with these genes or polynucleotides, using techniques such as AFLP analysis.

[0180] The present invention is further described in the following examples, which are provided to further illustrate the invention. These examples describe preferred modes currently contemplated for carrying out the invention and are intended to illustrate, but not limit, the invention. EXAMPLES

[0181] To generate a population of plants carrying random point mutations, M0 seeds of Nicotiana tabacum AA37 were treated with ethyl-methanesulfonate (EMS) at different concentrations and exposure times. Killing curves were predicted in the M1 generation for each treatment along with mortality, fertility and chimerism rates. M1 plants were self-pollinated to generate M2 lines of seeds, where recessive alleles were restored as homozygotes and lethal alleles were restored as heterozygotes. Genomic DNA from eight M2 plants per family of EMS-mutagenized populations was extracted and screened for mutants, and M2 plant material and M3 seeds were collected and stored for future analysis. To identify and characterize mutants, genomic DNA samples from M2 plants were pooled in groups and screened by sequencing of targeted gene fragments. Target gene fragments were amplified using primers specific for the tobacco NIA2 gene shown in Table 2. Mutations in the target genes were elicited by sequencing individual DNA fragments. The tobacco NIA2 mutation M527I is listed in Table 1. Mutant plants were crossed with AA37 wild type and the heterozygous F1 was self-pollinated. F2 segregants were analyzed by quantitative nucleic acid amplification (TaqMan).

[0182] In the field trial, three NIA2 M527I homozygous mutant lines were tested against five out-segregant homozygous wild-type lines and one AA37 control line that had not received EMS treatment. In the field design, plants were displayed in 20 randomly distributed plant plots to minimize position effects due to field heterogeneity. Plants were grown according to standard Swiss burley tobacco good agricultural practice. At harvest, the total wet leaf biomass was measured. Measurement of plant leaf biomass was performed by cutting the leaves at harvest and recording their wet weight.

[0183] The leaves of the second harvest (mid-stem position) were then air-dried and the samples analyzed for chemical profiling via the standard Skalar method.

[0184] The results are reported in Figure 2. The mutant plant line NtNIA2 M527I demonstrates a 37% reduction in nitrate levels in desiccated leaves compared to out-segregant wild-type control plants in field trials. No effects were observed on the total harvested plant biomass (expressed as wet leaf biomass per plant) and on the leaf contents of nicotine, total alkaloids, ammonia, and reducing sugars.

[0185] Any publications cited or described herein provide relevant information disclosed prior to the filing date of this application. Statements herein should not be construed as admissions that the inventors are not entitled to prior to such disclosure. All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the present invention will become 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 connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention that are apparent to those skilled in the art of cell biology, molecular biology, and plant biology, or related fields, are intended to be within the scope of the claims.

[0186] Sequence Listing SEQ ID NO:1 Wild-type polypeptide sequence of the Nicotiana tabacum NIA2 gene for nitrate reductase, GenBank accession number X14059. The sequence that acts as a recognition site for the binding of nitrate reductase kinase responsible for phosphorylation of NtNIA2 amino acid S523 is highlighted in bold and underlined.

[0187] TIFF2024180620000002.tif77150

[0188] SEQ ID NO:2 Wild-type polynucleotide sequence of the Nicotiana tabacum NIA2 gene for nitrate reductase, GenBank Accession No. X14059. The sequence that acts as a recognition site for the binding of nitrate reductase kinase responsible for phosphorylation of NtNIA2 amino acid S523 is highlighted in bold and underlined. TIFF2024180620000003.tif180170 TIFF2024180620000004.tif175170 TIFF2024180620000005.tif175170 TIFF2024180620000006.tif101170

[0189] SEQ ID NO: 3. Mutant polypeptide sequence of the Nicotiana tabacum NIA2 gene for nitrate reductase. The mutant polypeptide in the recognition site for the binding of nitrate reductase kinase is highlighted in bold and underlined. TIFF2024180620000007.tif77150

[0190] SEQ ID NO: 4. Mutant polynucleotide sequence of the Nicotiana tabacum NIA2 gene for nitrate reductase. The sequence that acts as a recognition site for the binding of nitrate reductase kinase involved in phosphorylation of NtNIA2 amino acid S523 is highlighted in bold. The g to a mutation is underlined. TIFF2024180620000008.tif180170 TIFF2024180620000009.tif175170 TIFF2024180620000010.tif175170 TIFF2024180620000011.tif101170

[0191] SEQ ID NO:5: A polypeptide sequence that acts as a recognition site for the binding of nitrate reductase kinase involved in phosphorylation of NtNIA2 amino acid S523 from the wild-type polypeptide sequence of the Nicotiana tabacum NIA2 gene for nitrate reductase, GenBank Accession No. X14059. LK(K or R)(S or T)(I or V or A)S(T or S)PFM

[0192] SEQ ID NO:6 Mutant polypeptide sequence of SEQ ID NO:5. LK(K or R)(S or T)(I or V or A)S(T or S)PFI

[0193] SEQ ID NO:7 Wild-type polypeptide sequence that acts as a recognition site for the binding of nitrate reductase kinase involved in phosphorylation of NtNIA2 amino acid S523 from the wild-type polypeptide sequence of the Nicotiana tabacum NIA2 gene for nitrate reductase, GenBank Accession No. X14059. LKKSISTPFM

[0194] SEQ ID NO:8 (mutated polypeptide sequence of SEQ ID NO:7). LKKSISTPFI

[0195] Table 1 Details of the NtNIA2 M527I mutation [Table 1] Sequence information for mutation NtNIA2 M527I is shown. The second and third columns show the 10 nucleotides immediately preceding the mutated nucleotide and the 10 nucleotides immediately following the mutated nucleotide. The fourth and fifth columns report the original codon and original amino acid (respectively) found in the wild-type sequence. The last two columns report the codon and amino acid (respectively) in the mutated sequence. The G to A mutation is shown in bold and underlined.

[0196] Table 2 Details of NtNIA2-specific primers [Table 2] Reported are detailed sequences of NtNIA2-specific primers. The second column shows the DNA sequence of the primers.

[0197] Yet another aspect of the present invention may be as follows. [1] A plant cell, (a) a polynucleotide sequence encoding a nitrate reductase polypeptide comprising the contiguous polypeptide sequence of SEQ ID NO:5 or SEQ ID NO:7, wherein a methionine has been replaced with an amino acid that reduces nitrate reductase activity in said plant cell compared to a control plant cell; (b) a polypeptide sequence encoded by the polynucleotide sequence described in (a); or (c) a construct, vector, or expression vector comprising the polynucleotide sequence described in (b). A plant cell comprising: [2] The plant cell of [1], wherein methionine is replaced with a different amino acid, preferably the replaced amino acid is isoleucine, and preferably the polypeptide comprises a continuous polypeptide sequence of SEQ ID NO: 6 or SEQ ID NO: 8. [3] The plant cell described in [1] or [2], wherein the nitrate reductase polypeptide contains an amino acid substitution at a position corresponding to position 527 of a sequence having at least 80% sequence identity to SEQ ID NO:1. [4] The plant cell according to any one of [1] to [3], wherein the nitrate reductase polypeptide comprises the polypeptide sequence set forth in SEQ ID NO: 3, consists of the polypeptide sequence set forth in SEQ ID NO: 3, or essentially consists of the polypeptide sequence set forth in SEQ ID NO: 3. [5] A plant cell described in any of [1] to [4] above, wherein the polynucleotide sequence comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 4, consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 4, or essentially consists of a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 4. [6] The plant cell according to any one of [1] to [5] above, wherein the polynucleotide sequence comprises the polynucleotide sequence set forth in SEQ ID NO: 4, consists of the polynucleotide sequence set forth in SEQ ID NO: 4, or essentially consists of the polynucleotide sequence set forth in SEQ ID NO: 4. [7] The plant cell according to any one of [1] to [6] above, wherein the plant cell is a Nicotiana tabacum plant cell. [8] The plant cell according to [7], wherein the plant cell is a plant cell of the Nicotiana tabacum AA37 cultivar. [9] A plant or part thereof comprising a plant cell according to any one of [1] to [8] above, wherein desiccated leaves of the plant or part thereof contain a lower level of nitrate compared to a control plant or part thereof, and preferably the level of nitrate is reduced compared to the control plant or part thereof.

[10] A plant material, a dried plant material, or a homogenized plant material derived from the plant or part thereof of [9], preferably the dried plant material is air-dried, sun-dried, or hot-air tube-dried, and preferably the plant material, the dried plant material, or the homogenized plant material comprises biomass, seeds, stems, flowers, or leaves of the plant or part thereof of [9].

[11] A tobacco product comprising the plant cell according to any one of [1] to [8], the part of the plant according to [9], or the plant material according to

[10] .

[12] A method for generating the plant according to [9], comprising: (a) providing a plant cell according to any one of [1] to [8] above; and (b) propagating the plant cell in a plant. A method comprising:

[13] A method for producing a dried processed plant material having an altered amount of nitrate compared to a control plant material, comprising the steps of: (a) providing a plant or a part thereof according to [9] above, or a plant material according to

[10] above; (b) optionally harvesting said plant material from said plant or part thereof; and (c) drying the plant material A method comprising:

[14] The method according to

[13] , wherein the plant material comprises dried leaves.

[15] The method according to

[14] , wherein the drying method is selected from the group consisting of air drying, fire drying, smoke drying, and hot air tube drying.

Claims

[Claim 1] A plant cell comprising: (a) a polynucleotide sequence encoding a nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO:5 or SEQ ID NO:7, wherein a methionine has been replaced with an amino acid that reduces nitrate reductase activity in said plant cell compared to a control plant cell; (b) a polypeptide sequence encoded by the polynucleotide sequence described in (a); or (c) a construct, vector, or expression vector comprising the polynucleotide sequence described in (b). A plant cell comprising:

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