Modulation of nitrate levels in tobacco via mutation of nitrate reductase

Abstract

There is disclosed herein a Nicotiana tabacum plant cell comprising: (a) a polynucleotide sequence encoding a NIA1 nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO: 4, wherein the serine at position 4 of SEQ ID NO: 4 is substituted for an amino acid that reduces nitrate levels 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). Double mutants are also disclosed containing, for example, a S521N mutation in a Nicotiana tabacum NIA1 nitrate reductase polypeptide and a M527I mutation in a Nicotiana tabacum NIA2 nitrate reductase polypeptide.

Description

[0001]MODULATION OF NITRATE LEVELS IN TOBACCO VIA MUTATION OF NITRATE REDUCTASE FIELD OF THE INVENTION The present invention relates to Nicotiana tabacum plants having reduced nitrate levels in cured leaves by mutating nitrate reductase (NIA), Nicotiana tabacum plant cells derived from the Nicotiana tabacum plants, products generated from the Nicotiana tabacum plants, and methods of modulating nitrate levels in Nicotiana tabacum plants by mutating nitrate reductase. BACKGROUND Tobacco plants accumulate high levels of free nitrate in their leaves, which is undesirable because high levels of nitrate have been associated with the formation of carcinogenic compounds referred to as tobacco-specific nitrosamines (TSNAs). TSNAs are a class of compounds that are predominantly produced during the curing of tobacco leaves, though additional formation can occur in the subsequent processing and storage of leaf, and possibly via pyrosynthesis during combustion. Two of the TSNAs found in the cured leaf, N- nitrosonornicotine (NNN) and 4-(methylnitrosamino)-l-(3-pyridyl)-l -butanone (NNK), are classified as Group I carcinogens (the highest designation) by the International Agency for Research on Cancer. Due to the volume of evidence implicating these compounds with various tobacco-associated cancers, the World Health organization has recommended that mandates be implemented to ensure that future tobacco products have reduced levels of these toxicants. TSNAs represent nitrosation products of tobacco alkaloids. In air-cured tobaccos there is a general consensus that nitrite is the agent directly responsible for TSNA formation. Due to its cellular toxicity, however, endogenous nitrite levels are typically very low in plant tissues. Instead, it is believed that the great majority of the nitrite involved in TSNA formation is derived from the nitrate reductase activity of microbes residing on the leaf surface during the 6 - 10 week curing process that converts a portion of the leaf nitrate pools to nitrite as cellular membranes and organelles become degraded during this period. TSNAs are formed primarily during the curing process of leaves and involve the nitrosation of tobacco alkaloids. Genetic strategies to lower TSNA content and levels in the cured leaf have focused on targeting either: (1) the alkaloid precursor(s); or (2) the nitrosating agent(s) involved. Most efforts to reduce TSNAs at the level of altering the genetics of tobacco have targeted the alkaloid precursors to TSNAs. Such strategies provide substantial reductions in NNN through the downregulation of the gene family responsible for the synthesis of its alkaloid precursor nomicotine. Modified tobacco plants having reduced nitrate levels are described by the present applicant in WO2016 / 046288. As described therein, the expression or activity of a Nicotiana tabacum nitrate reductase enzyme (NtNIA2) is deregulated. The deregulated NtNIA2 has an amino acid substitution at a position corresponding to position 523 of the polypeptide encoding NtNIA2. The mutation is referred to as S523D, meaning that serine (S) at amino acid position 523 is substituted for aspartic acid (D). Overexpression of the mutant NtNIA2 gave a dramatic increase in nitrate assimilation, resulting in low nitrate / TSNA content and higher amino acid levels. Plants displayed 90% less nitrate compared to the control. Modified tobacco plants having reduced nitrate levels are further described by the present applicant in WO2020 / 141062. As described therein, the expression or activity of a Nicotiana tabacum nitrate reductase enzyme (NtNIA2) is deregulated. The deregulated nitrate reductase enzyme has an amino acid substitution in the nitrate reductase kinase recognition site within the hinge 1 domain of NtNIA2. The amino acid substitution is at a position corresponding to position 527 of NtNIA2. The mutation is referred to as M527I, meaning that methionine (M) at amino acid position 527 of NtNIA2 is substituted for isoleucine (I). Mutant plant lines NtNIA2 M527I demonstrated a reduction in nitrate levels in cured leaves compared to out-segregant wild type control plants. WO2022 / 124361 reports that Nicotiana tabacum nitrate reductase 1 (NtNIA1) in which the amino acid corresponding to position 525 in the hinge region 1 of NtNIA1 is mutated from proline to leucine (P525L - proline (P) at amino acid position 525 of NtNIA1 is substituted for leucine (L)) or from proline to serine (P525S - proline (P) at amino acid position 525 of NtNIA1 is substituted for serine (S)) have reduced levels of nitrate as compared to a control. It can be desirable to develop non-genetically modified organism (non-GMO) approaches to reduce nitrate accumulation. Due to the difficulties of growing and commercialising genetically modified crops in countries, including Europe, it can be advantageous to work with mutants featuring single nucleotide polymorphisms rather than mutants obtained through the use of genetic engineering techniques. There remains a continuing need in the art for reducing nitrate levels in plants, especially via non-transgenic approaches. SUMMARY OF THE INVENTION The present invention is based, at least in part, on the finding that mutating Nicotiana tabacum nitrate reductase 1 (NtNIA1) via a non-transgenic approach can result in plants or plant material in which the cured leaves thereof contain modulated (for example, reduced) levels of nitrate as compared to cured leaves derived from a control plant. One such mutation that is described herein (S521N – meaning that serine at amino acid position 521 of NtNIA1 is substituted for asparagine) is located in the nitrate reductase kinase recognition site within the hinge 1 domain of NtNIA1. Advantageously, this S521N mutant gave a statistically significant 24.5 % reduction in a first trial and a statistically significant 30 % reduction in a second year trial. It is also observed that in a double mutant tobacco plant containing the S521N mutation in NtNIA1 and the previously described M527I mutation in NtNIA2 (see WO2020 / 141062), a statistically significant 35 % reduction in nitrate content cured leaves is obtained in a first trial and a statistically significant 52.8 % reduction in a second year trial. Accordingly, a double mutant containing a S521N mutation in a Nicotiana tabacum NIA1 nitrate reductase polypeptide and a M527I mutation in a Nicotiana tabacum NIA2 nitrate reductase polypeptide is also disclosed in one embodiment. It is surprisingly observed that the double mutations M527I and S521N act in a synergistic manner to further reduce nitrate content in cured leaves. Producing plants according to the present disclosure provides a number of other advantages in addition to reduced nitrate levels in cured leaves. For example, the plants described herein can, in certain embodiments, be non-genetically modified plants which overcomes the difficulties of growing and commercialising genetically modified crops. In a first aspect there is disclosed a Nicotiana tabacum plant cell comprising: (a) a polynucleotide sequence encoding a NIA1 nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO: 4, wherein the serine at position 4 of SEQ ID NO: 4 is substituted for an amino acid that reduces nitrate levels 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). Suitably, the serine at position 4 of SEQ ID NO: 4 is substituted for a polar uncharged aliphatic amino acid selected from the group consisting of cysteine, threonine, methionine, asparagine and glycine. Suitably, the serine at position 4 of SEQ ID NO: 4 is substituted for asparagine. Suitably, the polypeptide comprises the contiguous polypeptide sequence of SEQ ID NO: 8. Suitably, the polypeptide comprises, consists or consists essentially of the polypeptide sequence set forth in SEQ ID NO: 7. Suitably, the polynucleotide sequence comprises, consists or consists essentially of the polynucleotide sequence set forth in SEQ ID NO: 6. Suitably, the plant cell has reduced nitrate levels as compared to a control plant cell. Suitably, the plant cell further comprises at least one mutation in a NIA2 nitrate reductase, Suitably, the mutation is in the recognition site for binding of the nitrate reductase kinase. Suitably, the mutated NIA2 nitrate reductase polypeptide comprises a contiguous polypeptide sequence of SEQ ID NO: 13, wherein the methionine at position 10 of SEQ ID NO: 13 is substituted for an amino acid that reduces nitrate levels in the plant cell as compared to a control plant cell. Suitably, the methionine is substituted for a non-polar aliphatic amino acid selected from the group consisting of glycine, alanine, proline, isoleucine, leucine or valine. Suitably, the substituted amino acid is isoleucine. Suitably, the polypeptide comprises the contiguous polypeptide sequence of SEQ ID NO: 14. Suitably, the nitrate reductase NIA2 polypeptide comprises, consists or consists essentially of the polypeptide sequence set forth in SEQ ID NO: 11. Suitably, the mutated nitrate reductase NIA2 polynucleotide comprises, consists or consists essentially of SEQ ID NO: 12 or SEQ ID NO: 17. In another aspect, there is disclosed a Nicotiana tabacum plant or part thereof comprising the plant cell described herein. Suitably, cured leaves of the plant or part thereof contain lower levels of nitrate as compared to a control plant or part thereof. Suitably, the cured leaves are air-cured or sun-cured or flue-cured. In another aspect, there is disclosed Nicotiana tabacum plant material, cured plant material, or homogenized plant material, derived from the Nicotiana tabacum plant or part thereof that as described herein. Suitably, the plant material comprises biomass, seed, stem, flowers, or leaves from the plant or part thereof as described herein. In another aspect, there is disclosed a tobacco product comprising the Nicotiana tabacum plant cell described herein, a part of the Nicotiana tabacum plant described herein or the Nicotiana tabacum plant material described herein. In another aspect, there is disclosed a method for producing the Nicotiana tabacum plant described herein, comprising: (a) providing the Nicotiana tabacum plant cell described herein; and (b) propagating the Nicotiana tabacum plant cell into a Nicotiana tabacum plant. In another aspect, there is disclosed a method for producing cured Nicotiana tabacum plant material with an altered amount of nitrate as compared to control plant material, comprising the steps of: providing the Nicotiana tabacum plant or part thereof described herein or the Nicotiana tabacum plant material described herein; (b) harvesting the Nicotiana tabacum plant or Nicotiana tabacum plant material; and (c)curing the harvested Nicotiana tabacum plant or the harvested Nicotiana tabacum plant material. Suitably, the Nicotiana tabacum plant material comprises cured leaves. Suitably, the curing method is selected from the group consisting of air curing, fire curing, smoke curing, and flue curing. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the domains and consensus sequences of the NtNIA1 or NtNIA2 nitrate reductase protein sequence. (A) Secondary structure of the major domains of NtNIA1 or NtNIA2. The hinge 1 domain is shown in grey. (B) Primary structure of the NtNIA1 or NtNIA2 hinge 1 domain containing the nitrate reductase kinase recognition site (LKKSISTPFM) and 14-3-3 recognition site within the hinge 1 of NtNIA1 or NtNIA2. The positions of the M527I mutation of WO2020 / 141062 and the S521N mutation of the present invention within the nitrate reductase kinase recognition site are shown. Figure 2 is a graph showing nitrate levels in dry powder of total air cured leaves. The results represent an average of at least 7 lines per experimental point. Nitrate is expressed as % of dry weight. Results are shown for wild-type Nicotiana tabacum (wild type), the M527I mutant of NtNIA2 as described in WO2020 / 141062 (NIA2 M527I), the S521N mutant of NtNIA1 as described herein (NIA1 S521N), and the double mutant of NtNIA2 M527I and NtNIA1 S521N as described herein (double mutant). DEFINITIONS Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The term “and / or” means (a) or (b) or both (a) and (b). The present disclosure contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of” the embodiments or elements presented herein, whether explicitly set forth or not. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for 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 explicitly contemplated. As used throughout the specification and the claims, the following terms have the following meanings: “Coding sequence” or “polynucleotide encoding” means the nucleotides (RNA or DNA molecule) that comprise a polynucleotide which encodes a polypeptide. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered. The coding sequence may be codon optimized. “Complement” or “complementary” can mean Watson-Crick (for example, A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs. “Complementarity” refers to a property shared between two polynucleotides, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary. "Construct" refers to a double-stranded, recombinant polynucleotide fragment comprising one or more polynucleotides. The construct comprises a "template strand" base-paired with a complementary "sense or coding strand." A given construct can be inserted into a vector in two possible orientations, either in the same (or sense) orientation or in the reverse (or anti- sense) orientation with respect to the orientation of a promoter positioned within a vector - such as an expression vector. The term "control" in the context of a control plant or a control plant cell means a plant or plant cell in which the expression, function or activity of one or more genes or polypeptides has not been modified (for example, increased or decreased) and so it can provide a comparison with a plant in which the expression, function or activity of the same one or more genes or polypeptides has been modified. As used herein, a “control plant” is a plant that is substantially equivalent to a test plant or modified plant in all parameters with the exception of the test parameters. For example, when referring to a plant into which a mutation has been introduced, a control plant is an equivalent plant into which the mutation has not been introduced. The control plant can be an outsegregant control plant. "Expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment may refer to transcription of the polynucleotide fragment (for example, transcription resulting in mRNA or functional RNA) and / or translation of mRNA into a precursor or mature polypeptide. “Functional” and “full-functional” describes a polypeptide that has biological function or activity. A “functional gene” refers to a gene transcribed to mRNA, which is translated to a functional or active polypeptide. “Genetic construct" refers to DNA or RNA molecules that comprise a polynucleotide that encodes a polypeptide. The coding sequence can include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression. The terms "homology” or “similarity" refer to the degree of sequence similarity between two polypeptides or between two polynucleotide molecules compared by sequence alignment. The degree of homology between two discrete polynucleotides being compared is a function of the number of identical, or matching, nucleotides at comparable positions. "Identical" or "identity" in the context of two or more polynucleotides or polypeptides means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be determined manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA or Smith-Waterman. The popular multiple alignment program ClustalW (Nucleic Acids Research (1994) 22, 4673- 4680; Nucleic Acids Research (1997), 24, 4876-4882) is a suitable way for generating multiple alignments of polypeptides or polynucleotides. Suitable parameters for ClustalW maybe as follows: For polynucleotide alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For polypeptide alignments: Gap Open Penalty = 10. o, 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 it may be necessary to vary these and other parameters for optimal sequence alignment. Suitably, calculation of percentage identities is then calculated from such an alignment as (N / T), where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. The terms "isolated" or "purified" refer to material that is substantially or essentially free from components that normally accompany it as 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. The term "purified" as used herein denotes that a polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the polynucleotide or polypeptide is at least 85% pure, more suitably at least 95% pure, and most suitably at least 99% pure. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional polynucleotide purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides. "Modulate" or “modulating” refers to causing or facilitating a qualitative or quantitative change, alteration, or modification in a process, pathway, function or activity of interest. Without limitation, such a change, alteration, or modification may be an increase or decrease in the relative process, pathway, function or activity of interest. 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 comparison to a control. The term 'non-naturally occurring' describes an entity – such as a polynucleotide, a genetic mutation, a polypeptide, a plant, a plant cell and plant material - that is not formed by nature or that does not exist in nature. Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by methods described herein or that are known in the art. Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by man. By way of example, a non-naturally occurring entity can be an entity that has been mutated by methods known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide- directed mutagenesis, chemically-induced mutagenesis, irradiation-induced mutagenesis, mutagenesis utilizing modified bases, mutagenesis utilizing gapped duplex DNA, double- strand break mutagenesis, mutagenesis utilizing 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. “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, a polynucleotide also encompasses the complementary strand of a depicted single strand. Many variants of a polynucleotide may be used for the same purpose as a given polynucleotide. Thus, a polynucleotide also encompasses substantially identical polynucleotides and complements thereof. A single strand provides a probe that may hybridize to a given sequence under stringent hybridization conditions. Thus, a polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions. Polynucleotides may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods. The specificity of single-stranded DNA to hybridize 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 propensity to form DNA duplexes decreases. In polynucleotide hybridization reactions, the stringency can be chosen to favor specific hybridizations (high stringency), which can be used to identify, for example, full-length clones from a library. Less-specific hybridizations (low stringency) can be used to identify related, but not exact (homologous, but not identical), DNA molecules or segments. DNA duplexes are stabilised by: (1) the number of complementary base pairs; (2) the type of base pairs; (3) salt concentration (ionic strength) of the reaction mixture; (4) the temperature of the reaction; and (5) the presence of certain organic solvents, such as formamide, which decrease DNA duplex stability. In general, the longer the probe, the higher the temperature required for proper annealing. A common approach is to vary the temperature; higher relative temperatures result in more stringent reaction conditions. To hybridize under "stringent conditions" describes hybridization protocols in which polynucleotides at least 60% homologous to each other remain hybridized. Generally, stringent conditions are selected to be about 5ºC lower than the thermal melting point (Tm) for the specific 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 the given sequence hybridize to the given sequence at equilibrium. Since the given sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. "Stringent hybridization conditions" are conditions that enable a probe, primer, or oligonucleotide to hybridize only to its specific sequence. Stringent conditions are sequence-dependent and will differ. Stringent conditions typically comprise: (1) low ionic strength and high temperature washes, for example 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate, at 50ºC; (2) a denaturing agent during hybridization, for example, 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 42ºC; or (3) 50% formamide. Washes typically also comprise 5 x SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xDenhardt's solution, sonicated salmon sperm DNA (50 µg / mL), 0.1% SDS, and 10% dextran sulfate at 42ºC, with a wash at 42ºC in 0.2xSSC (sodium chloride / sodium citrate) and 50% formamide at 55ºC, followed by a high-stringency wash consisting of 0.1xSSC containing EDTA at 55ºC. Suitably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. "Moderately stringent conditions" use washing solutions and hybridization conditions that are less stringent, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide. One example comprises hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 µg / mL denatured salmon sperm DNA at 55ºC, followed by one or more washes in 1xSSC, 0.1% SDS at 37ºC. The temperature, ionic strength, etc., can be adjusted to accommodate 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, N.J. (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, N.Y. (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, N.Y. (1988)). "Low stringent conditions" use washing solutions and hybridization conditions that are less stringent than those for moderate stringency, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide. A non-limiting example of low stringency hybridization conditions includes hybridization in 35% formamide, 5xSSC, 50 mM Tris HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 µg / mL denatured salmon sperm DNA, 10% (wt / vol) dextran sulfate at 40ºC, followed by one or more washes in 2xSSC, 25 mM Tris HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50ºC. The term "plant" refers to any plant at any stage of its life cycle or development, and its progenies. 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 progeny of same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Suitable species, cultivars, hybrids and varieties of tobacco plant are described herein. "Polynucleotide", "polynucleotide sequence" or "polynucleotide fragment" are used interchangeably herein and refer to a polymer of RNA or DNA that is single- or double- stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (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 can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) thereof. Moreover, a polynucleotide can 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 with a mixture of single-stranded and double-stranded regions or a fragment(s) thereof. In addition, the polynucleotide can be composed of triple-stranded regions comprising DNA, RNA, or both or a fragment(s) thereof. A polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid (PNA). Generally, polynucleotides can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or a combination of the foregoing. Although the polynucleotides described herein are shown as DNA sequences, the polynucleotides include their corresponding RNA sequences, and their complementary (for example, completely complementary) DNA or RNA sequences, including the reverse complements thereof. The polynucleotides of the present disclosure are set forth in the accompanying sequence listing. "Polypeptide” or "polypeptide sequence" refer to a polymer of amino acids in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring polymers of amino acids. The terms are also inclusive of 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. “Recombinant" as used herein refers to an artificial combination of two otherwise separated segments of sequence – such as by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering 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 the cell or vector by naturally occurring events (for example, spontaneous mutation, natural transformation or transduction or transposition) such as those occurring without deliberate human intervention. The term “tobacco” is used in a collective sense to refer to tobacco crops (for example, a plurality of tobacco plants grown in the field and not hydroponically grown tobacco), tobacco plants and parts thereof, including but not limited to, roots, stems, leaves, flowers, and seeds prepared and / or obtained, as described herein. In one embodiment, the term excludes propagating material. It is understood that “tobacco” refers to Nicotiana tabacum plants and products thereof. The term “tobacco products” refers to consumer tobacco products, including but not limited to, smoking materials (for example, cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials and ingredients for manufacture of consumer tobacco products. Suitably, these tobacco products are manufactured from tobacco leaves and stems harvested from tobacco and cut, dried, cured, and / or fermented according to conventional techniques in tobacco preparation. “Variant” with respect to a peptide or polypeptide means a peptide or polypeptide that differs in sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological function or activity. Variant may also mean a polypeptide that retains at least one biological function or activity. A conservative substitution of an amino acid, that is, replacing an amino acid with a different amino acid of similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. The term "variety" refers to a population of plants that share constant characteristics which separate them from other plants of the same species. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individuals within that variety. A variety is often sold commercially. "Vector" refers to a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the transport of polynucleotides, polynucleotide constructs and polynucleotide conjugates and the like. A vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. Suitable vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other vectors of any origin. An "expression vector" as used herein is a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the expression of polynucleotide(s), polynucleotide constructs and polynucleotide conjugates and the like. Suitable expression vectors include episomes capable of extra-chromosomal replication such as circular, double- stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other functionally equivalent expression vectors of any origin. An expression vector comprises at least a promoter positioned upstream and operably-linked to a polynucleotide, polynucleotide constructs or polynucleotide conjugate, as defined below. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and polypeptide and polynucleotide chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. “Genome editing" refers to changing an endogenous gene that encodes an endogenous polypeptide, such that polypeptide expression of a truncated endogenous polypeptide or an endogenous polypeptide having an amino acid modification - such as a substitution - is obtained. Genome editing can include replacing the region of the endogenous gene to be targeted or replacing the entire endogenous gene with a copy of the gene that has a truncation or an amino acid substitution with a repair mechanism – such as homology- directed repair. Genome editing may also include generating an amino acid substitution in the endogenous gene by generating a double stranded break in the endogenous gene that is then repaired using a non-homologous end joining (NHEJ) pathway. NHEJ may add or delete at least one base pair during repair which may generate an amino acid substitution. Genome editing may also include deleting a gene segment by the simultaneous action of two nucleases on the same DNA strand in order to create a truncation between the two nuclease target sites and repairing the DNA break by NHEJ. DETAILED DESCRIPTION The polynucleotide(s) described herein encode an active nitrate reductase polypeptide that has at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the function or activity of the polypeptide(s) shown in the sequence listing. In certain embodiments, the polynucleotide sequence comprises, consists or consists essentially of a sequence having at least 80% sequence identity to any of the sequences described herein, including any of the polynucleotides shown in the sequence listing. Suitably, the isolated polynucleotide comprises, consists 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. There is disclosed a polynucleotide sequence encoding a NtNIA1 nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO: 3, wherein the serine at position 521 of SEQ ID NO: 3 is substituted for an amino acid that reduces nitrate levels in the plant cell as compared to a control plant cell. Suitably, the serine at position 521 of SEQ ID NO: 3 is substituted for a polar uncharged aliphatic amino acid selected from the group consisting of cysteine, threonine, methionine, asparagine and glycine. Suitably, the serine at position 521 of SEQ ID NO: 3 is substituted for asparagine. Suitably, the polynucleotide sequence comprises, consists or consists essentially of the polynucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6. There is also disclosed polynucleotide fragments of SEQ ID NO: 6 encoding a polypeptide including the S521N mutation with substantial homology (that is, sequence similarity) or substantial identity thereto that have 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 fragments of SEQ ID NO: 6 Suitably, the polynucleotide sequence encodes the polypeptide set forth in SEQ ID NO: 8. There is also disclosed a polynucleotide sequence encoding a NtNIA2 nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO: 11, wherein the methionine at position 527 of SEQ ID NO: 11 is substituted for an amino acid that reduces nitrate levels in the plant cell as compared to a control plant cell. The polynucleotide sequence encoding this mutant is set forth in SEQ ID NO: 12 or SEQ ID NO: 17. Suitably, the methionine at position 527 is substituted for a non-polar aliphatic amino acid selected from the group consisting of glycine, alanine, proline, isoleucine, leucine or valine. Suitably, the methionine at position 527 of SEQ ID NO: 11 is substituted for isoleucine. Suitably, the polynucleotide sequence comprises, consists or consists essentially of the polynucleotide sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 17. There is also disclosed polynucleotide fragments of SEQ ID NO: 11 encoding a polypeptide including the M527I mutation with substantial homology (that is, sequence similarity) or substantial identity thereto that have 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 fragments of SEQ ID NO: 11. Suitably, the polynucleotide sequence encodes the polypeptide set forth in SEQ ID NO: 14. According to the present disclosure, a polynucleotide sequence encoding the S521N mutation in NtNIA1 and a polynucleotide sequence encoding the M527I mutation in NtNIA2 is incorporated into a tobacco plant or cell thereof. The polynucleotide sequence encoding the S521N mutation in NtNIA1 is set forth in SEQ ID NO: 6. The polynucleotide sequence encoding the M527I mutation in NtNIA2 is set forth in SEQ ID NO: 12 or SEQ ID NO: 17.Fragments of the polynucleotides incorporating the mutation(s) described herein are also disclosed. Polynucleotide fragments typically comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 100 or at least 200 contiguous nucleotides. A polynucleotide as described herein can include a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingly, a polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) or truncate(s) thereof. Moreover, a polynucleotide can be single-stranded or double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, a hybrid molecule comprising DNA and RNA, or a hybrid molecule with a mixture of single-stranded and double-stranded regions or a fragment(s) thereof. In addition, the polynucleotide can be composed of triple-stranded regions comprising DNA, RNA, or both or a fragment(s) thereof. Generally, polynucleotides can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or a combination of the foregoing. Although the polynucleotides described herein are shown as DNA sequences, they include their corresponding RNA sequences, and their complementary (for example, completely complementary) DNA or RNA sequences, including the reverse complements thereof. A polynucleotide as described herein will generally contain phosphodiester bonds. Other analogue polynucleotides include those with positive backbones; non-ionic backbones, and non-ribose backbones. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring polynucleotides and analogues can be made; alternatively, mixtures of different polynucleotide analogues, and mixtures of naturally occurring polynucleotides and analogues may be made. Analogue polynucleotides can include those with positive backbones, non-ionic backbones and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also included. Other analogues include peptide polynucleotides which are peptide polynucleotide analogues. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring polynucleotides. This may result in advantages. First, the peptide polynucleotide backbone may exhibit improved hybridization kinetics. Peptide polynucleotides have larger changes in the melting temperature for mismatched versus perfectly matched base pairs. DNA and RNA typically exhibit a 2-4 °C drop in melting temperature for an internal mismatch. With the non-ionic peptide polynucleotide backbone, the drop is closer to 7-9 °C. Similarly, due to their non- ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. In addition, peptide polynucleotides may not be degraded or degraded to a lesser extent by cellular enzymes, and thus may be more stable. Among the uses of the disclosed polynucleotides, and fragments thereof, is the use of fragments as probes in hybridisation assays or primers for use in amplification assays. Exemplary primers are set forth in SEQ ID Nos: 18 and 19. Such fragments generally comprise at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least about 10, 15, 20, 30, 40, 50 or 60 or more contiguous nucleotides of a DNA sequence. Thus, in one aspect, there is also provided a method for detecting a polynucleotide comprising the use of the probes or primers or both. Exemplary primers are described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are described by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Using knowledge of the genetic code in combination with the polypeptide sequences described herein, sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, for example, in polymerase chain reactions (PCR), whereby 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 express sequence tag or DNA libraries. Homologous sequences identified by this method would then be used as probes to identify homologues of the sequences identified herein. Also of potential use are polynucleotides and oligonucleotides (for example, primers or probes) that hybridize under decreased stringency conditions, typically moderately stringent conditions, and commonly highly stringent conditions to the polynucleotide(s), as described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and can be readily determined by those having ordinary skill in the art based on, for example, the length or base composition of the polynucleotide. One way of achieving moderately and high stringent conditions is defined herein. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, for example, Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y). When hybridizing a polynucleotide to a polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10 °C less than the melting temperature of the hybrid, where melting temperature is determined according to the following equations. For hybrids less than 18 base pairs in length, melting temperature (°C)=2(number of A+T bases)+4(number of G+C bases). For hybrids above 18 base pairs in length, 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 concentration of sodium ions in the hybridization buffer ([Na+] for 1x Standard Sodium Citrate=0.165M). Typically, each such hybridizing polynucleotide has a length that is at least 25% (commonly at least 50%, 60%, or 70%, and most commonly at least 80%) of the length of a polynucleotide to which it hybridizes, and has at least 60% sequence identity (for example, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) with a polynucleotide to which it hybridizes. As will be understood by the person skilled in the art, a linear DNA has two possible orientations: the 5'-to-3' direction and the 3'-to-5' direction. For example, if a first sequence is positioned in the 5'-to-3' direction, and if a second sequence is positioned in the 5'-to-3' direction within the same polynucleotide molecule / strand, then the first sequence and the second sequence are orientated in the same direction, or have the same orientation. Typically, a promoter sequence and a gene of interest under the regulation of the given promoter are positioned in the same orientation. However, with respect to the first sequence positioned in the 5'-to-3' direction, if a second sequence is positioned in the 3'-to-5' direction within the same polynucleotide molecule / strand, then the first sequence and the second sequence are orientated in anti-sense direction, or have anti-sense orientation. Two sequences having anti-sense orientations with respect to each other can be alternatively described as having the same orientation, if the first sequence (5'-to-3' direction) and the reverse complementary sequence of the first sequence (first sequence positioned in the 5'- to-3') are positioned within the same polynucleotide molecule / strand. The sequences set forth herein are shown in the 5'-to-3' direction. Vectors containing recombinant polynucleotide constructs such as those described herein are also provided. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available. In one aspect, there is provided an isolated polypeptide comprising, consisting or consisting essentially of a polypeptide having at least 80% sequence identity to any of the polypeptides described herein, including any of the polypeptides shown in the sequence listing. Suitably, the isolated polypeptide comprises, consists 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. In another aspect, there is disclosed a polypeptide sequence encoded by the polynucleotide sequence described herein. In another aspect, there is disclosed a NtNIA1 nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO: 3, wherein the serine at position 521 of SEQ ID NO: 3 is substituted for an amino acid that reduces nitrate levels in the plant cell as compared to a control plant cell. Suitably, the serine at position 521 of SEQ ID NO: 3 is substituted for a polar uncharged aliphatic amino acid selected from the group consisting of cysteine, threonine, methionine, asparagine and glycine. Suitably, the serine at position 521 of SEQ ID NO: 3 is substituted for asparagine. Suitably, the polypeptide comprises the contiguous polypeptide sequence of SEQ ID NO: 7. Suitably, the polypeptide comprises, consists or consists essentially of the polypeptide sequence set forth in SEQ ID NO: 7. Suitably, the polypeptide comprises, consists or consists essentially of the polypeptide sequence set forth in SEQ ID NO: 8. In another aspect, there is disclosed a NtNIA2 nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO: 9, wherein the methionine at position 527 of SEQ ID NO: 9 is substituted for an amino acid that reduces nitrate levels in the plant cell as compared to a control plant cell. Suitably, the methionine at position 527 of SEQ ID NO: 9 is substituted for a non-polar aliphatic amino acid selected from the group consisting of glycine, alanine, proline, isoleucine, leucine or valine. Suitably, the methionine at position 527 of SEQ ID NO: 9 is substituted for isoleucine. Suitably, the polypeptide comprises the contiguous polypeptide sequence of SEQ ID NO: 11. Suitably, the polypeptide comprises, consists or consists essentially of the polypeptide sequence set forth in SEQ ID NO: 11. Suitably, the polypeptide comprises, consists or consists essentially of the polypeptide sequence set forth in SEQ ID NO: 14. According to the present disclosure, a polypeptide sequence encoding the S521N mutation in NtNIA1 and a polypeptide sequence encoding the M527I mutation in NtNIA2 is incorporated into a tobacco plant. The polypeptide sequence containing the S521N mutation in NtNIA1 is set forth in SEQ ID NO: 7. The polypeptide sequence containing the M527I mutation in NtNIA2 is set forth in SEQ ID NO: 11. Fragments of the polypeptides incorporating the mutation(s) described herein are also disclosed. The fragments of the polypeptide(s) typically retain some or all of the function or activity of the full length sequence. Polypeptide fragments typically comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 100 or at least 200 contiguous amino acids. The polypeptides disclosed herein include at least one mutation produced by introducing any type of one or more alterations, which can be isolated naturally. Suitably, the function or activity of the nitrate reductase polypeptide into which the mutation(s) is introduced is modulated (for example, reduced) by the introduction of the mutation. Suitably, the mutation is a substitution. A substitution refers to the replacement of at least one amino acid of the nitrate reductase polypeptide with another amino acid having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break alpha-helical structures or β-sheet structures and the like). A plant or plant cell comprising or carrying a mutation in one or more polynucleotides or polypeptides described herein is disclosed, wherein said mutation results in modulated function or activity of nitrate reductase. Mutations described herein can include man-made mutations or synthetic mutations. Mutations in the polynucleotides and polypeptides described herein can be mutations that are obtained or obtainable via a process which includes an in vitro or an in vivo manipulation step. Mutations in the polynucleotides and polypeptides described herein can be mutations that are obtained or obtainable via a process which includes intervention by man. There is provided a method for modulating the level of a polypeptide in a (cured) plant or in (cured) plant material said method comprising introducing into the genome of said plant one or more mutations that modulate expression of at least one gene, wherein said at least one mutation is selected from the sequences according to the present disclosure – such as S521N or S521N and M527I. Suitably, the gene encodes the Nicotiana tabacum nitrate reductase polypeptide as described herein. There is also disclosed a plant or plant cell that is heterozygous or homozygous for one or more mutations according to the present disclosure, wherein said mutation results in modulated expression of the gene or function or activity of the polypeptide encoded thereby. The function or activity of one or more polypeptides of the present disclosure in a plant is increased or decreased if the function or activity is lower or higher than the function or activity of the same polypeptide(s) in a plant that has not been modified to inhibit the function or activity of that polypeptide and which has been cultured, harvested and cured using the same protocols. Methods for obtaining mutant polynucleotides and polypeptides as described herein are also disclosed. A plant of interest – such as tobacco, including a 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, irradiation-induced mutagenesis, mutagenesis utilizing modified bases, mutagenesis utilizing gapped duplex DNA, double-strand break mutagenesis, mutagenesis utilizing repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling and other other methods as discussed below. Methods that introduce mutations randomly in a polynucleotide can include chemical mutagenesis and radiation mutagenesis. Methods that introduce one or more targeted mutations into a polynucleotide sequence include but are not limited to genome editing technology, particularly zinc finger nuclease- mediated mutagenesis, tilling (targeting induced local lesions in genomes), homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis. Such methods are well known in the art and are described in WO2020 / 141062, for example. Another method of genome editing involves the use of the bacterial CRISPR / Cas system. CRISPR / Cas technology was implemented in plants in WO2015 / 189693, which discloses a viral-mediated genome editing platform that is broadly applicable across plant species. In the context of the present disclosure, a guide RNA may be derived from any of the sequences disclosed herein and in the teaching of WO2015 / 189693 to edit the genome of a plant cell and obtain a desired mutant plant. The fast pace of the development of the technology has generated a great variety of protocols with broad applicability in plantae, which have been well catalogued in a number of recent scientific review articles (for example, Plant Methods (2016) 12:8; and Front Plant Sci. (2016) 7:506). A review of CRISPR / Cas systems with a particular focus on its application is described in Biotechnology Advances (2015) 33, 1, 41- 52). More recent developments in the use of CRISPR / Cas for manipulating plant genomes are discussed 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”, the non-profit plasmid repository (addgene.org), and CRISPR / Cas plasmids are commercially available. One or more introduced mutations – such as the mutation 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 impact gene expression or that interfere with the function of the encoded polypeptide can be determined using methods that are well known in the art. The plants or plant cells according to the present disclosure comprise the mutation described herein and optionally any combination of one or more further mutations in one or more genes – such as the M527I mutation. For example, the plants or plant cells may have a single mutation in a single gene – such as the S521N mutation in NtNIA1; multiple mutations in a single gene; a single mutation in two or more or three or more or four or more genes – such as the S521N mutation in NtNIA1 and the M527I mutation in NtNIA2; or multiple mutations in two or more or three or more or four or more genes - such as the S521N mutation in NtNIA1 and the M527I mutation in NtNIA2. In one embodiment, seeds from plants are mutagenised and then grown into first generation mutant plants. The first generation plants are then allowed to self-pollinate and seeds from the first generation plant are grown into second generation plants, which are then screened for mutations – such as the mutation described herein - in their loci. Though the mutagenized plant material can be screened for mutations, an advantage of screening the second generation plants is that all somatic mutations correspond to germline mutations. One of skill in the art will understand that a variety of plant materials, including but not limited to, seeds, pollen, plant tissue or plant cells, may be mutagenised to create the mutant plants. However, the type of plant material mutagenised may affect when the plant polynucleotide is screened for mutations. For example, when pollen is subjected to mutagenesis prior to pollination of a non-mutagenized plant the seeds resulting from that pollination are grown into first generation plants. Every cell of the first generation plants will contain mutations created in the pollen; thus these first generation plants may then be screened for mutations instead of waiting until the second generation. Prepared polynucleotide from individual plants, plant cells, or plant material can optionally be pooled in order to expedite screening for at least the mutation described herein in the population of plants originating from the mutagenized plant tissue, cells or material. One or more subsequent generations of plants, plant cells or plant material can be screened. The size of the optionally pooled group is dependent upon the sensitivity of the screening method used. 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 the gene or the sequences immediately adjacent to the gene may be utilized to amplify the sequences within the optionally pooled sample. Exemplary primers are set forth in SEQ ID Nos: 18 and 19. Suitably, the one or more primers or probes are designed to amplify the regions of the locus where useful mutations are most likely to arise. Most suitably, the primer is designed to detect mutations within regions of the polynucleotide. Suitably, the primer(s) and probe(s) avoid known polymorphic sites in order to ease screening for point mutations. To facilitate detection of amplification products, the one or more primers or probes may be labelled using any conventional labelling method. Primer(s) or probe(s) can be designed based upon the sequences described herein using methods that are well understood in the art. To facilitate detection of amplification products, the primer(s) or probe(s) may be labelled using any conventional labelling method. These can be designed based upon the sequences described herein using methods that are well understood in the art. Polymorphisms may be identified by means known in the art and some have been described in the literature. Accordingly, in a further aspect there is provided a method of preparing a plant comprising the mutation described herein. The method involves providing at least one cell of a plant comprising a gene encoding a functional nitrate reductase polynucleotide. Next, the at least one cell of the plant is treated under conditions effective to modulate the function of the nitrate reductase polynucleotide. The at least one mutant plant cell is then propagated into a mutant plant, where the mutant plant has a modulated level of nitrate reductase polypeptide as compared to that of a control plant. In one embodiment, the treating step involves subjecting the at least one cell to a chemical mutagenising agent as described herein and under conditions effective to yield at least one mutant plant cell. In another embodiment of this method, the treating step involves subjecting the at least one cell to a radiation source under conditions effective to yield at least one mutant plant cell. The term "mutant plant" includes mutant plants in which the genotype is modified as compared to a control plant, suitably by means other than genetic engineering or genetic modification. In certain embodiments, the mutant plant, mutant plant cell or mutant plant material may comprise one or more mutations that have occurred naturally in another plant, plant cell or plant material and confer a desired trait. This mutation can be incorporated (for example, introgressed) into another plant, plant cell or plant material (for example, a plant, plant cell or plant material with a different genetic background to the plant from which the mutation was derived) to confer the trait thereto. Thus, by way of example, a mutation that occurred naturally in a first plant may be introduced into a second plant – such as a second plant with a different genetic background to the first plant. The skilled person is therefore able to search for and identify a plant carrying naturally in its genome one or more mutant alleles of the genes described herein which confer a desired trait. The mutant allele(s) that occurs naturally can be transferred to the second plant by various methods including breeding, backcrossing and introgression to produce lines, varieties or hybrids that have one or more mutations in the genes described herein. The same technique can also be applied to the introgression of one or more non-naturally occurring mutation(s) from a first plant into a second plant. Plants showing a desired trait may be screened out of a pool of mutant plants. Suitably, the selection is carried out utilising the knowledge of the polynucleotide as described herein. Consequently, it is possible to screen for a genetic trait as compared to a control. Such a screening approach may involve the application of conventional 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 comprising the steps of: (a) providing a sample comprising polynucleotide from a plant; and (b) determining the sequence of the polynucleotide, wherein a difference in the sequence of the polynucleotide as compared to the polynucleotide of a control plant is indicative that said plant is a mutant plant. In another aspect there is provided a method for identifying a mutant plant which accumulates decreased levels of nitrate as compared to a control plant comprising the steps of: (a) providing a sample from a plant to be screened; (b) determining if said sample comprises one or more mutations as described herein in a nitrate reductase polynucleotide; and (c) determining the level of nitrate in said plant. Suitably the level of nitrate is determined in cured leaves. In another aspect there is provided a method for preparing a mutant plant which has decreased levels of nitrate as compared to a control plant comprising the steps of: (a) providing a sample from a first plant; (b) determining if said sample comprises one or more mutations as described herein in a nitrate reductase polynucleotide that results in decreased levels of nitrate; and (c) transferring the one or more mutations into a second plant. Suitably the level of nitrate is determined in cured leaves. The mutation(s) can be transferred into the second plant using various methods that are known in the art – such as by genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing and the like. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background to the first plant. In another aspect there is provided a method for preparing a mutant plant which has decreased levels of nitrate as compared to a control plant comprising the steps of: (a) providing a sample from a first plant; (b) determining if said sample comprises one or more mutations as described herein in a nitrate reductase polynucleotide that results in decreased levels of nitrate; and (c) introgressing the one or more mutations from the first plant into a second plant. Suitably the level of nitrate is determined in cured leaves. In one embodiment, the step of introgressing comprises plant breeding, optionally including backcrossing and the like. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background to the first plant. In one embodiment, the first plant is not a cultivar or an elite cultivar. In one embodiment, the second plant is a cultivar or an elite cultivar. A further aspect relates to a mutant plant (including a cultivar or elite cultivar mutant plant) obtained or obtainable by the methods described herein. In certain embodiments, the “mutant plants” may have one or more mutations localised only to a specific region of the plant – such as within the sequence of the one or more polynucleotide(s) described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be the same or substantially the same as the plant prior to the mutagenesis. In a further aspect there is provided a method of identifying a plant, a plant cell or plant material comprising a mutation in a gene encoding a nitrate reductase polynucleotide comprising: (a) subjecting a plant, a plant cell or plant material to mutagenesis; (b) obtaining a sample from said plant, plant cell or plant material or descendants thereof; and (c) determining the presence of a mutated polynucleotide sequence carrying the mutation described herein. The disclosed compositions and methods are applied to Nicotiana tabacum. The use of tobacco cultivars and elite tobacco cultivars is also contemplated herein. The plant may therefore be a tobacco variety or elite tobacco cultivar that comprises one or more genetic mutations. The genetic mutation(s) (for example, one or more polymorphisms) can be mutations that do not exist naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar) or can be genetic mutation(s) that do occur naturally provided that the mutation does not occur naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar). Nicotiana tabacum varieties include Burley type, dark type, flue-cured type, and Oriental type tobaccos. Non-limiting examples of varieties or cultivars are: 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, Speight G-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, Samsun 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, Galpão 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 subvarieties of the above, even if not specifically identified herein, are also contemplated. In one embodiment, the cultivar is AA37 which is generally understood to be a cross between a South American dark tobacco and American Burley germplasm. The Nicotiana tabacum may be propagatable or non-propagatable. The Nicotiana tabacum may not be obtained exclusively by an essentially biological process. The process may not consist exclusively of entirely natural phenomena - such as crossing or selection. The genome of the Nicotiana tabacum can be purposefully (genetically) modified or engineered. The Nicotiana tabacum described herein may be obtained by techniques which differ from conventional breeding techniques in that they work primarily through the purposeful insertion and / or modification of one or more genes or polynucleotides or polypeptides in a plant. Embodiments are also directed to compositions and methods for producing plants that have been modified to modulate the expression or function of a polynucleotide(s) described herein (or any combination thereof as described herein). Advantageously, the plants that are obtained may be similar or substantially the same in overall appearance to control plants. Various phenotypic characteristics such as degree of maturity, number of leaves per plant, stalk height, leaf insertion angle, leaf size (width and length), internode distance, and lamina- midrib ratio can be assessed by field observations. One aspect relates to a seed of a tobacco plant described herein. A further aspect relates to pollen or an ovule of a plant that is described herein. In addition, there is provided a plant as described herein which further comprises a polynucleotide conferring male sterility. Also provided is a tissue culture of regenerable cells of the plant as described herein, which culture regenerates plants capable of expressing all the morphological and physiological characteristics of the parent. The regenerable cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and a part thereof, ovules, shoots, stems, stalks, pith and capsules or callus or protoplasts derived therefrom. One object is to provide plants or parts thereof that exhibit modulated (for example, reduced) levels of nitrate in the plant material, for example, in cured leaves. Suitably, the plants or parts thereof exhibit modulated (for example, reduced) levels of nitrate as compared to a control plant. Suitably, the plants or parts thereof have substantially the same total harvest biomass (indicated as fresh leaf biomass per plant) as the control plant. Suitably, the plants or parts thereof have substantially the same level of nicotine leaf content as the control plant. Suitably, the plants or parts thereof have substantially the same level of total alkaloid leaf content as the control plant. Suitably, the plants or parts thereof have substantially the same level of ammonia leaf content as the control plant. Suitably, the plants or parts thereof have substantially the same level of reducing sugar leaf content as the control plant. Accordingly, there is described herein plants or parts thereof or plant cells that have modulated levels of nitrate as compared to control cells or control plants. The plants or plant cells are modified to modulate the synthesis or function of one or more of the polypeptides described herein by modulating the expression of one or more of the corresponding polynucleotides described herein. Suitably, the modulated levels of nitrate are observed in cured leaves. In certain embodiments, the level of nitrate in the plant – such as the cured leaves or cured tobacco – is reduced. A further aspect, relates to a plant or plant cell, wherein the expression or the function of one or more of the polypeptides described herein is modulated and a part of the plant (for example, the cured leaves or cured tobacco) have decreased levels of nitrate of at least about 20% therein as compared to a control plant in which the expression or the function of said polypeptide(s) has not been modulated. In certain embodiments, the level of nitrate in the plant – such as the cured leaves or cured tobacco – may be decreased, for example, by at least about 25% or more, or about 30% or more, or about 35% or more, or about 40% or more. A still further aspect, relates to a cured plant material – such as cured leaf or cured tobacco - derived or derivable from a plant or plant cell as described herein, wherein expression of one or more of the polynucleotides described herein or the function of the polypeptide encoded thereby is modulated (for example, increased) and wherein the level of nitrate is modulated (for example, decreased) by at least about 25% or more, or about 30% or more, or about 35% or more, or about 40% or more. For example, the S521N mutation may reduce the level of nitrate in the tobacco plant by increasing or constitutively activating the activity of NtNIA1. 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 the one or more of the polynucleotides or polypeptides described herein which can result in plants or plant components (for example, leaves – such as cured leaves – or tobacco) or plant cells with modulated nitrate content. An increase in function or activity as compared to a control may be from about 5 % to about 100 %, or 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 100 % or more - such as 200%, 300%, 500%, 1000% or more. A reduction in function or activity as compared to a control may be from about 5 % to about 100 %, or a reduction 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 100 %. A plant carrying the mutation described herein in a nitrate reductase polynucleotide can be used in a plant breeding program to create useful lines, varieties and hybrids. In particular, the mutant can be introgressed into the commercially important varieties described above. Thus, methods for breeding plants are provided, that comprise crossing plant as described herein with a plant comprising a different genetic identity. The method may further comprise crossing the progeny plant with another plant, and optionally repeating the crossing until a progeny with the desirable genetic traits or genetic background is obtained. One purpose served by such breeding methods is to introduce a desirable genetic trait into other varieties, breeding lines, hybrids or cultivars, particularly those that are of commercial interest. Another purpose is to facilitate stacking of genetic modifications of different genes in a single plant variety, lines, hybrids or cultivars. Intraspecific as well as interspecific matings are contemplated. The progeny plants that arise from such crosses, also referred to as breeding lines, are examples of plants of the disclosure. In one embodiment, a method is provided for producing a plant comprising: (a) crossing a plant of the present disclosure with a second plant to yield progeny tobacco seed; (b) growing the progeny tobacco seed, under plant growth conditions, to yield a non-naturally occurring plant. The method may further comprise: (c) crossing the previous generation of non-naturally occurring plant with itself or another plant to yield progeny tobacco seed; (d) growing the progeny tobacco seed of step (c) under plant growth conditions, to yield additional non-naturally occurring plants; and (e) repeating the crossing and growing steps of (c) and (d) multiple times to generate further generations of non-naturally occurring plants. The method may optionally comprises prior to step (a), a step of providing a parent plant which comprises a genetic identity that is characterized and that is not identical to the plant of the present disclosure. In some embodiments, depending on the breeding program, the crossing and growing steps are repeated from 0 to 2 times, from 0 to 3 times, from 0 to 4 times, 0 to 5 times, from 0 to 6 times, from 0 to 7 times, from 0 to 8 times, from 0 to 9 times or from 0 to 10 times, in order to generate generations of non-naturally occurring plants. Backcrossing is an example of such a method wherein a progeny is crossed with one of its parents or another plant genetically similar to its parent, in order to obtain a progeny plant in the next generation that has a genetic identity which is closer to that of one of the parents. Techniques for plant breeding, particularly plant breeding, are well known and can be used in the methods of the disclosure. Certain embodiments exclude the step of selecting a plant. According to the disclosure, in a breeding program, successful crosses yield F1 plants that are fertile. Selected F1 plants can be crossed with one of the parents, and the first backcross generation plants are self-pollinated to produce a population that is again screened for variant gene expression (for example, the null version of the gene). The process of backcrossing, self-pollination, and screening is repeated, for example, at least 4 times until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent. This plant, if desired, is self-pollinated and the progeny are subsequently screened again to confirm that the plant exhibits variant gene expression. In some embodiments, a plant population in the F2 generation is screened for variant gene expression, for example, a plant is identified that fails to express a polypeptide due to the absence of the gene according to standard methods, for example, by using a PCR method with primers based upon the polynucleotide sequence information for the polynucleotide(s) described herein (or any combination thereof as described herein). Aside from the mutations described herein, the plants or plant cells 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. Without limitation, the plants and parts thereof described herein can be modified either before or after the expression, function or activity of the one or more polynucleotides and / or polypeptides according to the present disclosure have been modulated. One or more of the following further genetic modifications (for example, mutations) can be present in the plants and parts thereof. Parts of the plants described herein, particularly the leaf lamina and midrib of such plants, can be incorporated into or used in making various consumable products including but not limited to aerosol forming materials, aerosol forming devices, smoking articles, smokable articles, smokeless products, medicinal or cosmetic products, intravenous preparations, tablets, powders, and tobacco products. Examples of aerosol forming materials include tobacco compositions, tobaccos, tobacco extract, cut tobacco, cut filler, cured tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobaccos. 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 comprise chewing tobaccos, and snuffs. In certain aerosol forming devices, rather than combustion, a tobacco composition or another aerosol forming material is heated by one or more electrical heating elements to produce an aerosol. In another type of heated aerosol forming device, an aerosol is produced by the transfer of heat from a combustible fuel element or heat source to a physically separate aerosol forming material, which 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 as dried particles, shreds, granules, powders, or a slurry, deposited on, mixed in, surrounded by, or otherwise combined with other ingredients in any format, such as flakes, films, tabs, foams, or beads. As used herein, the term ‘smoke’ is used to describe a type of aerosol that is produced by smoking articles, such as cigarettes, or by combusting an aerosol forming material. In one embodiment, there is also provided cured plant material from the plants described herein. Processes of curing green tobacco leaves are known by those having skills in the art and include without limitation air-curing, fire-curing, flue-curing and sun-curing as described herein. In another embodiment, there is described tobacco products including tobacco-containing aerosol forming materials comprising plant material – such as leaves, suitably cured leaves - from the tobacco plants described herein. The tobacco products described herein can be a blended tobacco product which may further comprise unmodified tobacco. The plants may have other uses in, for example, agriculture. For example, plants described herein can be used to make animal feed and human food products. The disclosure also provides methods for producing seeds comprising cultivating the plant described herein, and collecting seeds from the cultivated plants. Seeds from plants described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture. Packaging material such as paper and cloth are well known in the art. A package of seed can have a label, for example, a tag or label secured to the packaging material, a label printed on the package that describes the nature of the seeds therein. Compositions, methods and kits for genotyping plants for identification, selection, or breeding can comprise a means of detecting the presence of a polynucleotide (or any combination thereof as described herein) in a sample of polynucleotide. Accordingly, a composition is described comprising one or more primers for specifically amplifying at least a portion of one or more of the polynucleotides and optionally one or more probes and optionally one or more reagents for conducting the amplification or detection. Accordingly, gene specific oligonucleotide primers or probes comprising about 10 or more contiguous polynucleotides corresponding to the polynucleotide(s) described herein are disclosed. Said primers or probes may comprise or consist of about 15, 20, 25, 30, 40, 45 or 50 more contiguous polynucleotides that hybridise (for example, specifically hybridise) to the polynucleotide(s) described herein. In a further aspect, there is also provided a method of detecting a polynucleotide(s) described herein (or any combination thereof as described herein) in a sample comprising the step of: (a) providing a sample comprising, or suspected of comprising, a polynucleotide; (b) contacting said sample with one or more primers or one or more probes for specifically detecting at least a portion of the polynucleotide(s); and (c) detecting the presence of an amplification product, wherein the presence of an amplification product is indicative of the presence of the polynucleotide(s) in the sample. In a further aspect, there is also provided the use of one or more primers or probes for specifically detecting at least a portion of the polynucleotide(s). Kits for detecting at least a portion of the polynucleotide(s) are also provided which comprise one or more primers or probes for specifically detecting at least a portion of the polynucleotide(s). The kit may comprise reagents for polynucleotide amplification - such as PCR - or reagents for probe hybridization-detection technology - such as Southern Blots, Northern Blots, in-situ hybridization, or microarray. The kit may comprise reagents for antibody binding-detection technology such as Western Blots, ELISAs, SELDI mass spectrometry or test strips. The kit may comprise reagents for DNA sequencing. The kit may comprise reagents and instructions for using the kit. In some embodiments, a kit may comprise instructions for one or more of the methods described. The kits described may be useful for genetic identity determination, phylogenetic studies, genotyping, haplotyping, pedigree analysis or plant breeding particularly with co- dominant scoring. The present disclosure also provides a method of genotyping a plant, a plant cell or plant material comprising a polynucleotide as described herein. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. The specific method of genotyping may employ any number of molecular marker analytic techniques including amplification fragment length polymorphisms (AFLPs). AFLPs are the product of allelic differences between amplification fragments caused by polynucleotide variability. Thus, the present disclosure further provides a means to follow segregation of one or more genes or polynucleotides as well as chromosomal sequences genetically linked to these genes or polynucleotides using such techniques as AFLP analysis. The invention is further described in the Examples below, which are provided to describe the invention in further detail. These examples, which set forth a preferred mode presently contemplated for carrying out the invention, are intended to illustrate and not to limit the invention. EXAMPLE An AA37 EMS mutant population is screened for mutations in the hinge 1 site of the NtNIA1 gene (GeneBank X14058.1, EC 1.6.6.1) and the mutation g to a in the sequence ctaaagaagagtatctcaactccattcatg (site of mutation indicated) is identified, leading to the substitution of Serine 521 with Asparagine. NtNIA1 can be amplified using a forward primer (5’- gaaataacgctcttacactatgag – 3’) and a reverse primer (5’- gcaaatcgaaatttcctaacatcag – 3’). In a field trial, different BC2S2 AA37 x TN90 breeding lines are tested carrying the NtNIA2 M527I and the NtNIA1 S521N homozygous single mutation, against outsegregant homozygous wild type lines and homozygous double mutant lines. Field design displayed the plants in 20 plant plots randomly distributed to minimize positional effect due to field inhomogeneity. Plants are grown according to standard good agricultural practices for burley tobacco (according to Grandes cultures fiches techniques, agridea - developpement de l’agriculture et de l’espace rural, www.agridea.ch). Leaves are harvested at maturity and air cured. Samples are taken from the cured material and analyzed for chemical profiling (according to the Coresta recommended method No.36 - Determination of Nitrate in Tobacco and Smokeless Tobacco Products by Reduction to Nitrite and Continuous Flow Analysis, https: / / www.coresta.org / determination-nitrate-tobacco-and-smokeless- tobacco-products-reduction-nitrite-and-continuous-flow ). Results are reported in Figure 2, which reports nitrate levels in dry powder of total air cured leaves. The CORESTA recommendation for tobacco curing is described in CORESTA Guide N°17, April 2016, Sustainability in Leaf Tobacco Production. The results are the average of at least 7 lines per experimental point. Nitrate is expressed as % of dry weight. In the tested growing season using Burley tobacco background, the single S521N mutant gave a statistically significant 24.5 % reduction in nitrate content as measured in the outsegregant wild type (p values=0.00335 and 0.01012). The double M527I and S521N mutant gave a statistically significant 35 % reduction in nitrate content as measured in the outsegregant wild type (p value=0.00125). A 2-year field trial is carried under the same conditions as the first trial reported above in which nitrate levels in dry powder of total air cured leaves is measured. The results are the average of 10 or 12 plots of 8 plants each. Nitrate is expressed as % of dry weight. In the tested growing season using Burley tobacco background: the single M527I mutant gave a statistically significant 29.5 % reduction in nitrate content; the single S521N mutant gave a statistically significant 30 % reduction in nitrate content; and the double M527I and S521N mutant gave a statistically significant 52.8 % reduction in nitrate content (p value=0.008357). Any publication cited or described herein provides relevant information disclosed prior to the filing date of the present application. Statements herein are not to be construed as an admission that the inventors are not entitled to antedate such disclosures. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific 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 modes for carrying out the invention which are obvious to those skilled in cellular, molecular and plant biology or related fields are intended to be within the scope of the claims. SEQUENCE LISTING SEQ ID NO: 1 – genomic sequence of wild type NtNIA1, GenBank Accession number X14058.1, EC 1.6.6.1). The sequence coding for the nitrate reductase kinase recognition site is highlighted tctttgagtaatgtatacatttaagagctatatatatatatatatgaccctgcaatgaaagaggaagctaacctg tttgcctttgtcgtattctgcaaatttcagtttaaaagcccatttgagattgaattaattcgttataactaacga tatcaaagaaaacaattagttaaatgcttgtgtaatttgaagaatttttttggacgtggtcgctgaaaacagaga aatactttctgaaaagttggtcttgttcaaaaacgtaataagagagttgattctttttcgtaaaaagtcactttc tggaatttttcacttgatataccaggtaaatagttactgatatttaatatttataccaaacaaatgaaagtaaaa tatgtgtgtctttcatacatatatttatctatcatagttaatgatatatatatatatattttaaccttaaatttt gactactaaaatgtaattatatttaatttgggtagatatcagatgccactaaacatttacctagccactcctccg aaaataaattgagaaggaaattagagttagtggagccataataatgtttaatgtgaccataactcagtgaaacag ctgttagtcctaaccaacagctgcatatctttaagccatttgctattaccccaatcccgcatcttcctctgatcc cgaccctacgggcgtaaaaagtgtaaatcattagaattgttttattttgtgatgtcactatttttttaaaatcaa aattaaattggggtgtcgatttttttgggtcccacttatgtatagtatgggggctatggaggcattgagagagtc cgtaacgtttctatataaggccaccccacgcattcacaaacttcgttcgaaaatcaaatcttagagagagagaga gagaaatattttgagagagaaatacagaaaatctctctttcttttttagaataatctatggcggcatctgtcgaa aacaggcagttcagtcacatagaagccggtttatcccggtctttcaagcctcggtctgattccccggttcgtggc tgcaacttccctccgcccaacagtactaatttccaaaagaaaccaaattccaccattttccttgattactcgtcg agtgaagacgacgatgatgatgacgaaaaaaatgagtaccttcaaatgatcaaaaaagggaattcagaattagag ccatctgttcatgacagcagggacgaaggtaccgctgataactggattgaacgcaacttttccttgattcgtctc accggaaagcatccatttaactccgaaccgccgttgaaccgtctcatgcaccacggttttatcacaccggtccca cttcattacgttcgtaaccatggaccggttcccaagggcacatgggatgactggaccgtggaagtcacgggacta gtgaaacgtcctatgaaattcacaatggaccagttggttaacgaattcccttccagagaattgcccgttacgctt gtgtgtgctggcaaccgaaggaaagaacagaacatggttaaacaaaccattggtttcaactggggtgccgctgcc gtttcaacaactgtatggcgcggggtacccctacgcgctttgttaaaacggtacggtgtttttagcaagaataaa ggggcgcttaatgtttgcttcgaaggagctgatgtcttgcccggaggcggtggttcaaagtatggaaccagcatt aagaaggaatttgcaatggatccagcacgagatatcataatagcttacatgcagaacggagaaaaattggcaccc gaccacgggtttccagtacgaatgataattccaggattcattggaggaagaatggtgaaatggataaagaggatt atagtcaccacccaagaatcagacagctattatcatttcaaggacaatagagttcttcctccccatgttgatgct gaacttgcaaatactgaaggtaatttttattaagtggtcaatatattttaattagttgagacttatacatacaag ctaaatatttcttagaatttgaagaagcttacaaaatcaactgaaagtgaaaaggaaacaattatatatattcaa cgggtctatgtatatgttcctgtcattaatctcatctcaaatcaaatggtgacaaaggactttggaaacatagaa ttgtcagctttatatatagttataagttagctgtttgcagctattcattattggttaatctgtgtgcagcatggt ggtacaagccagagtacatcatcaatgagctcaatattaactctgtcattacgacgccgtgtcatgaagaaattt tgcctattaacgcctggacgactcagcgaccttacacgttgaggggctattcttattctggttagtatttttttt ttcttctctttttccgattttgctgaaaatttcatatttcttagtattgtcgaaatacatcgtatcctctaactc tgacgttttacttcgtccttatgcacccacttacttccctattttcgacccgataacctcagcgtccctaattaa atgtaaaatataattatagtagtaattaaagttcgtagatgtcttctttagaaagcgtgtaaaaacttttaaaac ggaatataatatgaatattatctaatacttacaaagtgtcaataattggtagccaatttaaactatatagataaa aagtctgtgaatacaagtattggtatagggattagggagaatcgaagtaaagtggagtaattggacgcatgagct tgggatgctgtcagctagtttgctaatgtgaaacagaatagtaagaaaaggccaacatggttttgtttattttat gctagtacacaaaaacctggggagctttcctagttctgaagagtcggtcgcaaaattaatactatagtataccaa gtgaatattaaattcaattgtctaaagcacggaatgtttttgactactttagttcctgcatcttgggttgcctcg acaacaacctttgctggattattatattaatgttcaatataatatgcaattagaaaactttcaagtggtcacttt atatggatgtagtcaatactatttcctctaacctacgtgcctaattacttcccactttccagtacaggaccacca ttaagtttgtcaattccttgtgcaattgacctttcacttcagctactattacaggattaaacatgttaggaaatt caagaattgatgaaaacattagaataattagccattgtattgattgaaatactgattgtgaacgtgtaacaggcg gagggaaaaaagtaacgcgagtagaagtgaccttggatggaggagaaacatggcaagtttgcacactagatcacc cagagaagcccaccaaatatggcaagtactggtgttggtgcttttggtcactcgaggttgaggtgttagacttgc tcagtgccaaagaaattgctgttcgagcttgggatgagaccctcaatactcaacctgagaagcttatttggaatg tcatggtaagttcacatctcctttacctttcttttaagttctatagactaatggtttaaactattttacaccata agtaacttacaataacatgtactaattatttatcctttcaacctttttctgattgtttcattatctagattcaca gagcacatgccaatacaaaaaacttttcactggttttagtctaagattcccttttgttttttggaggtgtgtggt ccatactccatagatcaattccagccactgacgtaccagccctgaaaattcctggtagttatagcaacgtacaat catttcatattacgtaagcagagacgtatcacatgaactacatgtgaataccacttgcccagtccattaggtcaa ttcatctagatatacagtaaatcttgacaccaacactggctcactgtttataacactagtagcgtttaacaacac tttcatccttgatcattacctgatctaattaagatttttttatgtactctaaaaattgtaattacataaataaat taaacttttataagctgacaccgttactaattccagttttatcatttaggtgaaataacgctcttacactatgag tgtattgataaaagttatatacattttctaaatattgtggtacgttgcaattttcagggaatgatgaacaattgc tggttccgagtaaagatgaatgtgtgcaagcctcacaagggagagattggaatagtgtttgaacacccgactcaa cctggaaaccaatcaggtggatggatggcaaaggagaggcatttggagatatcagcagaggcacctccaacacta aagaagagtatctcaactccattcatgaacacagcttccaagatgtactccatgtcggaggtgaggaaacacagc tctgctgactctgcttggatcatagtccatggtcatatctatgacgccacgcgtttcttgaaagatcaccccggt ggttctgacagcattctcatcaatgctggcactgattgcactgaggaatttgatgcaattcattctgataaggct aagaagctattggaggaattcaggattggtgaactcctaactactggttacacctctgactctcctggcaactcc gtccatggatcttcttccttcagcagctttctagcacctattaaggaacttgttccagcgcagaggagtgtggcc ctcattccaagagagaaaatcccatgcaaactcatcgacaaacaatccatctcccctgatgttaggaaatttcga tttgcattgccctctgaggatcaagtcttgggcttgcctgttggtaaacacatcttcctctgtgccgttattgac gataagctctgcatgcgcgcctacacgcctactagcacgatcgatgaggtggggtacttcgagttggttgtcaag atatacttcaaaggaattcaccctaaattccccaatggggggcaaatgtcacaataccttgattctctccaatta gggtcatttctcgacgtgaaaggtccattaggtcacattgaataccaaggaaagggcaatttcttagttcatggc aaacaaaagtttgccaagaagttggccatgatagcaggtggaacagggataactccagtttatcaagtcatgcag gcaattctgaaagatccagaagatgacacagaaatgtatgtggtctatgctaatagaacagaggatgatatttta cttaaggaagagcttgattcatgggctgagaaaattccagaaagggttaaagtttggtatgtggttcaagattct attaaagaaggatggaagtacagccttggttttatttcagaagccattttgagagaacatatccctgagccatct cacacaacactggctttggcttgtggaccacctcctatgattcaatttgctgttaatccaaacttggagaagatg ggctatgacattaaggattccttattggtgttctaattttcaaaacaaaacaatatctgcaggaataaacttttt gtttcccctatcagttgtacatattgtatttggtatatcacccccatgtactacgcactatttgtagttcttaca tcttcttcttctttttaatttttttttaaaaccttaggatataaaggtttaattttctcttcctacaaagtgagt ctttagggaagaaatgttgtactgtactagtatgtctgagtcaaaaggttgtaatgtttaccatgacaaattgta ttcaattcctcgtggaatagtaacattgtgctcatgtgtcttcctgtaagagattcttcaaaatatcaatgtgtg tgtatatatatatatatatatatatatatatatatatatatatatatatatatatagtaattgcaacggtttgtt ccttttccctatgtggttaactgctcttaccttagcttctagtctctggtgaatatttctttctttttctaaaac tctttaatacggccttaaataagagaaaagtataaaccacgaatatcattatgcagacgatatggtaattaatct actttttgaaaaaaaattttctttatttggtccttgaaaataatattctagaaccttttgtatattcccttttaa cttctatttagtttt SEQ ID NO: 2 - coding sequence of NtNIA1 wild type. The sequence coding for the nitrate reductase kinase recognition site is highlighted atggcggcatctgtcgaaaacaggcagttcagtcacatagaagccggtttatcccggtctttcaagcctcggtct gattccccggttcgtggctgcaacttccctccgcccaacagtactaatttccaaaagaaaccaaattccaccatt ttccttgattactcgtcgagtgaagacgacgatgatgatgacgaaaaaaatgagtaccttcaaatgatcaaaaaa gggaattcagaattagagccatctgttcatgacagcagggacgaaggtaccgctgataactggattgaacgcaac ttttccttgattcgtctcaccggaaagcatccatttaactccgaaccgccgttgaaccgtctcatgcaccacggt tttatcacaccggtcccacttcattacgttcgtaaccatggaccggttcccaagggcacatgggatgactggacc gtggaagtcacgggactagtgaaacgtcctatgaaattcacaatggaccagttggttaacgaattcccttccaga gaattgcccgttacgcttgtgtgtgctggcaaccgaaggaaagaacagaacatggttaaacaaaccattggtttc aactggggtgccgctgccgtttcaacaactgtatggcgcggggtacccctacgcgctttgttaaaacggtgcggt gtttttagcaagaataaaggggcgcttaatgtttgcttcgaaggagctgatgtcttgcccggaggcggtggttca aagtatggaaccagcattaagaaggaatttgcaatggatccagcacgagatatcataatagcttacatgcagaac ggagaaaaattggcacccgaccacgggtttccagtacgaatgataattccaggattcattggaggaagaatggtg aaatggataaagaggattatagtcaccacccaagaatcagacagctattatcatttcaaggacaatagagttctt cctccccatgttgatgctgaacttgcaaatactgaagcatggtggtacaagccagagtacatcatcaatgagctc aatattaactctgtcattacgacgccgtgtcatgaagaaattttgcctattaacgcctggacgactcagcgacct tacacgttgaggggctattcttattctggcggagggaaaaaagtaacgcgagtagaagtgaccttggatggagga gaaacatggcaagtttgcacactagatcacccagagaagcccaccaaatatggcaagtactggtgttggtgcttt tggtcactcgaggttgaggtgttagacttgctcagtgccaaagaaattgctgttcgagcttgggatgagaccctc aatactcaacctgagaagcttatttggaatgtcatgggaatgatgaacaattgctggttccgagtaaagatgaat gtgtgcaagcctcacaagggagagattggaatagtgtttgaacacccgactcaacctggaaaccaatcaggtgga tggatggcaaaggagaggcatttggagatatcagcagaggcacctccaacactaaagaagagtatctcaactcca ttcatgaacacagcttccaagatgtactccatgtcggaggtgaggaaacacagctctgctgactctgcttggatc atagtccatggtcatatctatgacgccacgcgtttcttgaaagatcaccccggtggttctgacagcattctcatc aatgctggcactgattgcactgaggaatttgatgcaattcattctgataaggctaagaagctattggaggaattc aggattggtgaactcataactactggttacacctctgactctcctggcaactccgtccatggatcttcttccttc agcagctttctagcacctattaaggaacttgttccagcgcagaggagtgtggccctcattccaagagagaaaatc ccatgcaaactcatcgacaaacaatccatctcccctgatgttaggaaatttcgatttgcattgccctctgaggat caagtcttgggcttgcctgttggtaaacacatcttcctctgtgccgttattgacgataagctctgcatgcgcgcc tacacgcctactagcacgatcgatgaggtggggtacttcgagttggttgtcaagatatacttcaaaggaattcac cctaaattccccaatggggggcaaatgtcacaataccttgattctctccaattagggtcatttctcgacgtgaaa ggtccattaggtcacattgaataccaaggaaagggcaatttcttagttcatggcaaacaaaagtttgccaagaag ttggccatgatagcaggtggaacagggataactccagtttatcaagtcatgcaggcaattctgaaagatccagaa gatgacacagaaatgtatgtggtctatgctaatagaacagaggatgatattttacttaaggaagagcttgattca tgggctgagaaaattccagaaagggttaaagtttggtatgtggttcaagattctattaaagaaggatggaagtac agccttggttttatttcagaagccattttgagagaacatatccctgagccatctcacacaacactggctttggct tgtggaccacctcctatgattcaatttgctgttaatccaaacttggagaagatgggctatgacattaaggattcc ttattggtgttctaa SEQ ID NO: 3 - protein sequence of NtNIA1 wild type. The sequence coding for the consensus nitrate reductase kinase recognition site is highlighted MAASVENRQFSHIEAGLSRSFKPRSDSPVRGCNFPPPNSTNFQKKPNSTIFLDYSSSEDDDDDDEKNEYLQMIKK GNSELEPSVHDSRDEGTADNWIERNFSLIRLTGKHPFNSEPPLNRLMHHGFITPVPLHYVRNHGPVPKGTWDDWT VEVTGLVKRPMKFTMDQLVNEFPSRELPVTLVCAGNRRKEQNMVKQTIGFNWGAAAVSTTVWRGVPLRALLKRCG VFSKNKGALNVCFEGADVLPGGGGSKYGTSIKKEFAMDPARDIIIAYMQNGEKLAPDHGFPVRMIIPGFIGGRMV KWIKRIIVTTQESDSYYHFKDNRVLPPHVDAELANTEAWWYKPEYIINELNINSVITTPCHEEILPINAWTTQRP YTLRGYSYSGGGKKVTRVEVTLDGGETWQVCTLDHPEKPTKYGKYWCWCFWSLEVEVLDLLSAKEIAVRAWDETL NTQPEKLIWNVMGMMNNCWFRVKMNVCKPHKGEIGIVFEHPTQPGNQSGGWMAKERHLEISAEAPPTLKKSISTP FMNTASKMYSMSEVRKHSSADSAWIIVHGHIYDATRFLKDHPGGSDSILINAGTDCTEEFDAIHSDKAKKLLEEF RIGELITTGYTSDSPGNSVHGSSSFSSFLAPIKELVPAQRSVALIPREKIPCKLIDKQSISPDVRKFRFALPSED QVLGLPVGKHIFLCAVIDDKLCMRAYTPTSTIDEVGYFELVVKIYFKGIHPKFPNGGQMSQYLDSLQLGSFLDVK GPLGHIEYQGKGNFLVHGKQKFAKKLAMIAGGTGITPVYQVMQAILKDPEDDTEMYVVYANRTEDDILLKEELDS WAEKIPERVKVWYVVQDSIKEGWKYSLGFISEAILREHIPEPSHTTLALACGPPPMIQFAVNPNLEKMGYDIKDS LLVF* SEQ ID NO: 4 - protein sequence of wild type nitrate reductase kinase recognition site in NtNIA1 LKKSISTPFM SEQ ID NO: 5 - genomic sequence encoding the NtNIA1 S521N mutant. The sequence coding for the NR kinase recognition site is highlighted and the specific mutation is underscored tctttgagtaatgtatacatttaagagctatatatatatatatatgaccctgcaatgaaagaggaagctaacctg tttgcctttgtcgtattctgcaaatttcagtttaaaagcccatttgagattgaattaattcgttataactaacga tatcaaagaaaacaattagttaaatgcttgtgtaatttgaagaatttttttggacgtggtcgctgaaaacagaga aatactttctgaaaagttggtcttgttcaaaaacgtaataagagagttgattctttttcgtaaaaagtcactttc tggaatttttcacttgatataccaggtaaatagttactgatatttaatatttataccaaacaaatgaaagtaaaa tatgtgtgtctttcatacatatatttatctatcatagttaatgatatatatatatatattttaaccttaaatttt gactactaaaatgtaattatatttaatttgggtagatatcagatgccactaaacatttacctagccactcctccg aaaataaattgagaaggaaattagagttagtggagccataataatgtttaatgtgaccataactcagtgaaacag ctgttagtcctaaccaacagctgcatatctttaagccatttgctattaccccaatcccgcatcttcctctgatcc cgaccctacgggcgtaaaaagtgtaaatcattagaattgttttattttgtgatgtcactatttttttaaaatcaa aattaaattggggtgtcgatttttttgggtcccacttatgtatagtatgggggctatggaggcattgagagagtc cgtaacgtttctatataaggccaccccacgcattcacaaacttcgttcgaaaatcaaatcttagagagagagaga gagaaatattttgagagagaaatacagaaaatctctctttcttttttagaataatctatggcggcatctgtcgaa aacaggcagttcagtcacatagaagccggtttatcccggtctttcaagcctcggtctgattccccggttcgtggc tgcaacttccctccgcccaacagtactaatttccaaaagaaaccaaattccaccattttccttgattactcgtcg agtgaagacgacgatgatgatgacgaaaaaaatgagtaccttcaaatgatcaaaaaagggaattcagaattagag ccatctgttcatgacagcagggacgaaggtaccgctgataactggattgaacgcaacttttccttgattcgtctc accggaaagcatccatttaactccgaaccgccgttgaaccgtctcatgcaccacggttttatcacaccggtccca cttcattacgttcgtaaccatggaccggttcccaagggcacatgggatgactggaccgtggaagtcacgggacta gtgaaacgtcctatgaaattcacaatggaccagttggttaacgaattcccttccagagaattgcccgttacgctt gtgtgtgctggcaaccgaaggaaagaacagaacatggttaaacaaaccattggtttcaactggggtgccgctgcc gtttcaacaactgtatggcgcggggtacccctacgcgctttgttaaaacggtacggtgtttttagcaagaataaa ggggcgcttaatgtttgcttcgaaggagctgatgtcttgcccggaggcggtggttcaaagtatggaaccagcatt aagaaggaatttgcaatggatccagcacgagatatcataatagcttacatgcagaacggagaaaaattggcaccc gaccacgggtttccagtacgaatgataattccaggattcattggaggaagaatggtgaaatggataaagaggatt atagtcaccacccaagaatcagacagctattatcatttcaaggacaatagagttcttcctccccatgttgatgct gaacttgcaaatactgaaggtaatttttattaagtggtcaatatattttaattagttgagacttatacatacaag ctaaatatttcttagaatttgaagaagcttacaaaatcaactgaaagtgaaaaggaaacaattatatatattcaa cgggtctatgtatatgttcctgtcattaatctcatctcaaatcaaatggtgacaaaggactttggaaacatagaa ttgtcagctttatatatagttataagttagctgtttgcagctattcattattggttaatctgtgtgcagcatggt ggtacaagccagagtacatcatcaatgagctcaatattaactctgtcattacgacgccgtgtcatgaagaaattt tgcctattaacgcctggacgactcagcgaccttacacgttgaggggctattcttattctggttagtatttttttt ttcttctctttttccgattttgctgaaaatttcatatttcttagtattgtcgaaatacatcgtatcctctaactc tgacgttttacttcgtccttatgcacccacttacttccctattttcgacccgataacctcagcgtccctaattaa atgtaaaatataattatagtagtaattaaagttcgtagatgtcttctttagaaagcgtgtaaaaacttttaaaac ggaatataatatgaatattatctaatacttacaaagtgtcaataattggtagccaatttaaactatatagataaa aagtctgtgaatacaagtattggtatagggattagggagaatcgaagtaaagtggagtaattggacgcatgagct tgggatgctgtcagctagtttgctaatgtgaaacagaatagtaagaaaaggccaacatggttttgtttattttat gctagtacacaaaaacctggggagctttcctagttctgaagagtcggtcgcaaaattaatactatagtataccaa gtgaatattaaattcaattgtctaaagcacggaatgtttttgactactttagttcctgcatcttgggttgcctcg acaacaacctttgctggattattatattaatgttcaatataatatgcaattagaaaactttcaagtggtcacttt atatggatgtagtcaatactatttcctctaacctacgtgcctaattacttcccactttccagtacaggaccacca ttaagtttgtcaattccttgtgcaattgacctttcacttcagctactattacaggattaaacatgttaggaaatt caagaattgatgaaaacattagaataattagccattgtattgattgaaatactgattgtgaacgtgtaacaggcg gagggaaaaaagtaacgcgagtagaagtgaccttggatggaggagaaacatggcaagtttgcacactagatcacc cagagaagcccaccaaatatggcaagtactggtgttggtgcttttggtcactcgaggttgaggtgttagacttgc tcagtgccaaagaaattgctgttcgagcttgggatgagaccctcaatactcaacctgagaagcttatttggaatg tcatggtaagttcacatctcctttacctttcttttaagttctatagactaatggtttaaactattttacaccata agtaacttacaataacatgtactaattatttatcctttcaacctttttctgattgtttcattatctagattcaca gagcacatgccaatacaaaaaacttttcactggttttagtctaagattcccttttgttttttggaggtgtgtggt ccatactccatagatcaattccagccactgacgtaccagccctgaaaattcctggtagttatagcaacgtacaat catttcatattacgtaagcagagacgtatcacatgaactacatgtgaataccacttgcccagtccattaggtcaa ttcatctagatatacagtaaatcttgacaccaacactggctcactgtttataacactagtagcgtttaacaacac tttcatccttgatcattacctgatctaattaagatttttttatgtactctaaaaattgtaattacataaataaat taaacttttataagctgacaccgttactaattccagttttatcatttaggtgaaataacgctcttacactatgag tgtattgataaaagttatatacattttctaaatattgtggtacgttgcaattttcagggaatgatgaacaattgc tggttccgagtaaagatgaatgtgtgcaagcctcacaagggagagattggaatagtgtttgaacacccgactcaa cctggaaaccaatcaggtggatggatggcaaaggagaggcatttggagatatcagcagaggcacctccaacacta aagaagaatatctcaactccattcatgaacacagcttccaagatgtactccatgtcggaggtgaggaaacacagc tctgctgactctgcttggatcatagtccatggtcatatctatgacgccacgcgtttcttgaaagatcaccccggt ggttctgacagcattctcatcaatgctggcactgattgcactgaggaatttgatgcaattcattctgataaggct aagaagctattggaggaattcaggattggtgaactcctaactactggttacacctctgactctcctggcaactcc gtccatggatcttcttccttcagcagctttctagcacctattaaggaacttgttccagcgcagaggagtgtggcc ctcattccaagagagaaaatcccatgcaaactcatcgacaaacaatccatctcccctgatgttaggaaatttcga tttgcattgccctctgaggatcaagtcttgggcttgcctgttggtaaacacatcttcctctgtgccgttattgac gataagctctgcatgcgcgcctacacgcctactagcacgatcgatgaggtggggtacttcgagttggttgtcaag atatacttcaaaggaattcaccctaaattccccaatggggggcaaatgtcacaataccttgattctctccaatta gggtcatttctcgacgtgaaaggtccattaggtcacattgaataccaaggaaagggcaatttcttagttcatggc aaacaaaagtttgccaagaagttggccatgatagcaggtggaacagggataactccagtttatcaagtcatgcag gcaattctgaaagatccagaagatgacacagaaatgtatgtggtctatgctaatagaacagaggatgatatttta cttaaggaagagcttgattcatgggctgagaaaattccagaaagggttaaagtttggtatgtggttcaagattct attaaagaaggatggaagtacagccttggttttatttcagaagccattttgagagaacatatccctgagccatct cacacaacactggctttggcttgtggaccacctcctatgattcaatttgctgttaatccaaacttggagaagatg ggctatgacattaaggattccttattggtgttctaattttcaaaacaaaacaatatctgcaggaataaacttttt gtttcccctatcagttgtacatattgtatttggtatatcacccccatgtactacgcactatttgtagttcttaca tcttcttcttctttttaatttttttttaaaaccttaggatataaaggtttaattttctcttcctacaaagtgagt ctttagggaagaaatgttgtactgtactagtatgtctgagtcaaaaggttgtaatgtttaccatgacaaattgta ttcaattcctcgtggaatagtaacattgtgctcatgtgtcttcctgtaagagattcttcaaaatatcaatgtgtg tgtatatatatatatatatatatatatatatatatatatatatatatatatatatagtaattgcaacggtttgtt ccttttccctatgtggttaactgctcttaccttagcttctagtctctggtgaatatttctttctttttctaaaac tctttaatacggccttaaataagagaaaagtataaaccacgaatatcattatgcagacgatatggtaattaatct actttttgaaaaaaaattttctttatttggtccttgaaaataatattctagaaccttttgtatattcccttttaa cttctatttagtttt SEQ ID NO: 6 - coding sequence encoding the NtNIA1 S521N mutant. The sequence coding for the nitrate reductase kinase recognition site is highlighted and the specific mutation is underscored atggcggcatctgtcgaaaacaggcagttcagtcacatagaagccggtttatcccggtctttcaagcctcggtct gattccccggttcgtggctgcaacttccctccgcccaacagtactaatttccaaaagaaaccaaattccaccatt ttccttgattactcgtcgagtgaagacgacgatgatgatgacgaaaaaaatgagtaccttcaaatgatcaaaaaa gggaattcagaattagagccatctgttcatgacagcagggacgaaggtaccgctgataactggattgaacgcaac ttttccttgattcgtctcaccggaaagcatccatttaactccgaaccgccgttgaaccgtctcatgcaccacggt tttatcacaccggtcccacttcattacgttcgtaaccatggaccggttcccaagggcacatgggatgactggacc gtggaagtcacgggactagtgaaacgtcctatgaaattcacaatggaccagttggttaacgaattcccttccaga gaattgcccgttacgcttgtgtgtgctggcaaccgaaggaaagaacagaacatggttaaacaaaccattggtttc aactggggtgccgctgccgtttcaacaactgtatggcgcggggtacccctacgcgctttgttaaaacggtgcggt gtttttagcaagaataaaggggcgcttaatgtttgcttcgaaggagctgatgtcttgcccggaggcggtggttca aagtatggaaccagcattaagaaggaatttgcaatggatccagcacgagatatcataatagcttacatgcagaac ggagaaaaattggcacccgaccacgggtttccagtacgaatgataattccaggattcattggaggaagaatggtg aaatggataaagaggattatagtcaccacccaagaatcagacagctattatcatttcaaggacaatagagttctt cctccccatgttgatgctgaacttgcaaatactgaagcatggtggtacaagccagagtacatcatcaatgagctc aatattaactctgtcattacgacgccgtgtcatgaagaaattttgcctattaacgcctggacgactcagcgacct tacacgttgaggggctattcttattctggcggagggaaaaaagtaacgcgagtagaagtgaccttggatggagga gaaacatggcaagtttgcacactagatcacccagagaagcccaccaaatatggcaagtactggtgttggtgcttt tggtcactcgaggttgaggtgttagacttgctcagtgccaaagaaattgctgttcgagcttgggatgagaccctc aatactcaacctgagaagcttatttggaatgtcatgggaatgatgaacaattgctggttccgagtaaagatgaat gtgtgcaagcctcacaagggagagattggaatagtgtttgaacacccgactcaacctggaaaccaatcaggtgga tggatggcaaaggagaggcatttggagatatcagcagaggcacctccaacactaaagaagaatatctcaactcca ttcatgaacacagcttccaagatgtactccatgtcggaggtgaggaaacacagctctgctgactctgcttggatc atagtccatggtcatatctatgacgccacgcgtttcttgaaagatcaccccggtggttctgacagcattctcatc aatgctggcactgattgcactgaggaatttgatgcaattcattctgataaggctaagaagctattggaggaattc aggattggtgaactcataactactggttacacctctgactctcctggcaactccgtccatggatcttcttccttc agcagctttctagcacctattaaggaacttgttccagcgcagaggagtgtggccctcattccaagagagaaaatc ccatgcaaactcatcgacaaacaatccatctcccctgatgttaggaaatttcgatttgcattgccctctgaggat caagtcttgggcttgcctgttggtaaacacatcttcctctgtgccgttattgacgataagctctgcatgcgcgcc tacacgcctactagcacgatcgatgaggtggggtacttcgagttggttgtcaagatatacttcaaaggaattcac cctaaattccccaatggggggcaaatgtcacaataccttgattctctccaattagggtcatttctcgacgtgaaa ggtccattaggtcacattgaataccaaggaaagggcaatttcttagttcatggcaaacaaaagtttgccaagaag ttggccatgatagcaggtggaacagggataactccagtttatcaagtcatgcaggcaattctgaaagatccagaa gatgacacagaaatgtatgtggtctatgctaatagaacagaggatgatattttacttaaggaagagcttgattca tgggctgagaaaattccagaaagggttaaagtttggtatgtggttcaagattctattaaagaaggatggaagtac agccttggttttatttcagaagccattttgagagaacatatccctgagccatctcacacaacactggctttggct tgtggaccacctcctatgattcaatttgctgttaatccaaacttggagaagatgggctatgacattaaggattcc ttattggtgttctaa SEQ ID NO 7 - polypeptide sequence of the NtNIA1 S521N mutant. The consensus for the nitrate reductase kinase recognition is highlighted and the specific mutation is underscored MAASVENRQFSHIEAGLSRSFKPRSDSPVRGCNFPPPNSTNFQKKPNSTIFLDYSSSEDDDDDDEKNEYLQMIKK GNSELEPSVHDSRDEGTADNWIERNFSLIRLTGKHPFNSEPPLNRLMHHGFITPVPLHYVRNHGPVPKGTWDDWT VEVTGLVKRPMKFTMDQLVNEFPSRELPVTLVCAGNRRKEQNMVKQTIGFNWGAAAVSTTVWRGVPLRALLKRCG VFSKNKGALNVCFEGADVLPGGGGSKYGTSIKKEFAMDPARDIIIAYMQNGEKLAPDHGFPVRMIIPGFIGGRMV KWIKRIIVTTQESDSYYHFKDNRVLPPHVDAELANTEAWWYKPEYIINELNINSVITTPCHEEILPINAWTTQRP YTLRGYSYSGGGKKVTRVEVTLDGGETWQVCTLDHPEKPTKYGKYWCWCFWSLEVEVLDLLSAKEIAVRAWDETL NTQPEKLIWNVMGMMNNCWFRVKMNVCKPHKGEIGIVFEHPTQPGNQSGGWMAKERHLEISAEAPPTLKKNISTP FMNTASKMYSMSEVRKHSSADSAWIIVHGHIYDATRFLKDHPGGSDSILINAGTDCTEEFDAIHSDKAKKLLEEF RIGELITTGYTSDSPGNSVHGSSSFSSFLAPIKELVPAQRSVALIPREKIPCKLIDKQSISPDVRKFRFALPSED QVLGLPVGKHIFLCAVIDDKLCMRAYTPTSTIDEVGYFELVVKIYFKGIHPKFPNGGQMSQYLDSLQLGSFLDVK GPLGHIEYQGKGNFLVHGKQKFAKKLAMIAGGTGITPVYQVMQAILKDPEDDTEMYVVYANRTEDDILLKEELDS WAEKIPERVKVWYVVQDSIKEGWKYSLGFISEAILREHIPEPSHTTLALACGPPPMIQFAVNPNLEKMGYDIKDS LLVF* SEQ ID NO 8 - polypeptide sequence of nitrate reductase kinase recognition site in NtNIA1 with S521N mutation highlighted LKKNISTPFM SEQ ID NO: 9 Wild type polypeptide sequence encoded by the NtNIA2 gene, GenBank Accession number X14059, EC 1.6.6.1. The sequence which acts as the recognition site for binding of the nitrate reductase kinase responsible for the phosphorylation of NtNIA2 amino acid S523 is highlighted in bold. MAASVENRQFSHLEAGLSRSFKPRSDSPVRGCNFPSPNSTNFQKKPNSTIYLDYSSSEDDDDDDEKNEYLQMIKK GNSELEPSVHDTRDEGTADNWIERNFSMIRLTGKHPFNSEPPLNRLMHHGFITPVPLHYVRNHGPVPKGTWDDWT VEVTGLVKRPMKFTMDQLVNEFPCRELPVTLVCAGNRRKEQNMVKQTIGFNWGAAAVSTTIWRGVPLRALLKRCG VFSKNKGALNVCFEGADVLPGGGGSKYGTSIKKEFAMDPARDIIVAYMQNGEKLAPDHGFPVRMIIPGFIGGRMV KWIKRIIVTTQESDSYYHFKDNRVLPPHVDAELANTEAWWYKPEYIINELNINSVITTPCHEEILPINAWTTQRP YTLRGYSYSGGGKKVTRVEVTLDGGETWQVSTLDHPEKPTKYGKYWCWCFWSLEVEVLDLLSAKEIAVRAWDETL NTQPEKLIWNVMGMMNNCWFRVKMNVCKPHKGEIGIVFEHPTQPGNQSGGWMAKERHLEISAEAPQTLKKSISTP FMNTASKMYSMSEVRKHSSADSAWIIVHGHIYDATRFLKDHPGGTDSILINAGTDCTEEFDAIHSDKAKKLLEDF RIGELITTGYTSDSPGNSVHGSSSFSSFLAPIKELVPAQRSVALIPREKIPCKLIDKQSISHDVRKFRFALPSED QVLGLPVGKHIFLCAVIDDKLCMRAYTPTSTIDEVGYFELVVKIYFKGIHPKFPNGGQMSQYLDSMPLGSFLDVK GPLGHIEYQGKGNFLVHGKQKFAKKLAMIAGGTGITPVYQVMQAILKDPEDDTEMYVVYANRTEDDILLKEELDS WAEKIPERVKVWYVVQDSIKEGWKYSIGFITEAILREHIPEPSHTTLALACGPPPMIQFAVNPNLEKMGYDIKDS LLVF SEQ ID NO: 10 Wild type polynucleotide sequence of NtNIA2, GenBank Accession number X14059. The sequence which acts as the recognition site for binding of the nitrate reductase kinase responsible for the phosphorylation of NtNIA2 amino acid S523 is highlighted in bold. 1 tacatacaag ggcgcgaata aacttttttt aaagtaaatg tatatgaact tgcaatgaaa 61 gaggacctta acttgtttgt ctttgttgct ttctgcaaat ttcaccttaa cagcccattt 121 gagattgatt tagttagtta taacaattag ttaaatgctt gtgtaatttg aagaaaatat 181 ttggacgtgc tcgctgaaaa cattatactc ctatataata gaaatacttt ctgaaaagtt 241 ggtcttgttc aaaaacgtat aagagagttg gtcttctcat aaatagtcac tagctttctg 301 attttttttc actttctata tcacgtaaat aggtactcaa atttgatatt tacaccaaac 361 aaatgaaaat aggatatgtg tttttcatac gtatatttat ctatcgtact taatgataca 421 tacatataca tataacctta ctttttgatt actaaaaatt taattatatt taatttgggt 481 aaatatcaga tgccacaaaa catttaccta gccactgttt ttgactacta aaaatttaat 541 tatgtttagc ttgggtaaat atcagatgtc actaaacatt ttacctagcc attcctccga 601 aaagaaattg agaaggaaat tagagttagt ggagccataa taatgtttaa tgtgaccata 661 actcggtgaa aaccacggca agaataagaa acagctgtta aggctaacca acagctgcat 721 atctttaagc catttgctat taccccaaca tcgcatcttc ctctgatccc gaccctacgg 781 gcgtaaaaag tgtaaatcgt tagaattgtt ttatttattt tatgatgtca ctatttttta 841 aaatcaaaat taaattgggg tgtcgatttt tttgggtcct gcttatgtat agtatggcgc 901 tatggaggca ctgagagagt ccgaaacgtt tctatataag gccaccccac gcattcacaa 961 acttcgttcc caaacagaac aagaaaatca aatctcggag agagagagag agaaatattt 1021 tgagagagaa atacagaaaa tctctcttcc ttctttcctt tttttttcaa tccccattca 1081 tattcttttt ttagaataat ctatggcggc atctgtcgaa aacaggcagt tcagtcacct 1141 agaagccggt ttatcccggt ctttcaagcc ccggtctgat tccccggttc gtggctgcaa 1201 cttcccttcg cccaacagta ctaatttcca aaagaaacca aattccacca tttaccttga 1261 ttactcgtcg agtgaagacg acgatgatga tgacgaaaaa aatgagtacc ttcaaatgat 1321 taaaaaaggg aattcagagt tagagccatc tgttcatgac actagggacg aaggtaccgc 1381 tgataattgg attgaacgca acttttccat gattcgtctc accggaaagc atccatttaa 1441 ctccgaacca ccgttgaacc ggctcatgca ccacggcttt atcacaccgg tcccacttca 1501 ttacgttcgt aaccatggac cggttcccaa gggcacgtgg gatgactgga ccgtggaagt 1561 cacgggacta gtgaagcgtc ctatgaaatt cacaatggac cagttggtta acgaattccc 1621 ttgtagagaa ttgcccgtta cgcttgtttg tgctggcaat cgaaggaaag aacagaacat 1681 ggttaaacaa accattggtt tcaactgggg cgccgctgcc gtttcaacaa cgatatggcg 1741 cggggtaccc ctccgcgctt tgctaaaacg gtgcggtgtt tttagcaaga ataaaggggc 1801 gcttaatgtt tgcttcgaag gagctgatgt gttgcccgga ggtggtggtt caaagtatgg 1861 aaccagcatt aagaaggaat ttgcaatgga tccagcacga gatatcatcg tagcctacat 1921 gcagaacgga gaaaaattgg cacccgacca cgggtttcca gtacgaatga taattccagg 1981 attcattgga ggaagaatgg tgaaatggat aaagaggatt atagtcacca cccaagaatc 2041 agacagctat tatcatttca aggacaatag agttcttcct ccccatgttg atgctgaact 2101 tgcaaatacc gaaggtacgt accgtaacta tttcaattta ttactccatt tgttccaatt 2161 tatgtgaacc tatttccttt ttggtccgtt caaaaaagaa tgaacccttt ctaaatttgg 2221 taacaattta gcttaaactt acaacttcac ccttaatgag aaacttttat aaccacacaa 2281 ataccctggg gcccatttgg acttgtttag gtcgacaaat tccaaaagtt ttattttttt 2341 cttaaacttc gtgctcagtc aaacaggttc acgtaaattg aaacggagag agtatcattt 2401 ttattaaggg gtataaatat attttaatta gttgagactt gcacatacaa gtaaaatatt 2461 tcttagaata caaaatcaac tgaaagctta cttctaatta tatggttttg aattttcctt 2521 tcaatgaagt aaataaaaag gaaacaatta tattcaacgc atgtaggtat atggtcctgt 2581 cattatctca aatcaaatgg tttaaagaca aaggactttg gaaacataga attgtcagct 2641 ttatagttat ggagtactat attagttagc tgtttgcatc tattcataat tggtctatct 2701 gtgtgcagca tggtggtaca agccagagta tatcatcaat gagcttaata ttaactctgt 2761 cattacgacg ccgtgtcatg aagaaatttt gccaattaac gcctggacga ctcagcgacc 2821 ttacacgttg aggggctatt cttattctgg ttagtatttt tatattttcc gattttgctg 2881 agaatatcat atttcttagt tttgtcgata catcgtatcc tctaactctg acgttttact 2941 tcgtccttat gcacccactt acgtccttac tttctcagac agtttattga tgaaaactac 3001 ttactatttt cgacccgata gcctcagcgt ccttaattaa atgtgatgtt ttgaaagaga 3061 tattctctcc cgtctatttt aattaatttt tggctgtttt tatacgtggg aatctatttt 3121 taacattaat taatatagaa atgaaccata ttaatattat taatttcttc attgaaaata 3181 caacaaatac tcttcggctc ttactacaat gacaattttg aagaaaaata attaattcct 3241 tcctaatatc tgaaaaatca aatattgtgg accataaaaa aaggtcaaaa aattaattaa 3301 aatgaactgg agagagtaaa ttagaaaata taattatagc actagtaatt aaagttatta 3361 gatgtcttct ttaaaaagcg tgtgaaaact ttaaagacga aatataatat gaatattatc 3421 taatacttag aaagtgtcaa taattggtag acaatttaaa ctatatacta gttaaaaagt 3481 ctgtcaatac aactattagt attggggatt agagagaata gtagtaaaat ggagtaattg 3541 gacgcatgag cttgggcatg ctgattgctg tcagcttgtt tgctaatgtg aaaaagaaaa 3601 tagtaagaaa aggccaacat ggttttgttt attttattat gtggtagtac acaaaaacct 3661 ggggagcttt cctagttctg aagagtcggt ctttggtagc acaaaattaa tagtatagta 3721 taccaagtga atattaaatt caattgtcta aagcacggaa tctttttgac tactttagtt 3781 cctgcatctt gggttgcctc aacaacaccc tttattgaat tattatagta atgttcaata 3841 taatatacaa ttagaaaaca ctctaagtgg tcactttata tggatctagt caatactatt 3901 tcttctaaac aacgtgccta attacttccc actttccagt acatgaccac cattaagttt 3961 aatttttgtc aattccttgt gcaattggcc cttcaaatga gcagaagtgt tacgtaggaa 4021 aactaacttc agctactatt ataggagtaa acctgttagg aaaagatgct cgaggaactg 4081 acaaaacttg tagaataatt agccattgta ttgattgaaa tactgattgt gaacgtgtaa 4141 caaacaggcg gagggaaaaa agtaacgcga gtagaagtga cgttggatgg aggagaaaca 4201 tggcaagtta gcacactaga tcacccagag aagcccacca aatatggcaa gtactggtgt 4261 tggtgctttt ggtcactcga ggttgaggtg ttagacttgc tcagtgctaa agaaattgct 4321 gttcgagctt gggatgagac cctcaatact caacccgaga agcttatttg gaacgtcatg 4381 gtacgttcac ttcttctttt acctttattt cttttaactt ctatatacta gcggtgtaaa 4441 gttattttac accataagtt aacttacaaa aatatgtaac tatttatact acgagtgatg 4501 agggcaagaa ggggtttaag tatttgacaa taaatgtaaa ccctgcaatt ttgttcctaa 4561 ttttttatcc tttcaactct ttgtgattgc ttcattatct agattcacag agcacatgtg 4621 ttcacatgcc aaaacaaaaa actacaaaca aaaaaacttt tcactagctt tagtctaaga 4681 ttcccctttt tttttttggg aggtgtgtgg tccatactcc atagatcaat tccagccact 4741 gacgtaccaa accctgaaaa ttcctagtag ttatagcgac gtacaatcat ttcatattat 4801 gtaagcagag acgtgatcac atgaactaga tgtgaatacc acttgcccag tccaccaggt 4861 caattcatct agatgtgtaa atcttgacac cagcactggg tcacttttat aacactagca 4921 tttaacaaca tttcatcctt gaacattact tgggctaatt aataagtatt tttttttata 4981 tactctaaaa attgtaatta cataaatgaa tttaacttat acacgctgac aatgttacta 5041 attccacttt ttacggacgg ttatctatag aaatcattta ggtgaaacaa ttctcttaca 5101 ctatgatcag tgttagtaca taatggttat tacattttct aaatattgtg ctatgttgca 5161 atgttcaggg aatgatgaat aattgctggt tccgagtaaa gatgaatgtg tgcaagcctc 5221 acaagggaga gattggaata gtgtttgagc atccgactca acctggaaac caatcaggtg 5281 gatggatggc gaaggagaga catttggaga tatcagcaga ggcacctcaa acactaaaga 5341 agagtatctc aactccattc atgaacacag cttccaagat gtactccatg tccgaggtca 5401 ggaaacacag ctctgctgac tctgcttgga tcatagtcca tggtcatatc tatgacgcca 5461 cgcgtttctt gaaagatcac cctggtggga ctgacagcat tctcatcaat gctggcactg 5521 attgcactga ggaatttgat gcaattcatt ctgataaggc taagaagctc ttggaggatt 5581 tcaggattgg tgaactcata actactggtt acacctctga ctctcctggc aactccgtgc 5641 acggatcttc ttccttcagc agctttctag cacctattaa ggaacttgtt ccagcgcaga 5701 ggagtgtggc cctaattcca agagagaaaa tcccatgcaa actcatcgac aagcaatcca 5761 tctcccatga tgttaggaaa tttcgatttg cattgccctc tgaggatcaa gtcttgggct 5821 tgcctgttgg aaaacatatc ttcctctgtg ccgttattga cgataagctc tgcatgcgcg 5881 cttacacgcc tactagcacg atcgatgagg tggggtactt cgagttggtt gtcaagatat 5941 acttcaaagg aattcaccct aaattcccca atggagggca aatgtcacag tatcttgatt 6001 ctatgccgtt agggtcattt ctcgacgtga aaggtccatt aggtcacatt gaataccaag 6061 gaaagggaaa tttcttagtt catggcaaac agaagtttgc caagaagttg gccatgatag 6121 caggtggaac aggaataact ccagtgtatc aagtcatgca ggcaattctg aaagatccag 6181 aagatgacac agaaatgtat gtggtgtatg ctaacagaac agaggatgat attttactta 6241 aggaagagct tgattcatgg gctgagaaaa ttccagagag ggttaaagtt tggtatgtgg 6301 ttcaggattc tattaaagaa ggatggaagt acagcattgg ttttattaca gaagccattt 6361 tgagagaaca tatccctgag ccatctcaca caacactggc tttggcttgt ggaccacctc 6421 ctatgattca atttgctgtt aatccaaact tggagaagat gggctatgac attaaggatt 6481 ccttattggt gttctaattt taaaaacaaa acaatatctg caggaataaa tttttttttt 6541 ccccctatca gttgtacata ttgtatttgg tttatcaccc ccatgtacta cgtagtgttt 6601 gtagttctta catttttatt ttttagaatt tttttaaacc ttaggatata aaggttttct 6661 cttccaacaa agtgattctt tagggaagaa atgtactgta ctgtactagt atgtctaagc 6721 cgaaagttgt aatgtttacc atgacaaatt gtattcaatt cctcatggaa tagtaacatt 6781 gtgttcatgt gtcttcctgt aagcgatctt caaaatatca atgtatatat atagtaattg 6841 caaaccattg ttccttttcc cgatgtagtt aactactctt tctttagctt ctagtctctg 6901 gtgaatattt ttttttctat aactctttaa ttaatacggc cttaaataag agaaaagttt 6961 aaaccacgaa tatcattatg cagacgtata ggtaattaat ctactttttg aaaaaaaatc 7021 tattttcttt atgtggtcct tcaaaataat attctagaac cttttgtata ttccctttta 7081 acttctattt agtttt SEQ ID NO: 11 Mutant M527I polypeptide of NtNIA2. The mutated polypeptide in the recognition site for binding of the nitrate reductase kinase is highlighted in bold and underline. MAASVENRQFSHLEAGLSRSFKPRSDSPVRGCNFPSPNSTNFQKKPNSTIYLDYSSSEDDDDDDEKNEYLQMIKK GNSELEPSVHDTRDEGTADNWIERNFSMIRLTGKHPFNSEPPLNRLMHHGFITPVPLHYVRNHGPVPKGTWDDWT VEVTGLVKRPMKFTMDQLVNEFPCRELPVTLVCAGNRRKEQNMVKQTIGFNWGAAAVSTTIWRGVPLRALLKRCG VFSKNKGALNVCFEGADVLPGGGGSKYGTSIKKEFAMDPARDIIVAYMQNGEKLAPDHGFPVRMIIPGFIGGRMV KWIKRIIVTTQESDSYYHFKDNRVLPPHVDAELANTEAWWYKPEYIINELNINSVITTPCHEEILPINAWTTQRP YTLRGYSYSGGGKKVTRVEVTLDGGETWQVSTLDHPEKPTKYGKYWCWCFWSLEVEVLDLLSAKEIAVRAWDETL NTQPEKLIWNVMGMMNNCWFRVKMNVCKPHKGEIGIVFEHPTQPGNQSGGWMAKERHLEISAEAPQTLKKSISTP FINTASKMYSMSEVRKHSSADSAWIIVHGHIYDATRFLKDHPGGTDSILINAGTDCTEEFDAIHSDKAKKLLEDF RIGELITTGYTSDSPGNSVHGSSSFSSFLAPIKELVPAQRSVALIPREKIPCKLIDKQSISHDVRKFRFALPSED QVLGLPVGKHIFLCAVIDDKLCMRAYTPTSTIDEVGYFELVVKIYFKGIHPKFPNGGQMSQYLDSMPLGSFLDVK GPLGHIEYQGKGNFLVHGKQKFAKKLAMIAGGTGITPVYQVMQAILKDPEDDTEMYVVYANRTEDDILLKEELDS WAEKIPERVKVWYVVQDSIKEGWKYSIGFITEAILREHIPEPSHTTLALACGPPPMIQFAVNPNLEKMGYDIKDS LLVF SEQ ID NO: 12 Mutant M527I polynucleotide sequence of NtNIA2. The sequence which acts as the recognition site for binding of the nitrate reductase kinase responsible for the phosphorylation of NtNIA2 amino acid S523 is highlighted in bold. The g to a mutation is underlined. 1 tacatacaag ggcgcgaata aacttttttt aaagtaaatg tatatgaact tgcaatgaaa 61 gaggacctta acttgtttgt ctttgttgct ttctgcaaat ttcaccttaa cagcccattt 121 gagattgatt tagttagtta taacaattag ttaaatgctt gtgtaatttg aagaaaatat 181 ttggacgtgc tcgctgaaaa cattatactc ctatataata gaaatacttt ctgaaaagtt 241 ggtcttgttc aaaaacgtat aagagagttg gtcttctcat aaatagtcac tagctttctg 301 attttttttc actttctata tcacgtaaat aggtactcaa atttgatatt tacaccaaac 361 aaatgaaaat aggatatgtg tttttcatac gtatatttat ctatcgtact taatgataca 421 tacatataca tataacctta ctttttgatt actaaaaatt taattatatt taatttgggt 481 aaatatcaga tgccacaaaa catttaccta gccactgttt ttgactacta aaaatttaat 541 tatgtttagc ttgggtaaat atcagatgtc actaaacatt ttacctagcc attcctccga 601 aaagaaattg agaaggaaat tagagttagt ggagccataa taatgtttaa tgtgaccata 661 actcggtgaa aaccacggca agaataagaa acagctgtta aggctaacca acagctgcat 721 atctttaagc catttgctat taccccaaca tcgcatcttc ctctgatccc gaccctacgg 781 gcgtaaaaag tgtaaatcgt tagaattgtt ttatttattt tatgatgtca ctatttttta 841 aaatcaaaat taaattgggg tgtcgatttt tttgggtcct gcttatgtat agtatggcgc 901 tatggaggca ctgagagagt ccgaaacgtt tctatataag gccaccccac gcattcacaa 961 acttcgttcc caaacagaac aagaaaatca aatctcggag agagagagag agaaatattt 1021 tgagagagaa atacagaaaa tctctcttcc ttctttcctt tttttttcaa tccccattca 1081 tattcttttt ttagaataat ctatggcggc atctgtcgaa aacaggcagt tcagtcacct 1141 agaagccggt ttatcccggt ctttcaagcc ccggtctgat tccccggttc gtggctgcaa 1201 cttcccttcg cccaacagta ctaatttcca aaagaaacca aattccacca tttaccttga 1261 ttactcgtcg agtgaagacg acgatgatga tgacgaaaaa aatgagtacc ttcaaatgat 1321 taaaaaaggg aattcagagt tagagccatc tgttcatgac actagggacg aaggtaccgc 1381 tgataattgg attgaacgca acttttccat gattcgtctc accggaaagc atccatttaa 1441 ctccgaacca ccgttgaacc ggctcatgca ccacggcttt atcacaccgg tcccacttca 1501 ttacgttcgt aaccatggac cggttcccaa gggcacgtgg gatgactgga ccgtggaagt 1561 cacgggacta gtgaagcgtc ctatgaaatt cacaatggac cagttggtta acgaattccc 1621 ttgtagagaa ttgcccgtta cgcttgtttg tgctggcaat cgaaggaaag aacagaacat 1681 ggttaaacaa accattggtt tcaactgggg cgccgctgcc gtttcaacaa cgatatggcg 1741 cggggtaccc ctccgcgctt tgctaaaacg gtgcggtgtt tttagcaaga ataaaggggc 1801 gcttaatgtt tgcttcgaag gagctgatgt gttgcccgga ggtggtggtt caaagtatgg 1861 aaccagcatt aagaaggaat ttgcaatgga tccagcacga gatatcatcg tagcctacat 1921 gcagaacgga gaaaaattgg cacccgacca cgggtttcca gtacgaatga taattccagg 1981 attcattgga ggaagaatgg tgaaatggat aaagaggatt atagtcacca cccaagaatc 2041 agacagctat tatcatttca aggacaatag agttcttcct ccccatgttg atgctgaact 2101 tgcaaatacc gaaggtacgt accgtaacta tttcaattta ttactccatt tgttccaatt 2161 tatgtgaacc tatttccttt ttggtccgtt caaaaaagaa tgaacccttt ctaaatttgg 2221 taacaattta gcttaaactt acaacttcac ccttaatgag aaacttttat aaccacacaa 2281 ataccctggg gcccatttgg acttgtttag gtcgacaaat tccaaaagtt ttattttttt 2341 cttaaacttc gtgctcagtc aaacaggttc acgtaaattg aaacggagag agtatcattt 2401 ttattaaggg gtataaatat attttaatta gttgagactt gcacatacaa gtaaaatatt 2461 tcttagaata caaaatcaac tgaaagctta cttctaatta tatggttttg aattttcctt 2521 tcaatgaagt aaataaaaag gaaacaatta tattcaacgc atgtaggtat atggtcctgt 2581 cattatctca aatcaaatgg tttaaagaca aaggactttg gaaacataga attgtcagct 2641 ttatagttat ggagtactat attagttagc tgtttgcatc tattcataat tggtctatct 2701 gtgtgcagca tggtggtaca agccagagta tatcatcaat gagcttaata ttaactctgt 2761 cattacgacg ccgtgtcatg aagaaatttt gccaattaac gcctggacga ctcagcgacc 2821 ttacacgttg aggggctatt cttattctgg ttagtatttt tatattttcc gattttgctg 2881 agaatatcat atttcttagt tttgtcgata catcgtatcc tctaactctg acgttttact 2941 tcgtccttat gcacccactt acgtccttac tttctcagac agtttattga tgaaaactac 3001 ttactatttt cgacccgata gcctcagcgt ccttaattaa atgtgatgtt ttgaaagaga 3061 tattctctcc cgtctatttt aattaatttt tggctgtttt tatacgtggg aatctatttt 3121 taacattaat taatatagaa atgaaccata ttaatattat taatttcttc attgaaaata 3181 caacaaatac tcttcggctc ttactacaat gacaattttg aagaaaaata attaattcct 3241 tcctaatatc tgaaaaatca aatattgtgg accataaaaa aaggtcaaaa aattaattaa 3301 aatgaactgg agagagtaaa ttagaaaata taattatagc actagtaatt aaagttatta 3361 gatgtcttct ttaaaaagcg tgtgaaaact ttaaagacga aatataatat gaatattatc 3421 taatacttag aaagtgtcaa taattggtag acaatttaaa ctatatacta gttaaaaagt 3481 ctgtcaatac aactattagt attggggatt agagagaata gtagtaaaat ggagtaattg 3541 gacgcatgag cttgggcatg ctgattgctg tcagcttgtt tgctaatgtg aaaaagaaaa 3601 tagtaagaaa aggccaacat ggttttgttt attttattat gtggtagtac acaaaaacct 3661 ggggagcttt cctagttctg aagagtcggt ctttggtagc acaaaattaa tagtatagta 3721 taccaagtga atattaaatt caattgtcta aagcacggaa tctttttgac tactttagtt 3781 cctgcatctt gggttgcctc aacaacaccc tttattgaat tattatagta atgttcaata 3841 taatatacaa ttagaaaaca ctctaagtgg tcactttata tggatctagt caatactatt 3901 tcttctaaac aacgtgccta attacttccc actttccagt acatgaccac cattaagttt 3961 aatttttgtc aattccttgt gcaattggcc cttcaaatga gcagaagtgt tacgtaggaa 4021 aactaacttc agctactatt ataggagtaa acctgttagg aaaagatgct cgaggaactg 4081 acaaaacttg tagaataatt agccattgta ttgattgaaa tactgattgt gaacgtgtaa 4141 caaacaggcg gagggaaaaa agtaacgcga gtagaagtga cgttggatgg aggagaaaca 4201 tggcaagtta gcacactaga tcacccagag aagcccacca aatatggcaa gtactggtgt 4261 tggtgctttt ggtcactcga ggttgaggtg ttagacttgc tcagtgctaa agaaattgct 4321 gttcgagctt gggatgagac cctcaatact caacccgaga agcttatttg gaacgtcatg 4381 gtacgttcac ttcttctttt acctttattt cttttaactt ctatatacta gcggtgtaaa 4441 gttattttac accataagtt aacttacaaa aatatgtaac tatttatact acgagtgatg 4501 agggcaagaa ggggtttaag tatttgacaa taaatgtaaa ccctgcaatt ttgttcctaa 4561 ttttttatcc tttcaactct ttgtgattgc ttcattatct agattcacag agcacatgtg 4621 ttcacatgcc aaaacaaaaa actacaaaca aaaaaacttt tcactagctt tagtctaaga 4681 ttcccctttt tttttttggg aggtgtgtgg tccatactcc atagatcaat tccagccact 4741 gacgtaccaa accctgaaaa ttcctagtag ttatagcgac gtacaatcat ttcatattat 4801 gtaagcagag acgtgatcac atgaactaga tgtgaatacc acttgcccag tccaccaggt 4861 caattcatct agatgtgtaa atcttgacac cagcactggg tcacttttat aacactagca 4921 tttaacaaca tttcatcctt gaacattact tgggctaatt aataagtatt tttttttata 4981 tactctaaaa attgtaatta cataaatgaa tttaacttat acacgctgac aatgttacta 5041 attccacttt ttacggacgg ttatctatag aaatcattta ggtgaaacaa ttctcttaca 5101 ctatgatcag tgttagtaca taatggttat tacattttct aaatattgtg ctatgttgca 5161 atgttcaggg aatgatgaat aattgctggt tccgagtaaa gatgaatgtg tgcaagcctc 5221 acaagggaga gattggaata gtgtttgagc atccgactca acctggaaac caatcaggtg 5281 gatggatggc gaaggagaga catttggaga tatcagcaga ggcacctcaa acactaaaga 5341 agagtatctc aactccattc ataaacacag cttccaagat gtactccatg tccgaggtca 5401 ggaaacacag ctctgctgac tctgcttgga tcatagtcca tggtcatatc tatgacgcca 5461 cgcgtttctt gaaagatcac cctggtggga ctgacagcat tctcatcaat gctggcactg 5521 attgcactga ggaatttgat gcaattcatt ctgataaggc taagaagctc ttggaggatt 5581 tcaggattgg tgaactcata actactggtt acacctctga ctctcctggc aactccgtgc 5641 acggatcttc ttccttcagc agctttctag cacctattaa ggaacttgtt ccagcgcaga 5701 ggagtgtggc cctaattcca agagagaaaa tcccatgcaa actcatcgac aagcaatcca 5761 tctcccatga tgttaggaaa tttcgatttg cattgccctc tgaggatcaa gtcttgggct 5821 tgcctgttgg aaaacatatc ttcctctgtg ccgttattga cgataagctc tgcatgcgcg 5881 cttacacgcc tactagcacg atcgatgagg tggggtactt cgagttggtt gtcaagatat 5941 acttcaaagg aattcaccct aaattcccca atggagggca aatgtcacag tatcttgatt 6001 ctatgccgtt agggtcattt ctcgacgtga aaggtccatt aggtcacatt gaataccaag 6061 gaaagggaaa tttcttagtt catggcaaac agaagtttgc caagaagttg gccatgatag 6121 caggtggaac aggaataact ccagtgtatc aagtcatgca ggcaattctg aaagatccag 6181 aagatgacac agaaatgtat gtggtgtatg ctaacagaac agaggatgat attttactta 6241 aggaagagct tgattcatgg gctgagaaaa ttccagagag ggttaaagtt tggtatgtgg 6301 ttcaggattc tattaaagaa ggatggaagt acagcattgg ttttattaca gaagccattt 6361 tgagagaaca tatccctgag ccatctcaca caacactggc tttggcttgt ggaccacctc 6421 ctatgattca atttgctgtt aatccaaact tggagaagat gggctatgac attaaggatt 6481 ccttattggt gttctaattt taaaaacaaa acaatatctg caggaataaa tttttttttt 6541 ccccctatca gttgtacata ttgtatttgg tttatcaccc ccatgtacta cgtagtgttt 6601 gtagttctta catttttatt ttttagaatt tttttaaacc ttaggatata aaggttttct 6661 cttccaacaa agtgattctt tagggaagaa atgtactgta ctgtactagt atgtctaagc 6721 cgaaagttgt aatgtttacc atgacaaatt gtattcaatt cctcatggaa tagtaacatt 6781 gtgttcatgt gtcttcctgt aagcgatctt caaaatatca atgtatatat atagtaattg 6841 caaaccattg ttccttttcc cgatgtagtt aactactctt tctttagctt ctagtctctg 6901 gtgaatattt ttttttctat aactctttaa ttaatacggc cttaaataag agaaaagttt 6961 aaaccacgaa tatcattatg cagacgtata ggtaattaat ctactttttg aaaaaaaatc 7021 tattttcttt atgtggtcct tcaaaataat attctagaac cttttgtata ttccctttta 7081 acttctattt agtttt SEQ ID NO: 13 Recognition site for binding of the nitrate reductase kinase responsible for the phosphorylation of NtNIA2 amino acid S523 from the wild type polypeptide sequence of NtNIA2, GenBank Accession number X14059. LKKSISTPFM SEQ ID NO: 14 Mutant M527I polypeptide sequence of NtNIA2, mutation is identified in bold LKKSISTPFI SEQ ID NO: 15 – polynucleotide sequence encoding the NtNIA1 S521N mutant in the hinge 1 site with the mutation indicated ctaaagaagagtatctcaactccattcatg SEQ ID NO: 16 Coding polynucleotide sequence of NtNIA2, GenBank Accession number X14059. The sequence which acts as the recognition site for binding of the nitrate reductase kinase responsible for the phosphorylation of NtNIA2 amino acid S523 is highlighted in bold and underline. atggcggcatctgtcgaaaacaggcagttcagtcacctagaagccggtttatcccggtctttcaagccc cggtctgattccccggttcgtggctgcaacttcccttcgcccaacagtactaatttccaaaagaaacca aattccaccatttaccttgattactcgtcgagtgaagacgacgatgatgatgacgaaaaaaatgagtac cttcaaatgattaaaaaagggaattcagagttagagccatctgttcatgacactagggacgaaggtacc gctgataattggattgaacgcaacttttccatgattcgtctcaccggaaagcatccatttaactccgaa ccaccgttgaaccggctcatgcaccacggctttatcacaccggtcccacttcattacgttcgtaaccat ggaccggttcccaagggcacgtgggatgactggaccgtggaagtcacgggactagtgaagcgtcctatg aaattcacaatggaccagttggttaacgaattcccttgtagagaattgcccgttacgcttgtttgtgct ggcaatcgaaggaaagaacagaacatggttaaacaaaccattggtttcaactggggcgccgctgccgtt tcaacaacgatatggcgcggggtacccctccgcgctttgctaaaacggtgcggtgtttttagcaagaat aaaggggcgcttaatgtttgcttcgaaggagctgatgtgttgcccggaggtggtggttcaaagtatgga accagcattaagaaggaatttgcaatggatccagcacgagatatcatcgtagcctacatgcagaacgga gaaaaattggcacccgaccacgggtttccagtacgaatgataattccaggattcattggaggaagaatg gtgaaatggataaagaggattatagtcaccacccaagaatcagacagctattatcatttcaaggacaat agagttcttcctccccatgttgatgctgaacttgcaaataccgaagcatggtggtacaagccagagtat atcatcaatgagcttaatattaactctgtcattacgacgccgtgtcatgaagaaattttgccaattaac gcctggacgactcagcgaccttacacgttgaggggctattcttattctggcggagggaaaaaagtaacg cgagtagaagtgacgttggatggaggagaaacatggcaagttagcacactagatcacccagagaagccc accaaatatggcaagtactggtgttggtgcttttggtcactcgaggttgaggtgttagacttgctcagt gctaaagaaattgctgttcgagcttgggatgagaccctcaatactcaacccgagaagcttatttggaac gtcatgggaatgatgaataattgctggttccgagtaaagatgaatgtgtgcaagcctcacaagggagag attggaatagtgtttgagcatccgactcaacctggaaaccaatcaggtggatggatggcgaaggagaga catttggagatatcagcagaggcacctcaaacactaaagaagagtatctcaactccattcatgaacaca gcttccaagatgtactccatgtccgaggtcaggaaacacagctctgctgactctgcttggatcatagtc catggtcatatctatgacgccacgcgtttcttgaaagatcaccctggtgggactgacagcattctcatc aatgctggcactgattgcactgaggaatttgatgcaattcattctgataaggctaagaagctcttggag gatttcaggattggtgaactcataactactggttacacctctgactctcctggcaactccgtgcacgga tcttcttccttcagcagctttctagcacctattaaggaacttgttccagcgcagaggagtgtggcccta attccaagagagaaaatcccatgcaaactcatcgacaagcaatccatctcccatgatgttaggaaattt cgatttgcattgccctctgaggatcaagtcttgggcttgcctgttggaaaacatatcttcctctgtgcc gttattgacgataagctctgcatgcgcgcttacacgcctactagcacgatcgatgaggtggggtacttc gagttggttgtcaagatatacttcaaaggaattcaccctaaattccccaatggagggcaaatgtcacag tatcttgattctatgccgttagggtcatttctcgacgtgaaaggtccattaggtcacattgaataccaa ggaaagggaaatttcttagttcatggcaaacagaagtttgccaagaagttggccatgatagcaggtgga acaggaataactccagtgtatcaagtcatgcaggcaattctgaaagatccagaagatgacacagaaatg tatgtggtgtatgctaacagaacagaggatgatattttacttaaggaagagcttgattcatgggctgag aaaattccagagagggttaaagtttggtatgtggttcaggattctattaaagaaggatggaagtacagc attggttttattacagaagccattttgagagaacatatccctgagccatctcacacaacactggctttg gcttgtggaccacctcctatgattcaatttgctgttaatccaaacttggagaagatgggctatgacatt aaggattccttattggtgttctaa SEQ ID NO: 17 Coding sequence of mutant M527I polypeptide of NtNIA2. The mutated polynucleotide in the recognition site for binding of the nitrate reductase kinase is highlighted in bold and underline. atggcggcatctgtcgaaaacaggcagttcagtcacctagaagccggtttatcccggtctttcaagccc cggtctgattccccggttcgtggctgcaacttcccttcgcccaacagtactaatttccaaaagaaacca aattccaccatttaccttgattactcgtcgagtgaagacgacgatgatgatgacgaaaaaaatgagtac cttcaaatgattaaaaaagggaattcagagttagagccatctgttcatgacactagggacgaaggtacc gctgataattggattgaacgcaacttttccatgattcgtctcaccggaaagcatccatttaactccgaa ccaccgttgaaccggctcatgcaccacggctttatcacaccggtcccacttcattacgttcgtaaccat ggaccggttcccaagggcacgtgggatgactggaccgtggaagtcacgggactagtgaagcgtcctatg aaattcacaatggaccagttggttaacgaattcccttgtagagaattgcccgttacgcttgtttgtgct ggcaatcgaaggaaagaacagaacatggttaaacaaaccattggtttcaactggggcgccgctgccgtt tcaacaacgatatggcgcggggtacccctccgcgctttgctaaaacggtgcggtgtttttagcaagaat aaaggggcgcttaatgtttgcttcgaaggagctgatgtgttgcccggaggtggtggttcaaagtatgga accagcattaagaaggaatttgcaatggatccagcacgagatatcatcgtagcctacatgcagaacgga gaaaaattggcacccgaccacgggtttccagtacgaatgataattccaggattcattggaggaagaatg gtgaaatggataaagaggattatagtcaccacccaagaatcagacagctattatcatttcaaggacaat agagttcttcctccccatgttgatgctgaacttgcaaataccgaagcatggtggtacaagccagagtat atcatcaatgagcttaatattaactctgtcattacgacgccgtgtcatgaagaaattttgccaattaac gcctggacgactcagcgaccttacacgttgaggggctattcttattctggcggagggaaaaaagtaacg cgagtagaagtgacgttggatggaggagaaacatggcaagttagcacactagatcacccagagaagccc accaaatatggcaagtactggtgttggtgcttttggtcactcgaggttgaggtgttagacttgctcagt gctaaagaaattgctgttcgagcttgggatgagaccctcaatactcaacccgagaagcttatttggaac gtcatgggaatgatgaataattgctggttccgagtaaagatgaatgtgtgcaagcctcacaagggagag attggaatagtgtttgagcatccgactcaacctggaaaccaatcaggtggatggatggcgaaggagaga catttggagatatcagcagaggcacctcaaacactaaagaagagtatctcaactccattcataaacaca gcttccaagatgtactccatgtccgaggtcaggaaacacagctctgctgactctgcttggatcatagtc catggtcatatctatgacgccacgcgtttcttgaaagatcaccctggtgggactgacagcattctcatc aatgctggcactgattgcactgaggaatttgatgcaattcattctgataaggctaagaagctcttggag gatttcaggattggtgaactcataactactggttacacctctgactctcctggcaactccgtgcacgga tcttcttccttcagcagctttctagcacctattaaggaacttgttccagcgcagaggagtgtggcccta attccaagagagaaaatcccatgcaaactcatcgacaagcaatccatctcccatgatgttaggaaattt cgatttgcattgccctctgaggatcaagtcttgggcttgcctgttggaaaacatatcttcctctgtgcc gttattgacgataagctctgcatgcgcgcttacacgcctactagcacgatcgatgaggtggggtacttc gagttggttgtcaagatatacttcaaaggaattcaccctaaattccccaatggagggcaaatgtcacag tatcttgattctatgccgttagggtcatttctcgacgtgaaaggtccattaggtcacattgaataccaa ggaaagggaaatttcttagttcatggcaaacagaagtttgccaagaagttggccatgatagcaggtgga acaggaataactccagtgtatcaagtcatgcaggcaattctgaaagatccagaagatgacacagaaatg tatgtggtgtatgctaacagaacagaggatgatattttacttaaggaagagcttgattcatgggctgag aaaattccagagagggttaaagtttggtatgtggttcaggattctattaaagaaggatggaagtacagc attggttttattacagaagccattttgagagaacatatccctgagccatctcacacaacactggctttg gcttgtggaccacctcctatgattcaatttgctgttaatccaaacttggagaagatgggctatgacatt aaggattccttattggtgttctaa SEQ ID NO: 18 NtNIA1 forward primer gaaataacgctcttacactatgag SEQ ID NO: 19 NtNIA1 reverse primer gcaaatcgaaatttcctaacatcag

Claims

CLAIMS 1. A Nicotiana tabacum plant cell comprising: (a) a polynucleotide sequence encoding a NIA1 nitrate reductase polypeptide comprising a contiguous polypeptide sequence of SEQ ID NO: 4, wherein the serine at position 4 of SEQ ID NO: 4 is substituted for an amino acid that reduces nitrate levels 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).

2. The Nicotiana tabacum plant cell according to claim 1, wherein the serine at position 4 of SEQ ID NO: 4 is substituted for a polar uncharged aliphatic amino acid selected from the group consisting of cysteine, threonine, methionine, asparagine and glycine or other amino acid, suitably, wherein the serine at position 4 of SEQ ID NO: 4 is substituted for asparagine.

3. The Nicotiana tabacum plant cell according to any of the preceding claims, wherein the polypeptide comprises the contiguous polypeptide sequence of SEQ ID NO: 8; optionally, wherein the polypeptide comprises, consists or consists essentially of the polypeptide sequence set forth in SEQ ID NO:

7.

4. The Nicotiana tabacum plant cell according to any of preceding claims, wherein the polynucleotide sequence comprises, consists or consists essentially of the polynucleotide sequence set forth in SEQ ID NO:

6.

5. The Nicotiana tabacum plant cell according to any of preceding claims, further comprising at least one mutation in a NIA2 nitrate reductase, suitably,wherein the mutation is in the recognition site for binding of the nitrate reductase kinase.

6. The Nicotiana tabacum plant cell according to claim 5, wherein the mutated NIA2 nitrate reductase polypeptide comprises a contiguous polypeptide sequence of SEQ ID NO: 13, wherein the methionine at position 10 of SEQ ID NO: 13 is substituted for an amino acid that reduces nitrate levels in the plant cell as compared to a control plant cell.

7. The Nicotiana tabacum plant cell according to claim 6, wherein the methionine is substituted for a non-polar aliphatic amino acid selected from the group consisting of glycine, alanine, proline, isoleucine, leucine or valine, suitably, wherein the substituted amino acid is isoleucine, suitably wherein the polypeptide comprises the contiguous polypeptide sequence of SEQ ID NO:

14.

8. The Nicotiana tabacum plant cell according to any of claims 5 to 7, wherein the nitrate reductase NIA2 polypeptide comprises, consists or consists essentially of the polypeptide sequence set forth in SEQ ID NO:

11.

9. The Nicotiana tabacum plant cell according to any of claims 5 to 8, wherein the mutated nitrate reductase NIA2 polynucleotide comprises, consists or consists essentially of the polynucleotide sequence set forth in SEQ ID NO: 12 or SEQ ID NO:

17.

10. A Nicotiana tabacum plant or part thereof comprising the plant cell according to any preceding claim.

11. The Nicotiana tabacum plant or part thereof according to claim 10, wherein cured leaves of the plant or part thereof contain lower levels of nitrate as compared to a control plant or part thereof; suitably, wherein the cured leaves are air-cured or sun-cured or flue-cured or fermented.

12. Nicotiana tabacum plant material, cured plant material, or homogenized plant material, derived from the Nicotiana tabacum plant or part thereof of claim 11; suitably,wherein the plant material comprises biomass, seed, stem, flowers, or leaves from the plant or part thereof of claim 10 or claim 11.

13. A tobacco product comprising the Nicotiana tabacum plant cell of any of claims 1 to 9, a part of the Nicotiana tabacum plant of claim 11 or the Nicotiana tabacum plant material according to claim 12.

14. A method for producing the Nicotiana tabacum plant of claim 10 or claim 11, comprising: (a) providing the Nicotiana tabacum plant cell according to any of claims 1 to 9; and (b) propagating the Nicotiana tabacum plant cell into a Nicotiana tabacum plant.

15. A method for producing cured Nicotiana tabacum plant material with an altered amount of nitrate as compared to control plant material, comprising the steps of: (a) providing the Nicotiana tabacum plant or part thereof according to claim 10 or claim 11 or the Nicotiana tabacum plant material according to claim 12; (b) harvesting the Nicotiana tabacum plant or Nicotiana tabacum plant material; and (c) curing the harvested Nicotiana tabacum plant or the harvested Nicotiana tabacum plant material; suitably, wherein the Nicotiana tabacum plant material comprises cured leaves; suitably, wherein the curing method is selected from the group consisting of air curing, fire curing, smoke curing, and flue curing.

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