Methods and compositions related to improved nitrogen utilization efficiency in tobacco

Abstract

To provide compositions and methods useful for making and identifying tobacco plants with improved nitrogen utilization efficiency via breeding, transgenic approaches and cisgenic approaches.SOLUTION: The present disclosure provides metabolic signatures and genetic markers for tracking enhanced nitrogen utilization efficiency phenotypes in tobacco plants and for introgressing enhanced nitrogen utilization efficiency phenotypes into tobacco plants. The disclosure also provides tobacco plants with enhanced nitrogen utilization efficiency, and methods for creation of the tobacco plants with enhanced nitrogen utilization efficiency. The disclosure also provides recombinant polynucleotides and polypeptides for enhancing nitrogen utilization efficiency in modified tobacco plants, and tobacco plants comprising the recombinant polynucleotides and polypeptides.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 553,501, filed on September 1, 2017, the entire disclosure of which is incorporated herein by reference.

[0002] Incorporation of Sequence Listing This application includes a sequence listing submitted in electronic form together with a file named "P34523WO00_SL.txt" having a size of 188,432 bytes (measured in MS - Windows®), created on August 31, 2018, the entire disclosure of which is incorporated herein by reference.

[0003] Field The present disclosure provides compositions and methods useful for producing and identifying tobacco plants with improved nitrogen use efficiency via breeding, transgenic approaches, and cisgenic approaches.

Background Art

[0004] Background Fertilizers are a major cost for tobacco growers, and increased fertilization is associated with higher levels of alkaloids and tobacco - specific nitrosamines (TSNAs) in plant tissues. Different tobacco varieties require different levels of nitrogen fertilizer input per variety to achieve maximum yield. For example, Maryland tobacco varieties typically require approximately 25% less nitrogen input to achieve maximum yield compared to burley tobacco varieties.

[0005] Improving nitrogen use efficiency (NUE) in tobacco would increase the harvestable amount of tobacco per unit of nitrogen fertilizer input. Improving nitrogen use efficiency would also enable a reduction in farm input costs, a reduction in the use and dependence on non - renewable energy sources required for nitrogen fertilizer production, and a reduction in the environmental impact of nitrogen fertilizer manufacturing and agricultural use.

[0006] Methods and compositions for improving the nitrogen use efficiency of tobacco are provided herein.

Summary of the Invention

[0007] Summary In one aspect, the present disclosure provides a method for determining the NUE of a tobacco line, and includes obtaining at least one metabolite from a tobacco plant of the tobacco line, determining the amount of the obtained metabolite, and determining the NUE of the tobacco line based on the amount of the identified metabolite.

[0008] In one aspect, the present specification provides a method for determining the NUE of a tobacco line using a metabolite signature, and includes isolating a metabolite signature from a tobacco plant of the tobacco line, determining the amount of each metabolite included in the metabolite signature, and determining the NUE of the tobacco line by comparing the metabolite signature with a control metabolite signature derived from a control tobacco line including a known NUE.

[0009] In one aspect, the present specification includes determining a metabolite signature of a first tobacco plant derived from a first tobacco line, wherein the first tobacco plant includes an increased NUE compared to a control tobacco plant lacking the metabolite signature; crossing the first plant with a second plant of a second tobacco line; and obtaining at least one progeny seed from the cross, wherein the progeny plant grown from the at least one progeny seed includes the metabolite signature and the progeny plant includes an increased NUE compared to a control plant lacking the metabolite signature, and provides a method for breeding a tobacco line including a metabolite signature associated with an increased NUE, and includes the same.

[0010] In one aspect, this specification provides and includes a method of selecting a tobacco plant, the method comprising obtaining a population of tobacco plants, isolating at least one metabolite associated with increased NUE from at least one tobacco plant from the population of tobacco plants, and selecting at least one tobacco plant that contains a greater amount of at least one metabolite compared to a control tobacco plant. In a further aspect of this method, the selected tobacco plant contains increased NUE compared to the control tobacco plant.

[0011] In one aspect, this specification provides and includes a method of selecting a tobacco plant, the method comprising obtaining a population of tobacco plants, isolating at least one metabolite associated with increased NUE from at least one tobacco plant from the population of tobacco plants, and selecting at least one tobacco plant that contains a lesser amount of at least one metabolite compared to a control tobacco plant.

[0012] In one aspect, this specification provides and includes a method of screening a tobacco plant for a first metabolite signature associated with increased NUE, the method comprising isolating the first metabolite signature associated with increased NUE from a tobacco plant, determining the amount of at least one metabolite comprising the first metabolite signature, comparing the first metabolite signature to a second metabolite signature of a control tobacco plant having a known NUE, and determining whether the first metabolite signature is associated with increased NUE.

[0013] In one aspect, this specification provides and includes a modified tobacco seed or a tobacco plant grown therefrom, the modified tobacco seed or tobacco plant comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a coding region, the modified tobacco plant having increased nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide grown under the same conditions.

[0014] In one aspect, the present specification provides and includes a recombinant DNA construct comprising a heterologous promoter functionally linked to a polynucleotide encoding a polypeptide that is at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8.

[0015] In one aspect, the present specification provides and includes a dried tobacco material or a tobacco product containing the dried tobacco material, which is produced from a tobacco plant containing a cisgenic polynucleotide comprising a heterologous promoter functionally linked to a coding region, wherein the modified tobacco plant has an increased nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide grown under the same conditions.

[0016] In one aspect, the present specification provides and includes a greenhouse, growth chamber or field containing the modified tobacco seeds or plants disclosed herein.

[0017] In one aspect, the present specification provides and includes a modified tobacco seed or a tobacco plant grown therefrom, which contains at least one mutation encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40 at an endogenous locus, and has an increased nitrogen use efficiency compared to an unmodified control tobacco plant lacking the at least one mutation grown under the same conditions.

[0018] In one aspect, the present specification provides and includes a recombinant DNA construct comprising a heterologous promoter functionally linked to a guide RNA containing at least 18 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40.

[0019] In one aspect, the present specification provides and includes a dried tobacco material or a tobacco product containing a dried tobacco material, which is produced from a tobacco plant having at least one mutation encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40 at an endogenous locus, wherein the modified tobacco seed or tobacco plant contains an increased NUE as compared to a non-modified control tobacco plant lacking the at least one mutation when grown under the same conditions.

[0020] In one aspect, the present specification provides and includes a modified tobacco seed or a tobacco plant grown therefrom, which contains a cisgenic polynucleotide comprising a heterologous promoter functionally linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41 to 56, wherein the modified tobacco seed or tobacco plant contains an increased NUE as compared to a non-modified control tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0021] In one aspect, the present specification provides and includes a recombinant DNA construct comprising a heterologous promoter functionally linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41 to 56.

[0022] In one aspect, the present specification provides and includes a dried tobacco material or a tobacco product containing the dried tobacco material, which is produced from a tobacco plant containing a cisgenic polynucleotide comprising a heterologous promoter functionally linked to a polynucleotide encoding an sRNA that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56, wherein the modified tobacco seed or tobacco plant comprises increased NUE as compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide grown under the same conditions.

[0023] In one aspect, the present specification provides and includes a method for increasing the NUE of a tobacco plant, which includes the steps of introducing a cisgenic nucleic acid molecule into a tobacco cell and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises increased NUE as compared to a tobacco plant lacking the cisgenic nucleic acid molecule.

[0024] In one aspect, the present specification provides and includes a method for increasing the NUE of a tobacco plant, which includes the steps of introducing a modification to a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41 to 56 into a tobacco cell and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises increased NUE as compared to a tobacco plant lacking the modification.

[0025] In one aspect, the present specification provides and includes a method for increasing the NUE of a tobacco plant, which includes the steps of introducing into a tobacco cell a nucleic acid encoding a small molecule RNA (sRNA) homologous to at least 18 consecutive nucleic acids of a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41 to 56 and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises increased NUE as compared to a tobacco plant lacking the sRNA.

[0026] In one aspect, the present specification provides a method comprising the steps of providing a first population of tobacco plants comprising increased NUE, genotyping the first population of tobacco plants for the presence of molecular markers within 20 cM of the locus of the increased NUE gene, and selecting one or more tobacco plants that have been genotyped and found to contain said molecular marker, and includes it.

[0027] In one aspect, the present specification provides a method comprising the steps of providing a first population of tobacco plants, genotyping the first population of tobacco plants for the presence of an increased NUE allele at a locus encoded by a sequence selected from the group consisting of SEQ ID NOs: 9-16, and selecting one or more genotyped tobacco plants that contain the increased NUE allele, and includes it.

[0028] In one aspect, the present specification provides a method for introgressing an increased NUE trait into a tobacco variety, comprising the steps of crossing a first tobacco variety comprising an increased nitrogen use efficiency trait with a second tobacco variety lacking the increased nitrogen use efficiency trait, obtaining progeny seeds from the cross, genotyping at least one progeny seed for a molecular marker associated with the increased nitrogen use efficiency trait, wherein the molecular marker is within 20 cM of a locus having a sequence selected from the group consisting of SEQ ID NOs: 9-16, and selecting progeny seeds that contain the increased nitrogen use efficiency trait, and includes it.

[0029] In one aspect, the present specification provides a method for selecting a tobacco plant having an increased NUE trait, comprising the steps of isolating nucleic acids from a collection of tobacco germplasm, assaying the isolated nucleic acids for one or more markers located within 20 cM of a locus selected from the group consisting of SEQ ID NOs: 9-16, and selecting a tobacco plant that contains the increased NUE trait, and includes it.

[0030] In one aspect, this specification provides and includes a method for selecting a tobacco plant having an increased NUE trait, the method including the steps of isolating nucleic acids from a collection of tobacco germplasm, assaying the isolated nucleic acids for one or more markers located within 20 cM of a marker selected from the group consisting of SEQ ID NO:57-64, and selecting a tobacco plant comprising the increased NUE trait. [Inventive concept 1001] A method for producing a tobacco plant comprising an increased nitrogen use efficiency (NUE) trait, the method comprising the following steps: (a) providing a first population of tobacco plants comprising the increased NUE trait; (b) genotyping the first population of tobacco plants for the presence of one or more molecular markers located within 20 cM of an SNP marker selected from the group consisting of SEQ ID NO:57, 58, 59, 60, 61, 62, 63, and 64; (c) selecting tobacco plants comprising the one or more molecular markers; (d) crossing the tobacco plants selected in step (c) with a second tobacco plant; and (e) obtaining progeny seeds from the cross of step (d), wherein plants grown from the progeny seeds comprise the increased NUE trait. [Inventive concept 1002] The method of Inventive concept 1001, wherein the increased NUE trait is selected from the group consisting of increased partial factor productivity (PFP), increased agronomic efficiency (AE), increased recovery efficiency (RE), increased physiological efficiency (PE), and increased internal efficiency (IE) as compared to growing a tobacco plant lacking the increased NUE trait under the same conditions. [Inventive concept 1003] The method of Inventive concept 1001, wherein the first population of tobacco plants is selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, and NC925. [Inventive concept 1004] The method of the present invention 1001, wherein the second tobacco plant is a burley tobacco variety. [The present invention 1005] The method of the present invention 1001, wherein the second tobacco plant is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC. [The present invention 1006] The method of the present invention 1001, wherein the one or more molecular markers are within 10 cM of the SNP marker. [The present invention 1007] The method of the present invention 1001, wherein the one or more molecular markers are within 5 cM of the SNP marker. [The present invention 1008] A method for producing a tobacco plant comprising an increased NUE trait, comprising the following steps: (a) providing a first population of tobacco plants comprising an increased NUE trait; (b) genotyping the first population of tobacco plants for the presence of one or more molecular markers located within 20 cM of an increased NUE locus having a sequence selected from the group consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, and 16; (c) selecting tobacco plants comprising the one or more molecular markers; (d) crossing the tobacco plants selected in step (c) with a second tobacco plant; and (e) obtaining progeny seeds from the cross of step (d), wherein plants grown from the progeny seeds comprise an increased NUE trait. [The present invention 1009] The method of the present invention 1008, wherein the increased NUE trait is selected from the group consisting of increased partial factor productivity (PFP), increased agronomic efficiency (AE), increased recovery efficiency (RE), increased physiological efficiency (PE), and increased internal efficiency (IE) compared to tobacco plants grown under the same conditions lacking the increased NUE trait. [The present invention 1010] The method of the present invention 1008, wherein the first population of tobacco plants is selected from the group consisting of MD609, MD601, Banquet A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, and NC925. [The present invention 1011] The method of the present invention 1008, wherein the second tobacco plant is a burley tobacco variety. [The present invention 1012] The method of the present invention 1008, wherein the second tobacco plant is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC. [The present invention 1013] The method of the present invention 1008, wherein the one or more molecular markers are within 10 cM of the locus. [The present invention 1014] The method of the present invention 1008, wherein the one or more molecular markers are within 5 cM of the locus. [The present invention 1015] A transgenic tobacco seed or a tobacco plant grown therefrom, comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a coding region, wherein the coding region encodes a polypeptide comprising at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8, and the transgenic tobacco plant comprises an increased NUE trait as compared to a non-modified control tobacco plant lacking the cisgenic polynucleotide grown under the same conditions. The transgenic tobacco seed or the tobacco plant grown therefrom. [The present invention 1016] The modified tobacco seeds of the present invention 1015 or tobacco plants grown therefrom, wherein the increased NUE trait is selected from the group consisting of increased partial factor productivity (PFP), increased agronomic efficiency (AE), increased recovery efficiency (RE), increased physiological efficiency (PE), and increased internal efficiency (IE) compared to the case of growing tobacco plants lacking the increased NUE trait under the same conditions. [The present invention 1017] The modified tobacco seeds of the present invention 1015 or tobacco plants grown therefrom, wherein the heterologous promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, and a tissue-specific promoter. [The present invention 1018] The modified tobacco seeds of the present invention 1015 or tobacco plants grown therefrom, wherein the tissue-preferred promoter is a leaf-preferred promoter comprising a sequence having at least 90% sequence identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs: 17, 18, and 19. [The present invention 1019] The modified tobacco seeds of the present invention 1015 or tobacco plants grown therefrom, wherein the tissue-preferred promoter is a root-preferred promoter comprising a sequence having at least 90% sequence identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, and 24. [The present invention 1020] The modified tobacco seeds of the present invention 1015 or tobacco plants grown therefrom, wherein the coding region comprises at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, and 16.

[0031] Brief Description of the Sequences SEQ ID NOs: 1 to 8 are the amino acid sequences of genes that are positively correlated with increased NUE in root tissue, leaf tissue, or both.

[0032] SEQ ID NOs: 9 to 16 are nucleotide sequences of genes that are positively correlated with increased NUE in root tissue, leaf tissue, or both.

[0033] SEQ ID NOs: 17 to 19 are nucleotide sequences of promoter regions for genes having leaf-preferential expression.

[0034] SEQ ID NOs: 20 to 24 are nucleotide sequences of promoter regions for genes having root-preferential expression.

[0035] SEQ ID NOs: 25 to 40 are amino acid sequences of genes that are negatively correlated with increased NUE in root tissue, leaf tissue, or both.

[0036] SEQ ID NOs: 41 to 56 are nucleotide sequences of genes that are negatively correlated with increased NUE in root tissue, leaf tissue, or both.

[0037] SEQ ID NOs: 57 to 64 are nucleotide sequences of SNP markers containing polymorphisms related to increased NUE.

[0038] SEQ ID NO: 65 is the backbone sequence for the expression vector p45-2-7.

Brief Description of the Drawings

[0039]

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Mode for Carrying Out the Invention

[0040] Detailed Description Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One of ordinary skill in the art will recognize that many methods can be used in the practice of this disclosure. Indeed, this disclosure is in no way limited to the methods and materials described. For the purposes of this disclosure, the following terms are defined below.

[0041] Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the technical field to which this disclosure pertains. When a term is provided in the singular, the inventors also contemplate aspects of this disclosure described by the plural of that term. If there are discrepancies in the terms and definitions used in incorporated references, the terms used in this application shall have the definitions given herein. Other technical terms used have the ordinary meaning in the technical field in which they are used, as exemplified in various specialized dictionaries, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to the mechanism or mode of action. References to them are provided for illustrative purposes only.

[0042] The practice of the present disclosure, unless otherwise indicated, involves conventional techniques in biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, biotechnology, metabolomics, plant breeding, and genetics, within the skill of the art. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols In Molecular Biology (F.M. Ausubel, et al. eds., (1987)); Plant Breeding Methodology (N.F. Jensen, Wiley-Interscience (1988)); the series Methods In Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (R.I. Freshney, ed. (1987)); Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences; C.N. Stewart, A. Touraev, V. Citovsky, T. Tzfira eds. (2011) Plant Transformation Technologies (Wiley-Blackwell); and R.H. Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.).

[0043] Any references cited herein (including, for example, all patents, published patent applications, and non-patent publications) are hereby incorporated by reference in their entirety.

[0044] As used herein, the singular forms "a," "an," and "the" include the plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds (including mixtures thereof).

[0045] As used herein, the terms "sequence identity" or "identity" refer to residues in two sequences that are the same when the two polynucleotide or polypeptide sequences are aligned for maximum correspondence over a specified comparison window. When the percentage of sequence identity is used with respect to a protein, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions (where an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), such that the functional properties of the molecule are not changed). When sequences differ by such conservative substitutions, the percentage of sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Alignment of two or more sequences can be performed using any suitable computer program. For example, a widely used and accepted computer program for performing sequence alignment is CLUSTALW v1.6 (Thompson, et al. (1994) Nucl. Acids Res., 22: 4673-4680).

[0046] As used herein, the term "complementary" with respect to a nucleic acid molecule refers to nucleotide base pairing such that adenine is complementary to thymine or uracil, and guanine is complementary to cytosine. Two complementary nucleic acid molecules can hybridize to each other. As an example, the two strands of double-stranded DNA are complementary to each other.

[0047] A specific polynucleotide having a length of at least 3 nucleotides can be referred to as an "oligonucleotide". The nucleic acid molecules provided herein include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and functional analogs thereof, such as complementary DNA (cDNA). The nucleic acid molecules provided herein can be single-stranded or double-stranded. Nucleic acid molecules include the nucleotide bases adenine (A), guanine (G), thymine (T), and cytosine (C). Uracil (U) replaces thymine in RNA molecules. The symbol "R" can be used to represent a purine (e.g., A or G) nucleotide base. The symbol "Y" can be used to represent a pyrimidine (e.g., C or T) nucleotide base. The symbol "W" can be used to represent an A or T nucleotide base. The symbol "S" can be used to represent a G or C nucleotide base. The symbol "M" can be used to represent an A or C nucleotide base. The symbol "K" can be used to represent a G or T nucleotide base. The symbol "B" can be used to represent a G, C, or T nucleotide base. The symbol "H" can be used to represent an A, C, or T nucleotide base. The symbol "D" can be used to represent an A, G, or T nucleotide base. The symbol "V" can be used to represent an A, G, or C nucleotide base. The symbol "N" can be used to represent any nucleotide base (e.g., A, G, C, T, or U).

[0048] The use of the term "polynucleotide" is not intended to limit the present disclosure to polynucleotides containing DNA. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules can include ribonucleotides, and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of the present disclosure also encompass all sequence forms, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc.

[0049] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide provided herein.

[0050] The nucleic acid molecules, polypeptides or proteins provided herein can be isolated or substantially purified. An "isolated" or "purified" nucleic acid molecule, polypeptide, protein or biologically active portion thereof is substantially or essentially free of components that are normally associated with or interact with the polynucleotide or protein as it is found in its natural environment. For example, an isolated or purified polynucleotide or protein produced by recombinant techniques is substantially free of other cellular material or culture medium, or a chemically synthesized polynucleotide or protein is substantially free of chemical precursors or other chemicals. In one aspect, an isolated polynucleotide provided herein can contain a nucleic acid sequence of less than 10,000 nucleotides, less than 5,000 nucleotides, less than 4,000 nucleotides, less than 3,000 nucleotides, less than 2,000 nucleotides, less than 1,000 nucleotides, less than 500 nucleotides, or less than 100 nucleotides that is naturally adjacent to the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. In one aspect, an isolated polynucleotide provided herein can contain a nucleic acid sequence of 100 - 10,000 nucleotides, 500 - 10,000 nucleotides, 1,000 - 10,000 nucleotides, 2,000 - 10,000 nucleotides, 3,000 - 10,000 nucleotides, 4,000 - 10,000 nucleotides, 1 - 500 nucleotides, 1 - 1,000 nucleotides, 1 - 2,000 nucleotides, 1 - 3,000 nucleotides, 1 - 4,000 nucleotides, 1 - 5,000 nucleotides, 1 - 10,000 nucleotides, 100 - 500 nucleotides, 100 - 1,000 nucleotides, 100 - 2,000 nucleotides, 100 - 3,000 nucleotides, or 100 - 4,000 nucleotides that is naturally adjacent to the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. In another aspect, an isolated polypeptide provided herein is substantially free of cellular material in a preparation having less than 30%, less than 20%, less than 10%, less than 5% or less than 1% (by dry weight) of chemical precursors or chemicals that are not the protein of interest.Fragments of the disclosed polynucleotides and the polypeptides encoded thereby are also encompassed by the present invention. Fragments of the polynucleotides may encode polypeptide fragments that retain the biological activity of the native polypeptide. Alternatively, fragments of the polynucleotides that are useful as hybridization probes or PCR primers using methods known in the art generally do not encode fragment polypeptides that retain biological activity. Fragments of the polynucleotides provided herein can range from at least 20 nucleotides, at least 50 nucleotides, at least 70 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, and up to the full-length polynucleotide, depending on the desired result.

[0051] Nucleic acids can be isolated using routine techniques in the art. For example, nucleic acids can be isolated using any method including, but not limited to, recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation which can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized either as single nucleic acid molecules or as a series of oligonucleotides. Polypeptides can be purified from natural sources (e.g., biological samples) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Polypeptides can also be purified, for example, by expressing nucleic acids in an expression vector. In addition, purified polypeptides can be obtained by chemical synthesis. The degree of purity of a polypeptide can be measured using any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0052] In one aspect, the disclosure provides methods for detecting recombinant nucleic acids and polypeptides in plant cells. Without limitation, nucleic acids can also be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0053] The polypeptide can be detected using an antibody. Techniques for detecting a polypeptide using an antibody include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. The antibodies provided herein can be polyclonal antibodies or monoclonal antibodies. Antibodies having specific binding affinity for the polypeptides provided herein can be prepared using methods well known in the art. The antibodies provided herein can be attached to a solid support, such as a microtiter plate, using methods known in the art.

[0054] Detection (e.g., of an amplification product, a hybridization complex, a polypeptide) can be accomplished using a detectable label. The term “label” is intended to encompass the use of both direct labels and indirect labels. Detectable labels include enzymes, moieties, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0055] As used herein, the phrases “associated with” or “related to” refer to a recognizable and / or assayable relationship between two entities. For example, the phrase “associated with increased NUE” refers to a trait, locus, gene, allele, marker, phenotype, etc., or the expression, presence or absence thereof, that can affect the extent, degree and / or ratio to which a plant or a part thereof of interest has the increased NUE trait. Thus, a marker is “associated with” a trait when the presence of the marker is an indicator of whether and / or to what extent a desired trait or form of a trait occurs in a plant / germplasm containing the marker. Similarly, a marker is “associated with” an allele when the presence of the marker is an indicator of whether the allele is present in a plant / germplasm containing the marker. For example, a “marker associated with an increased NUE allele” refers to a marker that can be used to predict whether and to what extent a plant exhibits an increased NUE phenotype by its presence or absence.

[0056] As used herein, "centimorgan" (cM) is a unit of measure of the recombination frequency and genetic distance between two loci. 1 cM is equal to a 1% probability that a marker at one locus will be separated from a marker at a second locus by a single generation of crossing over.

[0057] As used herein, "closely related" means that a marker or locus is within about 20 cM, 15 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM of another marker or locus. For example, 20 cM means that recombination occurs between the marker and the locus at a frequency of about 20% or less.

[0058] As used herein, "plant" refers to an entire plant. A cell or tissue culture obtained from a plant can include any plant component or plant organ (e.g., leaves, stems, roots, etc.), plant tissue, seeds, plant cells, and / or their progeny. Progeny plants can be derived from any hybrid generation, e.g., F1, F2, F3, F4, F5, F6, F7, etc. A plant cell is a living cell of a plant that has been taken from a plant or obtained through culturing of cells taken from a plant.

[0059] As used herein, tobacco plants include Nicotiana tabacum tabacum; Nicotiana tabacum amplexicaulis PI 271989; Nicotiana tabacum benthamiana PI 555478; Nicotiana tabacum bigelovii PI 555485; Nicotiana tabacum debneyi; Nicotiana tabacum excelsior PI 224063; Nicotiana tabacum glutinosa PI 555507; Nicotiana tabacum goodspeedii PI 241012; Nicotiana tabacum gossei PI 230953; Nicotiana tabacum hesperis PI 271991; Nicotiana tabacum knightiana PI 555527; Nicotiana tabacum maritima PI 555535; Nicotiana tabacum megalosiphon PI 555536; Nicotiana tabacum nudicaulis PI 555540; Nicotiana tabacum paniculata PI 555545; Nicotiana tabacum plumbaginifolia PI 555548; Nicotiana tabacum repanda PI 555552;It can be derived from any plant from the genus Nicotiana tabacum, including but not limited to Nicotiana tabacum rustica; Nicotiana tabacum suaveolens PI 230960; Nicotiana tabacum sylvestris PI 555569; Nicotiana tabacum tomentosa PI 266379; Nicotiana tabacum tomentosiformis; and Nicotiana tabacum trigonophylla PI 555572.

[0060] In one aspect, the plant components provided herein include, but are not limited to, leaves, stems, roots, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, sheaths, embryos, endosperms, explants, calli, tissue cultures, shoots, cells and protoplasts. In a further aspect, the disclosure provides tobacco plant cells, tissues and organs that are not reproductive materials and do not mediate natural reproduction of the plant. In another aspect, the disclosure also provides tobacco plant cells, tissues and organs that are reproductive materials and mediate natural reproduction of the plant. In another aspect, the disclosure provides tobacco plant cells, tissues and organs that cannot sustain themselves via photosynthesis. In another aspect, the disclosure provides somatic cells of a tobacco plant. Somatic cells, in contrast to germ cells, do not mediate reproduction of the plant.

[0061] The provided cells, tissues, and organs can be derived from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperms, roots, shoots, stems, sheaths, flowers, inflorescences, peduncles, pedicels, styles, stigmas, receptacles, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarp, phloem, and vascular tissue. In another aspect, the present disclosure provides tobacco plant chloroplasts. In a further aspect, the present disclosure provides epidermal cells, stroma cells, trichomes (hairy projections), root hairs, or storage roots. In another aspect, the present disclosure provides tobacco protoplasts.

[0062] One of ordinary skill in the art will understand that tobacco plants reproduce naturally via seeds rather than asexually or vegetatively. In one aspect, the present disclosure provides tobacco endosperm. In another aspect, the present disclosure provides tobacco endosperm cells. In a further aspect, the present disclosure provides male or female sterile tobacco plants that are incapable of reproducing without human intervention.

[0063] In one aspect, the present disclosure provides methods and compositions related to modified tobacco plants, seeds, plant components, plant cells, and products made from modified tobacco plants, seeds, plant parts, and plant cells. In one aspect, the modified seeds provided herein give rise to the modified plants provided herein. In one aspect, the modified plants, seeds, plant components, plant cells, or plant genomes provided herein contain the recombinant DNA constructs provided herein. In another aspect, the dried tobacco materials or tobacco products provided herein contain the modified tobacco plants, plant components, plant cells, or plant genomes provided herein.

[0064] As used herein, "modified" refers to plants, seeds, plant components, plant cells, and plant genomes that have undergone mutagenesis, genome editing, genetic transformation, or combinations thereof.

[0065] As used herein, "cisgenesis" or "cisgenic" refers to the genetic modification of a plant, plant cell or plant genome in which all components (e.g., promoter, donor nucleic acid, selectable gene) have only a plant origin (e.g., components other than those of plant origin are not used). In one aspect, the modified plants, plant cells or plant genomes provided herein are cisgenic. The cisgenic plants, plant cells and plant genomes provided herein can lead to immediately available tobacco lines. In another aspect, the modified tobacco plants provided herein do not contain genetic material or sequences other than those of tobacco.

[0066] As used herein, "functional fragment" or "the functional fragment thereof" refers to a nucleotide or amino acid sequence of any size that retains the function of the full-length sequence it refers to. In certain aspects, the functional fragment can be at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, or more than 5000 nucleotides in length. In certain aspects, the functional fragment can be at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, or more than 2000 amino acids in length. In certain aspects, the functional fragment can be between 5 and 5000 nucleotides, between 10 and 4000 nucleotides, between 25 and 3000 nucleotides, between 50 and 2000 nucleotides, between 75 and 1000 nucleotides, between 100 and 900 nucleotides, between 150 and 800 nucleotides, between 200 and 700 nucleotides, between 250 and 600 nucleotides, or between 300 and 500 nucleotides in length. In certain aspects, the functional fragment can be between 5 and 2000 amino acids, between 10 and 1000 amino acids, between 25 and 900 amino acids, between 50 and 800 amino acids, between 50 and 800 amino acids, between 75 and 700 amino acids, between 100 and 600 amino acids, between 150 and 500 amino acids, between 200 and 400 amino acids, or between 250 and 300 amino acids in length. In a further aspect, the polynucleotides described herein are contemplated as a whole and as any of its functional fragments.In a further aspect, the polypeptides described herein are contemplated as a whole and as any functional fragments thereof. In a further aspect, polynucleotides having the sequences of SEQ ID NOs: 9-24 and 41-56 are contemplated as a whole and as any functional fragments thereof. In a further aspect, polypeptides having the sequences of SEQ ID NOs: 1-8 and 25-40 are contemplated as a whole and as any functional fragments thereof.

[0067] As used herein, the term "nitrogen use efficiency" (NUE) refers to the ability of a plant to absorb, assimilate and / or use nitrogen (e.g., from soil, water and / or nitrogen fertilizer). NUE genes have utility for improving nitrogen use in crop plants and affecting yield. Increased nitrogen use efficiency can result not only from improved nitrogen fertilizer uptake and assimilation and / or subsequent remobilization and reuse of stored nitrogen reserves, but also from increased plant tolerance to stress situations such as low nitrogen environments. By using NUE genes to alter the genetic makeup of plants, it is possible to make plants more productive under current fertilization standards and to maintain their productivity with significantly reduced fertilizer or reduced nitrogen availability.

[0068] NUE is defined in various ways, but the yield per unit of nitrogen available in the soil integrates all the important parameters for evaluating the adaptability of crop varieties, and this is the common measure of NUE. See, for example, Ladha et al. 2005. Advances in Agronomy, 87:85-156 (which is incorporated herein by reference in its entirety). This metric is sometimes referred to as "agronomic NUE". As another measure of NUE, the ratio of plant product (e.g., tobacco leaf tissue) to aboveground nitrogen in the plant can be determined (sometimes referred to as "physiological NUE"). Increased NUE relates to three important components: 1) the yield does not differ significantly when grown at 25% normal nitrogen content compared to plants grown at 100% normal nitrogen content; 2) the chlorophyll loss rate is reduced compared to plants without increased NUE; and 3) the dry leaf quality does not differ significantly when grown at 25% normal nitrogen content compared to plants grown at 100% normal nitrogen content. In a preferred aspect, plants with increased NUE can provide similar yields and leaf quality when grown at 25% of the barley fertilization rate compared to barley plants grown at 100% of the normal barley fertilization rate.

[0069] At least five approaches and indices for NUE are used in the art and are discussed below.

[0070] (1) Partial factor productivity (PFP) from applied nitrogen (N) is a measure of how much yield is produced per unit of nitrogen applied: PFP N = kilograms of yield / kilograms of N applied PFP N = Y +N / FN Where Y +N is the yield (kilograms per hectare; kg / ha), and FN is the amount of fertilizer applied (kg / ha).

[0071] (2) The agronomic efficiency (AE) of applied nitrogen (N) is a measure of how much additional yield is produced per unit of applied nitrogen: AE N = Kilograms of yield increase / Kilograms of N applied AE N =(Y +N -Y 0N ) / FN Where Y +N is the yield (kg / ha) in the treatment with N application; Y 0N is the yield (kg / ha) in the control treatment without N application; FN is the amount of N fertilizer applied (kg / ha).

[0072] (3) The recovery efficiency (RE) of applied nitrogen (N) is a measure of how much of the applied nitrogen is recovered and taken up by the crop. RE N = Kilograms of N taken up / Kilograms of N applied RE N =(UN +N -UN 0N ) / FN Where UN +N is the total N uptake (kg / ha) of the plant measured in the aboveground biomass at physiological maturity in the plot receiving FN of applied N; UN 0N is the total N uptake in the control plot without N addition.

[0073] (4) The physiological efficiency (PE) of applied nitrogen (N) is a measure of how much additional yield is produced per additional unit of nitrogen uptake. PE N = Kilograms of yield increase / Kilograms of fertilizer N taken up PE N =(Y +N -Y 0N ) / (UN +N -UN 0N ) Where Y +N is the yield (kg / ha) in the treatment with N application; Y 0Nis the yield (kg / ha) in the control treatment without N application; UN +N is the total N uptake (kg / ha) in the treatment receiving fertilizer N; UN 0N is the total N uptake (kg / ha) in the treatment without fertilizer N application.

[0074] (5) The internal efficiency (IE) of nitrogen (N) is directed towards how much yield is produced per unit of N taken up from both fertilizer and indigenous (e.g., soil) nutrient sources: IE N = kilograms of yield / kilograms of N taken up IE N = Y / UN where Y is the yield (kg / ha); and UN is the total N uptake (kg / ha).

[0075] Nitrogen can be in any form including organic and / or inorganic forms. Without limitation, forms of nitrogen include nitrates (e.g., ammonium nitrate, calcium nitrate, potassium nitrate), nitrites, ammonia, aqueous ammonia, anhydrous ammonia, ammonium sulfate, diammonium phosphate, low-pressure nitrogen solutions, normal-pressure nitrogen solutions, urea, and urea-ammonium nitrate (UAN). In certain aspects, nitrogen is in a form readily available to plants (e.g., ammonia and / or nitrate) and / or in a form that can be readily converted to a form available to plants (e.g., urea).

[0076] In one aspect, a modified tobacco plant provided herein that includes increased NUE includes increased nitrogen uptake as compared to a control tobacco plant. In another aspect, a modified tobacco plant provided herein that includes increased NUE includes increased nitrogen assimilation as compared to a control tobacco plant. In a further aspect, a modified tobacco plant provided herein that includes increased NUE includes increased yield as compared to a control tobacco plant. In yet another aspect, a modified tobacco plant provided herein that includes increased NUE includes increased yield under low nitrogen conditions as compared to a control tobacco plant. In a preferred aspect, low nitrogen conditions such as those used in the field are approximately 25% nitrogen as compared to levels typically used by those of ordinary skill in the art. In another aspect, low nitrogen conditions such as those used in the field can be between approximately 5% and 50% nitrogen as compared to levels typically used by those of ordinary skill in the art. In a greenhouse environment, low nitrogen conditions are approximately 25 parts per million (ppm), and normal nitrogen conditions are approximately 100 ppm. In another aspect, low nitrogen conditions such as those used in the greenhouse can be between 5 ppm and 50 ppm.

[0077] In certain aspects, the modified tobacco plants provided herein that include increased NUE include a yield increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500% compared to control tobacco plants grown under similar growth conditions. In certain aspects, the modified tobacco plants provided herein that include increased NUE include a yield increase between 5% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 10% and 200%, between 10% and 300%, between 10% and 400%, between 10% and 500%, or between 5% and 500% compared to control tobacco plants grown under similar growth conditions.

[0078] In one aspect, a population of modified tobacco plants comprising increased NUE provided herein exhibits a yield increase of at least 0.25 kg / ha, at least 0.5 kg / ha, at least 0.75 kg / ha, at least 1 kg / ha, at least 2 kg / ha, at least 3 kg / ha, at least 4 kg / ha, at least 5 kg / ha, at least 6 kg / ha, at least 7 kg / ha, at least 8 kg / ha, at least 9 kg / ha, at least 10 kg / ha, at least 15 kg / ha, at least 20 kg / ha, at least 25 kg / ha, at least 30 kg / ha, at least 35 kg / ha, at least 40 kg / ha, at least 45 kg / ha, at least 50 kg / ha, at least 75 kg / ha, at least 100 kg / ha, at least 200 kg / ha, at least 300 kg / ha, at least 400 kg / ha, or at least 500 kg / ha compared to a population of control tobacco plants grown under similar growth conditions. In another aspect, a population of modified tobacco plants comprising increased NUE provided herein exhibits a yield increase between 0.25 kg / ha and 100 kg / ha, between 0.5 kg / ha and 100 kg / ha, between 0.75 kg / ha and 100 kg / ha, between 1 kg / ha and 100 kg / ha, between 2 kg / ha and 100 kg / ha, between 3 kg / ha and 100 kg / ha, between 4 kg / ha and 100 kg / ha, between 5 kg / ha and 100 kg / ha, between 6 kg / ha and 100 kg / ha, between 7 kg / ha and 100 kg / ha, between 8 kg / ha and 100 kg / ha, between 9 kg / ha and 100 kg / ha, between 10 kg / ha and 100 kg / ha, between 15 kg / ha and 100 kg / ha, between 20 kg / ha and 100 kg / ha, between 30 kg / ha and 100 kg / ha, between 40 kg / ha and 100 kg / ha, between 50 kg / ha and 100 kg / ha, between 75 kg / ha and 100 kg / ha, between 100 kg / ha and 500 kg / ha, between 100 kg / ha and 400 kg / ha, between 100 and 300 kg / ha, or between 100 kg / ha and 200 kg / ha compared to a population of control tobacco plants grown under similar growth conditions.As used herein, a "population" of tobacco plants can be of any size, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, 500, 1000, 5000, 10000, 25000, 50000, 100000, 500000 or more. The population can be derived from a single variety, cultivar or line. The population can be produced using any breeding technique known in the art.

[0079] In one aspect, a modified tobacco plant comprising increased NUE provided herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or at least 25 more leaves than a control tobacco plant grown under similar growth conditions. In another aspect, a modified tobacco plant comprising increased NUE provided herein comprises between 1 - 25, between 2 - 25, between 3 - 25, between 4 - 25, between 5 - 25, between 6 - 25, between 7 - 25, between 8 - 25, between 9 - 25, between 10 - 25, between 11 - 25, between 12 - 25, between 13 - 25, between 14 - 25, between 15 - 25, or between 20 - 25 more leaves than a control tobacco plant grown under similar growth conditions.

[0080] As used herein, "equivalent conditions," "similar conditions," or "similar growing conditions" refer to similar environmental conditions, farming methods, and / or drying processes for growing or drying tobacco to make a meaningful comparison between two or more plant genotypes such that neither the environmental conditions nor the farming methods (including the drying process) contribute or account for any observed differences between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, and nutrients (e.g., nitrogen and phosphorus). Farming methods include, for example, seeding, mowing, lower leaf removal, planting, topping, tillering, and drying. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp. 70-103.

[0081] In one aspect, the modified plants, seeds, plant parts, or plant cells provided herein contain one or more non-naturally occurring mutations. In one aspect, the mutations provided herein improve nitrogen use efficiency in plants. The types of mutations provided herein include, for example, substitutions (point mutations), deletions, insertions, duplications, and inversions. Such mutations preferably are present in the coding region of a gene; however, mutations in a promoter or other regulatory region, intron, intron-exon boundary, or untranslated region of a gene also may be desirable in some cases.

[0082] In one aspect, the methods provided herein can produce tobacco plants having increased nitrogen use efficiency compared to control tobacco plants. Mutagenesis methods include, but are not limited to, chemical mutagenesis, e.g., treatment of seeds with ethyl methanesulfonate (EMS) (Hildering and Verkerk, In, The use of induced mutations in plant breeding. Pergamon Press, pp. 317-320, 1965); or UV irradiation, X-rays, electron beams, ion beams (e.g., carbon ion beam, helium ion beam, neon ion beam), and fast neutron irradiation (see, e.g., Verkerk, Neth. J. Agric. Sci. 19:197-203, 1971; Poehlman, Breeding Field Crops, Van Nostrand Reinhold, New York (3.sup.rd ed.), 1987; and Tanaka, J. Radiat. Res. 51:223-233, 2010); transposon tagging (Fedoroff et al., 1984; U.S. Patent Nos. 4,732,856 and 5,013,658); and T-DNA insertion methodologies (Hoekema et al., 1983; U.S. Patent No. 5,149,645). EMS-induced mutagenesis consists of chemically inducing random point mutations across the genome length. Fast neutron mutagenesis consists of exposing seeds to neutron bombardment that causes large deletions through double-stranded DNA breaks. Transposon tagging involves inserting a transposon within an endogenous gene to reduce or eliminate gene expression.

[0083] In addition, a rapid and automatable screening method for chemically induced mutations using denaturing high performance liquid chromatography (HPLC) or selective endonuclease digestion of the selected PCR products, i.e., TILLING (Targeting Induced Local Lesions In Genomes), is also applicable to the present disclosure. See McCallum et al. (2000) Nat. Biotechnol. 18:455-457. Mutations that affect gene expression of the genes provided herein or interfere with their function can be determined using methods well known in the art. Insertion mutations in gene exons usually result in null mutants. Mutations at conserved residues can be particularly effective in inhibiting protein function.

[0084] Screening and selection of mutagenized tobacco plants can be by any methodology known to those skilled in the art. Examples of screening and selection methodologies include, but are not limited to, Southern blot, PCR amplification for detection of polynucleotides, Northern blot, RNase protection, primer extension, RT-PCR amplification for detection of RNA transcripts, Sanger sequencing, next generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting enzymatic or ribozyme activity of polypeptides and polynucleotides; as well as protein gel electrophoresis, Western blot, immunoprecipitation, and enzyme-linked immunosorbent assay for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining can also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods of performing all of the techniques mentioned are known in the art.

[0085] In one aspect, the plant genomes provided herein are mutated (edited) by a nuclease selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR / Cas9 nucleases, CRISPR / Cpf1 or CRISPR / Cmx1 nucleases. In another aspect, the plant genomes provided herein are mutated by CRISPR / CasX or CRISPR / CasY nucleases. As used herein, "editing" or "genome editing" refers to targeted mutagenesis of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an endogenous plant genomic nucleic acid sequence, or the removal or replacement of an endogenous plant genomic nucleic acid sequence.

[0086] Also provided herein is the transformation of tobacco plants with the recombinant constructs or expression cassettes described herein using any suitable transformation method known in the art. Methods for introducing a polynucleotide sequence into a tobacco plant are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. "Stable transformation" refers to a transformation in which a nucleotide construct of interest introduced into a plant is integrated into the genome of a plant cell and can be inherited by its progeny. "Transient transformation" is intended to mean that a sequence is introduced into a plant or plant cell and is simply expressed transiently or simply present transiently in that plant or plant cell.

[0087] In one aspect, the methods and compositions provided herein include the introduction of one or more polynucleotides into one or more plant cells. In one aspect, the plant genome provided herein is modified to include the introduced polynucleotide or recombinant DNA construct. As used herein, "plant genome" refers to the nuclear genome, mitochondrial genome, or plastid (e.g., chloroplast) genome of a plant cell. In another aspect, the polynucleotides provided herein are incorporated into artificial chromosomes. In one aspect, an artificial chromosome containing the polynucleotide provided herein is incorporated into a plant cell.

[0088] In one aspect, the modified plants, seeds, plant components, plant cells or plant genomes provided herein contain one or more transgenes. In one aspect, the transgenes provided herein improve nitrogen use efficiency in tobacco plants. As used herein, "transgene" refers to a polynucleotide transferred into the genome by any method known in the art. In one aspect, the transgene is an exogenous polynucleotide. In one aspect, the transgene is an endogenous polynucleotide incorporated into a novel genomic locus where it is not normally found. Thus, a transgene can also be a cis-gene under appropriate circumstances.

[0089] In one aspect, the transgenes provided herein comprise recombinant DNA constructs. In one aspect, the recombinant DNA constructs or expression cassettes provided herein can include a selectable marker gene for the selection of transgenic cells. Selectable marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NPTII) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidally active compounds such as glufosinate ammonium, bromoxynil, imidazolinones, triazolopyrimidines, sulfonylureas (e.g., chlorosulfuron and sulfometuron methyl), and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers, such as fluorescent proteins such as β-galactosidase and green fluorescent protein (GFP).

[0090] In one aspect, the methods and compositions provided herein include a vector. As used herein, the terms "vector" or "plasmid" are used interchangeably and refer to circular double-stranded DNA molecules that are physically separated from chromosomal DNA. In one aspect, the plasmids or vectors used herein can replicate in vivo. A "transformation vector" as used herein is a plasmid capable of transforming plant cells. In certain aspects, the plasmids provided herein are bacterial plasmids. In another aspect, the plasmids provided herein are Agrobacterium Ti plasmids or are derived from Agrobacterium Ti plasmids. In yet another aspect, the vectors provided herein are viral vectors.

[0091] In one aspect, the plasmids or vectors provided herein are recombinant vectors. As used herein, the term "recombinant vector" refers to a vector formed by laboratory techniques of genetic recombination such as molecular cloning. In another aspect, the plasmids provided herein are synthetic plasmids. As used herein, a "synthetic plasmid" is an artificially created plasmid that can perform the same functions (e.g., replication) as a natural plasmid (e.g., Ti plasmid). Without limitation, one of ordinary skill in the art can create synthetic plasmids de novo by synthesizing the plasmid with individual nucleotides or by splicing nucleic acid molecules together from different existing plasmids.

[0092] Vectors can be commercially available or produced by routine recombinant DNA techniques in the art. In one aspect, the vectors provided herein include all or a portion of SEQ ID NO:65. A vector containing nucleic acid can have an expression element operably linked to such nucleic acid and can further include a sequence encoding a selectable marker (e.g., an antibiotic resistance gene). A vector containing nucleic acid can encode a chimeric or fusion polypeptide (i.e., a polypeptide operably linked to a heterologous polypeptide that can be present at either the N-terminus or C-terminus of the polypeptide). Representative heterologous polypeptides are those that can be used for the purification of the encoded polypeptide (e.g., 6×His tag, glutathione S-transferase (GST)).

[0093] Suitable methods for introducing polynucleotides (e.g., transgenes, recombinant vectors, recombinant DNA constructs, expression constructs) into the plant cells of the present disclosure include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Shillito et al. (1987) Meth. Enzymol. 153:313-336; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Patent Nos. 5,104,310, 5,149,645, 5,177,010, 5,231,019, 5,463,174, 5,464,763, 5,469,976, 4,762,785, 5,004,863, 5,159,135, 5,563,055, and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (e.g., see U.S. Patent Nos. 4,945,050, 5,141,131, 5,886,244, 5,879,918, and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926).Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation). In one aspect, the bacterial cells provided herein contain the recombinant DNA constructs or recombinant vectors provided herein. It is recognized that many different species of bacterial cells can contain a recombinant DNA construct or recombinant vector. Non-limiting examples include, for instance, Agrobacterium tumefaciens, Escherichia coli, yeast cells (e.g., Saccharomyces cerevisiae) that contain the recombinant DNA constructs or recombinant vectors provided herein.

[0094] In another aspect, the recombinant constructs or expression cassettes provided herein can be introduced into plants by contacting the plants with a virus or viral nucleic acid. Generally, such methods involve incorporating the expression cassettes of the disclosure into viral DNA or RNA molecules. It is recognized that the promoters for use in the expression cassettes provided herein also include promoters that are utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing the proteins encoded therein (with viral DNA or RNA molecules) are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221.

[0095] Any plant tissue that can be subsequently propagated using a cloning method (whether by organogenesis or embryogenesis) can be transformed with the recombinant constructs or expression cassettes provided herein. "Organogenesis" is intended to mean the process by which shoots and roots develop continuously from meristem centers. "Embryogenesis" is intended to mean the process by which shoots and roots develop together in concert (not continuously), whether from somatic cells or gametes. Exemplary tissues suitable for the various transformation protocols described herein include, but are not limited to, callus tissue, existing meristems (e.g., apical meristems, axillary buds, and root meristems) and induced meristems (e.g., cotyledonary meristems and hypocotyl meristems), hypocotyls, cotyledons, leaf disks, pollen, embryos, etc.

[0096] As is generally understood in the art, the term "promoter" generally refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site, and / or a TATA box, and that aids or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter may be produced synthetically, altered, or may be derived from a known or native promoter sequence or other promoter sequences (e.g., as provided herein). A promoter may also include a chimeric promoter that comprises a combination of two or more heterologous sequences. Thus, the promoters of the present invention may include variants of promoter sequences that have a similar composition but are not identical or complementary to other promoter sequences known or provided herein. As used herein, "heterologous promoter" in the context of a DNA construct refers to either: (i) a promoter derived from a source different from the structural gene or coding region to which it is operably linked, or (ii) a promoter derived from the same source as the structural gene or coding region to which it is operably linked, but whose sequence has been modified from its original form. As used herein, the term "operably linked" refers to a functional linkage between a promoter or other regulatory element and an associated transcribable polynucleotide sequence or coding sequence of a gene (or transgene), such that the promoter, etc., functions to initiate, aid, act on, induce, and / or promote the transcription and expression of the associated coding sequence or transcribable polynucleotide sequence, at least in a particular tissue, developmental stage, and / or under certain conditions. A "plant-expressible promoter" refers to a promoter that can be used to express an associated coding sequence, transgene, or transcribable polynucleotide sequence operably linked to the promoter in a plant, plant cell, and / or plant tissue.

[0097] Promoters may be classified according to a variety of criteria regarding the expression pattern of a coding array or gene (including a transgene) that is operably linked to the promoter (e.g., constitutive, developmental, tissue-specific, inducible, etc.). A promoter that initiates transcription in all or most tissues of a plant is called a “constitutive” promoter. A promoter that initiates transcription during a certain period or stage of development is called a “developmental” promoter. A promoter whose expression is enhanced in certain tissues of a plant compared to other plant tissues is called a “tissue-enhanced” or “tissue-preferred” promoter. Thus, a “tissue-preferred” promoter causes relatively high or preferential expression in a particular tissue of a plant, but has a low expression level in other tissues of the plant. A promoter that is expressed within a particular tissue of a plant but is hardly or not at all expressed in other plant tissues is called a “tissue-specific” promoter. A promoter that is expressed in certain cell types of a plant is called a “cell-type-specific” promoter. An “inducible” promoter is a promoter that initiates transcription in response to environmental stimuli such as cold, drought or light, or other stimuli such as wounding or application of a chemical substance. Promoters may also be classified from the perspective of their origin, such as heterologous, homologous, chimeric, synthetic, etc. A “heterologous” promoter is a promoter sequence that has a different origin with respect to its associated transcribable sequence, coding sequence or gene (or transgene), and / or does not naturally occur in the plant species being transformed. The term “heterologous” may more broadly refer to a combination of two or more DNA molecules or sequences (when such a combination is not normally found in nature). For example, two or more DNA molecules or sequences are heterologous to each other if they are normally found in different genomes or at different loci within the same genome, or if they are not normally combined identically in nature.

[0098] Exemplary constitutive promoters include the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J 3:2723-2730); the ALS promoter (U.S. Patent No. 5,659,026), and the like.

[0099] Exemplary chemically inducible promoters include the tobacco PR-1a promoter that is activated by salicylic acid. Other chemically inducible promoters of interest include steroid-responsive promoters (see, e.g., the glucocorticoid-inducible promoters in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible promoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, as well as U.S. Pat. Nos. 5,814,618 and 5,789,156). Additional exemplary promoters that can be used herein include heat-regulated gene expression, light-regulated gene expression (e.g., pea rbcS-3A; maize rbcS promoter; the chlorophyll alb-binding protein gene found in pea; or the Arabssu promoter), hormone-regulated gene expression (e.g., the abscisic acid (ABA)-responsive sequence from the wheat Em gene; the ABA-inducible HVA1 and HVA22 of barley and Arabidopsis, and the rd29A promoter; as well as wound-inducible gene expression (e.g., wunl), organ-specific gene expression (e.g., of tuber-specific storage protein genes; the 23-kDa zein gene from maize as described; or responsive to the French bean (β-phaseolin gene), or pathogen-inducible promoters (e.g., the PR-1, prp-1 or β-1,3 glucanase promoters, the fungal-inducible wirla promoter of wheat, and the nematode-inducible promoters, TobRB7-5A and Hmg-1 of tobacco and parsley, respectively).

[0100] As used herein, a "leaf" promoter includes any promoter that initiates, induces, drives, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence in leaf tissue derived from any part of a plant. Such a "leaf" promoter can be further defined as initiating, inducing, driving, etc., the transcription or expression of its associated gene / transgene or transcribable DNA sequence in one or more tissues of a plant, such as one or more floral tissues. Such a "leaf" promoter can be further defined as a "leaf-preferred" promoter that initiates, induces, drives, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence at least preferentially or predominantly (if not exclusively) in leaf tissue (as contrasted with floral tissue) derived from any part of a plant. However, "leaf" and "leaf-preferred" promoters each can also permit, enable, induce, drive, etc., the transcription or expression of their associated gene, transgene, or transcribable DNA sequence during the reproductive period or stage of development in one or more cells or tissues of a plant, such as one or more vegetative or reproductive tissues. Indeed, a "leaf" promoter can even initiate, induce, drive, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence in one or more reproductive or vegetative tissues at a greater level or extent than in leaf tissue.

[0101] As used herein, a "root" promoter includes any promoter that initiates, induces, drives, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence in root tissue derived from any part of a plant. Such a "root" promoter can be further defined as one that initiates, induces, drives, etc., the transcription or expression of its associated gene / transgene or transcribable DNA sequence in one or more tissues of a plant, such as one or more floral tissues. Such a "root" promoter can be further defined as a "root-preferred" promoter that initiates, induces, drives, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence at least preferentially or predominantly (if not exclusively) in root tissue (as contrasted with floral tissue) derived from any part of a plant. However, "root" and "root-preferred" promoters each can also permit, enable, induce, drive, etc., the transcription or expression of their associated gene, transgene, or transcribable DNA sequence during the reproductive stage or phase of development in one or more cells or tissues of a plant, such as one or more vegetative or reproductive tissues. Indeed, a "root" promoter can even initiate, induce, drive, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence in one or more reproductive or vegetative tissues at a greater level or extent than in root tissue.

[0102] Additional exemplary tissue-preferred promoters include those disclosed in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.

[0103] As used herein, "functionally linked" refers to a functional linkage between two or more elements. For example, a functional linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional relationship that enables expression of the polynucleotide of interest. Functionally linked elements may be contiguous or non-contiguous.

[0104] As used herein, "heterologous" refers to a sequence that is of exogenous origin, or, if from the same species, a sequence whose composition and / or genomic locus has been substantially modified from its native form by intentional human intervention. This term also applies to nucleic acid constructs, also referred to herein as "polynucleotide constructs" or "nucleotide constructs". Thus, a "heterologous" nucleic acid construct is intended to mean a construct that is of exogenous origin, or, if from the same species, a construct whose composition and / or genomic locus has been substantially modified from its native form by intentional human intervention. Heterologous nucleic acid constructs include, but are not limited to, recombinant nucleotide constructs that have been introduced into a plant or a plant part thereof, for example, via a transformation method or subsequent crossing of a transgenic plant with another plant of interest.

[0105] In one aspect, inhibition of the expression of one or more polypeptides provided herein may be obtained by RNA interference (RNAi) by the expression of a polynucleotide provided herein. In one aspect, RNAi includes expressing a non-coding RNA. As used herein, "non-coding RNA" is selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), trans-acting siRNA (ta-siRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), intron, hairpin RNA (hpRNA), intron-containing hairpin RNA (ihpRNA), and guide RNA. In one aspect, a single non-coding RNA provided herein inhibits the expression of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more than 10 polypeptides. In one aspect, the non-coding RNA provided herein is stably transformed into the plant genome. In another aspect, the non-coding RNA provided herein is transiently transformed into the plant genome.

[0106] As used herein, the terms "down-regulate," "suppress," "inhibit," "inhibition," and "inhibiting" are defined as any method known in the art or described herein for reducing the expression or function of a gene product of interest (e.g., mRNA, protein, non-coding RNA). "Inhibition" can relate to a comparison between two plants, e.g., a modified plant and a control plant. Alternatively, inhibition of the expression or function of a target gene product can relate to a comparison within the same plant or between different plants, among plant cells, organelles, organs, tissues, or plant components, and can also include a comparison within the same plant or plant component or between plants or plant components at different developmental or temporal stages. "Inhibition" includes any relative decrease, including complete elimination, of the function or production of the gene product of interest up to and including complete elimination of the function or production of that gene product. The term "inhibition" encompasses any method or composition that down-regulates the translation and / or transcription of a target gene product or the functional activity of a target gene product.

[0107] The term "inhibitory sequence" encompasses any polynucleotide or polypeptide sequence that can inhibit the expression or function of a gene in a plant, e.g., a full-length polynucleotide or polypeptide sequence, a truncated polynucleotide or polypeptide sequence, a fragment of a polynucleotide or polypeptide sequence, a variant of a polynucleotide or polypeptide sequence, a sense-oriented nucleotide sequence, an antisense-oriented nucleotide sequence, a complement of a sense or antisense-oriented nucleotide sequence, an inverted region of a nucleotide sequence, a hairpin of a nucleotide sequence, a double-stranded nucleotide sequence, a single-stranded nucleotide sequence, combinations thereof, etc. The term "polynucleotide sequence" includes sequences such as RNA, DNA, chemically modified nucleic acids, nucleic acid analogs, combinations thereof, etc.

[0108] When the phrase "capable of inhibiting" is used in the context of a polynucleotide inhibitory sequence, does it mean that the inhibitory sequence itself exerts an inhibitory effect; or, when the inhibitory sequence encodes an inhibitory nucleotide molecule (e.g., hairpin RNA, miRNA or double-stranded RNA polynucleotide) or an inhibitory polypeptide (e.g., a polypeptide that inhibits the expression or function of a target gene product), is it intended that following its transcription (e.g., in the case of an inhibitory sequence encoding a hairpin RNA, miRNA or double-stranded RNA polynucleotide) or its transcription and translation (in the case of an inhibitory sequence encoding an inhibitory polypeptide), the transcription product or translation product respectively exerts an inhibitory effect (e.g., inhibits the expression or function of a target gene product) on the target gene product?

[0109] The inhibitory sequences provided herein can be sequences that cause gene silencing via any silencing pathway or mechanism known in the art, including but not limited to sense suppression / co-suppression, antisense suppression, double-stranded RNA (dsRNA) interference, hairpin RNA interference and intron-containing hairpin RNA interference, amplicon-mediated interference, ribozymes, small interfering RNAs, artificial or synthetic microRNAs, and artificial trans-acting siRNAs. The inhibitory sequences can range from at least 20 nucleotides, at least 50 nucleotides, at least 70 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, and full-length polynucleotides encoding the proteins of the present disclosure, depending on the desired result. In one aspect, the inhibitory sequences can be fragments that are between 50 and 400 nucleotides, between 70 and 350 nucleotides, between 90 and 325 nucleotides, between 90 and 300 nucleotides, between 90 and 275 nucleotides, between 100 and 400 nucleotides, between 100 and 350 nucleotides, between 100 and 325 nucleotides, between 100 and 300 nucleotides, between 125 and 300 nucleotides, or between 125 and 275 nucleotides in length.

[0110] MicroRNA (miRNA) is a non-protein-coding RNA of generally 19-25 nucleotides (usually 20-24 nucleotides in plants) that guides the trans cleavage of target transcripts and negatively regulates the expression of genes involved in various regulatory and developmental pathways (Bartel (2004) Cell, 116:281-297). In some cases, miRNA serves to guide the phased processing of siRNA primary transcripts (see Allen et al. (2005) Cell, 121:207-221).

[0111] A number of microRNA genes (MIR genes) have been identified and are publicly available in databases (see "miRBase", available online at microrna.sanger.ac.uk / sequences; also see Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to occur both singly and in clusters in intergenic regions within the genome, but may also be located wholly or in part within the introns of other genes (both protein-coding and non-protein-coding). Transcription of MIR genes can, in at least some cases, be under the regulatory control of a promoter of the MIR gene itself. The primary transcript, termed "pri-miRNA", can be extremely large (several kilobases) and polycistronic, containing the normal 5' "cap" and polyadenylation tail of an mRNA, together with one or more pre-miRNAs (folded structures containing the stem-loop arrangement that is processed into the mature miRNA).

[0112] Maturation of mature miRNAs from their corresponding precursors (pri-miRNAs and pre-miRNAs) differs greatly between animals and plants. For example, in plant cells, microRNA precursor molecules are thought to be almost entirely processed into mature miRNAs in the nucleus, whereas in animal cells, pri-miRNA transcripts are processed in the nucleus by the animal-specific enzyme Drosha, and subsequently the pre-miRNA is transported to the cytoplasm where it is further processed into the mature miRNA. Mature miRNAs in plants are typically 21 nucleotides in length.

[0113] Using transgenic expression of miRNAs (whether naturally occurring or artificial sequences), the expression of one or more target genes of a miRNA can be regulated. Inclusion of miRNA recognition sites within the transgenic expressed transcript is also useful in regulating the expression of the transcript; see, for example, Parizotto et al. (2004) Genes Dev., 18:2237-2242. miRNA recognition sites have been verified in all regions of mRNA including the 5' untranslated region, coding region and 3' untranslated region, and the position of the miRNA target site relative to the coding sequence has been shown not to necessarily affect repression. Since miRNAs are important regulatory elements in eukaryotes, transgenic repression of miRNAs is useful for manipulating biological pathways and responses. Finally, the promoters of MIR genes can have very specific expression patterns (e.g., cell-specific, tissue-specific, time-specific or inducible), and thus are useful for inducing such specific transcription of DNA sequences operably linked in a recombinant construct. Various uses of miRNAs, their precursors, their recognition sites and their promoters are known. Non-limiting examples of these uses include: (1) expression of native miRNA or miRNA precursor sequences to repress target genes; (2) expression of artificial miRNA or miRNA precursor sequences to repress target genes; (3) expression of a transgene having a miRNA recognition site (in which case the transgene is repressed when mature miRNA is expressed); (4) expression of a transgene driven by a miRNA promoter.

[0114] Designing artificial miRNA sequences can be as simple as replacing nucleotides in the miRNA stem region of the miRNA precursor with sequences complementary to the intended target. One non-limiting example of a general method for determining nucleotide changes in a native miRNA sequence to produce an engineered miRNA precursor involves the following steps: (a) selecting a unique target sequence of at least 18 nucleotides specific to the target gene, for example, using a sequence alignment tool such as BLAST® (see, e.g., Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402) for both the cDNA database and genomic DNA database of, for example, tobacco, identifying any potential matches with the target transcript ortholog and unrelated genes, thereby avoiding unintended silencing of non-target sequences; (b) analyzing the target gene for undesirable sequences (e.g., those that match sequences from non-target species), scoring each potential 19-mer segment for GC content, Reynolds score (see Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and functional asymmetry (".DELTA..DELTA.G" or "ΔΔG") characterized by a negative difference in free energy [preferably, a 19-mer having all or most of the following characteristics is selected: (1) Reynolds score >4, (2) GC content between 40% and 60%, (3) negative ΔΔG, (4) adenosine at the terminus, (5) no more than 4 consecutive identical nucleotides; (6) located near the 3' end of the target gene; (7) minimal difference from the miRNA precursor transcript).The positions every three nucleotides in siRNA have been reported to be particularly important with respect to the effect on the efficacy of RNAi, and the algorithm "siExplorer" is publicly available at rna.chem.t.u-tokyo.ac.jp / siexplorer.htm]; (c) determining the reverse complement of the selected 19-mer for use in producing the modified mature miRNA (the additional nucleotide at position 20 preferably matches the selected target sequence, and the nucleotide at position 21 is preferably selected not to pair to prevent expansion of silencing in the target transcript or is selected to pair with the target sequence to promote expansion of silencing in the target transcript); and (d) transforming a plant with the artificial miRNA.

[0115] In one aspect, the artificial miRNAs provided herein reduce or eliminate RNA transcription or protein translation of a target gene.

[0116] In one aspect, the miRNAs or artificial miRNAs provided herein are under the control of a tissue-specific promoter. In a further aspect, the miRNAs or artificial miRNAs provided herein are under the control of a tissue-preferred promoter. In a further aspect, the miRNAs or artificial miRNAs provided herein are under the control of a constitutive promoter.

[0117] Tobacco products can be manufactured using tobacco materials obtained from the disclosed modified tobacco lines, varieties or hybrids. As used herein, "tobacco product" is defined as any product manufactured or obtained from tobacco intended for human use or consumption. In one aspect, the tobacco products provided herein contain dried components derived from the modified tobacco plants provided herein. In another aspect, the tobacco products provided herein contain dried tobacco leaves derived from the modified tobacco plants provided herein.

[0118] The tobacco products provided herein include, without limitation, cigarette products (e.g., cigarettes, bidis, kreteks), cigar products (e.g., cigars, cigar-wrapped tobacco, cigarillos), pipe tobacco products, tobacco-derived products, nicotine products derived from tobacco, smokeless tobacco products (e.g., moist snuff, dry snuff, chewing tobacco, moist smokeless tobacco, fine-cut chewing tobacco, long-cut chewing tobacco, pouch-packaged chewing tobacco), films, chewables (e.g., gums), lozenges, dissolvable strips, tabs, tablets, shaped parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, etc. See, e.g., U.S. Patent Publication No. US 2006 / 0191548.

[0119] As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler". The "rod" of a cigarette includes cigarette paper, a filter, a plug wrap (used to hold the filter material), tipping paper that holds the cigarette paper (including the filler) to the filter, and all adhesives that hold these components together. The "filler" includes (1) all tobacco (including, but not limited to, reconstituted tobacco and expanded tobacco), (2) non-tobacco alternatives (including, but not limited to, herbs, non-tobacco plant materials, and other spices that may be associated with the tobacco wrapped in cigarette paper), (3) casing, (4) flavorings, and (5) all other additives (mixed into the tobacco and alternatives and wrapped in the cigarette).

[0120] In one aspect, the disclosure provides nicotine derived from the modified tobacco plants provided herein, and methods for producing nicotine from the modified tobacco plants provided herein for use in products.

[0121] Tobacco products derived from the plants of the present disclosure also include cigarettes and other smoking articles, particularly smoking articles including filter elements, in which case the rod-shaped portion of the smokable material contains dried tobacco within a tobacco blend. In certain aspects, the tobacco products of the present disclosure are selected from the group consisting of cigars, non-ventilated recess filter cigarettes, ventilated recess filter cigarettes, bidis, cigars, snuffs, pipe tobaccos, cigar tobaccos, cigarette tobaccos, chewing tobaccos, leaf tobaccos, water tobaccos, ground tobaccos, and cut tobaccos. In another aspect, the tobacco products of the present disclosure are smokeless tobacco products. Smokeless tobacco products do not burn and include, but are not limited to, chewing tobacco, moist smokeless tobacco, snus, and dry snuff. Chewing tobacco is coarsely shredded tobacco leaves, typically packaged in large pouch-like packages and used in lumps or twisted states. Moist smokeless tobacco is more finely shredded tobacco with moisture, provided in loose form or in bags, typically packaged in round cans and used by placing a pinch or in a bag between the cheek and gums of adult tobacco consumers. Snus is heat-treated smokeless tobacco. Dry snuff is finely powdered tobacco and is placed in the mouth or used nasally. In a further aspect, the tobacco products of the present disclosure are selected from the group consisting of loose leaf chewing tobacco, lump chewing tobacco, moist snuff, and nasal snuff. In yet another aspect, the tobacco products of the present disclosure are selected from the group consisting of electronic heated cigarettes, e-cigarettes, and electronic vaporization devices. In one aspect, the methods provided herein include preparing a tobacco product using dried tobacco leaves derived from the modified tobacco plants provided herein.

[0122] As used herein, "reconstituted tobacco" refers to a type of tobacco filler manufactured from tobacco dust and other tobacco scrap materials that is processed into a sheet and cut into strips to resemble tobacco. In addition to cost reduction, reconstituted tobacco is very important for its contribution to the taste of cigarettes by treating the generation of flavors using the reaction of ammonia and sugar.

[0123] As used herein, "expanded tobacco" refers to a type of tobacco filler that has been processed through the expansion of a suitable gas so as to "inflate" the tobacco to produce a reduction in density and greater filling performance. Expanded tobacco reduces the weight of the tobacco used in cigarettes.

[0124] Also provided herein is a dried tobacco material produced from a tobacco plant or plant component provided herein. "Drying" is an aging process that reduces moisture and causes chlorophyll destruction, turning the tobacco leaf golden, thereby converting starch to sugar. Thus, dried tobacco has a higher reducing sugar content and a lower starch content compared to harvested green leaves. In one aspect, the tobacco plant or plant component provided herein can be dried using conventional means, such as hot air drying, barn drying, fire drying, air drying, or sun drying. For an explanation of different types of drying methods, see, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Dried tobacco is typically aged for several years (e.g., 2 - 5 years) at a moisture content in the range of 10% - about 25% under compression conditions, either in a wooden drum (e.g., cask) or cardboard carton. See U.S. Pat. Nos. 4,516,590 and 5,372,149. The dried and aged tobacco can then be further processed. Further processing can include conditioning the tobacco under vacuum with or without introducing steam at various temperatures, pasteurization, and fermentation. Fermentation is typically characterized by a high initial moisture content, heat generation, and a 10 - 20% loss of dry weight. See, for example, U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S. Pub. No. 2005 / 0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, for example, U.S. Pat. Nos. 4,528,993; 4,660,577; and 4,987,907.In one aspect, the cured tobacco material of the present disclosure is cured by hot air curing, sun curing, air curing, or fire curing.

[0125] The present disclosure further provides a method of manufacturing a tobacco product comprising a tobacco material derived from a tobacco plant provided herein. In one aspect, the method provided herein comprises adjusting a cured tobacco material produced from a tobacco plant provided herein to increase its moisture content from between 12.5% and 13.5% to 21%, and blending the adjusted tobacco material to produce a desired blend. In one aspect, the method of manufacturing a tobacco product provided herein further comprises casing or flavoring the blend. Generally, during the casing process, casing or source materials are added to the blend and their quality is enhanced by balancing the chemical composition to develop certain desirable flavor characteristics. Further details regarding the casing process can be found in Tobacco Production, Chemistry and Technology, Edited by L. Davis and M. Nielsen, Blackwell Science, 1999.

[0126] The tobacco materials provided herein can also be processed using methods including, but not limited to, heat treatment (e.g., cooking, toasting), flavoring, enzymatic treatment, expansion, and / or curing. Both fermented and unfermented tobacco can be processed using these techniques. Examples of suitable processed tobaccos include dark air cured, dark fire cured, burley, hot air cured, and cigar filler or wrapper, as well as products from whole leaf stemming operations. In one aspect, the tobacco fiber comprises up to 70% dark tobacco on a fresh weight basis. For example, the tobacco can be conditioned by heat, leaching, and / or pasteurization processes as described in U.S. Publication Nos. 2004 / 0118422 or 2005 / 0178398.

[0127] The tobacco materials provided herein can be subjected to fermentation. Fermentation is typically characterized by a high initial moisture content, heat generation, and a loss of 10 to 20% of the dry weight. See, for example, U.S. Patent Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149. In addition to modifying the aroma of the leaf, fermentation can change one or both of the color and the feel of the leaf. Also, during the fermentation process, gas generation can occur, oxygen can be taken up, the pH can change, and the amount of water retained can vary. See, for example, U.S. Publication No. 2005 / 0178398 and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried tobacco or the dried and fermented tobacco can be further processed (e.g., cut, expanded, blended, ground, or pulverized) before being incorporated into oral products. The tobacco is, in some cases, long cut fermented dried moist tobacco having an oven volatile component content of 48 to 50 weight percent before being mixed with copolymers and optionally flavorings and other additives.

[0128] In one aspect, the tobacco materials provided herein can be processed to a desired size. In certain aspects, the tobacco fibers can be processed to have an average fiber size of less than 200 micrometers. In one aspect, the tobacco fibers are 75 to 125 micrometers. In another aspect, the tobacco fibers are processed to have a size of 75 micrometers or less. In one aspect, the tobacco fibers include long cut tobacco that can be cut or shredded to a width of from 10 cuts per inch to 110 cuts per inch and a length of from 0.1 inch to 1 inch. The double cut tobacco fibers can have a particle size range such that 70% of the double cut tobacco fibers are within a mesh size between -20 mesh and 80 mesh.

[0129] The tobacco materials provided herein can be processed to have a total oven volatile component content of 10 wt% or more; 20 wt% or more; 40 wt% or more; 15 wt% - 25 wt%; 20 wt% - 30 wt%; 30 wt% - 50 wt%; 45 wt% - 65 wt%; or 50 wt% - 60 wt%. One of ordinary skill in the art will understand that "moist" tobacco typically refers to tobacco having an oven volatile component content between 40 wt% and 60 wt% (e.g., 45 wt% - 55 wt%, or 50 wt%). As used herein, "oven volatile component" is determined by calculating the percentage of weight loss of a sample after drying the sample in a preheated forced draft oven at 110°C for 3.25 hours. Oral products can have an overall oven volatile component content different from that of the tobacco fibers used to manufacture the oral products. The processing steps described herein can reduce or increase the oven volatile component content.

[0130] In one aspect, the tobacco plants, seeds, plant components, plant cells, and plant genomes provided herein are derived from a tobacco type selected from the group consisting of hot air dried tobacco, sun cured tobacco, air dried tobacco, dark air dried tobacco, and dark fire cured tobacco. In another aspect, the tobacco plants, seeds, plant components, plant cells, and plant genomes provided herein are derived from a tobacco type selected from the group consisting of burley tobacco, maryland tobacco, bright tobacco, virginia tobacco, oriental tobacco, turkish tobacco, and galpao tobacco. In one aspect, the tobacco plant or seed provided herein is a hybrid plant or seed. As used herein, "hybrid" is produced by crossing two plants from different varieties or species such that the progeny contains genetic material from each parent. One of ordinary skill in the art will recognize that higher order hybrids can be produced in a similar manner. For example, a first hybrid can be produced by crossing variety C with variety D to produce a C×D hybrid, and a second hybrid can be produced by crossing variety E with variety F to produce an E×F hybrid. The first and second hybrids can be further crossed to produce a higher order hybrid (C×D)×(E×F) that contains all of the genetic information of the four parental varieties.

[0131] Flue-cured tobacco (also known as Bright Virginia tobacco) accounts for approximately 40% of the world's tobacco production. Flue-cured tobacco is also often called "Bright tobacco" because of the golden-yellow to dark orange color it reaches during drying. Flue-cured tobacco has a light and fragrant aroma and taste. Flue-cured tobacco generally has a high sugar content and a low oil content. The main flue-cured tobacco cultivating countries are Argentina, Brazil, China, India, Tanzania, and the United States. In one aspect, the modified tobacco plants or seeds provided herein are of a flue-cured tobacco background selected from the group consisting of CC 13, CC 27, CC 33, CC35, CC 37, CC 65, CC 67, CC 700, GF 318, GL 338, GL 368, GL 939, K 346, K 399, K326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471, NC 55, NC 606, NC 71, NC 72, NC 92, PVH 1118, PVH 1452, PVH 2110, Speight 168, Speight 220, Speight 225, Speight 227, Speight 236, and any variety essentially derived from any one of the foregoing varieties.In another aspect, the modified tobacco plants or seeds provided herein are of a hot air dried tobacco background selected from the group consisting of Coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K 149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC 37NF, NC 55, NC 60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC 2326, McNair 373, McNair 944, Ox 207, Ox 414 NF, Reams 126, Reams 713, Reams 744, RG 8, RG 11, RG 13, RG 17, RG 22, RG 81, RG H4, RG H51, Speight H-20, Speight G-28, Speight G-58, Speight G-70, Speight G-108, Speight G-l11, Speight G-l17, Speight 168, Speight 179, Speight NF-3, Va 116, Va 182, and any variety that is essentially derived from any one of the foregoing varieties. See WO 2004 / 041006 A1. In a further aspect, the modified tobacco plants, seeds, hybrids, varieties or lines provided herein are of any hot air dried background selected from the group consisting of K326, K346 and NC196.

[0132] Air-dried tobacco includes Burley, Maryland, and Dark tobacco. A common requirement is that drying mainly does not involve artificial heat sources and moisture sources. Burley tobacco is light brown to dark brown in color, high in oil content, and low in sugar content. Burley tobacco is air-dried in warehouses. The main Burley cultivating countries are Argentina, Brazil, Italy, Malawi, and the United States. Maryland tobacco is extremely soft, has good combustibility, low nicotine, and a neutral aroma. The main Maryland cultivating countries include the United States and Italy. In one aspect, the modified tobacco plants or seeds provided herein are of Burley tobacco background selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R 711, R 712, NCBH 129, HB4488PLC, PD 7319LC, Bu 21×Ky 10, HB04P, Ky 14×L 8, Kt 200, Newton 98, Pedigo 561, Pf561, and Va 509. In a further aspect, the modified tobacco plants, seeds, hybrids, varieties, or lines provided herein are of any Burley background selected from the group consisting of TN 90, KT 209, KT 206, KT212, and HB 4488. In another aspect, the modified tobacco plants or seeds provided herein are of Maryland tobacco background selected from the group consisting of Md 10, Md 40, Md 201, Md 609, Md 872, and Md 341.

[0133] Dark air-cured tobacco is distinguished from other types mainly by its medium to dark brown color and the drying process that gives it a distinct aroma. Dark air-cured tobacco is mainly used in the production of chewing tobacco and snuff. In one aspect, the modified tobacco plants or seeds provided herein are of a dark air-cured tobacco background selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Sun-cured Virginia, and Paraguan Passado.

[0134] Dark fire-cured tobacco is generally dried over a low-burning wood fire on the floor of a closed curing barn. Dark fire-cured tobacco is used to produce pipe blends, cigarettes, chewing tobacco, snuff, and full-bodied cigars. The main growing regions for dark fire-cured tobacco are Tennessee, Kentucky, and Virginia in the United States. In one aspect, the modified tobacco plants or seeds provided herein are of a dark fire-cured tobacco background selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TN D94, TN D950, VA 309, and VA 359.

[0135] Oriental tobacco is also known as Greek Aroma Turkish tobacco because it is typically cultivated in the Eastern Mediterranean region, such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. The small plant and leaf size characteristic of today's Oriental varieties, as well as their distinctive aromatic properties, are the result of the plant's adaptation over the past centuries in poor soil and stressful climatic conditions. In one aspect, the modified tobacco plants or seeds provided herein are of an Oriental tobacco background selected from the group consisting of Izmir, Katerini, Samsun, Basma and Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and any variety that is essentially derived from any one of the foregoing varieties.

[0136] In one aspect, the modified tobacco plants, seeds, hybrids, varieties or lines provided herein are BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC 37, CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, CC 1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao, GL 26H, GL 338, GL 350, GL 395, GL 600, GL 737, GL 939, GL 973, GF 157, GF 318, RJR 901, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326, NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA 116, VA 119, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY 907LC, KTY14×L8 LC, Little Crittenden, MacNeal 373, MacNeal 944, male sterile KY 14×L8, narrow leaf madol, MS KY171, narrow leaf madol (phph), MS narrow leaf madol, MS TND950, PD 7302LC, PD 7305LC, PD 7309LC, PD 7312LC, PD 7318LC, PD 7319LC, MSTKS 2002, TKF 2002, TKF 6400, TKF 4028, TKF 4024, KT206LC, KT209LC, KT210LC, KT212LC, 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, "Perique", PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R7 - 12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Spate 168, Spate 172, Spate 179, Spate 210, Spate 220, Spate 225, Spate 227, Spate 234, Spate G - 28, Spate G - 70, Spate H - 6, Spate H20, Spate NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madoll, VA 309, VA 359, or essentially derived from or of the genetic background of any commercially available tobacco variety by standard tobacco breeding techniques known in the art.

[0137] The foregoing specific varieties of dark air - dried, Burley, Maryland, dark fire - dried or Oriental types are all listed for illustrative purposes only. Any additional dark air - dried, Burley, Maryland, dark fire - dried, Oriental varieties are also contemplated herein.

[0138] Also provided herein is a population of tobacco plants. In one aspect, the population of tobacco plants provided herein has a planting density between 5,000 and 8,000 plants per acre, between 5,000 and 7,600 plants per acre, between 5,000 and 7,200 plants per acre, between 5,000 and 6,800 plants per acre, between 5,000 and 6,400 plants per acre, between 5,000 and 6,000 plants per acre, between 5,000 and 5,600 plants per acre, between 5,000 and 5,200 plants per acre, between 5,200 and 8,000 plants per acre, between 5,600 and 8,000 plants per acre, between 6,000 and 8,000 plants per acre, between 6,400 and 8,000 plants per acre, between 6,800 and 8,000 plants per acre, between 7,200 and 8,000 plants per acre, or between 7,600 and 8,000 plants per acre.

[0139] Also provided herein is a container for seeds derived from a tobacco plant described herein. The container for the tobacco seeds of the present disclosure may contain any number, weight or volume of seeds. For example, the container may contain at least 10 or more than 10 seeds; at least 25 or more than 25 seeds; at least 50 or more than 50 seeds; at least 100 or more than 100 seeds; at least 200 or more than 200 seeds; at least 300 or more than 300 seeds; at least 400 or more than 400 seeds; at least 500 or more than 500 seeds; at least 600 or more than 600 seeds; at least 700 or more than 700 seeds; at least 800 or more than 800 seeds; at least 900 or more than 900 seeds; at least 1000 or more than 1000 seeds; at least 1500 or more than 1500 seeds; at least 2000 or more than 2000 seeds; at least 2500 or more than 2500 seeds; at least 3000 or more than 3000 seeds; at least 3500 or more than 3500 seeds; at least 4000 or more than 4000 seeds; or at least 5000 or more than 5000 seeds. Alternatively, the container may contain at least 1 ounce or more than 1 ounce of seeds; at least 5 grams or more than 5 grams of seeds; at least 10 grams or more than 10 grams of seeds; at least 30 grams or more than 30 grams of seeds; at least 50 grams or more than 50 grams of seeds; at least 100 grams or more than 100 grams of seeds; at least 500 grams or more than 500 grams of seeds; at least 1 kilogram or more than 1 kilogram of seeds; at least 1.5 kilograms or more than 1.5 kilograms of seeds; at least 2 kilograms or more than 2 kilograms of seeds; at least 5 kilograms or more than 5 kilograms of seeds; or at least 10 kilograms or more than 10 kilograms of seeds. The container for the tobacco seeds may be any container available in the art. By way of non-limiting example, the container may be a box, bag, packet, pouch, tape roll, tube or bottle.

[0140] In one aspect, the present disclosure provides dried leaves from a modified tobacco plant comprising a reduced level of one or more TSNA. In one aspect, the reduced one or more TSNA are selected from the group consisting of N'-nitrosonornicotine (NNN), 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and any combination thereof. In one aspect, the level of total TSNA or individual TSNA is measured on a freeze-dried leaf sample using liquid chromatography tandem mass spectrometry (LC / MS / MS).

[0141] In one aspect, the present disclosure provides dried leaves from a modified tobacco plant comprising a reduced level of one or more alkaloids. In one aspect, the reduced one or more alkaloids are selected from the group consisting of nicotine, nornicotine, anabasine, anatabine.

[0142] The present disclosure also provides a method for breeding tobacco lines, cultivars or varieties that include increased nitrogen use efficiency. Breeding can be carried out via any known procedure. DNA fingerprinting, SNP mapping, haplotype mapping or similar techniques can be used in a marker-assisted selection (MAS) breeding program to transfer or cross desirable traits or alleles into tobacco plants. For example, a breeder can use the F1 hybrid plants provided herein or further cross the F1 hybrid plants with other donor plants having an agronomically desirable genotype to produce a segregating population of the F2 or backcross generations. Plants of the F2 or backcross generations can be screened for a desired agronomic trait or desired chemical profile using one of the techniques known in the art or listed herein. Depending on the expected inheritance pattern or MAS technique used, self-pollination of the selected plants can be carried out prior to each backcross cycle to assist in the identification of the desired individual plants. The backcross or other breeding procedures can be repeated until the desired phenotype of the recurrent parent is restored. In one aspect, the recurrent parent in the present disclosure can be a flue-cured variety, a burley variety, a dark air-cured variety, a dark fire-cured variety or an oriental variety. In another aspect, the recurrent parent can be a modified tobacco plant, line or variety. In one aspect, the recurrent parent provided herein is TN90. In another aspect, the recurrent parent provided herein is MD609. Other breeding techniques can be found, for example, in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y., which are hereby incorporated herein by reference in their entirety.

[0143] The results of plant breeding programs using the modified tobacco plants described herein include useful lines, cultivars, varieties, progeny, inbreds, and hybrids of the present disclosure. As used herein, the term "variety" refers to a group of plants that share constant characteristics that distinguish them from other plants of the same species. Varieties are often, but not always, commercially available. While possessing one or more unique traits, varieties are further characterized by very little overall variation among individuals within that variety. "Pure line" varieties can be produced by several generations of self-pollination and selection, or by vegetative propagation from a single parent using tissue or cell culture techniques. Varieties can be essentially derived from another line or variety. As defined by the International Convention for the Protection of New Varieties of Plants (December 2, 1961, Geneva; November 10, 1972; October 23, 1978; and March 19, 1991), a variety is a) essentially derived from the original variety or from a variety that is itself essentially derived from the original variety, while retaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the original variety; b) clearly distinguishable from the original variety; and c) "essentially derived" from the original variety when, except for differences that result from the act of derivation, it conforms to the original variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the original variety. Essentially derived varieties can be obtained, for example, by natural or induced mutants, somaclonal variants, selection of variant individuals from plants of the original variety, backcrossing, or transformation. A first tobacco variety and a second tobacco variety that is essentially derived from the first variety are considered to have an essentially identical genetic background. A "line," as distinguished from a variety, most often refers to a group of plants that are used, for example, in plant research, non-commercially. Lines typically show little overall variation among individuals for one or more traits of interest, but may show some variation among individuals for other traits.

[0144] In one aspect, the present disclosure provides a method for producing tobacco plants, the method including crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety, wherein the at least one tobacco plant of the first tobacco variety exhibits increased nitrogen use efficiency compared to control tobacco plants of the same variety grown under equivalent conditions; and selecting progeny tobacco plants that exhibit increased nitrogen use efficiency compared to control tobacco plants of the same hybrid grown under equivalent conditions. In one aspect, the first tobacco variety provided herein includes a modified tobacco plant. In another aspect, the second tobacco variety provided herein includes a modified tobacco plant. In one aspect, the first or second tobacco variety is male sterile. In another aspect, the first or second tobacco variety is cytoplasmic male sterile. In another aspect, the first or second tobacco variety is female sterile. In one aspect, the first or second tobacco variety is a superior variety. In another aspect, the first or second tobacco variety is a hybrid.

[0145] In one aspect, the present disclosure provides a method for introducing one or more transgenes into a tobacco variety, the method including: (a) crossing a first tobacco variety containing one or more transgenes with a second tobacco variety not containing one or more transgenes to produce one or more progeny tobacco plants; (b) genotyping the one or more progeny tobacco plants for the one or more transgenes; and (c) selecting progeny tobacco plants containing the one or more transgenes. In another aspect, these methods further include backcrossing the selected progeny tobacco plants with the second tobacco variety. In a further aspect, these methods further include: (d) crossing the selected progeny plants with themselves or the second tobacco variety to produce one or more additional progeny tobacco plants; and (e) selecting additional progeny tobacco plants containing the one or more transgenes. In one aspect, the second tobacco variety is a superior variety.

[0146] In one aspect, the present disclosure provides a method of introgressing one or more mutations into a tobacco variety, the method comprising: (a) crossing a first tobacco variety comprising one or more mutations with a second tobacco variety that does not comprise one or more mutations to produce one or more progeny tobacco plants; (b) genotyping the one or more progeny tobacco plants for the one or more mutations; and (c) selecting progeny tobacco plants that comprise the one or more mutations. In another aspect, these methods further comprise backcrossing the selected progeny tobacco plants with the second tobacco variety. In a further aspect, these methods further comprise: (d) crossing the selected progeny plants with themselves or the second tobacco variety to produce one or more additional progeny tobacco plants; and (e) selecting additional progeny tobacco plants that comprise the one or more mutations. In one aspect, the second tobacco variety is a elite variety.

[0147] In one aspect, the present disclosure provides a method of growing a population of modified tobacco plants comprising increased nitrogen use efficiency, the method comprising planting a population of tobacco seeds comprising one or more mutations, one or more transgenes, or both, wherein the one or more modified tobacco plants exhibit increased nitrogen use efficiency compared to control tobacco plants of the same variety grown under equivalent conditions.

[0148] In one aspect, the present disclosure provides a method for producing modified seeds, the method comprising: introducing a recombinant DNA construct provided herein into a plant cell; screening a population of plant cells for the recombinant DNA construct; selecting one or more plant cells from the population; generating one or more modified plants from the one or more plant cells; and harvesting one or more modified seeds from the one or more modified plants.

[0149] As used herein, "locus" is a chromosomal region where a polymorphic nucleic acid, a trait determinant, a gene or a marker is located. The loci of the present disclosure contain one or more polymorphisms in a population; for example, alternative alleles are present in some individuals. As used herein, "allele" refers to an alternative nucleic acid sequence at a particular locus. The length of an allele can be as small as 1 nucleotide base, but is typically larger. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome (e.g., occurs for different chromosomes of a heterozygous individual, or occurs between different homozygous or heterozygous individuals in a population). As used herein, the chromosomes of a diploid plant are "hemizygous" when only 1 copy of the locus is present. For example, a transgene to be inserted is hemizygous when it is inserted into one sister chromosome (e.g., the second sister chromosome does not contain the inserted transgene).

[0150] In one aspect, a modified plant, seed, plant component, plant cell or plant genome is homozygous for the transgene provided herein. In another aspect, a modified plant, seed, plant component, plant cell or plant genome is heterozygous for the transgene provided herein. In one aspect, a modified plant, seed, plant component, plant cell or plant genome is hemizygous for the transgene provided herein. In one aspect, a modified plant, seed, plant component, plant cell or plant genome is homozygous for the mutation provided herein. In another aspect, a modified plant, seed, plant component, plant cell or plant genome is heterozygous for the mutation provided herein. In one aspect, a modified plant, seed, plant component, plant cell or plant genome is hemizygous for the mutation provided herein.

[0151] As used herein, "introgress" or "introgressing" refers to the transfer of a desired allele at a locus from one genetic background to another genetic background.

[0152] As used herein, "crossed" or "crossing" means to produce progeny (e.g., cells, seeds or plants) via fertilization, including crossing (sexual) between different plants and self-fertilization (selfing).

[0153] As used herein, "backcross" and "backcrossing" refer to the process of repeatedly backcrossing progeny plants to one of their parents. In a backcrossing scheme, the "donor" parent refers to the parent plant having the desired gene or locus to be introgressed. The "recipient" parent (used once or more) or "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is introgressed. The initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, and "BC2" refers to the third use of the recurrent parent (and so on). In one aspect, backcrossing is performed repeatedly by backcrossing the progeny individuals of each successive backcross generation back to the same parental genotype.

[0154] As used herein, "elite variety" means any variety resulting from breeding and selection for superior agronomic performance.

[0155] As used herein, "selecting" or "selection" refers to the act of removing or picking out a desired individual, usually from a population, based on certain predetermined criteria in the context of breeding.

[0156] In one aspect, the tobacco plants provided herein are hybrid plants. Hybrids can be produced by preventing self-pollination of a female parent plant of a first variety (e.g., the seed parent), fertilizing the female parent plant with pollen from a male parent plant of a second variety, and enabling the formation of F1 hybrid seeds in the female plant. Self-pollination of the female plant can be prevented by emasculating the flower at an early stage of flower development. Alternatively, a form of male sterility can be used to prevent pollen formation in the female parent plant. For example, male sterility can be produced by cytoplasmic male sterility (CMS), or transgenic male sterility in which a transgene inhibits microspore formation and / or pollen formation, or self-incompatibility. Female parent plants containing CMS are particularly useful. In the aspect where the female parent plant is CMS, pollen can be harvested from a male-fertile plant and manually applied to the stigma of the CMS female parent plant, and the resulting F1 seeds are harvested. Additionally, female-sterile plants can be used to prevent self-fertilization.

[0157] Single-cross tobacco F1 hybrids can be made using plants. Pollen from the male parent plant is manually transferred to an emasculated female parent plant or a female parent plant that is male-sterile to form F1 seeds. Alternatively, triple crosses can be performed in which a single-cross F1 hybrid is used as the female parent and crossed with a different male parent. Or alternatively, double-cross hybrids can be produced that cross the F1 progeny of two different single-crosses with each other. The use of self-incompatibility can provide a particular benefit in preventing self-pollination of the female parent when making double-cross hybrids.

[0158] In one aspect, the tobacco varieties provided herein are male sterile. In another aspect, the tobacco varieties provided herein are cytoplasmic male sterile (CMS). Male sterile tobacco plants may be produced by any method known in the art. Methods for producing male sterile tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761 pp. In another aspect, the tobacco varieties provided herein are female sterile. As a non-limiting example, female sterile plants can be produced by mutating the STIG1 gene. See, for example, Goldman et al. 1994, EMBO Journal 13:2976-2984.

[0159] In one aspect, the present disclosure provides and includes a method for determining the NUE of a tobacco line, the method comprising the steps of obtaining at least one metabolite from a tobacco plant of the tobacco line, determining the amount of at least one obtained metabolite, and determining the NUE of the tobacco line based on the determined amount of at least one metabolite. In a further aspect, the at least one metabolite is obtained from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristematic tissue and stem tissue. In a further aspect of this method, at least two metabolites are obtained. In a further aspect of this method, at least three metabolites are obtained. In a further aspect of this method, at least four metabolites are obtained. In a further aspect of this method, at least five metabolites are obtained. In a further aspect of this method, at least six metabolites are obtained. In a further aspect of this method, at least seven metabolites are obtained. In a further aspect of this method, at least eight metabolites are obtained. In a further aspect of this method, at least nine metabolites are obtained. In a further aspect of this method, at least ten metabolites are obtained. In a further aspect of this method, the amounts of at least two metabolites are determined. In a further aspect of this method, the amounts of at least three metabolites are determined. In a further aspect of this method, the amounts of at least four metabolites are determined. In a further aspect of this method, the amounts of at least five metabolites are determined. In a further aspect of this method, the amounts of at least six metabolites are determined. In a further aspect of this method, the amounts of at least seven metabolites are determined. In a further aspect of this method, the amounts of at least eight metabolites are determined. In a further aspect of this method, the amounts of at least nine metabolites are determined. In a further aspect of this method, the amounts of at least ten metabolites are determined.

[0160] In another aspect of the methods provided herein, the amount of a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-oxoglutaric acid, D-23937, X-23937, X-23916, 1-methyladenine, 4-guanidinobutanoate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, X-23366, N-acetylphenylalanine, naringenin, X-23454, X-23580 and X-23852 is determined.

[0161] In another aspect of the methods provided herein, a tobacco plant having increased NUE comprises increased NUE compared to a tobacco plant that contains a lesser amount of at least one metabolite in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least one metabolite in two tissues. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least four metabolites in at least two tissues. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least five metabolites in at least two tissues. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least one metabolite in three tissues. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least two metabolites in at least three tissues. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least three metabolites in at least three tissues. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least four metabolites in at least three tissues. In a further aspect, a tobacco plant having increased NUE contains a lesser amount of at least five metabolites in at least three tissues.In a further aspect, a tobacco plant having increased NUE comprises at least one metabolite in a lesser amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least two metabolites in a lesser amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least three metabolites in a lesser amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least four metabolites in a lesser amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least five metabolites in a lesser amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least one metabolite in a lesser amount in five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least two metabolites in a lesser amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least three metabolites in a lesser amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least four metabolites in a lesser amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least five metabolites in a lesser amount in at least five tissues.

[0162] In another aspect of the methods provided herein, a tobacco plant having increased NUE comprises increased NUE compared to a tobacco line that contains a greater amount of at least one metabolite in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least one metabolite in two tissues. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least four metabolites in at least two tissues. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least five metabolites in at least two tissues. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least one metabolite in three tissues. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least two metabolites in at least three tissues. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least three metabolites in at least three tissues. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least four metabolites in at least three tissues. In a further aspect, a tobacco plant having increased NUE contains a greater amount of at least five metabolites in at least three tissues.In a further aspect, a tobacco plant having increased NUE contains at least one metabolite in a greater amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE contains at least two metabolites in a greater amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE contains at least three metabolites in a greater amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE contains at least four metabolites in a greater amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE contains at least five metabolites in a greater amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE contains at least one metabolite in a greater amount in five tissues. In a further aspect, a tobacco plant having increased NUE contains at least two metabolites in a greater amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE contains at least three metabolites in a greater amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE contains at least four metabolites in a greater amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE contains at least five metabolites in a greater amount in at least five tissues.

[0163] In another aspect of the methods provided herein, tobacco plants having increased NUE comprise increased NUE as compared to a tobacco line comprising equal amounts of at least one metabolite in at least one tissue. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least two metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least three metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least four metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least five metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least one metabolite in two tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least two metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least three metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least four metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least five metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least one metabolite in three tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least two metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least three metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least four metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least five metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE comprise equal amounts of at least one metabolite in four tissues.In a further aspect, a tobacco plant having increased NUE comprises at least two metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least three metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least four metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least five metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least one metabolite in equal amounts in five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least two metabolites in equal amounts in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least three metabolites in equal amounts in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least four metabolites in equal amounts in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least five metabolites in equal amounts in at least five tissues.

[0164] In another aspect of the methods provided herein, tobacco plants having increased NUE contain decreased NUE compared to a tobacco line that contains a lesser amount of at least one metabolite in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least two metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least three metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least four metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least five metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least one metabolite in two tissues. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least two metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least three metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least four metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least five metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least one metabolite in three tissues. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least two metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least three metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least four metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain a lesser amount of at least five metabolites in at least three tissues.In a further aspect, a tobacco plant having increased NUE comprises at least one metabolite in a lesser amount in four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least two metabolites in a lesser amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least three metabolites in a lesser amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least four metabolites in a lesser amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least five metabolites in a lesser amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least one metabolite in a lesser amount in five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least two metabolites in a lesser amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least three metabolites in a lesser amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least four metabolites in a lesser amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least five metabolites in a lesser amount in at least five tissues.

[0165] In another aspect of the methods provided herein, tobacco plants having increased NUE contain decreased NUE as compared to a tobacco line containing a greater amount of at least one metabolite in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least two metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least three metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least four metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least five metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least one metabolite in two tissues. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least two metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least three metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least four metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least five metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least one metabolite in three tissues. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least two metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least three metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least four metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain a greater amount of at least five metabolites in at least three tissues.In a further aspect, a tobacco plant having increased NUE contains at least one metabolite in a greater amount in four tissues. In a further aspect, a tobacco plant having increased NUE contains at least two metabolites in a greater amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE contains at least three metabolites in a greater amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE contains at least four metabolites in a greater amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE contains at least five metabolites in a greater amount in at least four tissues. In a further aspect, a tobacco plant having increased NUE contains at least one metabolite in a greater amount in five tissues. In a further aspect, a tobacco plant having increased NUE contains at least two metabolites in a greater amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE contains at least three metabolites in a greater amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE contains at least four metabolites in a greater amount in at least five tissues. In a further aspect, a tobacco plant having increased NUE contains at least five metabolites in a greater amount in at least five tissues.

[0166] In another aspect of the methods provided herein, tobacco plants having increased NUE contain decreased NUE compared to a tobacco line containing equal amounts of at least one metabolite in at least one tissue. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least two metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least three metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least four metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least five metabolites in at least one tissue. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least one metabolite in two tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least two metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least three metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least four metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least five metabolites in at least two tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least one metabolite in three tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least two metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least three metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least four metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least five metabolites in at least three tissues. In a further aspect, tobacco plants having increased NUE contain equal amounts of at least one metabolite in four tissues.In a further aspect, a tobacco plant having increased NUE comprises at least two metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least three metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least four metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least five metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant having increased NUE comprises at least one metabolite in equal amounts in five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least two metabolites in equal amounts in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least three metabolites in equal amounts in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least four metabolites in equal amounts in at least five tissues. In a further aspect, a tobacco plant having increased NUE comprises at least five metabolites in equal amounts in at least five tissues.

[0167] In another aspect, the methods provided herein include determining the amount of metabolites using a method selected from the group consisting of liquid chromatography / mass spectrometry (LC / MS), high performance liquid chromatography (HPLC), ultra-HPLC (UHPLC), mass spectrometry (MS), tandem mass spectrometry (MS / MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), X-ray fluorescence spectrometry (XRF), ion chromatography (IC), gas chromatography (GC), gas chromatography / mass spectrometry (GC / MS), capillary electrophoresis / mass spectrometry (CE-MS), ion mobility spectrometry / mass spectrometry (IMS / MS), X-ray diffraction, nuclear magnetic resonance (NMR), luminescence spectroscopy, polarography, ultraviolet-visible spectroscopy, infrared spectroscopy, and thin layer chromatography.

[0168] In one aspect, the present specification provides and includes a method for determining the NUE of a tobacco line using metabolite signatures, the method including the steps of isolating metabolite signatures from tobacco plants of a tobacco line, determining the amount of each metabolite included in the metabolite signatures, and determining the NUE of the tobacco line by comparing the metabolite signatures with control metabolite signatures from a control tobacco line including known NUE. In a further aspect of this method, the NUE includes an increased NUE compared to the control tobacco line. In another aspect of this method, the metabolite signatures are isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristematic tissue, and stem tissue.

[0169] In certain aspects of the methods provided herein, a metabolite signature comprises at least two metabolites. In a further aspect, a metabolite signature comprises at least three metabolites. In a further aspect, a metabolite signature comprises at least four metabolites. In a further aspect, a metabolite signature comprises at least five metabolites. In a further aspect, a metabolite signature comprises at least six metabolites. In a further aspect, a metabolite signature comprises at least seven metabolites. In a further aspect, a metabolite signature comprises at least eight metabolites. In a further aspect, a metabolite signature comprises at least nine metabolites. In a further aspect, a metabolite signature comprises at least ten metabolites. In a further aspect, a metabolite signature comprises at least eleven metabolites. In a further aspect, a metabolite signature comprises at least twelve metabolites. In a further aspect, a metabolite signature comprises at least thirteen metabolites. In a further aspect, a metabolite signature comprises at least fourteen metabolites. In a further aspect, a metabolite signature comprises at least fifteen metabolites. In a further aspect, a metabolite signature comprises at least twenty metabolites. In a further aspect, a metabolite signature comprises at least twenty-five metabolites. In a further aspect, a metabolite signature comprises at least thirty metabolites. In a further aspect, a metabolite signature comprises at least thirty-five metabolites. In a further aspect, a metabolite signature comprises at least forty metabolites. In a further aspect, a metabolite signature comprises at least forty-five metabolites. In a further aspect, a metabolite signature comprises at least fifty metabolites. In a further aspect, a metabolite signature comprises between two and fifty metabolites. In a further aspect, a metabolite signature comprises between three and forty-five metabolites. In a further aspect, a metabolite signature comprises between three and forty metabolites. In a further aspect, a metabolite signature comprises between four and thirty-five metabolites.In a further aspect, the metabolite signature comprises metabolites between 5 and 30. In a further aspect, the metabolite signature comprises metabolites between 6 and 25. In a further aspect, the metabolite signature comprises metabolites between 7 and 20. In a further aspect, the metabolite signature comprises metabolites between 8 and 15. In a further aspect, the metabolite signature comprises metabolites between 9 and 14. In a further aspect, the metabolite signature comprises metabolites between 10 and 13. In a further aspect, the metabolite signature comprises metabolites between 10 and 12.

[0170] In one aspect, this specification provides a method for breeding a tobacco line comprising a metabolite signature associated with increased NUE, and includes: a step of determining a metabolite signature of a first tobacco plant derived from a first tobacco line, wherein the first tobacco plant comprises increased NUE compared to a control tobacco plant lacking the metabolite signature; a step of crossing the first plant with a second plant of a second tobacco line; and a step of obtaining at least one progeny seed from the cross, wherein a progeny plant grown from the at least one progeny seed comprises the metabolite signature and the progeny plant comprises increased NUE compared to a control plant lacking the metabolite signature. In a further aspect of this method, the progeny plant is crossed with a third plant derived from the first tobacco line. In another aspect, the first tobacco line is selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925. In another aspect, the second tobacco line is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC. In a further aspect, the metabolite signature comprises a leaf metabolite signature. In a further aspect, the metabolite signature comprises a root metabolite signature. In another aspect, the metabolite signature comprises a greater amount of 4-guanidinobutanoate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23454, X-23580, X-23852, or any combination thereof compared to the metabolite signature of a control tobacco plant.In another aspect, the metabolite signature comprises a lesser amount of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-oxoglutaric acid, X-23937, X-23916, 1-methyladenine, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, naringenin, or any combination thereof as compared to the metabolite signature of a control tobacco plant.

[0171] In another aspect, the methods provided herein include tobacco plants having an increased NUE that includes an increased partial factor productivity (PFP) as compared to tobacco plants lacking an increased NUE grown under the same conditions. In a further aspect, the increased NUE includes an increased agronomic efficiency (AE) as compared to tobacco plants lacking an increased NUE grown under the same conditions. In a further aspect, the increased NUE includes an increased recovery efficiency (RE) as compared to tobacco plants lacking an increased NUE grown under the same conditions. In a further aspect, the increased NUE includes an increased physiological efficiency (PE) as compared to the tobacco plants lacking the increased NUE grown under the same conditions. In a further aspect, the increased NUE includes an increased internal efficiency (IE) as compared to the tobacco plants lacking the increased NUE grown under the same conditions.

[0172] In one aspect, this specification provides and includes a method of selecting a tobacco plant, the method including the steps of obtaining a population of tobacco plants, isolating at least one metabolite associated with increased NUE from at least one tobacco plant from the population of tobacco plants, and selecting at least one tobacco plant that contains a greater amount of at least one metabolite compared to a control tobacco plant. In a further aspect of this method, the selected tobacco plant contains increased NUE compared to the control tobacco plant. In a further aspect of this method, the at least one metabolite is selected from the group consisting of 4-guanidinobutanoate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23454, X-23580, X-23852, or any combination thereof. In a further aspect of this method, the metabolite is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristematic tissue, and stem tissue.

[0173] In one aspect, this specification provides and includes a method of selecting a tobacco plant, the method including the steps of obtaining a population of tobacco plants, isolating at least one metabolite associated with increased NUE from at least one tobacco plant from the population of tobacco plants, and selecting at least one tobacco plant that contains less of at least one metabolite compared to a control tobacco plant. In a further aspect of this method, the selected tobacco plant includes increased NUE compared to the control tobacco plant. In a further aspect of this method, the at least one metabolite is selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-oxoglutaric acid, X-23937, X-23916, 1-methyladenine, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, naringenin, or any combination thereof. In a further aspect of this method, the metabolite is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristematic tissue, and stem tissue.

[0174] In one aspect, this specification provides and includes a method of screening a tobacco plant for a first metabolite signature associated with increased NUE, the method including the steps of isolating a first metabolite signature from a tobacco plant, determining the amount of at least one metabolite included in the first metabolite signature, comparing the first metabolite signature to a second metabolite signature of a control tobacco plant that includes a known NUE, and determining whether the first metabolite signature is associated with increased NUE.

[0175] In one aspect, the present specification provides and includes a modified tobacco seed or a tobacco plant grown therefrom, the modified tobacco seed or tobacco plant including a cisgenic polynucleotide comprising a heterologous promoter operably linked to a coding region, the modified tobacco plant having an increased nitrogen use efficiency as compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seed or tobacco plant includes a heterologous promoter selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, and a tissue-specific promoter. In another aspect, the heterologous promoter includes a polynucleotide sequence derived from a tobacco genome. In another aspect, the heterologous promoter includes a polynucleotide sequence derived from a plant genome. In another aspect, the tissue-preferred promoter is a leaf-preferred promoter. In another aspect, the tissue-preferred promoter is a root-preferred promoter. In a further aspect, the modified tobacco seed or tobacco plant is of the Burley variety.

[0176] In a further aspect, the modified tobacco seeds or tobacco plants of this specification contain less amounts of TSNA compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less amounts of N'-nitrosonornicotine (NNN) compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less amounts of 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK) compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less amounts of N'-nitrosoanatabine (NAT) compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less amounts of N'-nitrosoanabasine (NAB) compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less amounts of alkaloids compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less amounts of nicotine compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less amounts of nor-nicotine compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less amounts of anabasine compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions.In a further aspect, the modified tobacco seeds or tobacco plants contain a lesser amount of anatabine as compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions.

[0177] In a further aspect, the modified tobacco seeds or tobacco plants of the present specification comprise a coding region encoding a polypeptide that is at least 70% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polypeptide that is at least 75% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polypeptide that is at least 80% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polypeptide that is at least 85% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polypeptide that is at least 90% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polypeptide that is at least 95% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polypeptide that is at least 96% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polypeptide that is at least 97% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polypeptide that is at least 98% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8.In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 99% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-8.

[0178] In a further aspect, the modified tobacco seeds or tobacco plants of the present specification comprise a coding region encoding a polynucleotide that is at least 70% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polynucleotide that is at least 75% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polynucleotide that is at least 80% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polynucleotide that is at least 85% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polynucleotide that is at least 90% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polynucleotide that is at least 95% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polynucleotide that is at least 96% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polynucleotide that is at least 97% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seeds or tobacco plants comprise a coding region encoding a polynucleotide that is at least 98% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16.In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polynucleotide that is at least 99% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polynucleotide that is identical to a sequence selected from the group consisting of SEQ ID NOs: 9-16.

[0179] In a further aspect, the modified tobacco seeds or tobacco plants comprise a leaf-preferred promoter encoded by a sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence selected from the group consisting of SEQ ID NOs: 17-19 or a functional fragment thereof.

[0180] In a further aspect, the modified tobacco seed or tobacco plant comprises a root-preferred promoter encoded by a sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence selected from the group consisting of SEQ ID NOs: 20-24 or a functional fragment thereof.

[0181] In a further aspect, the modified tobacco plants of the present specification comprising a cisgenic polynucleotide contain in their root tissue a higher level of a metabolite selected from the group consisting of 4-guanidinobutanoate, syringaldehyde, thiamine and p-hydroxybenzaldehyde as compared to unmodified tobacco plants lacking the cisgenic polynucleotide grown under the same conditions.

[0182] In a further aspect, the modified tobacco plants of the present specification comprising a cisgenic polynucleotide contain in their leaf tissue a higher level of a metabolite selected from the group consisting of 4-guanidinobutanoate, X-23454, X-23580 and X-23852 as compared to unmodified tobacco plants lacking the cisgenic polynucleotide grown under the same conditions.

[0183] In a further aspect, the modified tobacco plants of the present specification comprising a cisgenic polynucleotide contain in their root tissue a lower level of a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-oxoglutaric acid, X-23937, X-23916 and 1-methyladenine as compared to unmodified tobacco plants lacking the cisgenic polynucleotide grown under the same conditions.

[0184] In a further aspect, the modified tobacco plants of the present specification comprising a cisgenic polynucleotide contain in their leaf tissue a lower level of a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine and naringenin as compared to unmodified tobacco plants lacking the cisgenic polynucleotide grown under the same conditions.

[0185] In one aspect, the present specification provides and includes a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a polypeptide that is at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 75% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 80% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 85% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 90% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 95% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 96% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 97% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 98% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 99% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8.In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is 100% identical to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8.

[0186] In one aspect, the present disclosure provides and includes a dried tobacco material or a tobacco product comprising a dried tobacco material, produced from a tobacco plant comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a coding region, wherein the modified tobacco plant comprises an increased nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0187] In one aspect, the present disclosure provides and includes a greenhouse, growth chamber or field comprising a modified tobacco seed or plant disclosed herein. In one aspect, the present disclosure provides and includes a method of growing the tobacco plants of the present disclosure in a greenhouse, growth chamber or field.

[0188] In one aspect, the present disclosure provides and includes a modified tobacco seed or a tobacco plant grown therefrom, comprising at least one mutation in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco plant comprises an increased nitrogen use efficiency compared to an unmodified control tobacco plant lacking at least one mutation when grown under the same conditions. In a further aspect, the mutation within the endogenous locus is selected from the group consisting of an insertion, deletion, substitution and inversion. In another aspect, the mutation within the endogenous locus is a silent mutation, a non-silent mutation or a null mutation. In a further aspect, the modified tobacco seed or the modified tobacco plant is of the Burley variety.

[0189] In a further aspect, the modified tobacco plant contains in its root tissue a metabolite selected from the group consisting of 4-guanidinobutanoate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde at a higher level compared to an unmodified tobacco plant grown under the same conditions. In a further aspect, the modified tobacco plant contains in its leaf tissue a metabolite selected from the group consisting of 4-guanidinobutanoate, X-23454, X-23580, and X-23852 at a higher level compared to an unmodified tobacco plant grown under the same conditions. In a further aspect, the modified tobacco plant contains in its root tissue a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-oxoglutaric acid, X-23937, X-23916, and 1-methyladenine at a lower level compared to an unmodified tobacco plant grown under the same conditions. In a further aspect, the modified tobacco plant contains in its leaf tissue a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, and naringenin at a lower level compared to an unmodified tobacco plant grown under the same conditions.

[0190] In one aspect, the present specification provides and includes a recombinant DNA construct comprising a heterologous promoter operably linked to a guide RNA comprising at least 18 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40. In a further aspect, the guide RNA comprises at least 19 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40. In a further aspect, the guide RNA comprises at least 20 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40. In a further aspect, the guide RNA comprises at least 21 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40. In a further aspect, the guide RNA comprises at least 22 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40. In a further aspect, the guide RNA comprises at least 23 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40. In a further aspect, the guide RNA comprises at least 24 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40. In a further aspect, the guide RNA comprises at least 25 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40. In a further aspect, the guide RNA comprises at least 26 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 25 to 40.In a further aspect, the guide RNA comprises at least 27 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 28 consecutive nucleotides that are 100% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40.

[0191] In one aspect, the present specification provides and includes a dried tobacco material or a tobacco product containing the dried tobacco material, produced from a tobacco plant containing at least one mutation encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus, wherein the modified tobacco seeds or tobacco plants contain increased NUE compared to unmodified control tobacco plants lacking at least one mutation when grown under the same conditions. In a further aspect, the tobacco plant contains at least two mutations encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus. In a further aspect, the tobacco plant contains at least three mutations encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus. In a further aspect, the tobacco plant contains at least four mutations encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus. In a further aspect, the tobacco plant contains at least five mutations encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus. In a further aspect, the tobacco plant contains at least six mutations encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus. In a further aspect, the tobacco plant contains at least seven mutations encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus. In a further aspect, the tobacco plant contains at least eight mutations encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus. In a further aspect, the tobacco plant contains at least nine mutations encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus. In a further aspect, the tobacco plant contains at least ten mutations encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus.

[0192] In one aspect, the present specification provides and includes a modified tobacco seed or a tobacco plant grown therefrom, the modified tobacco seed or tobacco plant comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56, and comprising increased NUE compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 90% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 91% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 92% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 93% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 94% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 95% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56.In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 96% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 97% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 98% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 99% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is 100% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56. In a further aspect, the heterologous promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, and a tissue-specific promoter. In a further aspect, the tissue-preferred promoter is a leaf-preferred promoter. In a further aspect, the tissue-preferred promoter is a root-preferred promoter.

[0193] In a further aspect, the sRNA has at least 18 nucleotides. In a further aspect, the sRNA comprises at least 19 nucleotides. In a further aspect, the sRNA comprises at least 20 nucleotides. In a further aspect, the sRNA comprises at least 21 nucleotides. In a further aspect, the sRNA comprises at least 22 nucleotides. In a further aspect, the sRNA comprises at least 23 nucleotides. In a further aspect, the sRNA comprises at least 24 nucleotides. In a further aspect, the sRNA comprises at least 25 nucleotides. In a further aspect, the sRNA comprises at least 26 nucleotides. In a further aspect, the sRNA comprises at least 27 nucleotides. In a further aspect, the sRNA comprises at least 28 nucleotides. In a further aspect, the sRNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, and precursors thereof. In a further aspect, the sRNA down-regulates the expression or translation of a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56.

[0194] In one aspect, the present specification provides and includes a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 90% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 91% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 92% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 93% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 94% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 95% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 96% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 97% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 98% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 99% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is 100% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0195] In one aspect, the present specification provides and includes a dry tobacco material or a tobacco product comprising a dry tobacco material, which is produced from a tobacco plant comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding an sRNA that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41-56, wherein the modified tobacco seeds or tobacco plants comprise increased NUE compared to unmodified control tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 90% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41-56. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 91% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41-56. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 92% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41-56. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 93% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41-56. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 94% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41-56. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 95% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41-56. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 96% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 41-56.In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 97% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 98% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is at least 99% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide encodes an sRNA that is 100% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0196] In one aspect, the present disclosure provides and includes a method for increasing the NUE of a tobacco plant, the method including introducing a cisgenic nucleic acid molecule into a tobacco cell and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises increased NUE compared to a tobacco plant lacking the cisgenic nucleic acid molecule. In another aspect, the method further includes crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0197] In one aspect, the present specification provides a method for increasing the NUE of a tobacco plant, comprising introducing a modification to a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41 to 56 into a tobacco cell, and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises increased NUE compared to a tobacco plant lacking the modification, and includes the steps. In another aspect, the method further comprises crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant. In a further aspect, the modification is introduced via a method including the use of an RNA-guided nuclease. In a further aspect, the RNA-guided nuclease is selected from the group consisting of Cas9 nuclease, Cpf1 nuclease, CasX nuclease, CasY nuclease, and functional homologs thereof. In a further aspect, the modification is selected from the group consisting of insertion, substitution, inversion, and deletion.

[0198] In one aspect, the present specification provides a method for increasing the NUE of a tobacco plant, comprising introducing a nucleic acid encoding a small interfering RNA (sRNA) homologous to at least 18 consecutive nucleic acids of a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41 to 56 into a tobacco cell, and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises increased NUE compared to a tobacco plant lacking the sRNA, and includes the steps. In another aspect, the method further comprises crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant. In a further aspect, the method comprises introducing an sRNA selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, and precursors thereof.

[0199] In one aspect, the present specification provides a step of providing a first population of tobacco plants comprising increased NUE, a step of genotyping the first population of tobacco plants for the presence of molecular markers within 20 cM of the increased NUE locus; and a step of selecting one or more tobacco plants that have been genotyped and found to contain the molecular marker, and includes it. In a further aspect, the method disclosed herein includes a step of genotyping the first population of tobacco plants for the presence of molecular markers within 15 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes a step of genotyping the first population of tobacco plants for the presence of molecular markers within 10 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes a step of genotyping the first population of tobacco plants for the presence of molecular markers within 9 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes a step of genotyping the first population of tobacco plants for the presence of molecular markers within 8 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes a step of genotyping the first population of tobacco plants for the presence of molecular markers within 7 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes a step of genotyping the first population of tobacco plants for the presence of molecular markers within 6 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes a step of genotyping the first population of tobacco plants for the presence of molecular markers within 5 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes a step of genotyping the first population of tobacco plants for the presence of molecular markers within 4 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes a step of genotyping the first population of tobacco plants for the presence of molecular markers within 3 cM of the increased NUE locus.In a further aspect, the method disclosed herein includes genotyping a first population of tobacco plants for the presence of molecular markers within 2 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes genotyping a first population of tobacco plants for the presence of molecular markers within 1 cM of the increased NUE locus. In a further aspect, the method disclosed herein includes genotyping a first population of tobacco plants for the presence of molecular markers within 0.5 cM of the increased NUE locus. In a further aspect, the method includes crossing one or more selected tobacco plants with a second tobacco plant; and obtaining progeny seeds from the cross. In a further aspect, the molecular marker is selected from the group consisting of SNP markers, INDEL markers, RFLP markers, SSR markers, AFLP markers and RAPD markers.

[0200] In a further aspect, the method provided herein includes tobacco plants comprising an increased NUE locus comprising a polynucleotide encoding a polypeptide that is at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 75% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 80% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 85% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 90% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 95% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 96% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 97% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 98% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 99% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is 100% identical to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8.In a further aspect, the increased NUE locus is genetically related to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 57 to 64. In another aspect, the increased NUE locus is genetically related to the G nucleotide at position 57 of SEQ ID NO: 58. In another aspect, the increased NUE locus is genetically related to the C nucleotide at position 117 of SEQ ID NO: 58. In another aspect, the increased NUE locus is genetically related to the G nucleotide at position 57 and the C nucleotide at position 117 of SEQ ID NO: 58. In another aspect, the increased NUE locus is genetically related to the T nucleotide at position 147 of SEQ ID NO: 57. In another aspect, the increased NUE locus is genetically related to the G nucleotide at position 162 of SEQ ID NO: 59. In another aspect, the increased NUE locus is genetically related to the C nucleotide at position 36 of SEQ ID NO: 60. In another aspect, the increased NUE locus is genetically related to the T nucleotide at position 36 of SEQ ID NO: 61. In another aspect, the increased NUE locus is genetically related to the T nucleotide at position 36 of SEQ ID NO: 62. In another aspect, the increased NUE locus is genetically related to the G nucleotide at position 36 of SEQ ID NO: 63. In another aspect, the increased NUE locus is genetically related to the T nucleotide at position 36 of SEQ ID NO: 64.

[0201] In a further aspect of the methods provided herein, the first population of tobacco plants is of the Maryland variety. In a further aspect, the methods provided herein include a first population of tobacco plants of a variety selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925. In a further aspect, the methods provided herein include a second population of tobacco plants of the Burley variety. In a further aspect, the methods provided herein include a second population of tobacco plants of a variety selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0202] In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker. In a further aspect, the methods provided herein include progeny seeds comprising increased NUE. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 20 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 15 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 10 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 9 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 8 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 7 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 6 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 5 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 4 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 3 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 2 cM of the increased NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 1 cM of the increased NUE efficiency locus provided herein.In a further aspect, the methods provided herein include progeny seeds containing molecular markers within 0.5 cM of the increased NUE efficiency loci provided herein.

[0203] In one aspect, the present disclosure provides a method comprising: providing a first population of tobacco plants; genotyping the first population of tobacco plants for the presence of an increased NUE allele at a locus encoded by a sequence selected from the group consisting of SEQ ID NOs: 9-16; and selecting one or more genotyped tobacco plants that contain the increased NUE allele. In a further aspect, the method further comprises crossing the one or more selected tobacco plants with a second tobacco plant; and obtaining progeny seeds from the cross.

[0204] In one aspect, the present disclosure provides a method for introgressing an increased NUE trait into a tobacco variety, the method comprising: crossing a first tobacco variety having an increased nitrogen use efficiency trait with a second tobacco variety lacking the increased nitrogen use efficiency trait; obtaining progeny seeds from the cross; genotyping at least one progeny seed for a molecular marker associated with the increased nitrogen use efficiency trait, wherein the molecular marker is within 20 cM of a locus having a sequence selected from the group consisting of SEQ ID NOs: 9-16; and selecting progeny seeds that contain the increased nitrogen use efficiency trait.

[0205] In one aspect, the present disclosure provides a method for selecting a tobacco plant having an increased NUE trait, the method comprising: isolating nucleic acid from a collection of tobacco germplasm; assaying the isolated nucleic acid for one or more markers located within 20 cM of a locus having a sequence selected from the group consisting of SEQ ID NOs: 9-16; and selecting a tobacco plant that contains the increased NUE trait. In a further aspect, the method further comprises crossing the one or more selected tobacco plants with a second tobacco plant; and obtaining progeny seeds from the cross.

[0206] In one aspect, this specification provides and includes a method for selecting a tobacco plant having an increased NUE trait, the method comprising the steps of isolating nucleic acid from a collection of tobacco germplasm, assaying the isolated nucleic acid for one or more markers located within 20 cM of a marker selected from the group consisting of SEQ ID NOs: 57 - 64, and selecting a tobacco plant that includes the increased NUE trait. In a further aspect, the method disclosed herein includes the step of assaying the isolated nucleic acid for one or more markers located within 15 cM of a marker selected from the group consisting of SEQ ID NO: 58. In a further aspect, the method disclosed herein includes the step of assaying the isolated nucleic acid for one or more markers located within 10 cM of a marker selected from the group consisting of SEQ ID NOs: 57 - 64. In a further aspect, the method disclosed herein includes the step of assaying the isolated nucleic acid for one or more markers located within 9 cM of a marker selected from the group consisting of SEQ ID NOs: 57 - 64. In a further aspect, the method disclosed herein includes the step of assaying the isolated nucleic acid for one or more markers located within 8 cM of a marker selected from the group consisting of SEQ ID NOs: 57 - 64. In a further aspect, the method disclosed herein includes the step of assaying the isolated nucleic acid for one or more markers located within 7 cM of a marker selected from the group consisting of SEQ ID NOs: 57 - 64. In a further aspect, the method disclosed herein includes the step of assaying the isolated nucleic acid for one or more markers located within 6 cM of a marker selected from the group consisting of SEQ ID NOs: 57 - 64. In a further aspect, the method disclosed herein includes the step of assaying the isolated nucleic acid for one or more markers located within 5 cM of a marker selected from the group consisting of SEQ ID NOs: 57 - 64.In a further aspect, the methods disclosed herein include assaying isolated nucleic acids for one or more markers located within 4 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein include assaying isolated nucleic acids for one or more markers located within 3 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein include assaying isolated nucleic acids for one or more markers located within 2 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein include assaying isolated nucleic acids for one or more markers located within 1 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein include assaying isolated nucleic acids for one or more markers located within 0.5 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein include assaying isolated nucleic acids for a marker selected from the group consisting of SEQ ID NOs: 57-64. In another aspect, an allele associated with increased NUE comprises a G nucleotide at position 57 of SEQ ID NO: 58. In another aspect, an allele associated with increased NUE comprises a C nucleotide at position 117 of SEQ ID NO: 58. In another aspect, an allele associated with increased NUE comprises a G nucleotide at position 57 and a C nucleotide at position 117 of SEQ ID NO: 58. In another aspect, an allele associated with increased NUE comprises a T nucleotide at position 147 of SEQ ID NO: 57. In another aspect, an allele associated with increased NUE comprises a G nucleotide at position 162 of SEQ ID NO: 59. In another aspect, an allele associated with increased NUE comprises a C nucleotide at position 36 of SEQ ID NO: 60. In another aspect, an allele associated with increased NUE comprises a T nucleotide at position 36 of SEQ ID NO: 61.In another aspect, the allele associated with increased NUE contains a T nucleotide at position 36 of SEQ ID NO:62. In another aspect, the allele associated with increased NUE contains a G nucleotide at position 36 of SEQ ID NO:63. In another aspect, the allele associated with increased NUE contains a T nucleotide at position 36 of SEQ ID NO:64. In a further aspect, a tobacco plant comprising any combination of alleles associated with increased NUE disclosed herein can be selected.

[0207] The following are exemplary embodiments.

[0208] Aspect 1. A method for determining the nitrogen use efficiency (NUE) of a tobacco line, the method comprising the following steps: a. obtaining at least one metabolite from a tobacco plant of the tobacco line; b. determining the amount of the at least one metabolite; and c. determining the nitrogen use efficiency of the tobacco line based on the amount of the at least one metabolite identified in step (b).

[0209] Aspect 2. The method of Aspect 1, wherein the at least one metabolite is obtained from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristem tissue, and stem tissue.

[0210] Aspect 3. The method of Aspect 1 or 2, wherein the plant tissue comprises leaf tissue.

[0211] Aspect 4. The method according to any one of Aspects 1 to 3, wherein the plant tissue comprises root tissue.

[0212] Aspect 5. The method according to any one of Aspects 1 to 4, wherein at least one metabolite is selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-oxoglutaric acid, D-23937, X-23937, X-23916, 1-methyladenine, 4-guanidinobutanoate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, X-23366, N-acetylphenylalanine, naringenin, X-23454, X-23580 and X-23852.

[0213] Aspect 6. The method according to any one of Aspects 1 to 5, wherein the NUE comprises an increased NUE compared to a tobacco line that contains a lower amount of at least one metabolite in at least one tissue.

[0214] Aspect 7. The method according to any one of Aspects 1 to 6, wherein the NUE comprises an increased NUE compared to a tobacco line that contains an equal amount of at least one metabolite in at least one tissue.

[0215] Aspect 8. The method according to any one of Aspects 1 to 7, wherein the NUE comprises an increased NUE compared to a tobacco line that contains an equal amount of at least one metabolite in at least one tissue.

[0216] Aspect 9. The method according to any one of Aspects 1 to 8, wherein the NUE comprises a decreased NUE compared to a tobacco line that contains a lower amount of at least one metabolite in at least one tissue.

[0217] Aspect 10. The method according to any one of Aspects 1 to 9, wherein the NUE comprises a decreased NUE compared to a tobacco line that contains an equal amount of at least one metabolite in at least one tissue.

[0218] Aspect 11. Any of the methods of Aspects 1 to 10, wherein the NUE is reduced compared to a tobacco line containing an equal amount of at least one metabolite in at least one tissue.

[0219] Aspect 12. Any of the methods of Aspects 1 to 11, wherein the step of determining the amount of at least one metabolite comprises a method selected from the group consisting of liquid chromatography / mass spectrometry (LC / MS), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), mass spectrometry (MS), tandem mass spectrometry (MS / MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), X-ray fluorescence spectrometry (XRF), ion chromatography (IC), gas chromatography (GC), gas chromatography / mass spectrometry (GC / MS), capillary electrophoresis / mass spectrometry (CE-MS), ion mobility spectrometry / mass spectrometry (IMS / MS), X-ray diffraction, nuclear magnetic resonance (NMR), luminescence spectrometry, polarography, ultraviolet-visible spectrometry, infrared spectrometry, thin layer chromatography.

[0220] Aspect 13. Any of the methods of Aspects 1 to 12, wherein the at least one metabolite comprises at least two metabolites.

[0221] Aspect 14. Any of the methods of Aspects 1 to 13, wherein the at least one metabolite comprises at least five metabolites.

[0222] Aspect 15. Any of the methods of Aspects 1 to 14, wherein the at least one metabolite comprises at least ten metabolites.

[0223] Aspect 16. A method for determining the nitrogen use efficiency (NUE) of a tobacco line using a metabolite signature, the method comprising the following steps: a. Isolating a metabolite signature from a tobacco plant of the tobacco line; b. Determining the amount of each metabolite included in the metabolite signature; c. Determining the NUE of the tobacco line by comparing the metabolite signature with a control metabolite signature from a control tobacco line containing a known NUE.

[0224] Aspect 17. The method of aspect 16, wherein the metabolite signature comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 metabolites.

[0225] Aspect 18. The method of aspect 16 or 17, wherein the NUE comprises an increased NUE compared to a control tobacco line.

[0226] Aspect 19. The method according to any one of aspects 16 to 18, wherein the NUE comprises a reduced NUE compared to a control tobacco line.

[0227] Aspect 20. The method according to any one of aspects 16 to 19, wherein the metabolite signature is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristem tissue, and stem tissue.

[0228] Aspect 21. A method for breeding a tobacco line comprising a metabolite signature associated with an increased nitrogen use efficiency (NUE), the method comprising the following steps: a. Determining the metabolite signature of a first tobacco plant from a first tobacco line, wherein the first tobacco plant comprises an increased NUE compared to a control tobacco plant lacking the metabolite signature; b. Crossing the first plant with a second plant of a second tobacco line; and c. Obtaining at least one progeny seed from the cross of step (a), wherein the progeny plant grown from the at least one progeny seed comprises the metabolite signature and the progeny plant comprises an increased NUE compared to a control plant lacking the metabolite signature.

[0229] Aspect 22. The method of Aspect 21, further comprising the following steps: d. Crossing the progeny plants with tobacco plants derived from a first tobacco line.

[0230] Aspect 23. The method of Aspect 21 or 22, wherein the first tobacco line is selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925.

[0231] Aspect 24. The method according to any one of Aspects 21 to 23, wherein the second tobacco line is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0232] Aspect 25. The method according to any one of Aspects 21 to 24, wherein the metabolite signature includes a leaf metabolite signature.

[0233] Aspect 26. The method according to any one of Aspects 21 to 25, wherein the metabolite signature includes a root metabolite signature.

[0234] Aspect 27. The method according to any one of Aspects 21 to 26, wherein the increased NUE includes an increased partial factor productivity (PFP) as compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0235] Aspect 28. The method according to any one of Aspects 21 to 27, wherein the increased NUE includes an increased agronomic efficiency (AE) as compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0236] Aspect 29. The method according to any one of Aspects 21 to 28, wherein the increased NUE includes an increased recovery efficiency (RE) as compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0237] Aspect 30. Any of the methods of Aspects 21 - 29, wherein the increased NUE comprises an increased physiological efficiency (PE) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0238] Aspect 31. Any of the methods of Aspects 21 - 30, wherein the increased NUE comprises an increased internal efficiency (IE) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0239] Aspect 32. Any of the methods of Aspects 21 - 31, wherein the metabolite signature comprises an equal amount of 4-guanidinobutanoate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23454, X-23580, X-23852, or any combination thereof compared to the metabolite signature of control tobacco plants.

[0240] Aspect 33. Any of the methods of Aspects 21 - 32, wherein the metabolite signature comprises a lesser amount of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-oxoglutaric acid, X-23937, X-23916, 1-methyladenine, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, naringenin, or any combination thereof compared to the metabolite signature of control tobacco plants.

[0241] Aspect 34. Any of the methods of Aspects 21 - 33, wherein the metabolite signature comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 metabolites.

[0242] Aspect 35. A method for selecting a tobacco plant, the method comprising the following steps: a. obtaining a population of tobacco plants; b. isolating at least one metabolite related to increased nitrogen use efficiency (NUE) from at least one tobacco plant in the population of tobacco plants; c. selecting at least one tobacco plant containing an equal amount of the at least one metabolite compared to a control tobacco plant.

[0243] Aspect 36. The method of Aspect 35, wherein the tobacco plant selected in step (c) has an equal NUE compared to the control tobacco plant.

[0244] Aspect 37. The method of Aspect 35 or 36, wherein the increased NUE includes increased partial factor productivity (PFP) compared to a tobacco plant lacking increased NUE grown under the same conditions.

[0245] Aspect 38. The method according to any one of Aspects 35 to 37, wherein the increased NUE includes increased agronomic efficiency (AE) compared to a tobacco plant lacking increased NUE grown under the same conditions.

[0246] Aspect 39. The method according to any one of Aspects 35 to 38, wherein the increased NUE includes increased recovery efficiency (RE) compared to a tobacco plant lacking increased NUE grown under the same conditions.

[0247] Aspect 40. The method according to any one of Aspects 35 to 39, wherein the increased NUE includes increased physiological efficiency (PE) compared to a tobacco plant lacking increased NUE grown under the same conditions.

[0248] Aspect 41. The method according to any one of Aspects 35 to 40, wherein the increased NUE includes increased internal efficiency (IE) compared to a tobacco plant lacking increased NUE grown under the same conditions.

[0249] Aspect 42. The method of any one of Aspects 35 to 41, wherein at least one metabolite is selected from the group consisting of 4-guanidinobutanoate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23454, X-23580, X-23852, or any combination thereof.

[0250] Aspect 43. The method of any one of Aspects 35 to 42, wherein at least one metabolite is isolated from leaf tissue or root tissue.

[0251] Aspect 44. The method of any one of Aspects 35 to 43, wherein at least one metabolite is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristematic tissue, and stem tissue.

[0252] Aspect 45. A method of selecting a tobacco plant, the method comprising the following steps: a. obtaining a population of tobacco plants; b. isolating at least one metabolite related to increased nitrogen use efficiency (NUE) from at least one tobacco plant in the population of tobacco plants; c. selecting at least one tobacco plant that contains a lesser amount of the at least one metabolite compared to a control tobacco plant.

[0253] Aspect 46. The method of Aspect 45, wherein the tobacco plant selected in step (c) contains an equal NUE compared to the control tobacco plant.

[0254] Aspect 47. The method of Aspect 45 or 46, wherein the increased NUE includes an increased partial factor productivity (PFP) compared to a tobacco plant lacking increased NUE grown under the same conditions.

[0255] Aspect 48. The method of any one of Aspects 45 to 47, wherein the increased NUE includes an increased agronomic efficiency (AE) compared to a tobacco plant lacking increased NUE grown under the same conditions.

[0256] Aspect 49. A method according to any one of aspects 45 - 48, wherein the increased NUE comprises an increased recovery efficiency (RE) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0257] Aspect 50. A method according to any one of aspects 45 - 49, wherein the increased NUE comprises an increased physiological efficiency (PE) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0258] Aspect 51. A method according to any one of aspects 45 - 50, wherein the increased NUE comprises an increased internal efficiency (IE) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0259] Aspect 52. A method according to any one of aspects 45 - 51, wherein at least one metabolite is selected from the group consisting of X - 2357, N - acetylmuramate, X - 23319, X - 23852, X - 23330, alpha - ketoglutarate, X - 21756, 4 - hydroxy - 2 - oxoglutaric acid, X - 23937, X - 23916, 1 - methyladenine, X - 23453, X - 11429, X - 21796, N'-methylnicotinamide, cotinine, X - 23389, N - acetylarginine, N - 23366, N - acetylphenylalanine, naringenin, or any combination thereof.

[0260] Aspect 53. A method according to any one of aspects 45 - 52, wherein at least one metabolite is isolated from leaf tissue or root tissue.

[0261] Aspect 54. A method according to any one of aspects 45 - 53, wherein at least one metabolite is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristem tissue, and stem tissue.

[0262] Aspect 55. A method for screening tobacco plants for a metabolite signature associated with increased nitrogen use efficiency (NUE), the method comprising the following steps: a. Isolating a first metabolite signature associated with increased NUE from the tobacco plants; b. Determining the amount of at least one metabolite comprising the first metabolite signature; c. Comparing the first metabolite signature with a second metabolite signature of a control tobacco plant comprising a known NUE; and d. Determining whether the first metabolite signature is associated with an increased NUE.

[0263] Aspect 56. The method of aspect 55, wherein the increased NUE comprises an increased partial factor productivity (PFP) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0264] Aspect 57. The method of aspect 55 or 56, wherein the increased NUE comprises an increased agronomic efficiency (AE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0265] Aspect 58. The method according to any one of aspects 55 to 57, wherein the increased NUE comprises an increased recovery efficiency (RE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0266] Aspect 59. The method according to any one of aspects 55 to 58, wherein the increased NUE comprises an increased physiological efficiency (PE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0267] Aspect 60. The method according to any one of aspects 55 to 59, wherein the increased NUE comprises an increased internal efficiency (IE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0268] Aspect 61. A modified tobacco seed or a tobacco plant grown therefrom, comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a coding region, the modified tobacco seed or the tobacco plant grown therefrom having an increased nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0269] Aspect 62. The modified tobacco seed or tobacco plant of Aspect 61, wherein the coding region encodes a polypeptide that is at least 70% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1 to 8.

[0270] Aspect 63. The modified tobacco seed or tobacco plant of Aspect 61 or 62, wherein the coding region comprises a polynucleotide sequence that is at least 70% identical or complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 9 to 16.

[0271] Aspect 64. The modified tobacco seed or tobacco plant of any one of Aspects 61 to 63, wherein the heterologous promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, and a tissue-specific promoter.

[0272] Aspect 65. The modified tobacco seed or tobacco plant of any one of Aspects 61 to 64, wherein the heterologous promoter comprises a polynucleotide sequence derived from the tobacco genome.

[0273] Aspect 66. The modified tobacco seed or tobacco plant of any one of Aspects 61 to 65, wherein the heterologous promoter comprises a polynucleotide sequence derived from a plant genome.

[0274] Aspect 67. The modified tobacco seed or tobacco plant of any one of Aspects 61 to 66, wherein the tissue-preferred promoter is a leaf-preferred promoter.

[0275] Aspect 68. The modified tobacco seed or tobacco plant of any one of Aspects 61 to 67, wherein the tissue-preferred promoter is a root-preferred promoter.

[0276] Aspect 69. The modified tobacco seed or tobacco plant of any one of Aspects 61 to 68, wherein the leaf-preferred promoter is encoded by a sequence that is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 17 to 19 or a functional fragment thereof.

[0277] Aspect 70. A modified tobacco seed or tobacco plant of any of aspects 61 - 69, wherein the root - preferred promoter is encoded by a sequence that is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NO: 20 - 24 or a functional fragment thereof.

[0278] Aspect 71. A modified tobacco plant of any of aspects 61 - 70, which contains in its root tissue a metabolite selected from the group consisting of 4 - guanidinobutanoate, syringaldehyde, thiamine, and p - hydroxybenzaldehyde at an equal level compared to an unmodified tobacco plant lacking the cis - genic polynucleotide grown under the same conditions.

[0279] Aspect 72. A modified tobacco plant of any of aspects 61 - 71, which contains in its leaf tissue a metabolite selected from the group consisting of 4 - guanidinobutanoate, X - 23454, X - 23580, and X - 23852 at an equal level compared to an unmodified tobacco plant lacking the cis - genic polynucleotide grown under the same conditions.

[0280] Aspect 73. A modified tobacco plant of any of aspects 61 - 72, which contains in its root tissue a metabolite selected from the group consisting of X - 2357, N - acetylmuramate, X - 23319, X - 23852, X - 23330, alpha - ketoglutarate, X - 21756, 4 - hydroxy - 2 - oxoglutarate, X - 23937, X - 23916, and 1 - methyladenine at a lower level compared to an unmodified tobacco plant lacking the cis - genic polynucleotide grown under the same conditions.

[0281] Aspect 74. A modified tobacco plant according to any of Aspects 61 - 73, comprising in its leaf tissue a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, and naringenin, at a lower level compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0282] Aspect 75. A modified tobacco seed or plant according to any of Aspects 61 - 74, which is of the Burley variety.

[0283] Aspect 76. A modified tobacco seed or plant according to any of Aspects 61 - 75, containing a lesser amount of TSNA compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0284] Aspect 77. A recombinant DNA construct comprising a heterologous promoter functionally linked to a polynucleotide encoding a polypeptide that is at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1 - 8.

[0285] Aspect 78. A dried tobacco material or a tobacco product containing the dried tobacco material, produced from a tobacco plant containing a cisgenic polynucleotide functionally linked to a heterologous promoter in its coding region, wherein the modified tobacco plant has an increased nitrogen use efficiency compared to an unmodified control tobacco plant lacking said cisgenic polynucleotide when grown under the same conditions.

[0286] Aspect 79. A greenhouse, growth chamber, or field containing a modified tobacco seed or plant according to any of Aspects 61 - 76.

[0287] Aspect 80. A modified tobacco seed or a tobacco plant grown therefrom, comprising at least one mutation encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus, and having an increased nitrogen use efficiency as compared to an unmodified control tobacco plant lacking said at least one mutation when grown under the same conditions.

[0288] Aspect 81. The modified tobacco seed or tobacco plant of Aspect 80, wherein the at least one mutation is selected from the group consisting of insertion, deletion, substitution, and inversion.

[0289] Aspect 82. The modified tobacco seed or tobacco plant of Aspect 80 or 81, wherein the at least one mutation is a null mutation.

[0290] Aspect 83. The modified tobacco plant of any one of Aspects 80 to 82, comprising in its root tissue a metabolite selected from the group consisting of 4-guanidinobutanoate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde at an equal level as compared to an unmodified tobacco plant lacking said at least one mutation when grown under the same conditions.

[0291] Aspect 84. The modified tobacco plant of any one of Aspects 80 to 83, comprising in its leaf tissue a metabolite selected from the group consisting of 4-guanidinobutanoate, X-23454, X-23580, and X-23852 at an equal level as compared to an unmodified tobacco plant lacking said at least one mutation when grown under the same conditions.

[0292] Aspect 85. A modified tobacco plant according to any of aspects 80 - 84, comprising in its root tissue a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-oxoglutaric acid, X-23937, X-23916 and 1-methyladenine, at a lower level compared to an unmodified tobacco plant lacking at least one mutation when grown under the same conditions.

[0293] Aspect 86. A modified tobacco plant according to any of aspects 80 - 85, comprising in its leaf tissue a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine and naringenin, at a lower level compared to an unmodified tobacco plant lacking at least one mutation when grown under the same conditions.

[0294] Aspect 87. A modified tobacco seed or plant according to any of aspects 80 - 86, which is of the Burley variety.

[0295] Aspect 88. A modified tobacco seed or plant according to any of aspects 80 - 87, containing a lesser amount of TSNA compared to an unmodified tobacco plant lacking at least one mutation when grown under the same conditions.

[0296] Aspect 89. A recombinant DNA construct comprising a heterologous promoter functionally linked to a guide RNA comprising at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or at least 28 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO:25 - 40.

[0297] Aspect 90. A dried tobacco material or a tobacco product containing the dried tobacco material, produced from a tobacco plant having at least one mutation encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40 at an endogenous locus, wherein the modified tobacco seeds or tobacco plants have an increased nitrogen use efficiency compared to unmodified control tobacco plants lacking said at least one mutation when grown under the same conditions.

[0298] Aspect 91. A modified tobacco seed or a tobacco plant grown therefrom, comprising a cisgenic polynucleotide comprising a heterologous promoter functionally linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56, wherein the modified tobacco seeds or tobacco plants have an increased nitrogen use efficiency compared to unmodified control tobacco plants lacking said cisgenic polynucleotide when grown under the same conditions.

[0299] Aspect 92. The modified tobacco seed or tobacco plant of Aspect 91, wherein the sRNA comprises at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or at least 28 nucleotides.

[0300] Aspect 93. The modified tobacco seed or tobacco plant of Aspect 91 or 92, wherein the sRNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, and precursors thereof.

[0301] Aspect 94. The modified tobacco seed or tobacco plant of any one of Aspects 91 to 93, wherein the sRNA down-regulates the expression or translation of a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56.

[0302] Aspect 95. A modified tobacco seed or tobacco plant according to any of Aspects 91 - 94, wherein the heterologous promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, and a tissue-specific promoter.

[0303] Aspect 96. A modified tobacco seed or tobacco plant according to any of Aspects 91 - 95, wherein the tissue-preferred promoter is a leaf-preferred promoter.

[0304] Aspect 97. A modified tobacco seed or tobacco plant according to any of Aspects 91 - 96, wherein the tissue-preferred promoter is a root-preferred promoter.

[0305] Aspect 98. A modified tobacco seed or tobacco plant according to any of Aspects 91 - 97, wherein the leaf-preferred promoter is encoded by a sequence that is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 17 - 19 or a functional fragment thereof.

[0306] Aspect 99. A modified tobacco seed or tobacco plant according to any of Aspects 91 - 98, wherein the root-preferred promoter is encoded by a sequence that is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 20 - 24 or a functional fragment thereof.

[0307] Aspect 100. A modified tobacco seed or tobacco plant according to any of Aspects 91 - 99, wherein the heterologous promoter comprises a polynucleotide sequence derived from the tobacco genome.

[0308] Aspect 101. A modified tobacco seed or tobacco plant according to any of Aspects 91 - 100, wherein the heterologous promoter comprises a polynucleotide sequence derived from a plant genome.

[0309] Aspect 102. A modified tobacco plant according to any of Aspects 91-101, comprising in its root tissue a metabolite selected from the group consisting of 4-guanidinobutanoate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde at an equal level compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0310] Aspect 103. A modified tobacco plant according to any of Aspects 91-102, comprising in its leaf tissue a metabolite selected from the group consisting of 4-guanidinobutanoate, X-23454, X-23580, and X-23852 at an equal level compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0311] Aspect 104. A modified tobacco plant according to any of Aspects 91-103, comprising in its root tissue a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-oxoglutaric acid, X-23937, X-23916, and 1-methyladenine at a lower level compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0312] Aspect 105. A modified tobacco plant according to any of Aspects 91-104, comprising in its leaf tissue a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, and naringenin at a lower level compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0313] Aspect 106. A modified tobacco seed or plant according to any of Aspects 91-105, which is of the Burley variety.

[0314] Modified tobacco seeds or plants of any of aspects 91 - 106, containing less amount of TSNA compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions.

[0315] A recombinant DNA construct comprising a heterologous promoter functionally linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 - 56.

[0316] A dried tobacco material or a tobacco product containing the dried tobacco material, produced from a tobacco plant containing a cisgenic polynucleotide comprising a heterologous promoter functionally linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41 - 56, wherein the modified tobacco seeds or plants have an increased nitrogen use efficiency compared to unmodified control tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions.

[0317] A method for increasing the nitrogen use efficiency (NUE) of a tobacco plant, the method comprising the following steps: a. introducing a cisgenic nucleic acid molecule into tobacco cells; and b. regenerating a modified tobacco plant from the tobacco cells, wherein the modified tobacco plant has an increased NUE compared to a tobacco plant lacking the cisgenic nucleic acid molecule.

[0318] The method of aspect 110, further comprising the following step: c. crossing the modified tobacco plant with a second tobacco plant or self - pollinating the modified tobacco plant.

[0319] Aspect 112. The method of Aspect 110 or 111, wherein the increased NUE comprises increased partial factor productivity (PFP) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0320] Aspect 113. The method according to any one of Aspects 110 to 112, wherein the increased NUE comprises increased agronomic efficiency (AE) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0321] Aspect 114. The method according to any one of Aspects 110 to 113, wherein the increased NUE comprises increased recovery efficiency (RE) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0322] Aspect 115. The method according to any one of Aspects 110 to 114, wherein the increased NUE comprises increased physiological efficiency (PE) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0323] Aspect 116. The method according to any one of Aspects 110 to 115, wherein the increased NUE comprises increased internal efficiency (IE) compared to tobacco plants lacking the increased NUE grown under the same conditions.

[0324] Aspect 117. A method for increasing the nitrogen use efficiency (NUE) of a tobacco plant, the method comprising the following steps: a. Introducing a modification into a tobacco cell to a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41 to 56; b. Regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises increased NUE compared to a tobacco plant lacking the modification.

[0325] Aspect 118. The method of Aspect 117, wherein the introduction comprises the use of an RNA-guided nuclease.

[0326] Aspect 119. The method of Aspect 117 or 118, wherein the RNA-guided nuclease is selected from the group consisting of Cas9 nuclease, Cpf1 nuclease, CasX nuclease, CasY nuclease, and functional homologs thereof.

[0327] Aspect 120. The method according to any one of Aspects 117 to 119, wherein the modification is selected from the group consisting of insertion, substitution, inversion, and deletion.

[0328] Aspect 121. The method according to any one of Aspects 117 to 120, wherein the increased NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0329] Aspect 122. The method according to any one of Aspects 117 to 121, wherein the increased NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0330] Aspect 123. The method according to any one of Aspects 117 to 122, wherein the increased NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0331] Aspect 124. The method according to any one of Aspects 117 to 123, wherein the increased NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0332] Aspect 125. The method according to any one of Aspects 117 to 124, wherein the increased NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0333] Aspect 126. The method according to any one of Aspects 117 to 125, further comprising the following step: c. Crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0334] Aspect 127. A method for increasing the nitrogen use efficiency (NUE) of a tobacco plant, the method comprising the following steps: a. Introducing into a tobacco cell a nucleic acid encoding a small interfering RNA (sRNA) that is homologous to at least 18 consecutive nucleic acids of a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41 to 56; b. Regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant has an increased NUE as compared to a tobacco plant lacking the sRNA.

[0335] Aspect 128. The method of Aspect 127, wherein the sRNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, and precursors thereof.

[0336] Aspect 129. The method of Aspect 127 or 128, wherein the increased NUE comprises an increased partial factor productivity (PFP) as compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0337] Aspect 130. The method according to any one of Aspects 127 to 129, wherein the increased NUE comprises an increased agronomic efficiency (AE) as compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0338] Aspect 131. The method according to any one of Aspects 127 to 130, wherein the increased NUE comprises an increased recovery efficiency (RE) as compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0339] Aspect 132. The method according to any one of Aspects 127 to 131, wherein the increased NUE comprises an increased physiological efficiency (PE) as compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0340] Aspect 133. The method according to any one of Aspects 127 to 132, wherein the increased NUE comprises an increased internal efficiency (IE) as compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0341] Aspect 134. The method according to any one of Aspects 127 to 133, further comprising the following steps: c. Crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0342] Aspect 135. A method comprising the following steps: a. Providing a first population of tobacco plants having increased nitrogen use efficiency; b. Genotyping the first population of tobacco plants for the presence of molecular markers within 20 cM of the locus of increased nitrogen use efficiency; and c. Selecting one or more tobacco plants genotyped in step (b) that contain the molecular marker.

[0343] Aspect 136. The method according to Aspect 135, further comprising the following steps: d. Crossing one or more tobacco plants selected in step (c) with a second tobacco plant; and e. Obtaining progeny seeds from the cross in step (d).

[0344] Aspect 137. The method according to Aspect 135 or 136, wherein the molecular marker is selected from the group consisting of SNP markers, INDEL markers, RFLP markers, SSR markers, AFLP markers, and RAPD markers.

[0345] Aspect 138. The method according to any one of Aspects 135 to 137, wherein the locus of increased nitrogen use efficiency comprises a polynucleotide encoding a polypeptide that is at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1 to 8.

[0346] Aspect 139. The method according to any one of Aspects 135 to 138, wherein the locus of increased nitrogen use efficiency comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 9 to 16.

[0347] Aspect 140. The method according to any one of Aspects 135 to 139, wherein the molecular marker is selected from the group consisting of SEQ ID NOs: 57 to 64.

[0348] Aspect 141. The method according to any one of Aspects 135 to 140, wherein the molecular marker contains a G nucleotide at position 57 of SEQ ID NO: 58.

[0349] Aspect 142. The method according to any one of Aspects 135 to 141, wherein the molecular marker contains a C nucleotide at position 117 of SEQ ID NO: 58.

[0350] Aspect 143. The method according to any one of Aspects 135 to 142, wherein the molecular marker contains a G nucleotide at position 57 and a C nucleotide at position 117 of SEQ ID NO: 58.

[0351] Aspect 144. The method according to any one of Aspects 135 to 143, wherein the molecular marker contains a T nucleotide at position 14 of SEQ ID NO: 57.

[0352] Aspect 145. The method according to any one of Aspects 135 to 144, wherein the molecular marker contains a G nucleotide at position 162 of SEQ ID NO: 59.

[0353] Aspect 146. The method according to any one of Aspects 135 to 145, wherein the molecular marker contains a C nucleotide at position 36 of SEQ ID NO: 60.

[0354] Aspect 147. The method according to any one of Aspects 135 to 146, wherein the molecular marker contains a T nucleotide at position 36 of SEQ ID NO: 61.

[0355] Aspect 148. The method according to any one of Aspects 135 to 147, wherein the molecular marker contains a T nucleotide at position 36 of SEQ ID NO: 62.

[0356] Aspect 149. The method according to any one of Aspects 135 to 148, wherein the molecular marker contains a G nucleotide at position 36 of SEQ ID NO: 63.

[0357] Aspect 150. The method according to any one of Aspects 135 to 149, wherein the molecular marker contains a T nucleotide at position 36 of SEQ ID NO: 64.

[0358] Aspect 151. The method according to any one of Aspects 135 to 150, wherein the first population of tobacco plants is of the Maryland variety.

[0359] Aspect 152. The method according to any one of Aspects 135 to 151, wherein the first population of tobacco plants is of a variety selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925.

[0360] Aspect 153. The method according to any one of Aspects 135 to 152, wherein the second tobacco plant is of a variety selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0361] Aspect 154. The method according to any one of Aspects 135 to 153, wherein the progeny seeds contain the molecular marker.

[0362] Aspect 155. The method according to any one of Aspects 135 to 154, wherein the progeny seeds contain increased nitrogen use efficiency.

[0363] Aspect 156. The method according to any one of Aspects 135 to 155, wherein the molecular marker is within 15 cM of the increased nitrogen use efficiency locus.

[0364] Aspect 157. The method according to any one of Aspects 135 to 156, wherein the molecular marker is within 10 cM of the increased nitrogen use efficiency locus.

[0365] Aspect 158. The method according to any one of Aspects 135 to 157, wherein the molecular marker is within 5 cM of the increased nitrogen use efficiency locus.

[0366] Aspect 159. The method according to any one of Aspects 135 to 158, wherein the molecular marker is within 2 cM of the locus of the increased nitrogen use efficiency gene.

[0367] Aspect 160. The method according to any one of Aspects 135 to 159, wherein the molecular marker is within 1 cM of the locus of the increased nitrogen use efficiency gene.

[0368] Aspect 161. The method according to any one of Aspects 135 to 160, wherein the molecular marker is within 0.5 cM of the locus of the increased nitrogen use efficiency gene.

[0369] Aspect 162. A method comprising the following steps: a. providing a first population of tobacco plants; b. genotyping the first population of tobacco plants for the presence of the allele of the locus of increased nitrogen use efficiency encoded by a sequence selected from the group consisting of SEQ ID NOs: 9 to 16; and c. selecting one or more tobacco plants genotyped in step (b) that contain the allele of the increased nitrogen use efficiency.

[0370] Aspect 163. The method according to Aspect 161 or 162, further comprising the following steps: d. crossing the one or more tobacco plants selected in step (c) with a second tobacco plant; and e. obtaining progeny seeds from the cross in step (d).

[0371] Aspect 164. The method according to any one of Aspects 161 to 163, wherein the first population of tobacco plants is of the Maryland variety.

[0372] Aspect 165. The method according to any one of Aspects 161 to 164, wherein the first population of tobacco plants is of a variety selected from the group consisting of MD609, MD601, Banquet A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925.

[0373] Aspect 166. The method according to any one of Aspects 161 to 165, wherein the second tobacco plant is of a variety selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0374] Aspect 167. The method according to any one of Aspects 161 to 166, wherein the progeny seeds contain the molecular marker.

[0375] Aspect 168. The method according to any one of Aspects 161 to 167, wherein the progeny seeds contain increased nitrogen use efficiency.

[0376] Aspect 169. A method for introducing an increased nitrogen use efficiency trait into a tobacco variety, the method comprising the following steps: a. Crossing a first tobacco variety having the increased nitrogen use efficiency trait with a second tobacco variety lacking the increased nitrogen use efficiency trait; b. Obtaining progeny seeds from the cross in step (a); c. Genotyping at least one of the progeny seeds obtained in step (b) for a molecular marker related to the increased nitrogen use efficiency trait, wherein the molecular marker is within 20 cM of a locus selected from the group consisting of SEQ ID NO: 9 to 16; and d. Selecting progeny seeds containing the increased nitrogen use efficiency trait.

[0377] Aspect 170. The method according to Aspect 169, wherein the first tobacco variety is a Maryland tobacco variety.

[0378] Aspect 171. The method according to Aspect 169 or 170, wherein the first tobacco variety is selected from the group consisting of MD609, MD601, Banquet A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925.

[0379] Aspect 172. The method according to any one of Aspects 169 to 171, wherein the second tobacco variety is a burley tobacco variety.

[0380] Aspect 173. The method according to any one of Aspects 169 to 172, wherein the second tobacco variety is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0381] Aspect 174. The method according to any one of Aspects 169 to 173, wherein the increased NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0382] Aspect 175. The method according to any one of Aspects 169 to 174, wherein the increased NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0383] Aspect 176. The method according to any one of Aspects 169 to 175, wherein the increased NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0384] Aspect 177. The method according to any one of Aspects 169 to 176, wherein the increased NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0385] Aspect 178. The method according to any one of Aspects 169 to 177, wherein the increased NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0386] Aspect 179. The method according to any one of Aspects 169 to 178, wherein the molecular marker is within 15 cM of the locus.

[0387] Aspect 180. The method according to any one of Aspects 169 to 179, wherein the molecular marker is within 10 cM of the locus.

[0388] Aspect 181. The method according to any one of Aspects 169 to 180, wherein the molecular marker is within 5 cM of the locus.

[0389] Aspect 182. The method according to any one of Aspects 169 to 181, wherein the molecular marker is within 2 cM of the locus.

[0390] Aspect 183. The method according to any one of Aspects 169 to 182, wherein the molecular marker is within 1 cM of the locus.

[0391] Aspect 184. The method according to any one of Aspects 169 to 183, wherein the molecular marker is within 0.5 cM of the locus.

[0392] Aspect 185. A method for selecting a tobacco plant comprising an increased nitrogen use efficiency trait, the method comprising the following steps: a. Isolating nucleic acid from a collection of tobacco germplasm; b. Assaying the nucleic acid for one or more markers located within 20 cM of a locus selected from the group consisting of SEQ ID NOs: 9 to 16; and c. Selecting a tobacco plant comprising an increased nitrogen use efficiency trait.

[0393] Aspect 186. The method of Aspect 185, further comprising the following steps: d. Crossing the tobacco plant selected in step (c) with a second tobacco plant; and e. Obtaining progeny seeds from the cross of step (d).

[0394] Aspect 187. The method according to Aspect 185 or 186, wherein the molecular marker is within 15 cM of the locus.

[0395] Aspect 188. The method according to any one of Aspects 185 to 187, wherein the molecular marker is within 10 cM of the locus.

[0396] Aspect 189. The method according to any one of Aspects 185 to 188, wherein the molecular marker is within 5 cM of the locus.

[0397] Aspect 190. The method according to any one of Aspects 185 to 189, wherein the molecular marker is within 2 cM of the locus.

[0398] Aspect 191. The method according to any one of Aspects 185 to 190, wherein the molecular marker is within 1 cM of the locus.

[0399] Aspect 192. The method according to any one of Aspects 185 to 191, wherein the molecular marker is within 0.5 cM of the locus.

[0400] Aspect 193. A method for selecting a tobacco plant comprising an increased nitrogen use efficiency trait, the method comprising the following steps: a. isolating nucleic acid from a collection of tobacco germplasm; b. assaying the nucleic acid for one or more markers located within 20 cM of an SNP marker selected from the group consisting of SEQ ID NOs: 57 to 64; and c. selecting a tobacco plant comprising an increased nitrogen use efficiency trait.

[0401] Aspect 194. The method according to Aspect 193, wherein the assaying step comprises assaying for the G nucleotide at position 57 of SEQ ID NO: 58.

[0402] Aspect 195. The method according to Aspect 193 or 194, wherein the assaying step comprises assaying for the C nucleotide at position 117 of SEQ ID NO: 58.

[0403] Aspect 196. The method according to any one of Aspects 193 to 195, wherein the assaying step comprises assaying for the G nucleotide at position 57 and the C nucleotide at position 117 of SEQ ID NO: 58.

[0404] Aspect 197. The method according to any one of Aspects 193 to 196, wherein the assaying step comprises assaying for the T nucleotide at position 14 of SEQ ID NO:57.

[0405] Aspect 198. The method according to any one of Aspects 193 to 197, wherein the assaying step comprises assaying for the G nucleotide at position 162 of SEQ ID NO:59.

[0406] Aspect 199. The method according to any one of Aspects 193 to 198, wherein the assaying step comprises assaying for the C nucleotide at position 36 of SEQ ID NO:60.

[0407] Aspect 200. The method according to any one of Aspects 193 to 199, wherein the assaying step comprises assaying for the T nucleotide at position 36 of SEQ ID NO:61.

[0408] Aspect 201. The method according to any one of Aspects 193 to 200, wherein the assaying step comprises assaying for the T nucleotide at position 36 of SEQ ID NO:62.

[0409] Aspect 202. The method according to any one of Aspects 193 to 201, wherein the assaying step comprises assaying for the G nucleotide at position 36 of SEQ ID NO:63.

[0410] Aspect 203. The method according to any one of Aspects 193 to 202, wherein the assaying step comprises assaying for the T nucleotide at position 36 of SEQ ID NO:64.

Example

[0411] Example 1. Field production implementation method Tobacco plants grown in the field are produced using standard field production practices. Each test plot contains up to 40 rows of planted seedlings. The seedlings are germinated in the greenhouse before planting. To test the NUE trait, a nitrogen rate of 60 pounds of nitrogen per acre is applied to the test plots. When 50% of the plants in the test plot reach the stage of elongated buds, the plants are pinched using standard procedures. Pesticide application follows standard protocols. The leaves are harvested at maturity, bundled with one drying stick per five plants, and sorted into three sticks per plot. The leaves are harvested from the sticks at the takedown / uprooting stage. Five leaves are harvested from three different sticks per experimental variety to make 15 leaves per sample. The midrib half of the fourth leaf from the top of each plant is harvested for sampling. Analytical analysis of alkaloids, TSNA, and NO3 is performed using routine methods known in the art.

[0412] Example 2. Identification of Metabolites Associated with Increased Nitrogen Use Efficiency The Maryland tobacco variety requires approximately 25% less nitrogen fertilizer input compared to the Burley tobacco variety. To identify metabolites associated with high nitrogen efficiency tobacco varieties (Maryland) and low nitrogen efficiency tobacco varieties (Burley), differences in metabolite levels were examined in the Maryland tobacco variety MD609 and the Burley tobacco variety TN90.

[0413] MD609 and TN90 seedlings were germinated from seeds and grown for 6 weeks without added nitrogen. After 6 weeks, the seedlings from each variety were divided into two groups: Group A included plants provided with 100 parts per million of nitrogen, i.e., normal greenhouse fertilization; Group B included plants provided with 25 ppm, i.e., 25% of the normal greenhouse fertilization rate. At 10 weeks and 14 weeks after sowing, metabolites were extracted from the root and leaf tissues using methanol.

[0414] The isolated metabolites were analyzed using three different LC / MS approaches (UHPLC-MS / MS(+ESI), UHPLC-MS / MS(-ESI) and GC-MS(+EI)) to separate and identify the individual metabolites. The metabolites were identified by comparing the resulting mass spectra with a standard spectral database (Metabolon Inc, Morrisville, NC). Peaks were quantified using the area under the curve. Each compound was scaled (referred to as "partial correction") by setting the median equal to 1 (1.00) and normalizing each data point proportionally. The molecular masses of the unknown metabolites are provided in Table 1. The identified metabolites are shown in Tables 2 - 5 below along with the measurements of each scaled sample. The identified metabolites were determined by a Student's t-test comparison between TN90 and MD609 considering all time points. Metabolites with a p-value less than 0.01 were included in the analysis.

[0415] (Table 1) Molecular masses (in kilodaltons) of unknown metabolite compounds TIFF2025108651000002.tif71128

[0416] (Table 2) Metabolites negatively correlated with increased nitrogen efficiency identified in root tissues when comparing the MD609 tobacco line and the TN90 tobacco line at 10 and 14 weeks after sowing TIFF2025108651000003.tif74135

[0417] (Table 3) Metabolites positively correlated with increased nitrogen efficiency identified in root tissues when comparing the MD609 tobacco line and the TN90 tobacco line TIFF2025108651000004.tif38136

[0418] (Table 4) Metabolites negatively correlated with increased nitrogen efficiency identified in leaf tissues when comparing the MD609 tobacco line and the TN90 tobacco line TIFF2025108651000005.tif70133

[0419] (Table 5) Metabolites positively correlated with increased nitrogen efficiency identified in leaf tissue when comparing the MD609 tobacco line and the TN90 tobacco line TIFF2025108651000006.tif38133

[0420] Example 3. Identification of gene expression associated with increased nitrogen use efficiency The same plants used in Example 1 are also subjected to RNA extraction for use in RNAseq. RNA is extracted from leaf and root tissues at 10 and 14 weeks after sowing and used for Illumina sequencing. The RNAseq data is analyzed according to standard methods in the art. Candidate genes are then verified.

[0421] It was found that 17 genes (Tables 6 and 7) were negatively correlated with the increased nitrogen use efficiency phenotype of MD609, and 7 genes (Tables 8 and 9) were positively correlated with the increased nitrogen use efficiency phenotype of MD609. The negatively correlated genes are candidates for downregulation (via mutagenesis, cisgenic transformation or transgenic transformation) in burley tobacco varieties, and the positively correlated genes are candidates for overexpression in burley tobacco varieties that improve nitrogen use efficiency. Associated single nucleotide polymorphism (SNP) markers are provided to track each candidate gene (Tables 6 - 10). Polymorphisms associated with the MD609 allele, thus polymorphisms favorable for increased NUE, are provided (Table 10).

[0422] For each of the correlated genes, the genomic positions are identified to identify four clusters of genes related to increased NUE in the tobacco genome (Figure 1). Seven genes are also located on chromosome 1, four genes are also located on chromosome 11, three genes are also located on chromosome 14, and five genes are also located on chromosome 20 (Figure 1). These four positions are also hotspots of genes differentially expressed between low-nitrogen and normal-nitrogen conditions (Figure 1). SNP markers are created to identify the MD609 polymorphisms specific to each of these positions, and thus the increased NUE polymorphisms (Tables 6 - 10). Further characterizing the region on chromosome 11, it contains a total of 79 expressed genes, 46 of which are genes differentially expressed under low-nitrogen conditions (Figure 2).

[0423] (Table 6) Genes identified as negatively correlated with increased nitrogen use efficiency in root tissue TIFF2025108651000007.tif85144

[0424] (Table 7) Genes identified as negatively correlated with increased nitrogen use efficiency in leaf tissue TIFF2025108651000008.tif57144

[0425] (Table 8) Genes identified as positively correlated with increased nitrogen use efficiency in root tissue TIFF2025108651000009.tif46144

[0426] (Table 9) Genes identified as positively correlated with increased nitrogen use efficiency in leaf tissue TIFF2025108651000010.tif46144

[0427] (Table 10) SNP markers containing polymorphisms related to increased NUE TIFF2025108651000011.tif57144

[0428] Example 4. Identification of Tobacco Leaf-Preferred Promoter and Root-Preferred Promoter RNA samples are obtained from 10 tissue types (axillary buds before topping; axillary buds 2 hours after topping; axillary buds 6 hours after topping; axillary buds 24 hours after topping; axillary buds 72 hours after topping; roots before topping; roots 24 hours after topping; roots 72 hours after topping; young leaves at the time of topping; and shoot apical meristem) of 4-week-old TN90 tobacco plants. The obtained RNA samples (3 independently collected samples for each tissue type) are used as starting materials for Illumina 1×100bp sequencing.

[0429] Map the Illumina reads and use this to identify a list of candidate genes showing high root expression or leaf expression. Tables 11 and 12 provide the RPKM expression values of the genes identified as having leaf-preferred expression or root-preferred expression. These genes are candidates that possess a leaf-preferred promoter or a root-preferred promoter, respectively.

[0430] (Table 11) Genes with Leaf-Preferred Expression TIFF2025108651000012.tif22536

[0431] (Table 12) Genes with Root-Preferred Expression TIFF2025108651000013.tif22741

[0432] Example 5. Development of Modified Plants Using the expression vector p45-2-7 (SEQ ID NO:65) as a backbone, a plurality of transformation vectors are prepared (see Examples X - Y). p45-2-7 contains a cassette comprising a CsVMV promoter, a NOS terminator, and a kanamycin selection marker (NPT II) functionally linked to an actin 2 promoter and a NOS terminator. A nucleic acid vector containing the transgene of interest is introduced into tobacco leaf disks via Agrobacterium transformation. See, for example, Mayo et al., 2006, Nat Protoc. 1:1105-11 and Horsch et al., 1985, Science 227:1229-1231.

[0433] TN90 tobacco plants are grown in Magenta (trademark) GA-7 boxes, and leaf disks are cut and placed into Petri dishes. Agrobacterium tumefaciens cells containing the transformation vector are collected by centrifuging 20 mL of the cell suspension in a 50 mL centrifuge tube at 3500 RPM for 10 minutes. The supernatant is removed, and the Agrobacterium tumefaciens cell pellet is resuspended in 40 mL of liquid resuspension medium. Avoiding the midrib, the tobacco leaves are cut into eight 0.6 cm disks with a #15 scalpel blade, turned upside down, and placed into Petri dishes. A thin layer of Murashige & Skoog containing B5 vitamin liquid resuspension medium is added to the Petri dishes, and the leaf disks are pricked uniformly with a fine-tipped needle. Approximately 25 mL of the Agrobacterium tumefaciens suspension is added to the Petri dishes, and the leaf disks are incubated in the suspension for 10 minutes.

[0434] Transfer the leaf disks to a co-culture Petri dish (1 / 2 MS medium), turn the disks upside down, and contact them with filter paper laid on co-culture TOM medium (MS medium containing 20 g / L sucrose; 1 mg / L indole-3-acetic acid; and 2.5 mg / L 6-benzylaminopurine (BAP)). After sealing the Petri dish with parafilm, incubate it for 3 days at 24 °C with a light cycle of 18 hours of light and 6 hours of dark under dim light (60 - 80 mE / ms). After incubation, transfer the leaf disks to a regeneration / selection TOM K medium Petri dish (TOM medium + 300 mg / L kanamycin). Subculture the leaf disks in fresh TOM K medium at 24 °C with a light cycle of 18 hours of light and 6 hours of dark under dim light once every two weeks until shoots can be excised. Remove the shoots from the leaves with forceps and insert them into MS basal medium containing 100 mg / L kanamycin. Incubate the shoots on MS basal medium containing 100 mg / L kanamycin at 24 °C with a light cycle of 18 hours of light and 6 hours of dark under high-intensity illumination (60 - 80 mE / ms) to induce root formation.

[0435] Once the small plantlets containing both shoots and roots have grown large enough (e.g., until they reach approximately half the height of a Magenta™ GA-7 box), transfer them to soil. Transfer the rooted seedlings to a greenhouse for further analysis and to allow them to set seeds. Evaluate the increased nitrogen use efficiency phenotype by growing modified plants (T0, T1, T2, or later generations) and control plants. The control plants are either untransformed NLM plants or NLM plants transformed with an empty p45-2-7 vector.

[0436] Perform phenotypic screening for increased nitrogen use efficiency in the greenhouse using zero parts per million (ppm) nitrogen (no nitrogen), 25 ppm nitrogen (low nitrogen), and 100 ppm nitrogen (normal nitrogen). Begin the initial screening in the greenhouse using T1 plants. Next, evaluate the homozygous T2 population in the field using 60 pounds of fertilizer per acre (about 25% of the recommended rate for burley tobacco). Measure seedling growth, chlorophyll loss, and final yield and compare to control plants grown at normal nitrogen levels.

[0437] In the T1 generation, plants overexpressing G20580 (two independent transformants), plants overexpressing G42290 (four independent transformants), plants overexpressing G41446 (four independent transformants), plants overexpressing G53261 (two independent transformants), and plants overexpressing G30999 (three independent transformants) are grown in the greenhouse with the control under nitrogen-limiting conditions equivalent to 60 pounds of nitrogen per acre. Nine plants per transformant are harvested, and one line overexpressing G41446 shows a statistically significant increase in yield (grams of fresh weight per plant) compared to the control (see Figure 5).

[0438] Example 6. Generation of Cisgenic Tobacco Plants with Increased Nitrogen Use Efficiency Nitrogen use efficiency can be improved by modifying the expression of genes involved in genes identified as differentially expressed in Example 2. Similarly, nitrogen use efficiency can be improved by regulating genes involved in the biosynthesis or degradation of metabolites identified in Example 1. Genes positively associated with increased nitrogen use efficiency can be overexpressed using a general overexpression promoter or a tissue-preferred promoter to overexpress the gene in the desired tissue.

[0439] Construct a transformation vector to overexpress a protein positively related to increased nitrogen use efficiency. Incorporate separate transformation vectors containing one of SEQ ID NOs: 9 - 16 into the p45 - 2 - 7 transformation vector. Additionally, construct a transformation vector containing one of SEQ ID NOs: 9 - 16.

[0440] Using these transformation vectors, produce modified tobacco plants according to Example 4. Then, evaluate the modified tobacco plants (T1 generation) and control tobacco plants with respect to phenotype as described in Example 4. The modified tobacco plants show increased nitrogen use efficiency compared to the control tobacco plants grown under the same conditions.

[0441] Example 7. Production of transgenic tobacco plants with increased nitrogen use efficiency Also, nitrogen use efficiency can be increased by down - regulating the expression of genes identified as being negatively related to nitrogen use efficiency in Example 2.

[0442] Design a transformation vector containing an RNAi construct to inhibit a tobacco gene whose expression was identified as being negatively related to nitrogen use efficiency in Example 2. Incorporate separate transformation vectors containing one of SEQ ID NOs: 41 - 56 into the p45 - 2 - 7 transformation vector. Construct an additional transformation vector containing one of SEQ ID NOs: 41 - 56.

[0443] Using these transformation vectors, produce modified tobacco plants according to Example 4. Then, evaluate the modified tobacco plants (T1 generation) and control tobacco plants with respect to phenotype as described in Example 4. The modified tobacco plants show increased nitrogen use efficiency compared to the control tobacco plants grown under the same conditions.

[0444] Example 8. Additional methods for improving nitrogen use efficiency using gene editing techniques Using gene editing technologies such as CRISPR / Cas9, CRISPR / Cpf1, CRISPR / CasX, CRISPR / CasY, CRISPR / Csm1, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN), the coding regions of genes negatively associated with increased nitrogen use efficiency are modified so that the genes encode non-functional proteins or proteins with lower functionality. Also, using these gene editing technologies, the endogenous promoter sequences are edited or replaced so as to drive the expression of their cognate proteins in either the leaf tissue or the root tissue, thereby improving nitrogen use efficiency. For example, endogenous G64360 is edited or replaced so that the gene is expressed only in the leaf tissue, where it can act to improve the nitrogen use efficiency of the plant.

[0445] Separate CRISPR / Cas9 guide RNAs or CRISPR / Cpf1 guide RNAs are constructed to recognize and hybridize to each one of the promoter sequences of SEQ ID NOs: 9 to 40. When a tobacco plant is provided with the engineered guide RNA and a donor polynucleotide containing a promoter selected from the group consisting of SEQ ID NOs: 17 to 24, the selected promoter replaces the endogenous promoter of the selected gene, making it possible to restrict the endogenous expression to either the leaf tissue or the root tissue as desired. The edited tobacco plants show increased nitrogen use efficiency compared to control tobacco plants grown under similar conditions.

[0446] Example 9. Development of novel mutations for improving nitrogen use efficiency via random mutagenesis Random mutagenesis of tobacco plants is carried out using ethyl methanesulfonate (EMS) mutagenesis or fast neutron bombardment. EMS mutagenesis consists of chemically inducing random point mutations. Fast neutron mutagenesis consists of exposing seeds to neutron bombardment that causes large deletions through double-strand DNA breaks.

[0447] For EMS mutagenesis, 1 gram (approximately 10,000 seeds) of burley tobacco variety TN90 seeds are washed in 0.1% Tween for 15 minutes and then immersed in 30 mL of ddH2O for 2 hours. Then, 150 μL of 0.5% EMS (Sigma, Catalogue No. M-0880) is mixed into the seed / ddH2O solution and incubated at room temperature (RT; approximately 20 °C) for 8 - 12 hours (while rotating at 30 R.P.M.) under a hood. Next, the liquid is removed from the seeds and mixed overnight in 1 M NaOH for decontamination and disposal. Then, the seeds are washed twice with 100 mL of ddH2O over 2 - 4 hours. Then, the washed seeds are suspended in a 0.1% agar solution.

[0448] The EMS-treated seeds in the agar solution are evenly spread at approximately 2000 seeds / flat on Carolina’s Choice Tobacco Mix (Carolina Soil Company, Kinston, NC) soaked with water in flats. Then, the flats are covered with wrap and placed in a growth chamber. When seedlings emerge from the soil, holes are made in the wrap to gradually reduce the humidity. After 2 weeks, the wrap is completely removed. The flats are moved to a greenhouse and NPK fertilizer is applied. The seedlings are transplanted into floating trays and grown to transplant size. Then, the plants are transplanted to the field. During growth, the plants are self-pollinated to form M1 seeds. At the maturity stage, 5 capsules are harvested from each plant and individual names are given to each set of seeds from each plant. This forms the M1 population. The mixture of M1 seeds from each M0 plant is grown and the plants are evaluated for phenotype as described in Example 4 for increased nitrogen efficiency. M1 plants showing increased nitrogen efficiency are selected and screened for mutations using DNA sequencing and gene mapping techniques known in the art.

[0449] Example 10. Use of breeding to produce tobacco plants with increased nitrogen use efficiency Using conventional breeding techniques, the preferred alleles of NUE provided herein can be introduced into any tobacco variety to increase NUE. A population of tobacco plants can be produced by crossing a tobacco plant having at least one preferred NUE allele (see Table 10) with a tobacco plant lacking that preferred allele. Marker-assisted selection, or other techniques known in the art (e.g., direct sequencing), can be used to track the introgression of the preferred allele in the F1 generation and to determine heterozygosity or homozygosity in subsequent generations. The increased NUE of the progeny plants can be determined using methods known in the art or the methods described above. Preferred alleles of multiple different NUEs can be combined into a single line. Molecular phenotypes, such as those determined by metabolite signatures, can be used to track increased NUE during breeding. The metabolite signatures of the progeny plants can be determined using the methods described above. Progeny plants having the metabolite signature of the parent plant with increased NUE are crossed to produce subsequent populations of tobacco plants with increased NUE.

[0450] The introduction of the Maryland 609 locus into commercially available burley varieties can be carried out as described to develop burley lines with increased NUE. Screening of 23 burley lines and 6 MD609 lines identified 3 burley lines containing the MD609 allele at SNP marker S451 (SEQ ID NO:58) (Figure 3). Three burley lines with the MD609 allele were tested for chlorophyll loss, growth, and yield under nitrogen-limiting conditions and compared to the control TN90 burley line and a control MD609 line (MD609 having the MD609 allele at SNP marker S451) (Figure 4). Burley lines with the MD609 allele exhibit chlorophyll loss, growth, and yield more similar to the Maryland control (Figure 4). The TN90 burley control shows increased chlorophyll loss, decreased growth, and decreased yield compared to the MD609 control (Figure 4). These results indicate that the introduction of the MD609 allele at SNP marker S451 can increase NUE.

[0451] To introduce the MD609 allele into burley, MD609 was crossed with burley. F1 progeny from this cross were selected and then self-pollinated to produce F2 seeds. F2 and F3 plants were grown and self-pollinated to produce F4 seeds. Large quantities of F4 seeds from two independent crossing schemes identified as the NUE-2 line and the NUE-3 line were grown and harvested in the field. Genotypes for SNP markers S451, S317, S12385, S238, S3894, and S2237 were determined for F4 seeds of both the NUE-2 line and the NUE-3 line (see Table 13). F4 plants were grown using the reduced nitrogen production method described in Example 1. Both the NUE-2 line and the NUE-3 line show increased yield (in pounds) per acre compared to the burley control TN90 (see Figure 6).

[0452] Alternatively, a modified tobacco plant comprising an increased NUE phenotype can be produced using the methods described herein and crossed with an unmodified tobacco plant to transmit the modification to subsequent generations. Selection for the genetic modification can be followed using appropriate techniques known in the art. The increased NUE of the progeny plants can be determined using methods known in the art or the methods described above.

[0453] (Table 13) Genotypes of plants grown in the field derived from the F4 NUE-2 and NUE-3 lines and TN90. MD represents the MD609 allele, Barley represents the Barley allele, and HET represents the heterozygous MD609 / Barley. TIFF2025108651000014.tif35128

[0454] Sequence Information SEQUENCE LISTING <110> ALTRIA CLIENT SERVICES LLC <120> METHODS AND COMPOSITIONS RELATED TO IMPROVED NITROGEN UTILIZATION EFFICIENCY IN TOBACCO <150> US 62 / 553,501 <151> 2017-09-01 <160> 65 <170> PatentIn version 3.5 <210> 1 <211> 128 <212> PRT <213> Nicotiana tabacum <400> 1 Met Gly Leu Lys Gly Lys Leu Ile Ser Gln Met Glu Met Lys Cys Ala 1 5 10 15 Gly Asp Leu Leu His Glu His Phe Lys Ser Asn Pro His Gln Thr Ser 20 25 30 Thr Met Ser Pro Asp Lys Ile Thr Asn Phe Thr Leu His Glu Gly Gln 35 40 45 Leu Gly Asn Thr Gly Ser Val Val Ser Trp Lys Tyr Val Leu Gly Gly 50 55 60 Lys Glu Arg His Ala Lys Gln Ala Leu His Ile Asp Asp Ala Lys Lys 65 70 75 80 Ser Ile Thr Phe Asn Phe Leu Glu Gly Tyr Met Asn Glu Leu Tyr Lys 85 90 95 Ser Met Thr Pro Gln Tyr Arg Ile Asn Asn Asn Leu Glu Cys His Lys 100 105 110 Ser Arg Asn His Pro Met Gln Val Thr Ser Pro Asn His Thr Gln Ile 115 120 125 <210> 2 <211> 540 <212> PRT <213> Nicotiana tabacum <400> 2 Met Lys Ala Glu Gly Ser Ala Leu Ser Ser Ala Gly Ser Tyr His Arg 1 5 10 15 Leu Ala Tyr His Glu Val Ile Asn Asp Asp Asn Gln Asn Lys Ile Phe 20 25 30 Thr Ser Asp Asp Ser Arg Leu Arg Gln Leu Gly Tyr Lys Gln Glu Leu 35 40 45 Tyr Arg Gly Leu Ser Phe Ile Ala Asn Phe Ser Phe Thr Phe Ala Ile 50 55 60 Val Ser Val Leu Thr Gly Ile Ser Thr Leu Tyr Asn Gln Ala Leu Thr 65 70 75 80 Phe Gly Gly Pro Ile Thr Leu Val Tyr Gly Trp Pro Ile Val Ser Leu 85 90 95 Met Thr Leu Ile Val Gly Leu Ala Met Ala Glu Ile Cys Ser Ala Tyr 100 105 110 Pro Thr Ser Ala Gly Leu Tyr Tyr Trp Ser Ala Lys Leu Ser Gly Asn 115 120 125 Tyr Phe Gly Pro Phe Ala Ser Trp Ile Thr Gly Trp Phe Asn Ile Val 130 135 140 Gly Gln Trp Ala Val Thr Ala Ser Ile Asp Phe Ser Leu Ala Gln Leu 145 150 155 160 Val Gln Val Met Ile Leu Leu Ser Thr Gly Gly Leu Asn Gly Gly Gly 165 170 175 Tyr Gln Ala Ser Lys Tyr Val Val Ile Ala Leu His Gly Gly Ile Leu 180 185 190 Leu Leu His Ala Ile Leu Asn Ser Leu Pro Ile Ser Trp Leu Ser Phe 195 200 205 Phe Gly Gln Leu Ala Ala Ala Trp Asn Val Leu Gly Val Phe Leu Leu 210 215 220 Met Ile Leu Ile Pro Met Val Ser Thr Glu Arg Ala Ser Ala Lys Phe 225 230 235 240 Val Phe Thr Asn Phe Asn Thr Asp Asn Gly Asp Gly Ile Asn Asn Asn 245 250 255 Leu Tyr Ile Phe Val Leu Gly Leu Leu Met Ser Gln Tyr Thr Leu Thr 260 265 270 Gly Tyr Asp Ala Ser Ala His Met Thr Glu Glu Thr Lys Asn Ala Asp 275 280 285 Lys Asn Gly Pro Lys Gly Ile Val Ser Ala Ile Gly Ile Ser Val Leu 290 295 300 Ala Gly Trp Ala Tyr Ile Leu Gly Ile Thr Phe Ala Val Thr Asp Ile 305 310 315 320 Pro His Leu Leu Asn Lys Asn Asn Asp Ser Gly Gly Tyr Ala Ile Ala 325 330 335 Gln Ile Phe Tyr Asp Ala Phe Lys Asn Arg Tyr Gly Ser Gly Val Gly 340 345 350 Gly Ile Ile Cys Leu Gly Val Ile Ala Ile Ala Val Phe Phe Cys Gly 355 360 365 Met Ser Ser Leu Thr Ser Asn Ser Arg Met Ala Tyr Ala Phe Ser Arg 370 375 380 Asp Gly Ala Met Pro Tyr Ser Ser Phe Trp His Lys Val Asn Lys Gln 385 390 395 400 Glu Val Pro Leu Asn Ala Val Trp Met Ser Ala Phe Ile Ala Phe Cys 405 410 415 Met Ala Leu Thr Ser Leu Gly Ser Leu Val Ala Phe Gln Ala Met Thr 420 425 430 Ser Ile Ala Thr Ile Gly Leu Tyr Ile Ala Tyr Ala Leu Pro Ile Leu 435 440 445 Phe Arg Val Thr Leu Ala Arg Lys Ser Phe Thr Pro Gly Pro Phe Asn 450 455 460 Leu Gly Ser Tyr Gly Leu Val Val Gly Trp Val Ala Ile Phe Trp Val 465 470 475 480 Ala Leu Ile Ser Val Leu Phe Ser Leu Pro Val Ala Tyr Pro Ile Thr 485 490 495 Asp Gln Thr Leu Asn Tyr Thr Pro Val Ala Val Gly Gly Leu Leu Ile 500 505 510 Leu Val Val Ser Ser Trp Ile Phe Ser Ala Ile His Trp Phe Lys Gly 515 520 525 Pro Ile Thr Asn Leu Gly Asn Ser Ser Glu Glu Ala 530 535 540 <210> 3 <211> 145 <212> PRT <213> Nicotiana tabacum <400> 3 Met Ala Ser Thr Gln Gln Ala Val Ser Ser Gly Ser Asp Ala Asp Gln 1 5 10 15 Arg Tyr Ala Lys Phe Asp Glu Arg Lys Arg Lys Arg Met Glu Ser Asn 20 25 30 Arg Glu Ser Ala Arg Arg Ser Arg Met Arg Lys Gln Gln Arg Leu Gly 35 40 45 Glu Leu Met Ser Glu Thr Thr Gln Leu Gln Asn Gln Asn Ser Ile Cys 50 55 60 Arg Glu Arg Ile Asp Ser Val Glu Arg Asn Tyr Cys Ala Ile Asp Ala 65 70 75 80 Glu Asn Asn Val Leu Arg Ala Gln Ile Ala Glu Leu Thr Glu Arg Leu 85 90 95 Asn Ser Leu Asn Ser Leu Thr Gln Phe Trp Ala Asp Ala Thr Gly Phe 100 105 110 Pro Val Asp Leu Pro Glu Ile Pro Asp Thr Leu Leu Glu Pro Trp Gln 115 120 125 Leu Pro Cys Pro Ile Gln Pro Ile Asp Ala Ser Ser Asp Met Leu Leu 130 135 140 Phe 145 <210> 4 <211> 654 <212> PRT <213> Nicotiana tabacum <400> 4 Met Pro Gly Val Tyr Leu Glu Thr Ala Ser Leu Pro Lys Gly Arg Gly 1 5 10 15 Leu Glu Cys Gln Glu Ser Gln Ala Val Arg Tyr Phe Phe Arg Gly Arg 20 25 30 Asn Lys Val Asp Asp Ser Leu Thr Ile Glu Ile Phe Asn Leu Phe Pro 35 40 45 Trp Ile Phe Phe Thr Ile Leu Ala Met Asp Lys His His His Gln Leu 50 55 60 Pro Leu Thr Lys Ser Thr Ser Arg Gln Arg Tyr Asn Glu Trp Val Phe 65 70 75 80 Arg Asp Val Pro Ser Asp Ile Thr Ile Glu Val Asp Gly Gly Ile Phe 85 90 95 Ser Leu His Lys Phe Pro Leu Val Ser Arg Ser Gly Arg Ile Arg Arg 100 105 110 Leu Val Ala Glu His Arg Asp Ser Asp Ile Ser Arg Ile Glu Leu Val 115 120 125 Ser Leu Pro Gly Gly Thr Glu Ser Phe Glu Leu Ala Ala Lys Phe Cys 130 135 140 Tyr Gly Val Asn Phe Glu Ile Thr Ala Ala Asn Val Ala Gln Leu Cys 145 150 155 160 Cys Val Ser Asp Tyr Leu Glu Met Ser Glu Asp Tyr Ser Lys Asn Asn 165 170 175 Leu Gly Ser Arg Ala Glu Glu Tyr Leu Asp Ser Ile Val Cys Lys Asn 180 185 190 Leu Glu Met Cys Val Glu Val Leu Arg Gln Cys Glu Asn Leu Leu Pro 195 200 205 Leu Ala Asp Glu Leu Lys Val Val Ser Arg Cys Ile Asp Ala Val Ala 210 215 220 Ser Lys Ala Cys Val Glu Gln Ile Ala Ser Ser Phe Ser Arg Leu Glu 225 230 235 240 Tyr Ser Ile Ser Gly Gly Arg Leu His Met Ser Lys Gln Ala Asn Cys 245 250 255 Glu Leu Asp Trp Trp Ile Glu Asp Ile Ser Met Leu Arg Ile Asp Leu 260 265 270 Tyr Gln Arg Val Ile Thr Ala Met Lys Phe Arg Gly Val Arg Pro Glu 275 280 285 Ser Ile Ala Ala Ser Leu Val Asn Tyr Ala Gln Lys Glu Leu Ile Gln 290 295 300 Lys Thr Leu Ser Gly Ser Asn Ile Gln Glu Lys Leu Val Val Glu Thr 305 310 315 320 Ile Val Ser Leu Met Pro Val Glu Lys Phe Val Val Pro Leu Thr Phe 325 330 335 Leu Phe Gly Leu Leu Arg Ser Ala Val Met Leu Asp Cys Thr Val Ala 340 345 350 Cys Arg Leu Asp Leu Glu Arg Arg Ile Gly Ser Gln Leu Asp Thr Ala 355 360 365 Thr Leu Asp Asp Ile Leu Ile Pro Ser Phe Arg His Ala Gly Asp Thr 370 375 380 Leu Phe Asp Val Asp Thr Val His Arg Ile Leu Val Asn Phe Ser Gln 385 390 395 400 Gln Glu Gly Asp Ser Asp Asp Asp Met Glu Asp Val Ser Val Phe Glu 405 410 415 Ser Asp Ser Pro Thr Thr Thr Pro Ser Gln Thr Ala Leu Phe Lys Val 420 425 430 Ser Lys Leu Val Asp Asn Tyr Leu Ala Glu Ile Ala Leu Asp Ala Asn 435 440 445 Leu Lys Leu Asn Lys Phe Ile Ala Val Ala Glu Thr Leu Pro Ala His 450 455 460 Ala Arg Thr Val His Asp Gly Leu Tyr Arg Ala Ile Asp Leu Tyr Leu 465 470 475 480 Lys Ala His Gln Thr Leu Ser Asp Pro Asp Lys Arg Arg Leu Cys Lys 485 490 495 Leu Ile Asp Phe Gln Lys Leu Ser Gln Glu Ala Gly Ala Gln Ala Ala 500 505 510 Gln Asn Glu Arg Leu Pro Leu Gln Ser Ile Val Gln Val Leu Tyr Phe 515 520 525 Glu Gln Leu Arg Leu Arg Asn Ala Leu Phe Cys Ser Tyr Pro Asp Asp 530 535 540 Asp Ile Lys Pro Thr His Gln Ser Trp Arg Ile Asn Ser Gly Ala Leu 545 550 555 560 Ser Ala Ala Met Ser Pro Lys Asp Asn Tyr Ala Ser Leu Arg Arg Glu 565 570 575 Asn Arg Glu Leu Lys Leu Glu Leu Ala Arg Met Arg Met Arg Leu Asn 580 585 590 Asp Leu Glu Lys Asp His Val Cys Met Lys Arg Asn Met Gln Lys Ser 595 600 605 Ser Ser Arg Arg Phe Met Lys Ser Phe Ser Lys Arg Ile Gly Lys Lys 610 615 620 Phe Asn Ile Phe Gly His Asn Phe Ser Arg Asp Cys Ser Ser Pro Ser 625 630 635 640 Ser Gln Ser Glu Arg Thr Glu Ser Lys Ile Thr Glu Arg Thr 645 650 <210> 5 <211> 303 <212> PRT <213> Nicotiana tabacum <400> 5 Met Glu His Ser Ala Ala Asp Arg Asp Pro Lys Ala Val Glu Phe Ala 1 5 10 15 Lys Asp Lys Asn Gly Val Gly Gln Val Leu Leu Arg Asn Pro Arg Gly 20 25 30 Ala Ser Val Arg Val Ser Leu His Gly Gly Gln Val Leu Ser Trp Lys 35 40 45 Asn Asp His Gly Glu Glu Leu Leu Phe Ile Ser Ser Lys Ala Thr Phe 50 55 60 Lys Pro Pro Thr Ala Val Arg Gly Gly Ile Pro Ile Cys Phe Pro Gln 65 70 75 80 Phe Gly Asn Arg Gly Ser Leu Glu Gln His Gly Phe Ala Arg Asn Arg 85 90 95 Met Trp Ile Ile Asp Asp Asn Pro Pro Pro Leu His Pro Asn Asp Ser 100 105 110 Asn Gly Lys Ala Phe Thr Asp Leu Leu Leu Lys Ser Ser Asp Asp Asp 115 120 125 Leu Lys Val Trp Pro His Gly Phe Glu Phe Arg Leu Arg Val Thr Leu 130 135 140 Ala Val Asp Gly Ser Leu Thr Leu Ile Ser Arg Ile Arg Asn Val Asn 145 150 155 160 Cys Lys Pro Phe Ser Phe Ser Ile Ala Tyr His Thr Tyr Phe Ala Leu 165 170 175 Ser Asp Ile Ser Glu Val Arg Val Glu Gly Leu Glu Thr Leu Asp Tyr 180 185 190 Leu Asp Asn Leu Cys Asn Arg Glu Arg Phe Thr Glu Gln Gly Asp Ala 195 200 205 Leu Thr Phe Glu Thr Glu Val Asp Arg Val Tyr Leu Ser Ser Ser Asp 210 215 220 Val Ile Ala Ile Phe Asp His Glu Lys Lys Arg Thr Phe Val Ile Lys 225 230 235 240 Arg Glu Gly Leu Pro Asp Val Va...

Claims

**Claim 1** A method for producing a tobacco plant comprising an increased nitrogen use efficiency (NUE) trait, comprising the following steps: (a) providing a first population of tobacco plants comprising an increased NUE trait; (b) genotyping the first population of tobacco plants for the presence of one or more molecular markers located within 20 cM of an SNP marker selected from the group consisting of SEQ ID NOs: 57, 58, 59, 60, 61, 62, 63, and 64; (c) selecting tobacco plants comprising the one or more molecular markers; (d) crossing the tobacco plants selected in step (c) with a second tobacco plant; and (e) obtaining progeny seeds from the cross of step (d), wherein the plants grown from the progeny seeds comprise an increased NUE trait. **Claim 2** The method according to claim 1, wherein the increased NUE trait is selected from the group consisting of increased partial factor productivity (PFP), increased agronomic efficiency (AE), increased recovery efficiency (RE), increased physiological efficiency (PE), and increased internal efficiency (IE) compared to tobacco plants lacking the increased NUE trait grown under the same conditions. **Claim 3** The method according to claim 1, wherein the first population of tobacco plants is selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, and NC925. **Claim 4** The method according to claim 1, wherein the second tobacco plant is a burley tobacco variety. **Claim 5** The method according to claim 1, wherein the second tobacco plant is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC. **Claim 6** The method according to claim 1, wherein the one or more molecular markers are within 10 cM of the SNP marker. **Claim 7** The method according to claim 1, wherein the one or more molecular markers are within 5 cM of the SNP marker. **Claim 8** A method for producing a tobacco plant comprising an increased NUE trait, comprising the following steps: (a) providing a first population of tobacco plants comprising an increased NUE trait; Step of genotyping a first population of tobacco plants for the presence of one or more molecular markers located within 20 cM of an increased NUE locus having a sequence selected from the group consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, and 16; Step of selecting a tobacco plant comprising the one or more molecular markers; Step of crossing the tobacco plant selected in step (c) with a second tobacco plant; and Step of obtaining progeny seeds from the cross in step (d), wherein the plants grown from the progeny seeds comprise the increased NUE trait. **Claim 9** The method according to claim 8, wherein the increased NUE trait is selected from the group consisting of increased partial factor productivity (PFP), increased agronomic efficiency (AE), increased recovery efficiency (RE), increased physiological efficiency (PE), and increased internal efficiency (IE) as compared to tobacco plants lacking the increased NUE trait grown under the same conditions. **Claim 10** The method according to claim 8, wherein the first population of tobacco plants is selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, and NC925. **Claim 11** The method according to claim 8, wherein the second tobacco plant is a burley tobacco variety. **Claim 12** The method according to claim 8, wherein the second tobacco plant is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC. **Claim 13** The method according to claim 8, wherein the one or more molecular markers are within 10 cM of the locus. **Claim 14** The method according to claim 8, wherein the one or more molecular markers are within 5 cM of the locus. **Claim 15** A modified tobacco seed or a tobacco plant grown therefrom, comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a coding region, wherein the coding region encodes a polypeptide having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8, and The modified tobacco plant comprises an increased NUE trait as compared to the case where an unmodified control tobacco plant lacking the cisgenic polynucleotide is grown under the same conditions. The modified tobacco seeds or tobacco plants grown therefrom.

16. The modified tobacco seeds or tobacco plants grown therefrom according to claim 15, wherein the increased NUE trait is selected from the group consisting of increased partial factor productivity (PFP), increased agronomic efficiency (AE), increased recovery efficiency (RE), increased physiological efficiency (PE), and increased internal efficiency (IE) as compared to the case where a tobacco plant lacking the increased NUE trait is grown under the same conditions.

17. The modified tobacco seeds or tobacco plants grown therefrom according to claim 15, wherein the heterologous promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, and a tissue-specific promoter.

18. The modified tobacco seeds or tobacco plants grown therefrom according to claim 15, wherein the tissue-preferred promoter is a leaf-preferred promoter comprising a sequence having at least 90% sequence identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs: 17, 18, and 19.

19. The modified tobacco seeds or tobacco plants grown therefrom according to claim 15, wherein the tissue-preferred promoter is a root-preferred promoter comprising a sequence having at least 90% sequence identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, and 24.

20. The modified tobacco seeds or tobacco plants grown therefrom according to claim 15, wherein the coding region comprises at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, and 16.

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