Methods and compositions for improved nitrogen use 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 September 1, 2017, the entire contents of which are incorporated herein by reference.

[0002] Incorporating a sequence listing This application contains a Sequence Listing, which has been submitted herewith in electronic format, including a file entitled "P34523WO00_SL.txt" created on August 31, 2018, having a size of 188,432 bytes (as measured in MS-Windows®), the entire contents of which are 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 through breeding, transgenic and cisgenic approaches. [Background technology]

[0004] background Fertilizer is 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 to achieve maximum yield. For example, Maryland tobacco varieties typically require approximately 25% less nitrogen input to achieve maximum yield than burley tobacco varieties.

[0005] Improving nitrogen use efficiency (NUE) in tobacco will increase the yield of tobacco per unit of nitrogen fertilizer applied. Improved nitrogen use efficiency also reduces farm input costs, reduces the use and reliance on non-renewable energy sources required for nitrogen fertilizer production, and reduces the environmental impact of nitrogen fertilizer production and agricultural use.

[0006] Provided herein are methods and compositions for improving nitrogen use efficiency in tobacco. Summary of the Invention

[0007] overview In one aspect, the present disclosure provides and includes a method for determining the NUE of a tobacco line, comprising 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 and includes a method for determining the NUE of a tobacco line using a metabolite signature, the method comprising: isolating a metabolite signature from a tobacco plant of the tobacco line; determining the amount of each metabolite comprising the metabolite signature; and determining the NUE of the tobacco line by comparing the metabolite signature to a control metabolite signature from a control tobacco line with a known NUE.

[0009] In one aspect, the present specification provides and includes a method of breeding a tobacco line that includes a metabolic signature associated with increased NUE, the method comprising: determining a metabolic signature of a first tobacco plant derived from a first tobacco line, wherein the first tobacco plant includes increased NUE compared to a control tobacco plant lacking the metabolic 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 metabolic signature and the progeny plant includes increased NUE compared to the control plant lacking the metabolic signature.

[0010] In one aspect, the present disclosure provides and includes a method for 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 the at least one metabolite compared to a control tobacco plant. In a further aspect of the method, the selected tobacco plant contains increased NUE compared to the control tobacco plant.

[0011] In one aspect, the present specification provides and includes a method for 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 includes a reduced amount of the at least one metabolite compared to a control tobacco plant.

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

[0013] In one aspect, the present specification provides and includes 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 modified tobacco plant comprises an increased nitrogen utilization 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 operably linked to a polynucleotide encoding a polypeptide 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 cured tobacco material or a tobacco product comprising the cured 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 utilization efficiency compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide grown under the same conditions.

[0016] In one aspect, the present description 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, the modified tobacco seed or plant comprising at least one mutation at an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or plant comprises increased nitrogen utilization 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 operably linked to a guide RNA comprising at least 18 contiguous nucleotides identical to 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 cured tobacco material or a tobacco product comprising the cured tobacco material produced from a tobacco plant comprising at least one mutation at an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or tobacco plant comprises an increased NUE compared to an unmodified control tobacco plant lacking the at least one mutation grown under the same conditions.

[0020] In one aspect, the description provides and includes a modified tobacco seed or a tobacco plant grown therefrom, comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, wherein the modified tobacco seed or tobacco plant comprises increased NUE compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide grown under the same conditions.

[0021] 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 to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0022] In one aspect, the present specification provides and includes a cured tobacco material or a tobacco product comprising the cured tobacco material 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 to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, wherein the modified tobacco seed or tobacco plant comprises an increased NUE 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 NUE in a tobacco plant, comprising 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.

[0024] In one aspect, the present specification provides and includes a method for increasing NUE in a tobacco plant, comprising introducing into a tobacco cell a modification to a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41-56, 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.

[0025] In one aspect, the present specification provides and includes a method for increasing NUE in a tobacco plant, comprising introducing into a tobacco cell a nucleic acid encoding a small RNA (sRNA) that is homologous to at least 18 contiguous nucleic acids of a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41-56, 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.

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

[0027] In one aspect, the present specification provides and includes 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 comprising the increased NUE allele.

[0028] In one aspect, the present specification provides and includes a method for introgressing an increased nitrogen utilization efficiency trait into a tobacco variety, comprising: crossing a first tobacco variety comprising the increased nitrogen utilization efficiency trait with a second tobacco variety lacking the increased nitrogen utilization efficiency trait; obtaining progeny seeds from the cross; genotyping at least one progeny seed for a molecular marker associated with the increased nitrogen utilization 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 the progeny seeds comprising the increased nitrogen utilization efficiency trait.

[0029] In one aspect, the present specification provides and includes a method for selecting tobacco plants having an enhanced NUE trait, 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 selected from the group consisting of SEQ ID NOs:9-16, and selecting tobacco plants comprising the enhanced NUE trait.

[0030] In one aspect, the present specification provides and includes a method for selecting tobacco plants having an enhanced NUE trait, 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 marker selected from the group consisting of SEQ ID NOs:57-64, and selecting tobacco plants comprising the enhanced NUE trait. [The present invention 1001] 1. A method for producing a tobacco plant comprising an increased nitrogen use efficiency (NUE) trait, comprising the steps of: (a) providing a first population of tobacco plants comprising an enhanced NUE trait; (b) genotyping the first population of tobacco plants for the presence of one or more molecular markers located within 20 cM of a SNP marker selected from the group consisting of SEQ ID NOs: 57, 58, 59, 60, 61, 62, 63, and 64; (c) selecting tobacco plants containing the one or more molecular markers; (d) crossing the tobacco plant selected in step (c) with a second tobacco plant; and (e) obtaining progeny seed from the cross of step (d), wherein plants grown from said progeny seed contain the increased NUE trait. [The present invention 1002] 1001. The method of claim 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) compared to a tobacco plant lacking the increased NUE trait grown under the same conditions. [The present invention 1003] 1001. The method of claim 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. [The present invention 1004] The method of claim 1001, wherein the second tobacco plant is a Burley tobacco variety. [The present invention 1005] 1001. The method of claim 1001, wherein the second tobacco plant is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC. [The present invention 1006] 1001. The method of claim 1001, wherein said one or more molecular markers are within 10 cM of said SNP marker. [The present invention 1007] 1001. The method of claim 1001, wherein said one or more molecular markers are within 5 cM of said SNP marker. [The present invention 1008] 1. A method for producing a tobacco plant containing an enhanced NUE trait, comprising the steps of: (a) providing a first population of tobacco plants comprising an enhanced NUE trait; (b) genotyping the first population of tobacco plants for the presence of one or more molecular markers located within 20 cM of the amplified NUE locus having a sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, and 16; (c) selecting tobacco plants containing the one or more molecular markers; (d) crossing the tobacco plant selected in step (c) with a second tobacco plant; and (e) obtaining progeny seed from the cross of step (d), wherein plants grown from said progeny seed contain the increased NUE trait. [The present invention 1009] The method of claim 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 a tobacco plant lacking the increased NUE trait grown under the same conditions. [The present invention 1010] 1008. The method of claim 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 claim 1008, wherein the second tobacco plant is a Burley tobacco variety. [The present invention 1012] The method of claim 1008, wherein the second tobacco plant is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC. [The present invention 1013] 1008. The method of claim 10, wherein said one or more molecular markers are within 10 cM of said locus. [The present invention 1014] 1008. The method of claim 10, wherein said one or more molecular markers are within 5 cM of said locus. [The present invention 1015] 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 coding region encodes a polypeptide comprising at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8; and the modified tobacco plant comprises an increased NUE trait compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide grown under the same conditions. The modified tobacco seeds or tobacco plants grown therefrom. [The present invention 1016] The modified tobacco seed of the present invention or a tobacco plant 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 a tobacco plant lacking the increased NUE trait grown under the same conditions. [The present invention 1017] The modified tobacco seed or tobacco plant grown therefrom of the present invention 1015, 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 seed of the present invention or a tobacco plant grown therefrom, wherein the tissue-preferred promoter is a leaf-preferred promoter comprising a sequence having at least 90% sequence identity or complementarity to a sequence selected from the group consisting of SEQ ID NO: 17, 18, and 19. [The present invention 1019] The modified tobacco seed of the present invention or a tobacco plant grown therefrom, wherein the tissue-preferred promoter is a root-preferred promoter comprising a sequence having at least 90% sequence identity or complementarity to a sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, and 24. [The present invention 1020] 1015. The modified tobacco seed of the present invention or a tobacco plant 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] A brief description of arrays SEQ ID NOs: 1-8 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:9-16 are the nucleotide sequences of genes that are positively correlated with increased NUE in root tissue, leaf tissue, or both.

[0033] SEQ ID NOs:17-19 are the nucleotide sequences of promoter regions for genes with leaf-preferred expression.

[0034] SEQ ID NOs:20-24 are the nucleotide sequences of promoter regions for genes with root-preferred expression.

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

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

[0037] SEQ ID NOs:57-64 are the nucleotide sequences of SNP markers containing polymorphisms associated with increased NUE.

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

[0039] [Figure 1] Four gene clusters related to NUE in the tobacco genome are depicted. Genes differentially expressed between low- and normal-nitrogen conditions in plants with NUE metabolite fingerprints (correlated genes) are displayed. The total number of differentially expressed genes (DEGs) regardless of NUE metabolite fingerprinting is also displayed. [Figure 2]Depicts a 2-megabase region of tobacco chromosome 11 covered by Superscaffold 1. Superscaffold 1 is a contig of Scaffold A and Scaffold B. Of the 79 expressed genes located in this region, 56 genes are differentially expressed. [Figure 3] The allele composition for 23 burley and 6 Maryland varieties at a locus correlated with NUE on tobacco chromosome 11 is depicted. Lines 1, 2, and 3 are burley lines containing the favorable Maryland allele for SEQ ID NO:58, and line 4 is a standard burley line containing the unfavorable burley allele for SEQ ID NO:58. [Figure 4] Chlorophyll loss, growth, and yield are depicted for lines 1-4 and the MD609 control (C) described in Figure 3. Lines 1, 2, and 3 are burley lines containing the favorable Maryland allele for SEQ ID NO:58, and line 4 is a standard burley line containing the unfavorable burley allele for SEQ ID NO:58. [Figure 5] Figure 1 depicts the yield per plant (in grams fresh weight) of greenhouse-grown T1 plants overexpressing genes that are positively correlated with increased yield under nitrogen stress. Means and standard deviations based on 9 plants per sample are shown. [Figure 6] Post-harvest yields (in pounds) per acre are depicted for two independent field-grown F4 lines, NUE-2 and NUE-3. The test lines were developed from crosses of MD609 to burley TN90 as indicated. Means and standard deviations are provided relative to the control TN90 burley tobacco. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Those skilled in the art will recognize that many methods can be used in the practice of the present disclosure. Indeed, the present 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 art to which this disclosure belongs. Where a term is provided in the singular, the inventors also contemplate aspects of the present disclosure described by the plural of that term. Where there are discrepancies in terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions provided herein. Other technical terms used have their common meaning in the art in which they are used, as exemplified in various technical dictionaries, for example, "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 by mechanism or mode of action. References thereto are provided for illustrative purposes only.

[0042] The practice of the present disclosure involves, unless otherwise indicated, conventional techniques of biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, bioengineering, metabolomics, plant breeding and genetics, which are within the skill of the art. For example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols In Molecular Biology (FM Ausubel, et al. eds., (1987)); Plant Breeding Methodology (NF Jensen, Wiley-Interscience (1988)); the series Methods In Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (RI Freshney, ed. (1987)); Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences; CN 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] All references cited herein (including, for example, all patents, published patent applications, and non-patent publications) are incorporated by reference in their entirety.

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

[0045] As used herein, the term "sequence identity" or "identity," in the context of two polynucleotide or polypeptide sequences, refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When the percentage of sequence identity is used in relation to proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions (amino acid residues are replaced with other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), thereby not changing the functional properties of the molecule). When sequences differ by conservative substitutions, the percent 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 alignments is CLUSTALW v1.6 (Thompson, et al. (1994) Nucl. Acids Res., 22: 4673-4680).

[0046] As used herein, the term "complementary" in relation to nucleic acid molecules refers to the pairing of nucleotide bases, such as adenine being complementary to thymine or uracil, and guanine being complementary to cytosine.Two complementary nucleic acid molecules can hybridize with each other.As an example, the two strands of double-stranded DNA are complementary to each other.

[0047] Certain polynucleotides at least three nucleotides in length may be referred to as "oligonucleotides." Nucleic acid molecules provided herein include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and their functional analogs, such as complementary DNA (cDNA). Nucleic acid molecules provided herein can be single-stranded or double-stranded. Nucleic acid molecules contain the nucleotide bases adenine (A), guanine (G), thymine (T), and cytosine (C). Uracil (U) substitutes for 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 contain ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. 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, and the like.

[0049] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. Polypeptides can be encoded by the polynucleotides 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 from components that normally accompany or interact with the polynucleotide or protein as found in its natural environment. For example, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one aspect, the isolated polynucleotides provided herein can contain nucleic acid sequences 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 naturally flank 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 from 100 to 10,000 nucleotides, 500 to 10,000 nucleotides, 1,000 to 10,000 nucleotides, 2,000 to 10,000 nucleotides, 3,000 to 10,000 nucleotides, 4,000 to 10,000 nucleotides, 1 to 500 nucleotides, 1 to 1,000 nucleotides, 1 to 2,000 nucleotides, 1 to 3,000 nucleotides, 1 to 4,000 nucleotides, 1 to 5,000 nucleotides, 1 to 10,000 nucleotides, 100 to 500 nucleotides, 100 to 1,000 nucleotides, 100 to 2,000 nucleotides, 100 to 3,000 nucleotides, or 100 to 4,000 nucleotides naturally contiguous 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. Polynucleotide fragments may encode polypeptide fragments that retain the biological activity of the native polypeptide. Alternatively, polynucleotide fragments useful as hybridization probes or PCR primers using methods known in the art generally do not encode fragment polypeptides that retain biological activity. The polynucleotide fragments 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 encoding the polypeptide of the present invention, depending on the desired results.

[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 technology 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 technology includes, 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 a single nucleic acid molecule 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 expression vectors. In addition, purified polypeptides can be obtained by chemical synthesis. The degree of purity of a polypeptide can be measured using any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0052] In one aspect, the present disclosure provides a method for detecting recombinant nucleic acids and polypeptides in plant cells.Nucleic acids can also be detected using hybridization, but this is not limited to this.Nucleic acid hybridization 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] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. The antibodies provided herein can be polyclonal or monoclonal. Antibodies with specific binding affinity to the polypeptides provided herein can be produced 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 well known in the art.

[0054] Detection (e.g., of amplification products, of hybridization complexes, of polypeptides) can be achieved using a detectable label. The term "label" is intended to encompass the use of both direct and indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0055] As used herein, the phrases "associated with" or "related to" refer to a discernible 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 their expression, presence, or absence, that can affect the degree, level, and / or proportion of a plant or portion thereof of interest that has an increased NUE trait. Thus, a marker is "associated with" a trait when it is associated with the trait and when the presence of the marker is an indicator of whether and / or to what extent a desired trait or trait morphology occurs in a plant / germplasm containing the marker. Similarly, a marker is "associated with" an allele when it is associated with an allele and when the presence of the marker is an indicator of whether or not 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 whose presence or absence can be used to predict whether and to what extent a plant will exhibit an increased NUE phenotype.

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

[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 between the marker and locus occurs at a frequency of about 20% or less.

[0058] As used herein, "plant" refers to the whole plant. Cell or tissue culture obtained from a plant can include any plant component or plant organ (e.g., leaf, stem, root, etc.), plant tissue, seed, plant cell, and / or their progeny. The progeny plant can be derived from any hybrid generation, for example, F1, F2, F3, F4, F5, F6, F7, etc. Plant cells are living plant cells that are harvested from a plant or obtained through culturing from cells harvested 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 555486; 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, pods, embryos, endosperm, explants, callus, tissue cultures, buds, cells, and protoplasts. In a further aspect, the present disclosure provides tobacco plant cells, tissues, and organs that are not reproductive materials and do not mediate the natural reproduction of plants. In another aspect, the present disclosure also provides tobacco plant cells, tissues, and organs that are reproductive materials and mediate the natural reproduction of plants. In another aspect, the present disclosure provides tobacco plant cells, tissues, and organs that cannot sustain themselves through photosynthesis. In another aspect, the present disclosure provides somatic cells of tobacco plants. In contrast to reproductive cells, somatic cells do not mediate the reproduction of plants.

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

[0062] Those skilled in the art will understand that tobacco plants reproduce naturally through seeds, rather than through asexual or vegetative propagation. 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 cannot reproduce without human intervention.

[0063] In one aspect, the present disclosure provides methods and compositions relating to modified tobacco plants, seeds, plant components, plant cells, and products produced from the modified tobacco plants, seeds, plant parts, and plant cells. In one aspect, the modified seeds provided herein produce 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 cured tobacco material 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 been subjected to mutagenesis, genome editing, genetic transformation or a combination 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., promoters, donor nucleic acids, selection genes) have only plant origin (e.g., no components other than those of plant origin are 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 ready-to-use tobacco lines. In another aspect, the modified tobacco plants provided herein do not contain genetic material or sequences other than tobacco.

[0066] As used herein, "functional fragment" or "functional fragment thereof" refers to a nucleotide or amino acid sequence of any size that retains the function of the full-length sequence to which it refers. In some aspects, a 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, functional fragments 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, functional fragments 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, functional fragments 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 certain further aspects, the polynucleotides described herein are contemplated in their entirety and any functional fragments thereof.In certain further aspects, the polypeptides described herein are contemplated in their entirety and any functional fragments thereof. In certain further aspects, polynucleotides having the sequences of SEQ ID NOs: 9-24 and 41-56 are contemplated in their entirety and any functional fragments thereof. In certain further aspects, polypeptides having the sequences of SEQ ID NOs: 1-8 and 25-40 are contemplated in their entirety and any functional fragments thereof.

[0067] As used herein, the term "nitrogen use efficiency" (NUE) refers to a plant's ability to absorb, assimilate, and / or use nitrogen (e.g., from soil, water, and / or nitrogen fertilizer). NUE genes affect yield and are useful for improving nitrogen use in crop plants. Increased nitrogen use efficiency can result not only from improved nitrogen fertilizer uptake and assimilation and / or subsequent remobilization and recycling of accumulated nitrogen stores, but also from increased plant tolerance to stress situations, such as low-nitrogen environments. NUE genes can be used to change the genetic makeup of plants to make them more productive with current fertilization standards or to maintain their production rates with significantly reduced fertilizer or reduced nitrogen availability.

[0068] Although NUE has been defined in various ways, yield per unit of soil-available nitrogen integrates all important parameters for evaluating the suitability of crop varieties and is a common measure of NUE. See, for example, Ladha et al. 2005. Advances in Agronomy, 87:85-156 (incorporated herein in its entirety). This index 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 a plant can be determined (sometimes referred to as "physiological NUE"). Increased NUE relates to three important components: 1) yield is not significantly different when grown at 25% normal nitrogen content compared to plants grown at 100% normal nitrogen content; 2) chlorophyll loss is reduced compared to plants without increased NUE; and 3) dry leaf quality is not significantly different when grown at 25% normal nitrogen content compared to plants grown at 100% normal nitrogen content. In one preferred aspect, plants with increased NUE can produce similar yield and leaf quality when grown under a 25% burley fertilization rate compared to burley plants grown under a 100% normal burley fertilization rate.

[0069] At least five approaches and indices of 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 In the formula, 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 nitrogen applied: AE N = Kilograms of yield increase / Kilograms of N applied AE N =(Y +N -Y 0N ) / FN In the formula, Y +N is the yield (kg / ha) in the treatment with N application; Y 0N is the yield in the control treatment without N application (kg / ha); FN is the amount of N fertilizer applied (kg / ha).

[0072] (3) 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 incorporated / kilograms of N applied RE N =(UN +N -UN 0N ) / FN During the ceremony, UN +N is the total plant N uptake (kg / ha) measured in aboveground biomass at physiological maturity in plots receiving applied N at the rate of FN (kg / ha); UN 0N is the total N uptake of 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 = kg of yield increase / kg of fertilizer N incorporated PE N =(Y +N -Y 0N ) / (UN +N -UN 0N ) In the formula, 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 application; UN 0N is the total N uptake (kg / ha) in the treatment without fertilizer N application.

[0074] (5) Nitrogen (N) internal efficiency (IE) refers to how much yield is produced per unit of N taken up from both fertilizer and existing (e.g., soil) nutrient sources: IE N = kg of yield / kg of N incorporated IE N =Y / UN where Y is yield (kg / ha); UN is total N uptake (kg / ha).

[0075] Nitrogen can be in any form, including organic and / or inorganic forms. Forms of nitrogen include, but are not limited to, nitrates (e.g., ammonium nitrate, calcium nitrate, potassium nitrate), nitrites, ammonia, aqueous ammonia, anhydrous ammonia, ammonium sulfate, diammonium phosphate, low-pressure nitrogen solutions, atmospheric-pressure nitrogen solutions, urea, and urea-ammonium nitrate (UAN). In some aspects, the nitrogen is in a form that is readily available to plants (e.g., ammonia and / or nitrate) and / or is in a form that can be easily converted to a plant-available form (e.g., urea).

[0076] In one aspect, modified tobacco plants comprising the increased NUE provided herein comprise increased nitrogen uptake compared to control tobacco plants. In another aspect, modified tobacco plants comprising the increased NUE provided herein comprise increased nitrogen assimilation compared to control tobacco plants. In a further aspect, modified tobacco plants comprising the increased NUE provided herein comprise increased yield compared to control tobacco plants. In yet another aspect, modified tobacco plants comprising the increased NUE provided herein comprise increased yield under low-nitrogen conditions compared to control tobacco plants. In a preferred aspect, low-nitrogen conditions as used in the field are approximately 25% nitrogen compared to levels typically used by those skilled in the art. In another aspect, low-nitrogen conditions as used in the field can be between approximately 5% and 50% nitrogen compared to levels typically used by those skilled 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 greenhouses, can be between 5 ppm and 50 ppm.

[0077] In certain aspects, modified tobacco plants comprising increased NUE provided herein comprise an increase in yield 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 a control tobacco plant grown under similar growth conditions. In certain aspects, modified tobacco plants comprising increased NUE provided herein comprise a yield increase of 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%, relative to a control tobacco plant grown under similar growth conditions.

[0078] In certain aspects, a population of modified tobacco plants comprising increased NUE provided herein comprises an increased yield 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 relative to a population of control tobacco plants grown under similar growing conditions. In another aspect, a population of modified tobacco plants comprising increased NUE provided herein exhibits an increased NUE of 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, or between 8 kg / ha and 100 kg / ha, compared to a population of control tobacco plants when grown under similar growth conditions. ha, between 8kg / ha and 100kg / ha, between 9kg / ha and 100kg / ha, between 10kg / ha and 100kg / ha, between 15kg / ha and 100kg / ha, between 20kg / ha and 100kg / ha, between 30kg / ha and 100kg / ha, between 40kg / ha and 100kg / ha, between 50kg / ha and 100kg / ha, between 75kg / ha and 100kg / ha, between 100kg / ha and 500kg / ha, between 100kg / ha and 400kg / ha, between 100 and 300kg / ha, or between 100kg / ha and 200kg / ha.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, 10,000, 25,000, 50,000, 100,000, 500,000, or more. A population can be derived from a single variety, cultivar, or line. A population can be produced using any breeding technique known in the art.

[0079] In certain aspects, modified tobacco plants comprising increased NUE provided herein contain 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, modified tobacco plants comprising increased NUE provided herein comprise between 1 and 25, between 2 and 25, between 3 and 25, between 4 and 25, between 5 and 25, between 6 and 25, between 7 and 25, between 8 and 25, between 9 and 25, between 10 and 25, between 11 and 25, between 12 and 25, between 13 and 25, between 14 and 25, between 15 and 25, or between 20 and 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, agricultural practices, and / or curing processes for growing or curing tobacco to allow meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agricultural practices (including the curing process) contribute to or account for the differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, and nutrients (e.g., nitrogen and phosphorus). Agronomic practices include, for example, sowing, pruning, headcutting, planting, topping, dividing, and curing. 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 comprise one or more non-naturally occurring mutations. In one aspect, the mutations provided herein improve nitrogen utilization efficiency in plants. The types of mutations provided herein include, for example, substitutions (point mutations), deletions, insertions, duplications, and inversions. Such mutations are preferably present in the coding region of a gene; however, mutations in promoters or other regulatory regions, introns, intron-exon boundaries, or untranslated regions of a gene may also be desirable.

[0082] In one aspect, the methods provided herein can produce tobacco plants with increased nitrogen use efficiency relative to control tobacco plants. Mutagenesis methods include, but are not limited to, chemical mutagenesis, e.g., seed treatment with ethyl methyl sulfate (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 beams, helium ion beams, neon ion beams), 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 (3rd 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 methodology (Hoekema et al., 1983; U.S. Patent No. 5,149,645). EMS-induced mutagenesis consists of chemically inducing random point mutations throughout the genome. Rapid neutron mutagenesis consists of exposing seeds to neutron bombardment, which causes large deletions through double-stranded DNA breaks. Transposon tagging involves inserting a transposon into an endogenous gene to reduce or eliminate gene expression.

[0083] In addition, a rapid and automatable screening method for chemically induced mutations, i.e., TILLING (Targeting Induced Local Lesions In Genomes), using denaturing high-performance liquid chromatography (HPLC) or selective endonuclease digestion of selected PCR products, is also applicable to the present disclosure. See McCallum et al. (2000) Nat. Biotechnol. 18:455-457. Mutations that affect gene expression or interfere with the function of the genes provided herein can be determined using methods well known in the art. Insertion mutations in gene exons usually result in null mutants. Mutations in 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 detecting polynucleotides, Northern blot, RNase protection, primer extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, next-generation sequencing technology (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assay for detecting the enzyme or ribozyme activity of polypeptides and polynucleotides; and 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 for performing all of the techniques mentioned are known in the art.

[0085] In one aspect, the plant genome provided herein is mutated (edited) by a nuclease selected from the group consisting of meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR / Cas9 nuclease, CRISPR / Cpf1 or CRISPR / Cmx1 nuclease.In another aspect, the plant genome provided herein is mutated by CRISPR / CasX or CRISPR / CasY nuclease.As used herein, "editing" or "genome editing" refers to the 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 endogenous plant genome nucleic acid sequence, or the removal or replacement of endogenous plant genome 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 polynucleotide sequences into tobacco plants are known in the art, including, but not limited to, stable transformation, transient transformation, and virus-mediated methods."Stable transformation" refers to the transformation in which the nucleotide construct of interest introduced into a plant is integrated into the genome of the 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 merely expressed or exists transiently in the plant or plant cell.

[0087] In one aspect, the methods and compositions provided herein comprise the introduction of one or more polynucleotides into one or more plant cells.In one aspect, the plant genome provided herein is modified to contain 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 polynucleotide provided herein is incorporated into an artificial chromosome.In one aspect, the artificial chromosome comprising 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 comprise one or more transgenes.In one aspect, the transgenes provided herein improve nitrogen utilization efficiency in tobacco plants.As used herein, "transgene" refers to a polynucleotide that is transferred into a genome by any method known in the art.In one aspect, a transgene is an exogenous polynucleotide.In one aspect, a transgene is an endogenous polynucleotide that is integrated into a new genomic locus where it is not normally found.Therefore, a transgene can also be a cis-gene under appropriate circumstances.

[0089] In one aspect, the transgene provided herein comprises a recombinant DNA construct. In one aspect, the recombinant DNA construct or expression cassette provided herein can comprise a selectable marker gene for 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 herbicidal compounds such as glufosinate ammonium, bromoxynil, imidazolinones, triazolopyrimidines, sulfonylureas (e.g., chlorsulfuron and sulfometuron methyl), and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers, such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP).

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

[0091] In one aspect, the plasmid or vector provided herein is a recombinant vector. 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 plasmid provided herein is a synthetic plasmid. As used herein, a "synthetic plasmid" is an artificially created plasmid that can perform the same function (e.g., replication) as a natural plasmid (e.g., Ti plasmid). Without being limited thereto, those skilled in the art can create a synthetic plasmid de novo by synthesizing a plasmid by individual nucleotides or by splicing together nucleic acid molecules from different existing plasmids.

[0092] Vectors are commercially available or can be produced by routine recombinant DNA techniques in the art. In one aspect, the vectors provided herein comprise all or a portion of SEQ ID NO:65. A vector containing a nucleic acid can have expression elements 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 a nucleic acid can encode a chimeric or fusion polypeptide (i.e., a polypeptide operably linked to a heterologous polypeptide, which can be present at either the N-terminus or C-terminus of the polypeptide). Exemplary heterologous polypeptides are those that can be used to purify the encoded polypeptide (e.g., a 6xHis tag, glutathione S-transferase (GST)).

[0093] Suitable methods for introducing polynucleotides (e.g., transgenes, recombinant vectors, recombinant DNA constructs, expression constructs) into 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 (see, e.g., U.S. Pat. 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, 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, NY), 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 bacterial cells of many different species can contain the recombinant DNA constructs or recombinant vectors. Also provided are, for example, but not limited to, Agrobacterium tumefaciens, Escherichia coli, and yeast cells (e.g., Saccharomyces cerevisiae) containing 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 viruses or viral nucleic acids. Generally, such methods involve incorporating the expression cassettes of the present disclosure into viral DNA or RNA molecules. It is recognized that the promoters for use in the expression cassettes provided herein also encompass promoters utilized for transcription by viral RNA polymerase. Methods for introducing polynucleotides into plants to express proteins encoded therein (involving 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 subsequently be propagated using clonal methods (whether by organogenesis or embryogenesis) can be transformed with the recombinant constructs or expression cassettes provided herein. By "organogenesis" is intended the process by which shoots and roots develop sequentially from a meristem center. By "embryogenesis" is intended the process by which shoots and roots develop together in a concerted (rather than sequential) manner, 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 meristem, axillary bud, and root meristem) and induced meristems (e.g., cotyledonary meristem and hypocotyl meristem), hypocotyls, cotyledons, leaf discs, pollen, embryos, etc.

[0096] As commonly understood in the art, the term "promoter" generally refers to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and / or a TATA box and that supports or facilitates the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). Promoters may be synthetically produced, modified, or derived from known or naturally occurring promoter sequences or other promoter sequences (e.g., as provided herein). Promoters may also include chimeric promoters comprising a combination of two or more heterologous sequences. Thus, promoters of the present invention may include variants of promoter sequences that are similar in composition but are not identical to or complementary to other promoter sequences known or provided herein. As used herein, a "heterologous promoter," in the context of a DNA construct, refers to either: (i) a promoter derived from a source different from that of an operably linked structural gene or coding region, or (ii) a promoter derived from the same source as an operably linked structural gene or coding region, where the 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), whereby the promoter etc. operates to initiate, support, affect, induce and / or promote the transcription and expression of the associated coding or transcribable polynucleotide sequence, at least in particular tissues, developmental stages and / or under certain conditions. A "plant-expressible promoter" refers to a promoter that can be used to express in plants, plant cells and / or plant tissues an associated coding sequence, transgene or transcribable polynucleotide sequence operably linked to the promoter.

[0097] Promoters may be classified according to a variety of criteria regarding the expression pattern of a coding sequence or gene (including a transgene) 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 referred to as a "constitutive" promoter. A promoter that initiates transcription during a certain period or stage of development is referred to as a "developmental" promoter. A promoter whose expression is enhanced in certain tissues of a plant relative to other plant tissues is referred to as a "tissue-enhanced" or "tissue-preferred" promoter. Thus, a "tissue-preferred" promoter causes relatively high or preferential expression in specific plant tissues, but low levels of expression in other plant tissues. A promoter that is expressed in certain plant tissues but little or no expression in other plant tissues is referred to as a "tissue-specific" promoter. A promoter that is expressed in certain plant cell types is referred to as 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 the application of chemicals. Promoters may also be classified in terms of their origin, such as heterologous, homologous, chimeric, or synthetic. A "heterologous" promoter is a promoter sequence that has a different origin from 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" can refer more broadly to the 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. Pat. 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. Pat. No. 5,659,026), and the like.

[0099] Exemplary chemical inducible promoters include the tobacco PR-1a promoter, which is activated by salicylic acid.Other chemical inducible promoters of interest include steroid-responsive promoters (see, for example, Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257 for glucocorticoid-inducible promoters) and tetracycline-inducible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237 and U.S. Patent Nos. 5,814,618 and 5,789,156). Additional exemplary promoters that can be used herein include those responsive to heat-regulated gene expression, light-regulated gene expression (e.g., pea rbcS-3A; maize rbcS promoter; chlorophyll alb-binding protein gene found in pea; or Arabssu promoter), hormone-regulated gene expression (e.g., abscisic acid (ABA)-responsive sequences from the Em gene in wheat; ABA-inducible HVA1 and HVA22, and rd29A promoters in barley and Arabidopsis; and wound-inducible gene expression (e.g., of wunl), organ-specific gene expression (e.g., of tuber-specific storage protein genes; the 23-kDa zein gene from maize described; or French bean (β-phaseolin gene)), or pathogen-inducible promoters (e.g., PR-1, prp-1, or β-1,3 glucanase promoter, the fungus-inducible wirla promoter in wheat, and the nematode-inducible promoters, TobRB7-5A and Hmg-1 in 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 from any part of the plant. Such a "leaf" promoter may 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 the plant, such as one or more floral tissues. Such a "leaf" promoter may be further defined as a "leaf-preferential" promoter that at least preferentially or primarily (if not exclusively) initiates, induces, drives, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence in leaf tissue (as opposed to floral tissue) from any part of the plant. However, "leaf" and "leaf-preferential" promoters may each also permit, enable, induce, drive, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence in one or more cells or tissues of the plant, such as one or more vegetative or reproductive tissues, during the reproductive season or stage of development. Indeed, a "leaf" promoter may even initiate, induce, drive, etc., transcription or expression of its associated gene, transgene or transcribable DNA sequence in one or more reproductive or vegetative tissues to 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 from any part of the plant. Such a "root" promoter may 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 the plant, such as one or more floral tissues. Such a "root" promoter may be further defined as a "root-preferential" promoter that at least preferentially or primarily (if not exclusively) initiates, induces, drives, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence in root tissue (as opposed to floral tissue) from any part of the plant. However, "root" and "root-preferential" promoters may each also permit, enable, induce, drive, etc., the transcription or expression of its associated gene, transgene, or transcribable DNA sequence in one or more cells or tissues of the plant, such as one or more vegetative or reproductive tissues, during reproductive periods or stages of development. Indeed, a "root" promoter may even initiate, induce, drive, etc., transcription or expression of its associated gene, transgene, or transcribable DNA sequence in one or more reproductive or vegetative tissues to a greater level or extent than in root tissue.

[0102] Additional exemplary tissue-preferred promoters are those described 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, " operably linked " refers to the functional link between two or more elements.For example, the functional link between the polynucleotide of interest and the regulatory sequence (for example, promoter) is the functional relationship that allows the expression of the polynucleotide of interest.The operably linked elements can be contiguous or discontinuous.

[0104] As used herein, "heterologous" refers to a sequence originating from a foreign species, or, if derived from the same species, a sequence whose composition and / or genomic loci have been substantially altered from their native form by deliberate 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 originating from a foreign species, or, if derived from the same species, a construct whose composition and / or genomic loci have been substantially altered from their native form by deliberate human intervention. Heterologous nucleic acid constructs include, but are not limited to, recombinant nucleotide constructs that have been introduced into a plant or plant part thereof, for example, via transformation methods or subsequent crossing of the transgenic plant with another plant of interest.

[0105] In one aspect, the inhibition of the expression of one or more polypeptides provided herein may be achieved by RNA interference (RNAi) through 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 a plant genome. In another aspect, the non-coding RNA provided herein is transiently transformed into a 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 that reduces the expression or function of a gene product of interest (e.g., mRNA, protein, non-coding RNA). "Inhibition" can refer to a comparison between two plants, for example, between a modified plant and a control plant. Alternatively, inhibition of target gene product expression or function can refer to a comparison between plant cells, organelles, organs, tissues, or plant components, within the same plant or between different plants, and also includes comparisons between developmental or temporal stages, within the same plant or plant component, or between plants or plant components. "Inhibition" includes any relative reduction in the function or production of a 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 capable of inhibiting the expression or function of a gene in a plant, such as 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 of RNA, DNA, chemically modified nucleic acids, nucleic acid analogs, combinations thereof, etc.

[0108] The phrase "capable of inhibiting," when used in the context of a polynucleotide inhibitory sequence, is intended to mean either that the inhibitory sequence itself exerts an inhibitory effect; or, if the inhibitory sequence encodes an inhibitory nucleotide molecule (e.g., a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or encodes an inhibitory polypeptide (e.g., a polypeptide that inhibits the expression or function of a target gene product), 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 or translation product, respectively, exerts an inhibitory effect on the target gene product (e.g., inhibits the expression or function of the target gene product).

[0109] The inhibitory sequence provided herein can be the sequence that causes gene silencing through 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, ribozyme, small interfering RNA, artificial or synthetic microRNA and artificial trans-acting siRNA.Depending on the desired result, inhibitory sequence can be 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 up to the full-length polynucleotide that encodes the protein of the present disclosure. In one aspect, the inhibitory sequence can be a fragment 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] MicroRNAs (miRNAs) are non-protein-coding RNAs, generally 19-25 nucleotides (usually 20-24 nucleotides in plants), that guide the trans cleavage of target transcripts to negatively regulate the expression of genes involved in various regulatory and developmental pathways (Bartel (2004) Cell, 116:281-297). In some cases, miRNAs serve to guide the in-phase processing of siRNA primary transcripts (see Allen et al. (2005) Cell, 121:207-221).

[0111] Many microRNA genes (MIR genes) have been identified and are publicly available in databases ("miRBase," available online at microrna.sanger.ac.uk / sequences; see also Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to occur both singly and in clusters within the genome, in intergenic regions, but may also be located wholly or partially within introns of other genes (both protein-coding and non-protein-coding). Transcription of MIR genes may, at least in some cases, be under the stimulatory control of the MIR gene's own promoter. Primary transcripts, termed "pri-miRNAs," can be quite large (several kilobases) and polycistronic, containing the usual 5' "cap" and polyadenylated tail of mRNA, along with one or more pre-miRNAs (folded structures containing stem-loop arrangements that are processed into mature miRNAs).

[0112] The maturation of mature miRNA from its corresponding precursor (pri-miRNA and pre-miRNA) is significantly different between animals and plants.For example, in plant cells, microRNA precursor molecules are mostly considered to be completely processed into mature miRNA in the nucleus, while in animal cells, pri-miRNA transcripts are processed in the nucleus by the animal-specific enzyme Drosha, and then pre-miRNA is transported to the cytoplasm, where it is further processed into mature miRNA.The mature miRNA in plants is typically 21 nucleotides long.

[0113] Transgenic expression of miRNAs (whether naturally occurring or artificial) can be used to regulate the expression of one or more target genes of the miRNA. The inclusion of miRNA recognition sites within transgenically expressed transcripts is also useful in regulating transcript expression; see, for example, Parizotto et al. (2004) Genes Dev., 18:2237-2242. Recognition sites for miRNAs have been validated in all regions of mRNAs, including the 5' untranslated, coding, and 3' untranslated regions, suggesting that the location of the miRNA target site relative to the coding sequence does not necessarily affect repression. Because miRNAs are important regulatory elements in eukaryotes, transgenic repression of miRNAs is useful for manipulating biological pathways and responses. Finally, promoters of miRNA genes can have highly specific expression patterns (e.g., cell-specific, tissue-specific, temporally specific, or inducible) and are therefore useful in recombinant constructs for directing such specific transcription of operably linked DNA sequences. 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 transgenes bearing miRNA recognition sites, in which case the transgene is repressed when the mature miRNA is expressed; and (4) expression of transgenes driven by miRNA promoters.

[0114] Designing an artificial miRNA sequence can be as simple as replacing nucleotides in the miRNA stem region of a miRNA precursor with a sequence 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 includes the following steps: (a) selecting a unique target sequence of at least 18 nucleotides specific to the target gene by using a sequence alignment tool (e.g., of both tobacco cDNA and genomic DNA databases), such as, for example, BLAST® (see, e.g., Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402), to identify 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 undesired sequences (e.g., matches to sequences from non-target species) and determining the sequence identity based on GC content, Reynolds score (Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and scoring each potential 19-mer segment for functional asymmetry, characterized by a negative free energy difference (".DELTA..DELTA.G" or "ΔΔG") [preferably, 19-mers with all or most of the following characteristics are selected: (1) Reynolds score >4, (2) GC content between 40% and 60%, (3) negative ΔΔG, (4) terminal adenosine, (5) no more than four consecutive identical nucleotides; (6) located near the 3' end of the target gene; and (7) minimal divergence from the miRNA precursor transcript.The position of every third nucleotide in the siRNA has been reported to be particularly important with respect to its influence on RNAi efficacy, and the algorithm "siExplorer" is publicly available at rna.chem.tu-tokyo.ac.jp / siexplorer.htm; (c) determining the reverse complement of the selected 19-mer for use in producing a modified mature miRNA (the additional nucleotide at position 20 preferably matches the selected target sequence, and the nucleotide at position 21 is preferably selected to either not pair to prevent the spread of silencing in the target transcript, or to pair with the target sequence to promote the spread of silencing in the target transcript); and (d) transforming plants 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 miRNA or artificial miRNA provided herein is under the control of a tissue-specific promoter.In a further aspect, the miRNA or artificial miRNA provided herein is under the control of a tissue-preferential promoter.In a further aspect, the miRNA or artificial miRNA provided herein is under the control of a constitutive promoter.

[0117] Tobacco products can be produced using tobacco materials obtained from the modified tobacco lines, varieties, or hybrids of the present disclosure.As used herein, "tobacco product" is defined as any product made or obtained from tobacco intended for human use or consumption.In one aspect, the tobacco products provided herein comprise dry ingredients derived from the modified tobacco plants provided herein.In another aspect, the tobacco products provided herein comprise cured tobacco leaves derived from the modified tobacco plants provided herein.

[0118] Tobacco products provided herein include, but are not limited to, cigarette products (e.g., cigarettes, bidi cigarettes, kreteks), cigar products (e.g., cigars, cigar wrappers, cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco), films, chewables (e.g., gums), lozenges, dissolving 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, a "cigarette" refers to a tobacco product having a "rod" and a "filler." The "rod" of a cigarette includes the cigarette paper, filter, plug wrap (used to contain the filtration material), tipping paper that holds the cigarette paper (including the filler) to the filter, and any 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 substitutes (including, but not limited to, herbs, non-tobacco plant materials, and other spices that may accompany tobacco wrapped in cigarette paper), (3) casing, (4) flavorings, and (5) all other additives (mixed with the tobacco and substitutes and wrapped into the cigarette).

[0120] In one aspect, the present disclosure provides nicotine 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 plants of the present disclosure also include cigarettes and other smoking articles, particularly smoking articles including filter elements, where the rod of smokable material contains dried tobacco within a tobacco blend. In one aspect, the tobacco product of the present disclosure is selected from the group consisting of cigarillos, non-ventilation recess filter cigarettes, ventilation recess filter cigarettes, bidi cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah, shredded tobacco, and cut tobacco. In another aspect, the tobacco product of the present disclosure is a smokeless tobacco product. Smokeless tobacco products are not combusted and include, but are not limited to, chewing tobacco, moist smokeless tobacco, snus, and dry snuff. Chewing tobacco is coarsely divided tobacco leaves, typically packaged in large pouch-like packages and used in a loose or twisted form. Moist smokeless tobacco is a moist, more finely divided tobacco, provided in loose or pouch form, typically packaged in a round can and consumed as a pinch or in a pouch between the cheek and gum of an adult tobacco consumer. Snus is heat-treated smokeless tobacco. Dry snuff is finely powdered tobacco that is placed in the mouth or nasally. In a further aspect, the tobacco product of the present disclosure is selected from the group consisting of loose-leaf chewing tobacco, chunk chewing tobacco, moist snuff, and nasal snuff. In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of an electronically heated cigarette, an e-cigarette, and an electronic vaporization device. In one aspect, the method provided herein comprises preparing a tobacco product using cured tobacco leaves from the modified tobacco plant provided herein.

[0122] As used herein, "reconstituted tobacco" refers to a type of tobacco filler made from tobacco dust and other tobacco scrap materials that have been processed into sheets and cut into strips to resemble tobacco. In addition to cost savings, reconstituted tobacco is highly valued for its contribution to cigarette taste due to its processing of flavor development using ammonia and sugar reactions.

[0123] As used herein, "expanded tobacco" refers to a type of tobacco filler that has been processed through expansion with a suitable gas to "expand" the tobacco, resulting in reduced density and greater fill capacity. Expanded tobacco reduces the weight of the tobacco used in a cigarette.

[0124] Also provided herein are cured tobacco materials produced from the tobacco plants or plant components provided herein. "Curing" is a aging process that reduces moisture and causes chlorophyll destruction, thereby golden-coloring tobacco leaves and converting starch to sugars. Cured tobacco therefore has a higher reducing sugar content and a lower starch content than harvested green leaves. In one aspect, the tobacco plants or plant components provided herein can be dried using conventional means, such as hot air drying, barn drying, flame drying, air drying, or sun drying. For a description of different types of curing methods, see, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco is typically aged in wooden drums (e.g., vats) or cardboard cartons under compressed conditions for several years (e.g., 2-5 years) at a moisture content ranging from 10% to about 25%. See U.S. Patent Nos. 4,516,590 and 5,372,149. The cured and aged tobacco can then be further processed. Further processing includes conditioning the tobacco under vacuum with or without the introduction of steam at various temperatures, pasteurization, and fermentation. Fermentation is typically characterized by a high initial moisture content, heat generation, and a loss of 10-20% of the dry weight. See, e.g., U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, and 5,372,149; U.S. Publication No. 2005 / 0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The cured, aged, and fermented tobacco can then be further processed (e.g., cut, shredded, expanded, or blended). See, for example, U.S. Patent Nos. 4,528,993; 4,660,577; and 4,987,907.In one aspect, the cured tobacco material of the present disclosure is flue-cured, sun-cured, air-dried, or flame-cured.

[0125] The present disclosure further provides a method for producing a tobacco product comprising tobacco material derived from a tobacco plant provided herein. In one aspect, the method provided herein includes conditioning aged 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 conditioned tobacco material to produce a desired blend. In one aspect, the method for producing a tobacco product provided herein further includes casing or flavoring the blend. Typically, during the casing process, casing or source materials are added to the blend to enhance its quality by balancing its chemical composition and develop specific desirable flavor characteristics. Further details about 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, enzyme treatment, expansion, and / or curing. Both fermented and non-fermented tobaccos can be processed using these techniques. Examples of suitable processed tobaccos include dark air-cured, dark flue-cured, burley, flue-cured, and cigar filler or wrapper, as well as products from whole-leaf stemming operations. In one aspect, the tobacco fiber contains up to 70% dark tobacco on a fresh weight basis. For example, the tobacco can be conditioned by heating, infusion, and / or pasteurization processes, such as those 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 10-20% of 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 leaf's aroma, fermentation can change either or both the leaf's color and texture. Also, during the fermentation process, gas evolution occurs, oxygen is taken up, pH changes occur, and the amount of water retained can change. 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 cured tobacco or cured and fermented tobacco may be further processed (e.g., cut, expanded, blended, ground, or crushed) before being incorporated into an oral product. The tobacco is, in some cases, long-cut, fermented, cured, moist tobacco having an oven volatiles content of 48-50 percent by weight before being mixed with the copolymer 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, 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, which can be cut or chopped to widths of 10 cuts per inch to 110 cuts per inch and lengths of 0.1 inch to 1 inch. Double-cut tobacco fibers can have a particle size range such that 70% of the double-cut tobacco fibers have a mesh size between -20 mesh and 80 mesh.

[0129] The tobacco materials provided herein can be processed to have a total oven volatiles content of 10% by weight or more; 20% by weight or more; 40% by weight or more; 15% to 25% by weight; 20% to 30% by weight; 30% to 50% by weight; 45% to 65% by weight; or 50% to 60% by weight. Those skilled in the art will understand that "moist" tobacco typically refers to tobacco having an oven volatiles content of between 40% and 60% by weight (e.g., 45% to 55% by weight, or 50% by weight). As used herein, "oven volatiles" is determined by calculating the percentage weight loss of a sample after drying the sample in a preheated 110°C forced air oven for 3.25 hours. Oral products can have an overall oven volatiles content that differs from the oven volatiles content of the tobacco fiber used to make the oral product. The processing steps described herein can reduce or increase the oven volatiles 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 flue-cured, sun-cured, air-cured, dark air-cured, and dark flue-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, Maryland, bright, Virginia, Oriental, Turkish, and Galpao tobacco. In one aspect, the tobacco plants or seeds provided herein are hybrid plants or seeds. As used herein, a "hybrid" is produced by crossing two plants from different varieties or species so that the offspring contain genetic material from each parent. Those skilled in the art will recognize that higher-order hybrids can be produced as well. For example, a first hybrid can be produced by crossing variety C with variety D to produce a C x D hybrid, and a second hybrid can be produced by crossing variety E with variety F to produce an E x F hybrid. The first and second hybrids can be further crossed to produce a higher order hybrid (C x D) x (E x F) that contains the genetic information of all four parent 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" due to the golden yellow to deep orange color it achieves during curing. Flue-cured tobacco has a light, vibrant aroma and taste. Flue-cured tobacco is generally high in sugar and low in oil. The major flue-cured tobacco-growing countries are Argentina, Brazil, China, India, Tanzania, and the United States. In one aspect, the modified tobacco plants or seeds provided herein are 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 1518, PVH 1618, PVH 1718, PVH 1818, PVH 1918, PVH 2018, PVH 2118, PVH 2218, PVH 2318, PVH 2418, PVH 2518, PVH 2618, PVH 2718, PVH 2818, PVH 2918, PVH 3018, PVH 3118, PVH 3218, PVH 3318, PVH 3418, PVH 3518, PVH 3618, PVH 3718, PVH 3818, PVH 3918, PVH 4018, PVH 4118, PVH 4218, PVH 4318, PVH 4418, PVH 4518, PVH 4618, PVH 4718, PVH 4718, PVH 4818, 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 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-111, Speight G-117, Speight 168, Speight 179, Speight NF-3, Va 116, Va 182, and any variety essentially derived from any one of the aforementioned varieties. See WO 2004 / 041006 A1. In a further aspect, the modified tobacco plant, seed, hybrid, variety, or line provided herein is of any flue-cured background selected from the group consisting of K326, K346, and NC196.

[0132] Air-cured tobaccos include burley, Maryland, and dark tobacco. A common requirement is that the curing be primarily free of artificial heat and moisture sources. Burley tobacco is light to dark brown in color, high in oil, and low in sugar. Burley tobacco is air-cured in warehouses. The major burley-growing countries are Argentina, Brazil, Italy, Malawi, and the United States. Maryland tobacco is extremely soft, has good burning properties, low nicotine, and a neutral aroma. The major Maryland-growing countries include the United States and Italy. In one aspect, the modified tobacco plants or seeds provided herein are of a 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 x Ky 10, HB04P, Ky 14 x 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 a 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 primarily by its medium-to-dark brown color and its curing process, which imparts a distinct aroma to dark air-cured tobacco. Dark air-cured tobacco is primarily 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 flue-cured tobacco is typically cured over low-burning wood fires on the floor of enclosed drying warehouses. Dark flue-cured tobacco is used to make pipe blends, cigarettes, chewing tobacco, snuff, and strong-tasting cigars. The primary growing areas for dark flue-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 flue-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-aromatic Turkish tobacco because it is typically grown 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 unique aromatic characteristics, are the result of the plant's adaptations, which have evolved over the past centuries in poor soils and stressful climatic conditions. In one aspect, the modified tobacco plants or seeds provided herein are selected from the Izmir, Katerini, Samsun, Basma and Krumovgrad varieties. and of an oriental tobacco background selected from the group consisting of Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and any variety essentially derived from any one of the aforementioned varieties.

[0136] In one aspect, the modified tobacco plants, seeds, hybrids, varieties, or lines provided herein are selected from the group consisting of 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, McNair 373, McNair 944, male sterile KY 14×L8, Fine Leaf Madol, MS KY171, Fine Leaf Madol (phph), MS Fine 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, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madore, VA 309, VA 359, or any commercially available tobacco variety bred by standard tobacco breeding techniques known in the art.

[0137] All of the above specific varieties of dark air-cured, burley, Maryland, dark-fired, or Oriental type are listed for illustrative purposes only. Any additional dark air-cured, burley, Maryland, dark-fired, or Oriental varieties are also contemplated herein.

[0138] Also provided herein are populations of tobacco plants described herein. In one aspect, the populations of tobacco plants provided herein have a plant density of 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 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 of seeds from tobacco plants described herein.The container of tobacco seeds of the present disclosure can contain any number, weight or volume of seeds.For example, the container can 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 1500 or more seeds; at least 2000 or more seeds; at least 2500 or more seeds; at least 3000 or more seeds; at least 3500 or more seeds; at least 4000 or more seeds; or at least 5000 or more seeds. Alternatively, the container can contain at least 1 ounce or more of seeds; at least 5 grams or more of seeds; at least 10 grams or more of seeds; at least 30 grams or more of seeds; at least 50 grams or more of seeds; at least 100 grams or more of seeds; at least 500 grams or more of seeds; at least 1 kilogram or more of seeds; at least 1.5 kilograms or more of seeds; at least 2 kilograms or more of seeds; at least 5 kilograms or more of seeds; or at least 10 kilograms or more of seeds. The container of tobacco seeds can be any container available in the art. As a non-limiting example, the container can be a box, a bag, a packet, a pouch, a tape roll, a tube, or a bottle.

[0140] In one aspect, the present disclosure provides cured leaves from modified tobacco plants containing reduced levels of one or more TSNAs. In one aspect, the reduced one or more TSNAs are selected from the group consisting of N'-nitrosonornicotine (NNN), 4-methylnitrosamino-l-(3-pyridyl)-l-butanone (NNK), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and any combination thereof. In one aspect, the levels of total TSNAs or individual TSNAs are measured on freeze-dried leaf samples using liquid chromatography with tandem mass spectrometry (LC / MS / MS).

[0141] In one aspect, the present disclosure provides cured leaves from modified tobacco plants containing reduced levels of one or more alkaloids, wherein the reduced one or more alkaloids are selected from the group consisting of nicotine, nornicotine, anabasine, and anatabine.

[0142] The present disclosure also provides methods for breeding tobacco lines, cultivars, or varieties with increased nitrogen utilization efficiency. Breeding can be performed via any known procedure. DNA fingerprinting, SNP mapping, haplotype mapping, or similar techniques may be used in marker-assisted selection (MAS) breeding programs to transfer or cross desirable traits or alleles into tobacco plants. For example, breeders can use the F1 hybrid plants provided herein or further cross the F1 hybrid plants with other donor plants having agronomically desirable genotypes to produce segregating populations in the F2 or backcross generation. The F2 or backcross generation plants can be screened for desired agronomic traits or desirable chemical profiles 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 selected plants can be performed before each backcross cycle to aid in identifying desired individual plants. Backcrossing 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 flue-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, EA, and Rufty, RC 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. WH Fehr (ed.), MacMillan Publishing Go., Inc., New York, NY, the entire contents of which are incorporated herein by reference.

[0143] The results of plant breeding programs using the modified tobacco plants described herein include the 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 consistent characteristics that separate them from other plants of the same species. Varieties are often, but not necessarily, commercially available. While possessing one or more unique traits, varieties are further characterized by very little overall variation between individuals within the variety. "Pure-bred" 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. A variety 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, amended at Geneva November 10, 1972; October 23, 1978; and March 19, 1991), a variety is "essentially derived" from an original variety if: a) it is derived predominantly from the original variety or is derived from a variety derived predominantly from the original variety while retaining the expression of essential characteristics resulting from the genotype or combination of genotypes of the original variety; b) it is clearly distinguishable from the original variety; and c) it matches the original variety in the expression of essential characteristics resulting from the genotype or combination of genotypes of the original variety, except for differences resulting from acts of derivation. Essentially derived varieties can be obtained, for example, by natural or induced mutations, somaclonal variants, selection of variant individuals from plants of the original variety, backcrossing, or transformation. A first tobacco variety and a second tobacco variety from which the first variety is essentially derived are considered to have essentially the same genetic background. A "line," as distinguished from a variety, most often refers to a group of plants used non-commercially, for example, in botanical research. A line typically exhibits little overall variation among individuals for one or more traits of interest, although there may be some variation among individuals for other traits.

[0144] In one aspect, the present disclosure provides a method for producing a tobacco plant, comprising: 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 utilization efficiency compared to a control tobacco plant of the same variety grown under equivalent conditions; and selecting progeny tobacco plants that exhibit increased nitrogen utilization efficiency compared to a control tobacco plant of the same hybrid grown under equivalent conditions. In one aspect, the first tobacco variety provided herein comprises a modified tobacco plant. In another aspect, the second tobacco variety provided herein comprises 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 an elite variety. In another aspect, the first or second tobacco variety is a hybrid.

[0145] In one aspect, the present disclosure provides methods for introgressing one or more transgenes into tobacco varieties, the methods comprising: (a) crossing a first tobacco variety containing one or more transgenes with a second tobacco variety that does not contain the 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 the progeny tobacco plants containing the one or more transgenes. In another aspect, the methods further comprise backcrossing the selected progeny tobacco plants with the second tobacco variety. In a further aspect, the 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 the additional progeny tobacco plants containing the one or more transgenes. In one aspect, the second tobacco variety is an elite variety.

[0146] In one aspect, the present disclosure provides methods for introgressing one or more mutations into a tobacco variety, the methods comprising: (a) crossing a first tobacco variety containing one or more mutations with a second tobacco variety that does not contain the 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 the progeny tobacco plants containing the one or more mutations. In another aspect, the methods further comprise backcrossing the selected progeny tobacco plants with the second tobacco variety. In a further aspect, the 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 the additional progeny tobacco plants containing the one or more mutations. In one aspect, the second tobacco variety is an elite variety.

[0147] In one aspect, the present disclosure provides a method for 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 a control tobacco plant of the same variety when grown under equivalent conditions.

[0148] In one aspect, the present disclosure provides a method for producing modified seeds, comprising the steps of 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; producing 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" refers to a chromosomal region where a polymorphic nucleic acid, a trait determinant, a gene or a marker is located. The locus of the present disclosure includes one or more polymorphisms in a population; for example, alternative alleles exist in some individuals. As used herein, "allele" refers to alternative nucleic acid sequences at a specific locus. The length of an allele can be as small as one nucleotide base, but is typically longer. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome (for example, occurring on different chromosomes in a heterozygous individual, or occurring between different homozygous or heterozygous individuals in a population). As used herein, a chromosome of a diploid plant is "hemizygous" when only one copy of a locus exists. For example, an inserted transgene is hemizygous when it is inserted into one sister chromosome (for example, the second sister chromosome does not contain the inserted transgene).

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

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

[0152] As used herein, "hybridized" or "to hybridize" means to produce offspring (e.g., cells, seeds, or plants) through fertilization, and includes crosses between different plants (sexual) and self-fertilization (selfing).

[0153] As used herein, "backcrossing" and "backcrossing" refer to the process of repeatedly crossing a progeny plant back to one of its parents. In a backcrossing scheme, the "donor" parent refers to the parent plant that has the desired gene or locus to be introgressed. The "recipient" parent (used one or more times) or "recurrent" parent (used more than once) 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, "BC2" refers to the third use of the recurrent parent, and so on. In one aspect, backcrossing is performed repeatedly, with each successive backcross generation of progeny individuals themselves being crossed back to the same parent genotype.

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

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

[0156] In one aspect, the tobacco plants provided herein are hybrid plants. Hybrids can be produced by preventing self-pollination of a first variety of female parent plant (e.g., seed parent), fertilizing the female parent plant with pollen from a second variety of male parent plant, and allowing the female plant to form F1 hybrid seeds. Self-pollination of female plants can be prevented by emasculating flowers early in 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 male sterility (MS), or transgenic male sterility in which an introduced gene inhibits microspore formation and / or pollen formation, or self-incompatibility. Female parent plants containing MS are particularly useful. In situations where the female parent plant is MS, pollen may be harvested from the male fertile plants and manually applied to the stigmas of the MS female parent plants, and the resulting F1 seeds are harvested. Additionally, female sterile plants may be used to prevent self-fertilization.

[0157] The plants can be used to create single-cross tobacco F1 hybrids. Pollen from a male parent plant is manually transferred to an emasculated or male-sterile female parent plant to form F1 seeds. Alternatively, a tri-cross can be performed in which a single-cross F1 hybrid is used as the female parent to cross with a different male parent. Alternatively, the F1 progeny of two different single crosses can be crossed with themselves to produce a double-cross hybrid. Self-incompatibility can be used to provide particular benefits in preventing self-pollination of the female parent when creating 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, EA, and Rufty, RC 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. WH Fehr (ed.), MacMillan Publishing Go., Inc., New York, NY 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, comprising obtaining at least one metabolite from a tobacco plant of the tobacco line, determining the amount of the at least one obtained metabolite, and determining the NUE of the tobacco line based on the determined amount of the 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 with increased NUE comprises increased NUE compared to a tobacco plant comprising a lower amount of at least one metabolite in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a lower amount of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a lower amount of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a lower amount of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a lower amount of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a lower amount of at least one metabolite in two tissues. In a further aspect, a tobacco plant with increased NUE comprises a lower amount of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least four metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least five metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least one metabolite in three tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least two metabolites in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least three metabolites in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least four metabolites in at least three tissues. In certain further aspects, tobacco plants with increased NUE contain reduced amounts of at least five metabolites in at least three tissues.In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least one metabolite in four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least two metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least three metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least four metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least five metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least one metabolite in five tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least two metabolites in at least five tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least three metabolites in at least five tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least four metabolites in at least five tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least five metabolites in at least five tissues.

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

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

[0164] In another aspect of the methods provided herein, a tobacco plant with increased NUE comprises reduced NUE compared to a tobacco line comprising a reduced amount of at least one metabolite in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least one metabolite in two tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least four metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least five metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least one metabolite in three tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least two metabolites in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least three metabolites in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least four metabolites in at least three tissues. In certain further aspects, tobacco plants with increased NUE contain reduced amounts of at least five metabolites in at least three tissues.In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least one metabolite in four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least two metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least three metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least four metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least five metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least one metabolite in five tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least two metabolites in at least five tissues. In a further aspect, a tobacco plant with increased NUE comprises a reduced amount of at least three metabolites in at least five tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least four metabolites in at least five tissues. In a further aspect, a tobacco plant with increased NUE comprises reduced amounts of at least five metabolites in at least five tissues.

[0165] In another aspect of the methods provided herein, a tobacco plant with increased NUE comprises reduced NUE compared to a tobacco line comprising a greater amount of at least one metabolite in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a greater amount of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a greater amount of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a greater amount of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a greater amount of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises a greater amount of at least one metabolite in two tissues. In a further aspect, a tobacco plant with increased NUE comprises a greater amount of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least four metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least five metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least one metabolite in three tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least two metabolites in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least three metabolites in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least four metabolites in at least three tissues. In certain further aspects, tobacco plants with increased NUE contain greater amounts of at least five metabolites in at least three tissues.In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least one metabolite in four tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least two metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least three metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least four metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least five metabolites in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least one metabolite in five tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least two metabolites in at least five tissues. In a further aspect, a tobacco plant with increased NUE comprises increased amounts of at least three metabolites in at least five tissues. In a further aspect, a tobacco plant with increased NUE contains increased amounts of at least four metabolites in at least five tissues. In a further aspect, a tobacco plant with increased NUE contains increased amounts of at least five metabolites in at least five tissues.

[0166] In another aspect of the methods provided herein, a tobacco plant with increased NUE comprises reduced NUE compared to a tobacco line comprising an equal amount of at least one metabolite in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises equal amounts of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises equal amounts of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises equal amounts of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises equal amounts of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with increased NUE comprises equal amounts of at least one metabolite in two tissues. In a further aspect, a tobacco plant with increased NUE comprises equal amounts of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises equal amounts of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises at least four metabolites in equal amounts in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises at least five metabolites in equal amounts in at least two tissues. In a further aspect, a tobacco plant with increased NUE comprises at least one metabolite in equal amounts in three tissues. In a further aspect, a tobacco plant with increased NUE comprises at least two metabolites in equal amounts in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises at least three metabolites in equal amounts in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises at least four metabolites in equal amounts in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises at least five metabolites in equal amounts in at least three tissues. In a further aspect, a tobacco plant with increased NUE comprises at least one metabolite in equal amounts in four tissues.In a further aspect, a tobacco plant with increased NUE comprises at least two metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises at least three metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises at least four metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises at least five metabolites in equal amounts in at least four tissues. In a further aspect, a tobacco plant with increased NUE comprises at least one metabolite in equal amounts in five tissues. In a further aspect, a tobacco plant with increased NUE comprises at least two metabolites in equal amounts in at least five tissues. In a further aspect, a tobacco plant with increased NUE comprises at least three metabolites in equal amounts in at least five tissues. In a further aspect, a tobacco plant with increased NUE comprises at least four metabolites in equal amounts in at least five tissues. In a further aspect, the tobacco plant with increased NUE contains equal amounts of at least five metabolites in at least five tissues.

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

[0168] In one aspect, the present disclosure provides and includes a method for determining the NUE of a tobacco line using a metabolite signature, comprising: isolating a metabolite signature from a tobacco plant of the tobacco line; determining the amount of each metabolite comprising the metabolite signature; and determining the NUE of the tobacco line by comparing the metabolite signature with a control metabolite signature from a control tobacco line with a known NUE. In a further aspect of this method, the NUE comprises an increased NUE compared to the control tobacco line. In another aspect of this method, the metabolite signature is isolated from plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristem and stem tissue.

[0169] In certain aspects of the methods provided herein, the metabolite signature comprises at least two metabolites. In certain further aspects, the metabolite signature comprises at least three metabolites. In certain further aspects, the metabolite signature comprises at least four metabolites. In certain further aspects, the metabolite signature comprises at least five metabolites. In certain further aspects, the metabolite signature comprises at least six metabolites. In certain further aspects, the metabolite signature comprises at least seven metabolites. In certain further aspects, the metabolite signature comprises at least eight metabolites. In certain further aspects, the metabolite signature comprises at least nine metabolites. In certain further aspects, the metabolite signature comprises at least ten metabolites. In certain further aspects, the metabolite signature comprises at least eleven metabolites. In certain further aspects, the metabolite signature comprises at least twelve metabolites. In some further aspects, the metabolite signature includes at least 13 metabolites. In some further aspects, the metabolite signature includes at least 14 metabolites. In some further aspects, the metabolite signature includes at least 15 metabolites. In some further aspects, the metabolite signature includes at least 20 metabolites. In some further aspects, the metabolite signature includes at least 25 metabolites. In some further aspects, the metabolite signature includes at least 30 metabolites. In some further aspects, the metabolite signature includes at least 35 metabolites. In some further aspects, the metabolite signature includes at least 40 metabolites. In some further aspects, the metabolite signature includes at least 45 metabolites. In some further aspects, the metabolite signature includes at least 50 metabolites. In some further aspects, the metabolite signature includes between 2 and 50 metabolites. In some further aspects, the metabolic signature comprises between 3 and 45 metabolites. In some further aspects, the metabolic signature comprises between 3 and 40 metabolites. In some further aspects, the metabolic signature comprises between 4 and 35 metabolites.In some further aspects, the metabolite signature includes between 5 and 30 metabolites. In some further aspects, the metabolite signature includes between 6 and 25 metabolites. In some further aspects, the metabolite signature includes between 7 and 20 metabolites. In some further aspects, the metabolite signature includes between 8 and 15 metabolites. In some further aspects, the metabolite signature includes between 9 and 14 metabolites. In some further aspects, the metabolite signature includes between 10 and 13 metabolites. In some further aspects, the metabolite signature includes between 10 and 12 metabolites.

[0170] In one aspect, the present disclosure provides and includes a method for breeding a tobacco line that includes a metabolic signature associated with increased NUE, the method comprising: determining a metabolic signature of a first tobacco plant derived from a first tobacco line, wherein the first tobacco plant includes increased NUE compared to a control tobacco plant lacking the metabolic 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 metabolic signature and includes increased NUE compared to the control plant lacking the metabolic signature. In a further aspect of the 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, and NC925. In another aspect, the second tobacco line is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and 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 reduced 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, relative to the metabolite signature of a control tobacco plant.

[0171] In another aspect, the methods provided herein include a tobacco plant comprising an increased NUE, 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. In a further aspect, the increased NUE comprises an increased agronomic efficiency (AE) compared to a tobacco plant lacking the increased NUE grown under the same conditions. In a further aspect, the increased NUE comprises an increased recovery efficiency (RE) compared to a tobacco plant lacking the increased NUE grown under the same conditions. In a further aspect, the increased NUE comprises an increased physiological efficiency (PE) compared to a tobacco plant lacking the increased NUE grown under the same conditions. In a further aspect, the increased NUE comprises an increased internal efficiency (IE) compared to a tobacco plant lacking the increased NUE grown under the same conditions.

[0172] In one aspect, the present disclosure provides and includes a method for 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 having a greater amount of the at least one metabolite relative to a control tobacco plant. In a further aspect of this method, the selected tobacco plant has increased NUE relative 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 plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristematic tissue, and stem tissue.

[0173] In one aspect, the present disclosure provides and includes a method for 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 reduced amount of the at least one metabolite compared to a control tobacco plant. In a further aspect of the 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 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 plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristematic tissue, and stem tissue.

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

[0175] In one aspect, the present disclosure provides and includes 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 modified tobacco plant comprises an increased nitrogen utilization efficiency 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 comprises 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 comprises a polynucleotide sequence derived from the tobacco genome. In another aspect, the heterologous promoter comprises 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 a burley variety.

[0176] In a further aspect, the modified tobacco seeds or tobacco plants herein contain less TSNAs than unmodified tobacco plants lacking a cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less N'-nitrosonornicotine (NNN) than unmodified tobacco plants lacking a cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less 4-methylnitrosamino-l-(3-pyridyl)-l-butanone (NNK) than unmodified tobacco plants lacking a cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain less N'-nitrosoanatabine (NAT) than unmodified tobacco plants lacking a cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain a lower amount of N'-nitrosoanabasine (NAB) than an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain a lower amount of alkaloids than an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain a lower amount of nicotine than an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain a lower amount of nornicotine than an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions. In a further aspect, the modified tobacco seeds or tobacco plants contain a lower amount of anabasine than an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.In certain further aspects, the modified tobacco seeds or tobacco plants contain a lower amount of anatabine compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0177] In a further aspect, the modified tobacco seeds or tobacco plants herein comprise a coding region encoding a polypeptide 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 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 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 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 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 seed or tobacco plant comprises a coding region encoding a polypeptide 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 seed or tobacco plant comprises a coding region encoding a polypeptide 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 seed or tobacco plant comprises a coding region encoding a polypeptide 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 seed or tobacco plant comprises a coding region encoding a polypeptide 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 that encodes 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 that encodes 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 herein comprise a coding region encoding a polynucleotide 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 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 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 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 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 seed or tobacco plant comprises a coding region encoding a polynucleotide 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 seed or tobacco plant comprises a coding region encoding a polynucleotide 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 seed or tobacco plant comprises a coding region encoding a polynucleotide 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 seed or tobacco plant comprises a coding region encoding a polynucleotide 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 that encodes a polynucleotide that is at least 99% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9 to 16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region that encodes a polynucleotide that is identical to a sequence selected from the group consisting of SEQ ID NOs: 9 to 16.

[0179] In a further aspect, the modified tobacco seed or tobacco plant comprises a leaf-preferred promoter encoded by a sequence 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 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 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 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 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 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 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 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 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 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 herein comprising the cisgenic polynucleotide comprise higher levels of a metabolite selected from the group consisting of 4-guanidinobutanoate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde in root tissue compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions.

[0182] In certain further aspects, the modified tobacco plants herein comprising the cisgenic polynucleotide comprise higher levels of a metabolite selected from the group consisting of 4-guanidinobutanoate, X-23454, X-23580, and X-23852 in leaf tissue compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions.

[0183] In a further aspect, the modified tobacco plants herein comprising the cisgenic polynucleotide comprise lower levels 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-oxoglutarate, X-23937, X-23916, and 1-methyladenine in root tissue compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions.

[0184] In a further aspect, the modified tobacco plants herein comprising the cisgenic polynucleotide comprise lower levels 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 in leaf tissue compared to unmodified tobacco plants lacking the cisgenic polynucleotide when grown under the same conditions.

[0185] In one aspect, the description provides and includes a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a polypeptide 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 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 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 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 at least 90% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In a further aspect, a recombinant DNA construct comprises a polynucleotide encoding a polypeptide at least 95% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In a further aspect, a recombinant DNA construct comprises a polynucleotide encoding a polypeptide at least 96% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In a further aspect, a recombinant DNA construct comprises a polynucleotide encoding a polypeptide at least 97% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In a further aspect, a recombinant DNA construct comprises a polynucleotide encoding a polypeptide at least 98% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In a further aspect, a recombinant DNA construct comprises a polynucleotide encoding a polypeptide 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 that encodes 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 specification provides and includes a cured tobacco material or a tobacco product comprising the cured 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 utilization efficiency compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0187] In one aspect, the present specification provides and includes a greenhouse, growth chamber, or field containing the modified tobacco seeds or plants disclosed herein. In one aspect, the present specification provides and includes a method of growing the tobacco plants of the present specification 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, the modified tobacco seed or plant comprising at least one mutation at an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or plant comprises increased nitrogen utilization efficiency compared to an unmodified control tobacco plant lacking the at least one mutation when grown under the same conditions. In a further aspect, the mutation in the endogenous locus is selected from the group consisting of an insertion, a deletion, a substitution, and an inversion. In another aspect, the mutation in the endogenous locus is a silent mutation, a non-silent mutation, or a null mutation. In a further aspect, the modified tobacco seed or plant is of a Burley variety.

[0189] In a further aspect, the modified tobacco plant comprises a higher level of a metabolite selected from the group consisting of 4-guanidinobutanoate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde in root tissue compared to an unmodified tobacco plant when grown under the same conditions. In a further aspect, the modified tobacco plant comprises a higher level of a metabolite selected from the group consisting of 4-guanidinobutanoate, X-23454, X-23580, and X-23852 in leaf tissue compared to an unmodified tobacco plant when grown under the same conditions. In a further aspect, the modified tobacco plants contain lower levels 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 in root tissue compared to unmodified tobacco plants grown under the same conditions. In a further aspect, the modified tobacco plants contain lower levels 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 in leaf tissue compared to unmodified tobacco plants 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 contiguous nucleotides identical to 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 19 contiguous nucleotides identical to 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 20 contiguous nucleotides identical to 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 21 contiguous nucleotides identical to 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 22 contiguous nucleotides identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In some further aspects, the guide RNA comprises at least 23 contiguous nucleotides that are identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In some further aspects, the guide RNA comprises at least 24 contiguous nucleotides that are identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In some further aspects, the guide RNA comprises at least 25 contiguous nucleotides that are identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In some further aspects, the guide RNA comprises at least 26 contiguous nucleotides that are identical to 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 27 contiguous nucleotides that are identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40. In a further aspect, the guide RNA comprises at least 28 contiguous nucleotides that are 100% identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25 to 40.

[0191] In one aspect, the description provides and includes a cured tobacco material or a tobacco product comprising the cured tobacco material produced from a tobacco plant comprising at least one mutation at an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or tobacco plant comprises an increased NUE compared to an unmodified control tobacco plant lacking the at least one mutation when grown under the same conditions. In a further aspect, the tobacco plant comprises at least two mutations at the endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the tobacco plant comprises at least three mutations at the endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the tobacco plant comprises at least four mutations at the endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the tobacco plant comprises at least five mutations at the endogenous locus that encode a polypeptide selected from the group consisting of SEQ ID NOs:25-40. In a further aspect, the tobacco plant comprises at least six mutations at the endogenous locus that encode a polypeptide selected from the group consisting of SEQ ID NOs:25-40. In a further aspect, the tobacco plant comprises at least seven mutations at the endogenous locus that encode a polypeptide selected from the group consisting of SEQ ID NOs:25-40. In a further aspect, the tobacco plant comprises at least eight mutations at the endogenous locus that encode a polypeptide selected from the group consisting of SEQ ID NOs:25-40. In a further aspect, the tobacco plant comprises at least nine mutations at the endogenous locus that encode a polypeptide selected from the group consisting of SEQ ID NOs:25-40. In a further aspect, the tobacco plant comprises at least ten mutations at the endogenous locus that encode a polypeptide selected from the group consisting of SEQ ID NOs:25-40.

[0192] In one aspect, the description provides and includes a modified tobacco seed or a tobacco plant grown therefrom, comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, wherein the modified tobacco seed or plant comprises 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 to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 91% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 92% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 93% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 94% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 95% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 96% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 97% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 98% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is at least 99% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgenic polynucleotide comprises a polynucleotide encoding an sRNA that is 100% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. 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 some further aspects, the sRNA has at least 18 nucleotides. In some further aspects, the sRNA includes at least 19 nucleotides. In some further aspects, the sRNA includes at least 20 nucleotides. In some further aspects, the sRNA includes at least 21 nucleotides. In some further aspects, the sRNA includes at least 22 nucleotides. In some further aspects, the sRNA includes at least 23 nucleotides. In some further aspects, the sRNA includes at least 24 nucleotides. In some further aspects, the sRNA includes at least 25 nucleotides. In some further aspects, the sRNA includes at least 26 nucleotides. In some further aspects, the sRNA includes at least 27 nucleotides. In some further aspects, the sRNA includes at least 28 nucleotides. In some further aspects, the sRNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, and precursors thereof. In some further aspects, the sRNA downregulates expression or translation of a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0195] In one aspect, the description provides and includes a cured tobacco material or a tobacco product comprising the cured tobacco material produced from a tobacco plant comprising a cisgenic polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding an sRNA at least 85% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, wherein the modified tobacco seed or tobacco plant comprises an 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 encodes an sRNA at least 90% identical to 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 at least 91% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In some further aspects, the cisgenic polynucleotide encodes an sRNA that is at least 92% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In some further aspects, the cisgenic polynucleotide encodes an sRNA that is at least 93% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In some further aspects, the cisgenic polynucleotide encodes an sRNA that is at least 94% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In some further aspects, the cisgenic polynucleotide encodes an sRNA that is at least 95% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In some further aspects, the cisgenic polynucleotide encodes an sRNA that is at least 96% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.In some further aspects, the cisgenic polynucleotide encodes an sRNA that is at least 97% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In some further aspects, the cisgenic polynucleotide encodes an sRNA that is at least 98% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In some further aspects, the cisgenic polynucleotide encodes an sRNA that is at least 99% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In some further aspects, the cisgenic polynucleotide encodes an sRNA that is 100% identical to 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 NUE in a tobacco plant, the method comprising: 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 comprises crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0197] In one aspect, the present disclosure provides and includes a method for increasing NUE in a tobacco plant, the method comprising: introducing into a tobacco cell a modification to a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41-56; 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. 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 comprising the use of an RNA-guided nuclease. In a further aspect, the RNA-guided nuclease is selected from the group consisting of Cas9 nuclease, Cpfl nuclease, CasX nuclease, CasY nuclease, and functional homologs thereof. In a further aspect, the modification is selected from the group consisting of an insertion, a substitution, an inversion, and a deletion.

[0198] In one aspect, the present disclosure provides and includes a method for increasing NUE in a tobacco plant, comprising: introducing into a tobacco cell a nucleic acid encoding a small RNA (sRNA) homologous to at least 18 contiguous nucleic acids of a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41-56; 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. 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 disclosure provides and includes a method comprising providing a first population of tobacco plants containing an increased NUE, genotyping the first population of tobacco plants for the presence of a molecular marker within 20 cM of the increased NUE locus, and selecting one or more tobacco plants that have been genotyped and found to contain the molecular marker. In a further aspect, the method disclosed herein comprises genotyping the first population of tobacco plants for the presence of a molecular marker within 15 cM of the increased NUE locus. In a further aspect, the method disclosed herein comprises genotyping the first population of tobacco plants for the presence of a molecular marker within 10 cM of the increased NUE locus. In a further aspect, the method disclosed herein comprises genotyping the first population of tobacco plants for the presence of a molecular marker within 9 cM of the increased NUE locus. In a further aspect, the methods disclosed herein include genotyping a first population of tobacco plants for the presence of a molecular marker within 8 cM of the augmented NUE locus. In a further aspect, the methods disclosed herein include genotyping a first population of tobacco plants for the presence of a molecular marker within 7 cM of the augmented NUE locus. In a further aspect, the methods disclosed herein include genotyping a first population of tobacco plants for the presence of a molecular marker within 6 cM of the augmented NUE locus. In a further aspect, the methods disclosed herein include genotyping a first population of tobacco plants for the presence of a molecular marker within 5 cM of the augmented NUE locus. In a further aspect, the methods disclosed herein include genotyping a first population of tobacco plants for the presence of a molecular marker within 4 cM of the augmented NUE locus. In a further aspect, the methods disclosed herein include genotyping the first population of tobacco plants for the presence of a molecular marker within 3 cM of the amplified NUE locus.In a further aspect, the method disclosed herein comprises genotyping a first population of tobacco plants for the presence of a molecular marker within 2 cM of the augmented NUE locus. In a further aspect, the method disclosed herein comprises genotyping a first population of tobacco plants for the presence of a molecular marker within 1 cM of the augmented NUE locus. In a further aspect, the method disclosed herein comprises genotyping a first population of tobacco plants for the presence of a molecular marker within 0.5 cM of the augmented NUE locus. In a further aspect, the method comprises crossing one or more selected tobacco plants with a second tobacco plant; and obtaining progeny seed from the cross. In a further aspect, the molecular marker is selected from the group consisting of a SNP marker, an INDEL marker, an RFLP marker, an SSR marker, an AFLP marker, and a RAPD marker.

[0200] In a further aspect, the methods provided herein include a tobacco plant comprising an augmented NUE locus comprising a polynucleotide encoding a polypeptide 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 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 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 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 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 at least 95% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In some further aspects, the polynucleotide encodes a polypeptide at least 96% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In some further aspects, the polynucleotide encodes a polypeptide at least 97% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In some further aspects, the polynucleotide encodes a polypeptide at least 98% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In some further aspects, the polynucleotide encodes a polypeptide at least 99% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs:1-8. In some further aspects, 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 expanded NUE locus is genetically linked to a polynucleotide sequence selected from the group consisting of SEQ ID NOs:57-64. In another aspect, the expanded NUE locus is genetically linked to a G nucleotide at position 57 of SEQ ID NO:58. In another aspect, the expanded NUE locus is genetically linked to a C nucleotide at position 117 of SEQ ID NO:58. In another aspect, the expanded NUE locus is genetically linked to a G nucleotide at position 57 and a C nucleotide at position 117 of SEQ ID NO:58. In another aspect, the expanded NUE locus is genetically linked to a T nucleotide at position 147 of SEQ ID NO:57. In another aspect, the expanded NUE locus is genetically linked to a G nucleotide at position 162 of SEQ ID NO:59. In another aspect, the expanded NUE locus is genetically linked to a C nucleotide at position 36 of SEQ ID NO:60. In another aspect, the expanded NUE locus is genetically linked to the T nucleotide at position 36 of SEQ ID NO:61. In another aspect, the expanded NUE locus is genetically linked to the T nucleotide at position 36 of SEQ ID NO:62. In another aspect, the expanded NUE locus is genetically linked to the G nucleotide at position 36 of SEQ ID NO:63. In another aspect, the expanded NUE locus is genetically linked 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 a 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, Banquet A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, and NC925. In a further aspect, the methods provided herein include a second population of tobacco plants of a 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, and 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 an 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 an 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 an 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 an 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 an 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 an 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 certain further aspects, the methods provided herein include progeny seed that include a molecular marker within 0.5 cM of the increased NUE efficiency locus provided herein.

[0203] In one aspect, the description provides and includes 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 enhanced 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 enhanced NUE allele. In a further aspect, the method further comprises crossing the one or more selected tobacco plants to a second tobacco plant, and obtaining progeny seed from the cross.

[0204] In one aspect, the present specification provides and includes a method for introgressing an increased nitrogen utilization efficiency trait into a tobacco variety, comprising crossing a first tobacco variety comprising the increased nitrogen utilization efficiency trait with a second tobacco variety lacking the increased nitrogen utilization efficiency trait, obtaining progeny seeds from the cross, genotyping at least one progeny seed for a molecular marker associated with the increased nitrogen utilization 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 comprising the increased nitrogen utilization efficiency trait.

[0205] In one aspect, the description provides and includes a method for selecting tobacco plants having an enhanced NUE trait, 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 containing the enhanced NUE trait. In a further aspect, the method further comprises crossing one or more selected tobacco plants to a second tobacco plant; and obtaining progeny seed from the cross.

[0206] In one aspect, the description provides and includes a method for selecting tobacco plants with an enhanced NUE trait, 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 marker selected from the group consisting of SEQ ID NOs:57-64, and selecting tobacco plants containing the enhanced NUE trait. In a further aspect, the method disclosed herein comprises 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 comprises 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 comprises 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 methods disclosed herein comprise 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 methods disclosed herein comprise 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 methods disclosed herein comprise 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 methods disclosed herein comprise 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 comprise assaying the isolated nucleic acid 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 comprise assaying the isolated nucleic acid 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 comprise assaying the isolated nucleic acid 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 comprise assaying the isolated nucleic acid 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 comprise assaying the isolated nucleic acid 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 the isolated nucleic acid 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, the allele associated with increased NUE comprises a T nucleotide at position 36 of SEQ ID NO:61.In another aspect, an allele associated with increased NUE comprises a T nucleotide at position 36 of SEQ ID NO:62. In another aspect, an allele associated with increased NUE comprises a G nucleotide at position 36 of SEQ ID NO:63. In another aspect, an allele associated with increased NUE comprises a T nucleotide at position 36 of SEQ ID NO:64. In certain further aspects, tobacco plants can be selected that contain any combination of alleles associated with increased NUE disclosed herein.

[0207] The following are exemplary embodiments.

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

[0209] Embodiment 2. The method of embodiment 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, meristematic tissue, and stem tissue.

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

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

[0212] Aspect 5. 5. The method of any of embodiments 1-4, wherein 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, 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] Embodiment 6 The method of any of embodiments 1-5, wherein the NUE comprises increased NUE relative to a tobacco line comprising a lower amount of the at least one metabolite in at least one tissue.

[0214] Embodiment 7 The method of any of embodiments 1-6, wherein the NUE comprises increased NUE relative to a tobacco line comprising an equivalent amount of the at least one metabolite in at least one tissue.

[0215] Embodiment 8 The method of any of embodiments 1-7, wherein the NUE comprises increased NUE relative to a tobacco line comprising an equivalent amount of the at least one metabolite in at least one tissue.

[0216] Embodiment 9 The method of any of embodiments 1-8, wherein the NUE comprises reduced NUE relative to a tobacco line that comprises a lower amount of the at least one metabolite in at least one tissue.

[0217] Embodiment 10 The method of any of embodiments 1-9, wherein the NUE comprises reduced NUE relative to a tobacco line comprising an equivalent amount of the at least one metabolite in at least one tissue.

[0218] Embodiment 11 The method of any of embodiments 1-10, wherein the NUE comprises reduced NUE relative to a tobacco line comprising an equivalent amount of the at least one metabolite in at least one tissue.

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

[0220] Embodiment 13 The method of any of embodiments 1-12, wherein the at least one metabolite comprises at least two metabolites.

[0221] Embodiment 14 The method of any of embodiments 1 to 13, wherein the at least one metabolite comprises at least five metabolites.

[0222] Embodiment 15 The method of any of embodiments 1-14, wherein the at least one metabolite comprises at least 10 metabolites.

[0223] Embodiment 16. A method for determining nitrogen use efficiency (NUE) of a tobacco line using a metabolite signature, the method comprising the steps of: a. isolating a metabolite signature from tobacco plants of said tobacco line; b. determining the amount of each metabolite comprising said metabolite signature; c. Determining the NUE of the tobacco line by comparing the metabolite signature to a control metabolite signature from a control tobacco line with a known NUE.

[0224] Embodiment 17. The method of embodiment 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] Embodiment 18 The method of embodiment 16 or 17, wherein the NUE comprises increased NUE relative to a control tobacco line.

[0226] Embodiment 19 The method of any of embodiments 16-18, wherein the NUE comprises reduced NUE relative to a control tobacco line.

[0227] Embodiment 20. The method of any of embodiments 16-19, wherein the metabolite signature is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristematic tissue, and stem tissue.

[0228] Embodiment 21. A method of breeding a tobacco line comprising a metabolite signature associated with increased nitrogen use efficiency (NUE), the method comprising the steps of: a. determining a metabolic signature of a first tobacco plant derived from a first tobacco line, wherein the first tobacco plant comprises increased NUE relative to a control tobacco plant lacking the metabolic 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 a progeny plant grown from said at least one progeny seed comprises said metabolite signature, and said progeny plant comprises increased NUE compared to a control plant lacking said metabolite signature.

[0229] Embodiment 22 The method of embodiment 21, further comprising the steps of: d. Crossing the progeny plant to a tobacco plant derived from the first tobacco line.

[0230] Embodiment 23. The method of embodiment 21 or 22, wherein the first tobacco line 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.

[0231] Embodiment 24 The method of any of Embodiments 21-23, wherein the second tobacco line is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC.

[0232] Embodiment 25 The method of any of embodiments 21 to 24, wherein the metabolite signature comprises a leaf metabolite signature.

[0233] Embodiment 26 The method of any one of embodiments 21 to 25, wherein the metabolite signature comprises a root metabolite signature.

[0234] Embodiment 27 The method of any of embodiments 21-26, wherein the increased NUE comprises increased partial factor productivity (PFP) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0235] Embodiment 28 The method of any of embodiments 21-27, wherein the increased NUE comprises increased agronomic efficiency (AE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0236] Embodiment 29 The method of any of embodiments 21-28, wherein the increased NUE comprises an increased recovery efficiency (RE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0237] Embodiment 30 The method of any of embodiments 21-29, wherein the increased NUE comprises increased physiological efficiency (PE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0238] Embodiment 31 The method of any of embodiments 21-30, wherein the increased NUE comprises increased internal efficiency (IE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0239] Embodiment 32 The method of any of embodiments 21-31, wherein the metabolite signature comprises an equivalent amount of 4-guanidinobutanoate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23454, X-23580, X-23852, or any combination thereof, relative to the metabolite signature of a control tobacco plant.

[0240] Embodiment 33. The method of any of embodiments 21-32, wherein the metabolite signature comprises a reduced 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, relative to a metabolite signature of a control tobacco plant.

[0241] Embodiment 34. The method of any of embodiments 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] Embodiment 35. A method for selecting a tobacco plant, comprising the steps of: a. obtaining a population of tobacco plants; b. isolating at least one metabolite associated with increased nitrogen use efficiency (NUE) from at least one tobacco plant of said population of tobacco plants; c. Selecting at least one tobacco plant that contains an equivalent amount of said at least one metabolite compared to a control tobacco plant.

[0243] Embodiment 36 The method of embodiment 35, wherein the tobacco plants selected in step (c) comprise equal NUE compared to the control tobacco plants.

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

[0245] Embodiment 38 The method of any of embodiments 35-37, wherein the increased NUE comprises increased agronomic efficiency (AE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0246] Embodiment 39 The method of any of embodiments 35-38, wherein the increased NUE comprises an increased recovery efficiency (RE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0247] Embodiment 40 The method of any of embodiments 35-39, wherein the increased NUE comprises increased physiological efficiency (PE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0248] Embodiment 41 The method of any of embodiments 35-40, wherein the increased NUE comprises increased internal efficiency (IE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0249] Embodiment 42 The method of any of embodiments 35-41, wherein 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.

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

[0251] Embodiment 44 The method of any of embodiments 35 to 43, wherein the 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] Embodiment 45. A method for selecting a tobacco plant, comprising the steps of: a. obtaining a population of tobacco plants; b. isolating at least one metabolite associated with increased nitrogen use efficiency (NUE) from at least one tobacco plant of said population of tobacco plants; c. Selecting at least one tobacco plant that contains a lower amount of said at least one metabolite compared to a control tobacco plant.

[0253] Embodiment 46 The method of embodiment 45, wherein the tobacco plants selected in step (c) comprise equal NUE compared to the control tobacco plants.

[0254] Embodiment 47 The method of embodiment 45 or 46, wherein the increased NUE comprises increased partial factor productivity (PFP) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0255] Embodiment 48 The method of any of embodiments 45-47, wherein the increased NUE comprises increased agronomic efficiency (AE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0256] Embodiment 49 The method of any of embodiments 45-48, wherein the increased NUE comprises an increased recovery efficiency (RE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0257] Embodiment 50 The method of any of embodiments 45-49, wherein the increased NUE comprises increased physiological efficiency (PE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0258] Embodiment 51 The method of any of embodiments 45-50, wherein the increased NUE comprises increased internal efficiency (IE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0259] Embodiment 52. The method of any of embodiments 45-51, wherein 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.

[0260] Embodiment 53 The method of any of embodiments 45 to 52, wherein at least one metabolite is isolated from leaf tissue or root tissue.

[0261] Embodiment 54 The method of any of embodiments 45 to 53, wherein the 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.

[0262] Embodiment 55. A method of screening tobacco plants for a metabolite signature associated with increased nitrogen use efficiency (NUE), comprising the steps of: a. isolating a first metabolite signature associated with increased NUE from said tobacco plant; b. determining the amount of at least one metabolite comprising said first metabolite signature; c. comparing the first metabolite signature to a second metabolite signature of a control tobacco plant containing a known NUE; and d. Determining whether the first metabolite signature is associated with increased NUE.

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

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

[0265] Embodiment 58 The method of any of embodiments 55-57, wherein the increased NUE comprises an increased recovery efficiency (RE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0266] Embodiment 59 The method of any of embodiments 55-58, wherein the increased NUE comprises increased physiological efficiency (PE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0267] Embodiment 60 The method of any of embodiments 55-59, wherein the increased NUE comprises increased internal efficiency (IE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0268] Embodiment 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, wherein the modified tobacco seed or the tobacco plant grown therefrom comprises increased nitrogen utilization efficiency compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0269] Embodiment 62. The modified tobacco seed or tobacco plant of embodiment 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: 1-8.

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

[0271] Embodiment 64. The modified tobacco seed or tobacco plant of any of Embodiments 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] Embodiment 65. The modified tobacco seed or tobacco plant of any of Embodiments 61 to 64, wherein the heterologous promoter comprises a polynucleotide sequence derived from the tobacco genome.

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

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

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

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

[0277] Embodiment 70. The modified tobacco seed or tobacco plant of any of embodiments 61 to 69, wherein the root-preferred promoter is encoded by a sequence that is at least 70% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 20 to 24, or a functional fragment thereof.

[0278] Embodiment 71. The modified tobacco plant of any of embodiments 61-70, comprising an equivalent level of a metabolite selected from the group consisting of 4-guanidinobutanoate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde in root tissue compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0279] Embodiment 72. The modified tobacco plant of any of Embodiments 61-71, comprising an equivalent level of a metabolite selected from the group consisting of 4-guanidinobutanoate, X-23454, X-23580, and X-23852 in leaf tissue compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0280] Embodiment 73. The modified tobacco plant of any of Embodiments 61-72, comprising 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-oxoglutarate, X-23937, X-23916, and 1-methyladenine in root tissue compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0281] Embodiment 74. The modified tobacco plant of any of Embodiments 61-73, comprising 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 in leaf tissue compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0282] Embodiment 75. The modified tobacco seed or plant of any of embodiments 61 to 74, wherein the tobacco seed or plant is of a Burley variety.

[0283] Embodiment 76 The modified tobacco seed or plant of any of embodiments 61-75, wherein the modified tobacco seed or plant comprises a reduced amount of TSNAs compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

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

[0285] Embodiment 78. A cured tobacco material or a tobacco product comprising the cured 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 utilization efficiency compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0286] Embodiment 79. A greenhouse, growth chamber, or field comprising the modified tobacco seeds or plants of any of embodiments 61-76.

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

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

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

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

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

[0292] Embodiment 85. The modified tobacco plant of any of Embodiments 80-84, comprising 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-oxoglutarate, X-23937, X-23916, and 1-methyladenine in root tissue compared to an unmodified tobacco plant lacking the at least one mutation when grown under the same conditions.

[0293] Embodiment 86. The modified tobacco plant of any of embodiments 80-85, comprising 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 in leaf tissue relative to an unmodified tobacco plant lacking the at least one mutation when grown under the same conditions.

[0294] Embodiment 87. The modified tobacco seed or plant of any of embodiments 80 to 86, wherein the tobacco seed or plant is of a Burley variety.

[0295] Embodiment 88 The modified tobacco seed or plant of any of embodiments 80-87, wherein the modified tobacco seed or plant comprises a reduced amount of TSNAs compared to an unmodified tobacco plant lacking the at least one mutation when grown under the same conditions.

[0296] Embodiment 89. A recombinant DNA construct comprising a heterologous promoter operably 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 contiguous nucleotides identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40.

[0297] Embodiment 90. A cured tobacco material or a tobacco product comprising the cured tobacco material produced from a tobacco plant comprising at least one mutation at an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or tobacco plant comprises an increased nitrogen utilization efficiency compared to an unmodified control tobacco plant lacking said at least one mutation when grown under the same conditions.

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

[0299] Embodiment 92. The modified tobacco seed or tobacco plant of embodiment 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] Embodiment 93. The modified tobacco seed or tobacco plant of embodiment 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] Embodiment 94. The modified tobacco seed or tobacco plant of any of embodiments 91 to 93, wherein the sRNA downregulates the expression or translation of a polynucleotide selected from the group consisting of SEQ ID NOs: 41 to 56.

[0302] Embodiment 95. The modified tobacco seed or tobacco plant of any of Embodiments 91 to 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] Embodiment 96 The modified tobacco seed or tobacco plant of any of Embodiments 91 to 95, wherein the tissue-preferred promoter is a leaf-preferred promoter.

[0304] Embodiment 97 The modified tobacco seed or tobacco plant of any of Embodiments 91 to 96, wherein the tissue-preferred promoter is a root-preferred promoter.

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

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

[0307] Embodiment 100. The modified tobacco seed or tobacco plant of any of Embodiments 91 to 99, wherein the heterologous promoter comprises a polynucleotide sequence derived from the tobacco genome.

[0308] Embodiment 101. The modified tobacco seed or tobacco plant of any of Embodiments 91 to 100, wherein the heterologous promoter comprises a polynucleotide sequence derived from the plant genome.

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

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

[0311] Embodiment 104. The modified tobacco plant of any of Embodiments 91-103, comprising 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-oxoglutarate, X-23937, X-23916, and 1-methyladenine in root tissue compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0312] Embodiment 105. The modified tobacco plant of any of Embodiments 91-104, comprising 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 in leaf tissue relative to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0313] Embodiment 106. The modified tobacco seed or plant of any of embodiments 91 to 105, wherein the tobacco seed or plant is of a Burley variety.

[0314] Embodiment 107. The modified tobacco seed or plant of any of Embodiments 91-106, wherein the modified tobacco seed or plant comprises a reduced amount of TSNAs compared to an unmodified tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0315] Embodiment 108. A recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0316] Embodiment 109. A cured tobacco material or a tobacco product comprising the cured tobacco material, produced from a 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 to or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, wherein the modified tobacco seed or tobacco plant comprises an increased nitrogen utilization efficiency compared to an unmodified control tobacco plant lacking the cisgenic polynucleotide when grown under the same conditions.

[0317] Embodiment 110. A method for increasing nitrogen use efficiency (NUE) in a tobacco plant, comprising the steps of: a. introducing a cisgenic nucleic acid molecule into a tobacco cell; and b. Regenerating a modified tobacco plant from said tobacco cell, wherein said modified tobacco plant comprises increased NUE relative to a tobacco plant lacking said cisgenic nucleic acid molecule.

[0318] Embodiment 111 The method of embodiment 110, further comprising the steps of: c. Crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0319] Embodiment 112 The method of embodiment 110 or 111, wherein the increased NUE comprises increased partial factor productivity (PFP) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0320] Embodiment 113 The method of any of embodiments 110-112, wherein the increased NUE comprises increased agronomic efficiency (AE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0321] Embodiment 114 The method of any of embodiments 110-113, wherein the increased NUE comprises an increased recovery efficiency (RE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0322] Embodiment 115 The method of any of embodiments 110-114, wherein the increased NUE comprises increased physiological efficiency (PE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0323] Embodiment 116 The method of any of embodiments 110-115, wherein the increased NUE comprises increased internal efficiency (IE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

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

[0325] Embodiment 118. The method of embodiment 117, wherein the introducing comprises use of an RNA-guided nuclease.

[0326] Embodiment 119. The method of embodiment 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] Embodiment 120. The method of any of embodiments 117 to 119, wherein the modification is selected from the group consisting of an insertion, a substitution, an inversion, and a deletion.

[0328] Embodiment 121 The method of any of embodiments 117-120, wherein the increased NUE comprises increased partial factor productivity (PFP) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0329] Embodiment 122 The method of any of embodiments 117-121, wherein the increased NUE comprises increased agronomic efficiency (AE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0330] Embodiment 123 The method of any of embodiments 117-122, wherein the increased NUE comprises an increased recovery efficiency (RE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0331] Embodiment 124 The method of any of embodiments 117-123, wherein the increased NUE comprises increased physiological efficiency (PE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0332] Embodiment 125 The method of any of embodiments 117-124, wherein the increased NUE comprises increased internal efficiency (IE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0333] Embodiment 126 The method of any one of embodiments 117 to 125, further comprising the steps of: c. Crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0334] Embodiment 127. A method for increasing nitrogen use efficiency (NUE) in a tobacco plant, comprising the steps of: a. introducing into a tobacco cell a nucleic acid encoding a small RNA (sRNA) that is homologous to at least 18 contiguous 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 said tobacco cell, wherein said modified tobacco plant comprises increased NUE compared to a tobacco plant lacking said sRNA.

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

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

[0337] Embodiment 130 The method of any of embodiments 127-129, wherein the increased NUE comprises increased agronomic efficiency (AE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0338] Embodiment 131 The method of any of embodiments 127-130, wherein the increased NUE comprises an increased recovery efficiency (RE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0339] Embodiment 132 The method of any of embodiments 127-131, wherein the increased NUE comprises increased physiological efficiency (PE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0340] Embodiment 133 The method of any of embodiments 127-132, wherein the increased NUE comprises increased internal efficiency (IE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

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

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

[0343] Embodiment 136 The method of embodiment 135, further comprising the steps of: d. crossing one or more tobacco plants selected in step (c) to a second tobacco plant; and e. Obtaining progeny seeds from the hybrid of step (d).

[0344] Embodiment 137 The method of embodiment 135 or 136, wherein the molecular marker is selected from the group consisting of a SNP marker, an INDEL marker, an RFLP marker, an SSR marker, an AFLP marker, and a RAPD marker.

[0345] Embodiment 138 The method of any of embodiments 135-137, wherein the increased nitrogen utilization efficiency locus comprises a polynucleotide encoding a polypeptide at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8.

[0346] Embodiment 139 The method of any of embodiments 135-138, wherein the increased nitrogen utilization efficiency locus comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs:9-16.

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

[0348] Embodiment 141 The method of any of embodiments 135 to 140, wherein the molecular marker comprises a G nucleotide at position 57 of SEQ ID NO:58.

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

[0350] Embodiment 143. The method of any of embodiments 135 to 142, wherein the molecular marker comprises a G nucleotide at position 57 and a C nucleotide at position 117 of SEQ ID NO:58.

[0351] Embodiment 144. The method of any of embodiments 135 to 143, wherein the molecular marker comprises a T nucleotide at position 14 of SEQ ID NO:57.

[0352] Embodiment 145. The method of any of embodiments 135 to 144, wherein the molecular marker comprises a G nucleotide at position 162 of SEQ ID NO:59.

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

[0354] Embodiment 147 The method of any of embodiments 135 to 146, wherein the molecular marker comprises a T nucleotide at position 36 of SEQ ID NO:61.

[0355] Embodiment 148 The method of any of embodiments 135 to 147, wherein the molecular marker comprises a T nucleotide at position 36 of SEQ ID NO:62.

[0356] Embodiment 149. The method of any of embodiments 135 to 148, wherein the molecular marker comprises a G nucleotide at position 36 of SEQ ID NO:63.

[0357] Embodiment 150. The method of any of embodiments 135 to 149, wherein the molecular marker comprises a T nucleotide at position 36 of SEQ ID NO:64.

[0358] Embodiment 151 The method of any of embodiments 135-150, wherein the first population of tobacco plants is of the Maryland variety.

[0359] Embodiment 152. The method of any of embodiments 135-151, 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, and NC925.

[0360] Embodiment 153 The method of any of embodiments 135-152, wherein the second tobacco plant is of a variety selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC.

[0361] Embodiment 154 The method of any one of embodiments 135 to 153, wherein the progeny seed comprises the molecular marker.

[0362] Embodiment 155 The method of any of embodiments 135-154, wherein the progeny seed comprises increased nitrogen utilization efficiency.

[0363] Embodiment 156 The method of any of embodiments 135 to 155, wherein the molecular marker is within 15 cM of an increased nitrogen use efficiency locus.

[0364] Embodiment 157 The method of any of embodiments 135 to 156, wherein the molecular marker is within 10 cM of an increased nitrogen use efficiency locus.

[0365] Embodiment 158 ​​The method of any of embodiments 135 to 157, wherein the molecular marker is within 5 cM of an increased nitrogen use efficiency locus.

[0366] Embodiment 159 The method of any of embodiments 135 to 158, wherein the molecular marker is within 2 cM of an increased nitrogen use efficiency locus.

[0367] Embodiment 160 The method of any of embodiments 135 to 159, wherein the molecular marker is within 1 cM of an increased nitrogen use efficiency locus.

[0368] Embodiment 161 The method of any of embodiments 135 to 160, wherein the molecular marker is within 0.5 cM of an increased nitrogen use efficiency locus.

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

[0370] Embodiment 163. The method of embodiment 161 or 162, further comprising the steps of: d. crossing one or more tobacco plants selected in step (c) to a second tobacco plant; and e. Obtaining progeny seeds from the hybrid of step (d).

[0371] Embodiment 164 The method of any of Embodiments 161-163, wherein the first population of tobacco plants is of the Maryland variety.

[0372] Embodiment 165. The method of any of embodiments 161-164, 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, and NC925.

[0373] Embodiment 166 The method of any of Embodiments 161-165, wherein the second tobacco plant is of a variety selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC.

[0374] Embodiment 167 The method of any one of embodiments 161 to 166, wherein the progeny seed comprises the molecular marker.

[0375] Embodiment 168 The method of any of embodiments 161-167, wherein the progeny seed comprises increased nitrogen utilization efficiency.

[0376] Embodiment 169. A method for introgressing an increased nitrogen use efficiency trait into a tobacco variety, comprising the steps of: a. crossing a first tobacco variety containing said increased nitrogen utilization efficiency trait with a second tobacco variety lacking said increased nitrogen utilization efficiency trait; b. obtaining progeny seeds from the hybrid of step (a); c. genotyping at least one of the progeny seeds obtained in step (b) for a molecular marker associated with 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 NOs: 9-16; and d. Selecting progeny seeds containing said increased nitrogen use efficiency trait.

[0377] Embodiment 170. The method of embodiment 169, wherein the first tobacco variety is a Maryland tobacco variety.

[0378] Embodiment 171. The method of embodiment 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, and NC925.

[0379] Embodiment 172 The method of any one of embodiments 169-171, wherein the second tobacco variety is a Burley tobacco variety.

[0380] Embodiment 173. The method of any of Embodiments 169-172, wherein the second tobacco variety is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC.

[0381] Embodiment 174 The method of any of embodiments 169-173, wherein the increased NUE comprises increased partial factor productivity (PFP) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0382] Embodiment 175 The method of any of embodiments 169-174, wherein the increased NUE comprises increased agronomic efficiency (AE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0383] Embodiment 176 The method of any of embodiments 169-175, wherein the increased NUE comprises an increased recovery efficiency (RE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0384] Embodiment 177 The method of any of embodiments 169-176, wherein the increased NUE comprises increased physiological efficiency (PE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

[0385] Embodiment 178 The method of any of embodiments 169-177, wherein the increased NUE comprises increased internal efficiency (IE) relative to a tobacco plant lacking the increased NUE grown under the same conditions.

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

[0387] Embodiment 180 The method of any of embodiments 169 to 179, wherein the molecular marker is within 10 cM of the locus.

[0388] Embodiment 181 The method of any of embodiments 169 to 180, wherein the molecular marker is within 5 cM of the locus.

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

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

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

[0392] Embodiment 185. A method for selecting a tobacco plant comprising an increased nitrogen use efficiency trait, the method comprising the steps of: a. isolating nucleic acids 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: 9-16; and c. Selecting tobacco plants containing increased nitrogen use efficiency traits.

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

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

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

[0396] Embodiment 189 The method of any of embodiments 185 to 188, wherein the molecular marker is within 5 cM of the locus.

[0397] Embodiment 190. The method of any of embodiments 185 to 189, wherein the molecular marker is within 2 cM of the locus.

[0398] Embodiment 191 The method of any of embodiments 185 to 190, wherein the molecular marker is within 1 cM of the locus.

[0399] Embodiment 192 The method of any of embodiments 185 to 191, wherein the molecular marker is within 0.5 cM of the locus.

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

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

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

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

[0404] Embodiment 197. The method of embodiments 193 to 196, wherein the assaying step comprises assaying for a T nucleotide at position 14 of SEQ ID NO:57.

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

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

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

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

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

[0410] Embodiment 203. The method of any of embodiments 193 to 202, wherein the assaying step comprises assaying for a T nucleotide at position 36 of SEQ ID NO:64. [Example]

[0411] Example 1. Field production implementation method Field-grown tobacco plants are produced using standard field production practices. Each test plot contains up to 40 rows of planted seedlings. Seedlings are germinated in a greenhouse prior to planting. To test for the NUE trait, test plots are treated with a nitrogen rate of 60 pounds of nitrogen per acre. When 50% of the plants in the test plot reach the elongated bud stage, plants are topped using standard procedures. Pesticide applications follow standard protocols. Leaves are harvested at maturity and sorted into three sticks per plot, with five plants per drying stick. Leaves are collected from the sticks at the takedown / uprooting stage. Five leaves are harvested from three different sticks per experimental variety for a total of 15 leaves per sample. Half the blade of the fourth leaf from the top of each plant is harvested for sampling. Analytical analysis of alkaloids, TSNAs, and NO3 is performed using routine methods known in the art.

[0412] Example 2. Identification of metabolites associated with increased nitrogen utilization efficiency Maryland tobacco varieties require approximately 25% less nitrogen fertilizer input than burley tobacco varieties. To identify metabolites associated with high-nitrogen efficiency (Maryland) and low-nitrogen efficiency (Burley) tobacco varieties, differences in metabolite levels were examined in Maryland tobacco variety MD609 and burley tobacco variety TN90.

[0413] MD609 and TN90 seedlings were germinated from seeds and grown for six weeks without added nitrogen. After six weeks, seedlings from each variety were divided into two groups: Group A contained plants provided with 100 parts per million nitrogen, i.e., normal greenhouse fertilization; Group B contained plants provided with 25 ppm, i.e., 25% normal greenhouse fertilization. At 10 and 14 weeks after sowing, metabolites were extracted from root and leaf tissue 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 individual metabolites. 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 by setting the median equal to 1 (1.00) and proportionally normalizing each data point (referred to as "partial correction"). The molecular masses of unknown metabolites are provided in Table 1. Identified metabolites are listed below in Tables 2-5, along with the scaled values ​​for each sample. Identified metabolites were determined by Student's t-test comparison between TN90 and MD609, taking all time points into account. Metabolites with a p-value of less than 0.01 were included in the analysis.

[0415] (Table 1) Molecular masses (in kilodaltons) of unknown metabolite compounds TIFF0007671803000001.tif71128

[0416] Table 2. Metabolites negatively correlated with increased nitrogen efficiency identified in root tissues of MD609 and TN90 tobacco lines at 10 and 14 weeks after sowing. TIFF0007671803000002.tif74135

[0417] Table 3. Metabolites positively correlated with increased nitrogen efficiency identified in root tissues when comparing MD609 and TN90 tobacco lines. TIFF0007671803000003.tif38136

[0418] Table 4. Metabolites negatively correlated with increased nitrogen efficiency identified in leaf tissues when comparing MD609 and TN90 tobacco lines. TIFF0007671803000004.tif70133

[0419] Table 5. Metabolites positively correlated with increased nitrogen efficiency identified in leaf tissue when comparing MD609 and TN90 tobacco lines. TIFF0007671803000005.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. At 10 and 14 weeks after sowing, RNA is extracted from leaf and root tissues, and used for Illumina sequencing. RNAseq data is analyzed according to standard methods in the art. Candidate genes are then verified.

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

[0422] Identifying the genomic location of each of the correlated genes identified four clusters of genes associated with increased NUE in the tobacco genome (Figure 1). Seven genes co-locate on chromosome 1, four genes co-locate on chromosome 11, three genes co-locate on chromosome 14, and five genes co-locate on chromosome 20 (Figure 1). These four locations are also hotspots for differentially expressed genes between low- and normal-nitrogen conditions (Figure 1). SNP markers were generated to identify the MD609 polymorphisms, and thus the increased NUE polymorphisms, specific to each of these locations (Tables 6–10). Further characterization of the region on chromosome 11 revealed that it contained a total of 79 expressed genes, 46 of which were differentially expressed under low-nitrogen conditions (Figure 2).

[0423] Table 6. Genes identified as negatively correlated with increased nitrogen use efficiency in root tissue. TIFF0007671803000006.tif85144

[0424] Table 7. Genes identified as negatively correlated with increased nitrogen use efficiency in leaf tissue. TIFF0007671803000007.tif57144

[0425] Table 8. Genes identified as positively correlated with increased nitrogen use efficiency in root tissue. TIFF0007671803000008.tif46144

[0426] Table 9. Genes identified as positively correlated with increased nitrogen use efficiency in leaf tissue. TIFF0007671803000009.tif46144

[0427] Table 10. SNP markers containing polymorphisms associated with increased NUE TIFF0007671803000010.tif57144

[0428] Example 4. Identification of leaf- and root-preferred promoters in tobacco RNA samples from 4-week-old TN90 tobacco plants were obtained from 10 tissue types (axillary buds before pruning, axillary buds 2 hours after pruning, axillary buds 6 hours after pruning, axillary buds 24 hours after pruning, axillary buds 72 hours after pruning, roots before pruning, roots 24 hours after pruning, roots 72 hours after pruning, young leaves at the time of pruning, and shoot apical meristem). The resulting RNA samples (three independently collected samples per tissue type) were used as starting material for Illumina 1x100bp sequencing.

[0429] The Illumina reads were mapped and used to identify a list of candidate genes that exhibited high root or leaf expression. Tables 11 and 12 provide the RPKM expression values ​​of genes identified as having leaf- or root-preferential expression. These genes are candidates for possessing leaf- or root-preferential promoters, respectively.

[0430] Table 11. Genes with leaf-preferred expression TIFF0007671803000011.tif22536

[0431] Table 12. Genes with root-preferred expression TIFF0007671803000012.tif22741

[0432] Example 5. Development of modified plants The expression vector p45-2-7 (SEQ ID NO:65) is used as a backbone to generate multiple transformation vectors (see Examples X-Y). p45-2-7 contains a cassette comprising the CsVMV promoter, the NOS terminator, and a kanamycin selectable marker (NPT II) operably linked to the actin 2 promoter and the NOS terminator. The nucleic acid vector containing the transgene of interest is introduced into tobacco leaf discs 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] Leaf discs were cut from TN90 tobacco plants grown in Magenta™ GA-7 boxes and placed in Petri dishes. Agrobacterium tumefaciens cells containing the transformation vector were collected by centrifuging 20 mL of cell suspension in a 50 mL centrifuge tube at 3500 RPM for 10 minutes. The supernatant was removed and the Agrobacterium tumefaciens cell pellet was resuspended in 40 mL of liquid resuspension medium. Cut tobacco leaves, avoiding the midrib, into eight 0.6 cm discs with a #15 razor blade and place them upside down in a Petri dish. A thin layer of Murashige & Skoog B5 Vitamin Liquid Resuspension Medium was added to the Petri dish, and the leaf discs were poked evenly with a fine-tipped needle. Approximately 25 mL of Agrobacterium tumefaciens suspension was added to the Petri dish, and the leaf discs were incubated in the suspension for 10 minutes.

[0434] Transfer the leaf discs to a co-cultivation Petri dish (1 / 2 MS medium) and place the discs upside down on a filter paper placed on co-cultivation 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 the discs at 24°C under dim light (60-80 mE / ms) with a 18-hour light:6-hour dark photoperiod for 3 days. After incubation, transfer the discs to a regeneration / selection TOM K medium Petri dish (TOM medium + 300 mg / L kanamycin). Subculture the discs to fresh TOM K medium every 2 weeks under dim light at 24°C under a 18-hour light:6-hour dark photoperiod until the shoots are ready for cutting. 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 under high-intensity lighting (6080 mE / ms) with a photoperiod of 18 hours on and 6 hours off to induce rooting.

[0435] Once the plantlets containing both shoots and roots have grown large enough (e.g., until they reach approximately half the height of the Magenta™ GA-7 box), they are transferred to soil. Rooted seedlings are transferred to a greenhouse for further analysis and seed setting. The enhanced nitrogen use efficiency phenotype is evaluated by growing modified plants (T0, T1, T2, or later generations) and control plants. Control plants are either untransformed NLM plants or NLM plants transformed with the empty p45-2-7 vector.

[0436] Phenotypic screening for increased nitrogen use efficiency is conducted in greenhouses using zero parts per million (ppm) nitrogen (no nitrogen), 25 ppm nitrogen (low nitrogen), and 100 ppm nitrogen (normal nitrogen). Initial screening begins with T1 plants in the greenhouse. Homozygous T2 populations are then evaluated in the field using 60 pounds of fertilizer per acre (approximately 25% of the recommended rate for burley tobacco). Seedling growth, chlorophyll loss, and final yield are measured and compared to control plants grown at normal nitrogen levels.

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

[0438] Example 6. Production of cisgenic tobacco plants with increased nitrogen use efficiency Nitrogen utilization efficiency can be improved by modifying the expression of genes involved in genes identified as differentially expressed in Example 2. Similarly, genes involved in the biosynthesis or degradation of metabolites identified in Example 1 can be regulated to improve nitrogen utilization efficiency. Genes positively associated with increased nitrogen utilization efficiency can be overexpressed using general overexpression promoters or tissue-preferred promoters to overexpress the genes in desired tissues.

[0439] Transformation vectors are engineered to overexpress proteins positively associated with increased nitrogen utilization efficiency. A separate transformation vector containing one of SEQ ID NOs: 9-16 is integrated into the p45-2-7 transformation vector. Additional transformation vectors are engineered to contain one of SEQ ID NOs: 9-16.

[0440] These transformation vectors are used to generate modified tobacco plants according to Example 4. The modified tobacco plants (T1 generation) and control tobacco plants are then evaluated for phenotype as described in Example 4. The modified tobacco plants exhibit increased nitrogen use efficiency compared to control tobacco plants grown under the same conditions.

[0441] Example 7. Production of transgenic tobacco plants with increased nitrogen use efficiency Nitrogen utilization efficiency can also be increased by downregulating the expression of genes identified in Example 2 as being negatively associated with nitrogen utilization efficiency.

[0442] Transformation vectors containing RNAi constructs are designed to inhibit tobacco genes whose expression is negatively associated with nitrogen utilization efficiency in Example 2. Separate transformation vectors containing one of SEQ ID NOs: 41-56 are integrated into the p45-2-7 transformation vector. Additional transformation vectors containing one of SEQ ID NOs: 41-56 are created.

[0443] These transformation vectors are used to generate modified tobacco plants according to Example 4. The modified tobacco plants (T1 generation) and control tobacco plants are then evaluated for phenotype as described in Example 4. The modified tobacco plants exhibit increased nitrogen use efficiency compared to control tobacco plants grown under the same conditions.

[0444] Example 8. Additional methods for improving nitrogen use efficiency using gene editing techniques Gene editing techniques such as CRISPR / Cas9, CRISPR / Cpf1, CRISPR / CasX, CRISPR / CasY, CRISPR / Csm1, zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) are used to modify the coding region of the gene that is negatively related to increased nitrogen utilization efficiency, so that the gene encodes non-functional protein or a protein that functions less.These gene editing techniques can also be used to edit or replace endogenous promoter sequences so that their homologous protein expression is driven in either leaf tissue or root tissue, thereby improving nitrogen utilization efficiency.For example, endogenous G64360 is edited or replaced so that the gene is only expressed in leaf tissue, where it can work to improve plant nitrogen utilization efficiency.

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

[0446] Example 9. Development of novel mutations to improve nitrogen utilization efficiency via random mutagenesis Random mutagenesis of tobacco plants is performed 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, which causes large deletions through double-stranded DNA breaks.

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

[0448] The EMS-treated seeds in agar solution are spread evenly on water-soaked Carolina's Choice Tobacco Mix (Carolina Soil Company, Kinston, NC) in flats, approximately 2,000 seeds per flat. The flats are then covered with plastic wrap and placed in a growth chamber. As seedlings emerge from the soil, holes are made in the plastic wrap to allow for a gradual reduction in humidity. After two weeks, the plastic wrap is completely removed. The flats are moved to a greenhouse and NPK fertilizer is applied. The seedlings are planted in floating trays and grown to transplanting size. The plants are then transplanted to the field. During growth, the plants are self-pollinated to form M1 seeds. At maturity, five capsules are harvested from each plant, and the set of seeds from each plant is given an individual designation. This forms the M1 population. A mixture of M1 seeds from each M0 plant is grown, and the plants are evaluated phenotypically as described in Example 4 for increased nitrogen efficiency. M1 plants that exhibit 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 the 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 with 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 progeny plants can be determined using methods known in the art or described above. Multiple different preferred NUE alleles can be combined into a single line. Molecular phenotypes, such as those determined by metabolic signatures, can be used to track increased NUE during breeding. The metabolic signatures of progeny plants can be determined using the methods described above. Progeny plants with the metabolic signature of a parent plant with increased NUE can be crossed to produce subsequent populations of tobacco plants with increased NUE.

[0450] Introduction of the Maryland 609 locus into commercial burley varieties can be performed as described to develop burley lines with increased NUE. Screening of 23 burley lines and six MD609 lines identified three burley lines containing the MD609 allele at SNP marker S451 (SEQ ID NO:58) (Figure 3). The three burley lines with the MD609 allele were tested for chlorophyll loss, growth, and yield under nitrogen-limited conditions and compared to a control TN90 burley line and a control MD609 line (MD609 with the MD609 allele at SNP marker S451) (Figure 4). The burley lines with the MD609 allele exhibit chlorophyll loss, growth, and yield more similar to the Maryland control (Figure 4). The TN90 burley control exhibits increased chlorophyll loss, decreased growth, and decreased yield compared to the MD609 control (Figure 4). These results indicate that 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 seed. F2 and F3 plants were grown and self-pollinated to produce F4 seed. Bulk F4 seed from two independent crossing schemes, identified as lines NUE-2 and NUE-3, was grown and harvested in the field. F4 seed from both lines NUE-2 and NUE-3 was genotyped for SNP markers S451, S317, S12385, S238, S3894, and S2237 (see Table 13). F4 plants were grown using the reduced nitrogen production practices described in Example 1. Both lines NUE-2 and NUE-3 showed increased yield (in pounds) per acre compared to the burley control TN90 (see Figure 6).

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

[0453] Table 13: Genotypes of field-grown plants derived from F4 lines NUE-2 and NUE-3 and TN90. MD represents the MD609 allele, Burley represents the burley allele, and HET represents heterozygous MD609 / burley. TIFF0007671803000013.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 Ph 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 Val Val Trp Asn Pro Trp Glu Lys Lys 245 250 255 Ser Lys Thr Ile Ala Asp Phe Gly Asp Asp Glu Tyr Arg His Met Leu 260 265 270 Cys Val Asp Gly Ala Ala Ile Glu Lys Pro Ile Thr Leu Lys Pro Gly 275 280 285 Glu Glu Trp Thr Gly Arg Leu Glu Leu Ser Val Met Pro Ser Ser 290 295 300 <210> 6 <211> 578 <212> PRT <213> Nicotiana tabacum <400> 6 Met Glu Val Met Lys Lys Lys Glu Asn Thr Ser Arg Lys Met Lys Gly 1 5 10 15 Gly Met Ile Thr Met Pro Phe Ile Phe Ala Asn Glu Ile Cys Glu Lys 20 25 30 Leu Ala Val Val Gly Phe Gly Ala Asn Met Ile Ile Tyr Leu Thr Asn 35 40 45 Glu Leu His Leu Pro Leu Thr Lys Ala Ala Asn Thr Leu Thr Asn Phe 50 55 60 Gly Gly Thr Ala Ser Leu Thr Pro Leu Leu Gly Ala Phe Ile Ala Asp 65 70 75 80 Thr Phe Ala Gly Arg Phe Trp Thr Ile Thr Ile Ala Ser Ile Ile Tyr 85 90 95 Gln Ile Gly Met Ile Ile Leu Thr Val Ser Ala Ile Leu Pro Gln Leu 100 105 110 Arg Pro Pro Ser Cys Lys Gly Asp Glu Phe Cys Lys Glu Ala Asn Ser 115 120 125 Gly Gln Leu Ala Ile Leu Tyr Ile Ser Leu Leu Leu Thr Ala Phe Gly 130 135 140 Ser Gly Gly Ile Arg Pro Cys Val Val Ala Phe Gly Ala Glu Gln Phe 145 150 155 160 Asp Glu Thr Asp Pro Asn Gln Lys Thr Gln Thr Trp Lys Phe Phe Asn 165 170 175 Trp Tyr Tyr Phe Ser Met Gly Phe Ser Met Leu Ile Ala Val Thr Val 180 185 190 Ile Val Tyr Ile Gln Asp Asn Ile Gly Trp Gly Ile Gly Phe Gly Val 195 200 205 Pro Thr Ile Ala Met Leu Ile Ser Ile Ile Val Phe Ile Phe Gly Tyr 210 215 220 Pro Leu Tyr Arg Asn Leu Asp Pro Ala Gly Ser Pro Phe Thr Arg Leu 225 230 235 240 Leu Gln Val Cys Val Ala Ala Tyr Lys Lys Arg Lys Leu Asp Met Val 245 250 255 Ser Asp Pro Ser Phe Leu Tyr Gln Asn Glu Glu Leu Asp Ser Ala Ile 260 265 270 Ser Thr Ala Gly Lys Leu Val His Thr Lys Gln Met Lys Phe Leu Asp 275 280 285 Arg Ala Ala Ile Val Thr Glu Glu Asp Asn Arg Lys Ser Pro Asn Leu...

Claims

1. 1. A method for producing a tobacco plant, comprising the steps of: (a) providing a first population of tobacco plants comprising an increased nitrogen utilization efficiency (NUE) trait; (b) genotyping the first population of tobacco plants for the presence of a SNP marker comprising the sequence of SEQ ID NO:57 and having a T at polymorphic position 147 of an allele associated with an enhanced NUE trait; (c) selecting tobacco plants containing the SNP markers; (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), wherein a plant grown from the progeny seeds comprises the enhanced NUE trait and the SNP markers.

2. 2. The method of 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 a tobacco plant lacking the increased NUE trait grown under the same conditions.

3. 2. The method of 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.

4. 2. The method of claim 1, wherein the second tobacco plant is a Burley tobacco variety.

5. 2. The method of claim 1, wherein the second tobacco plant is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC.

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