Methods and compositions for controlling alkaloids in tobacco

Abstract

The present disclosure relates to tobacco plants, plant parts, seeds, compositions, and methods related to modulating the expression of novel genes in tobacco to control alkaloid levels.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 395,459, filed August 5, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Field The present disclosure relates to tobacco plants, plant parts, seeds, compositions, and methods related to modulating the expression of novel genes in tobacco to control alkaloid levels.

[0003] Sequence Listing Reference A Sequence Listing conforming to the rules of WIPO Standard ST.26 is incorporated herein by reference in its entirety. The Sequence Listing has been submitted via the Patent Center as an electronic document encoded as XML in UTF-8 text. The electronic document is named "P34753WO00_SL.xml", is 341,150 bytes in size (measured in MS-Windows), and was created on July 26, 2023.

[0004] Table 1 provides a list of nucleic acid and amino acid sequences.

[0005] Table 1: Nucleic acid and amino acid sequences TIFF2025525933000001.tif74128TIFF2025525933000002.tif22193TIFF2025525933 000003.tif22193TIFF2025525933000004.tif22193TIFF2025525933000005.tif25591 [Background technology]

[0006] background Four major alkaloids are found in tobacco: nicotine, nornicotine, anabasine, and anatabine. Nicotine is the predominant alkaloid, typically accounting for more than 90% of the total alkaloids in commercial tobacco cultivars. Nicotine biosynthesis occurs primarily in tobacco roots. The tobacco plant then transports nicotine through vascular bundles to leaves, where it is then stored in vacuoles. The cumulative levels of nicotine and related alkaloids in tobacco leaves depend on biosynthesis and transport, both of which can be affected by environmental conditions, plant hormones, growth conditions, and genetic regulatory factors.

[0007] Provided herein are methods and compositions for regulating novel tobacco genes to regulate alkaloid levels in tobacco. Summary of the Invention

[0008] overview In one aspect, the present disclosure provides a modified tobacco plant or portion thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0009] In one aspect, the present disclosure provides a modified tobacco plant, or portion thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0010] In one aspect, the present disclosure provides a modified tobacco plant or portion thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0011] In one aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) inducing a non-naturally occurring mutation in at least one tobacco cell in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell comprising the non-naturally occurring mutation from step (a); and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

[0012] In one aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

[0013] In one aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b).

[0014] In one aspect, the disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence, and the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0015] In one aspect, the disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0016] In one aspect, the disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0017] In one aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0018] In one aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0019] In one aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, wherein the non-naturally occurring mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one tobacco seed or plant germinated therefrom comprises the non-naturally occurring mutation.

[0020] In one aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, wherein the recombinant DNA construct is not present in an endogenous nucleic acid sequence in a control tobacco plant of the same variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein the at least one tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct.

[0021] In one aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, wherein the recombinant DNA construct is absent from the nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one tobacco seed or plant germinated therefrom comprises the recombinant DNA construct.

[0022] In one aspect, the disclosure provides a modified tobacco plant or portion thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence that regulates the expression or functional activity of a gene, wherein the gene encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 depicts the clustering of genes based on the dissimilarity of topological overlap measures. The bars at the bottom of the figure indicate the different network modules. [Figure 2]The figure depicts subnetworks extracted from wgcna and visualized in Cytoscape. Genes (nodes) labeled at the center of each subnetwork are known nicotine biosynthesis genes. Abbreviations: AO3 (L-ascorbic acid oxidase 3); AO-2a (L-ascorbic acid oxidase 2a); BBL-3 (berberine cross-linking enzyme-like 3); ADC-1b (arginine decarboxylase 1b); BBL-4 (berberine cross-linking enzyme-like 4); QS1b (quinolinic acid synthase 1b); QS1a (quinolinic acid synthase 1a); ADC-1a (arginine decarboxylase 1a); PMT1b (putrescine methyltransferase 1b); BBL-1( Berberine cross-linking enzyme-like 1; PMT3 (putrescine methyltransferase 3); AO7 (L-ascorbic acid oxidase 7); ODC-1a (ornithine decarboxylase 1a); AO11 (L-ascorbic acid oxidase 11); ODC-1b (ornithine decarboxylase 1b); PMT1a (putrescine methyltransferase 1a); AIC-1b (agmatine deiminase 1b); A622-1 (isoflavone reductase homolog A622 1); PMT4 (putrescine methyltransferase 4); MPO1a (N-methylputrescine oxidase 1a); AO12 (L-ascorbate oxidase 12); ADC-1c (arginine decarboxylase 1c); LDC (lysine decarboxylase); AO4 (L-ascorbate oxidase 4); AO17 (L-ascorbate oxidase 17); A622-2 (isoflavone reductase homolog A622 2); A622-3 (isoflavone reductase homolog A622 3); BBL-2 (berberine bridging enzyme-like 2); and A622-4 (isoflavone reductase homolog A622 4). [Figure 3] 1 depicts a heatmap showing expression in terms of log2 fold change between before deflowering (BT), 3 days after deflowering (3D), and 2 weeks after deflowering (2W) in TN90 tobacco root samples. [Figure 4]Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g95400 (SEQ ID NO: 58). [Figure 5] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g82124 (SEQ ID NO: 42). [Figure 6] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g23499 (SEQ ID NO: 7). [Figure 7] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g69182 (SEQ ID NO: 26). [Figure 8] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g14663 (SEQ ID NO: 2). [Figure 9] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g71598 (SEQ ID NO: 28). [Figure 10] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g74782 (SEQ ID NO: 30). [Figure 11] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g92662 (SEQ ID NO: 55). [Figure 12] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g91334 (SEQ ID NO: 51). [Figure 13] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g93191 (SEQ ID NO: 56). [Figure 14] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g74738 (SEQ ID NO: 29). [Figure 15] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g66301 (SEQ ID NO: 24). [Figure 16] Figure 1 depicts the relative expression (compared to tobacco eukaryotic translation elongation factor 1 alpha (EF1α)) of 14 novel tobacco genes in root tissue of tobacco lines TN90 and CS118. Expression is examined before flower thinning (BT), 3 days after flower thinning (3d), and 1 week after flower thinning (1w). Error bars, if present, represent one standard deviation. Figure 2 depicts the relative expression of g86407 (SEQ ID NO: 47). [Figure 17] Depicts the relative expression of g22648 (compared to EF1α) in overexpression (OX) and knockdown (RNAi) lines compared to three vector control lines (VC-1, VC-2, and VC-3). [Figure 18] Depicts the relative expression (compared to EF1α) of the putrescine N-methyltransferase (PMT) and quinolinate phosphoribosyltransferase (QPT) gene families in g22648 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC-1, VC-2, and VC-3). [Figure 19] 1 depicts nicotine levels in g22648 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC). [Figure 20] 1 depicts nornicotine, anatabine, and anabasine levels in g22648 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC). [Figure 21] Depicts the relative expression of g95400 (compared to EF1α) in RNAi knockdown tobacco lines compared to vector control lines (VC). [Figure 22] 1 depicts the relative expression of the PMT and QPT gene families (compared to EF1α) in g95400 RNAi knockdown tobacco lines compared to the vector control line (VC). [Figure 23] Depicts nicotine levels in g95400 RNAi knockdown strains compared to vector control strains (VC). [Figure 24] 1 depicts nornicotine, anatabine, and anabasine levels in g95400 RNAi knockdown tobacco lines compared to vector control lines (VC). [Figure 25] Depicts the relative expression of g74912 (relative to EF1α) in RNAi knockdown tobacco lines compared to three vector control lines (VC). [Figure 26] 1 depicts the relative expression of the PMT and QPT gene families (compared to EF1α) in g74912 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 27] 1 depicts nicotine levels in g74912 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 28] 1 depicts nornicotine, anatabine, and anabasine levels in g74912 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 29] Depicts the relative expression of g12941 (compared to EF1α) in overexpression (OX) and knockdown (RNAi) lines compared to three vector control lines (VC-1, VC-2, and VC-3). [Figure 30] Depicts the relative expression (compared to EF1α) of putrescine N-methyltransferase (PMT) and quinolinate phosphoribosyltransferase (QPT) in g12941 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC-1, VC-2, and VC-3). [Figure 31] 1 depicts nicotine levels in g12941 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC-1, VC-2, and VC-3). [Figure 32] 1 depicts nornicotine, anatabine, and anabasine levels in g12941 overexpression (OX) and knockdown (RNAi) tobacco lines compared to three vector control lines (VC-1, VC-2, and VC-3). [Figure 33] Depicts the relative expression of g75446 (relative to EF1α) in RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 34] 1 depicts the relative expression (compared to EF1α) of the PMT and QPT gene families in g75446 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 35] 1 depicts nicotine levels in g75446 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 36] 1 depicts nornicotine, anatabine, and anabasine levels in g75446 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 37] Depicts the relative expression of g31724 (relative to EF1α) in two RNAi knockdown tobacco lines compared to the vector control line (VC). [Figure 38]1 depicts the relative expression (compared to EF1α) of the PMT and QPT gene families in two g31724 RNAi knockdown tobacco lines compared to the vector control line (VC). [Figure 39] 1 depicts nicotine levels in two g31724 RNAi knockdown tobacco lines compared to a vector control line (VC). [Figure 40] 1 depicts nornicotine, anatabine, and anabasine levels in two g31724 RNAi knockdown tobacco lines compared to a vector control line (VC). [Figure 41] Depicts the relative expression (compared to EF1α) of g14663 overexpression (OX) in two tobacco lines compared to the vector control line (VC). [Figure 42] 1 depicts the relative expression of the PMT and QPT gene families (compared to EF1α) in two g14663-overexpressing (OX) tobacco lines compared to a vector control line (VC). [Figure 43] 1 depicts nicotine levels in two g14663-overexpressing (OX) tobacco lines compared to a vector control line (VC). [Figure 44] 1 depicts nornicotine, anatabine, and anabasine levels in two g14663-overexpressing (OX) tobacco lines compared to a vector control line (VC). [Figure 45] Depicts the relative expression (compared to EF1α) of g71598 overexpression (OX) in two tobacco lines compared to the vector control line (VC). [Figure 46] 1 depicts the relative expression of the PMT and QPT gene families (compared to EF1α) in two g71598 overexpressing (OX) tobacco lines compared to the vector control line (VC). [Figure 47] 1 depicts nicotine levels in two g71598 overexpressing (OX) tobacco lines compared to a vector control line (VC). [Figure 48]1 depicts nornicotine, anatabine, and anabasine levels in two g71598 overexpressing (OX) tobacco lines compared to a vector control line (VC). [Figure 49] Depicts the relative expression of g82744 (relative to EF1α) in two RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 50] 1 depicts the relative expression of the PMT and QPT gene families (compared to EF1α) in two g82744 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 51] 1 depicts nicotine levels in two g82744 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 52] 1 depicts nornicotine, anatabine, and anabasine levels in two g82744 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 53] Depicts the relative expression of g79774 (relative to EF1α) in three RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 54] 1 depicts the relative expression of the PMT and QPT gene families (compared to EF1α) in three g79774 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 55] Depicts nicotine levels in three g79774 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 56] 1 depicts nornicotine, anatabine, and anabasine levels in three g79774 RNAi knockdown tobacco lines compared to two vector control lines (VC). [Figure 57] Depicts the relative expression of g82124 (relative to EF1α) in three RNAi knockdown tobacco lines compared to three vector control lines (VC). [Figure 58]1 depicts the relative expression of the PMT and QPT gene families (compared to EF1α) in three g82124 RNAi knockdown tobacco lines compared to three vector control lines (VC). [Figure 59] 1 depicts nicotine levels in three g82124 RNAi knockdown tobacco lines compared to the vector control line (VC). [Figure 60] 1 depicts nornicotine, anatabine, and anabasine levels in three g82124 RNAi knockdown tobacco lines compared to the vector control line (VC).

[0024] For Figures 17, 18, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, and 58, the error bars represent the range of the observed data. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description 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 ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York).

[0026] Any references cited herein, including, for example, all patents, published patent applications, and non-patent literature, are hereby incorporated by reference in their entirety.

[0027] Where a grouping of alternatives is presented, any and all combinations of the members comprising that grouping of alternatives are specifically contemplated. For example, if an item is selected from the group consisting of A, B, C, and D, the inventors specifically contemplate each alternative individually (e.g., A alone, B alone, etc.) as well as combinations such as A, B, and D; A and C; B and C. The term "and / or," when used in a list of two or more items, means any one of the listed items alone or in combination with any one or more of the other listed items. For example, the phrase "A and / or B" is intended to mean either or both A and B, i.e., A alone, B alone, or a combination of A and B. The phrase "A, B, and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0028] Where numerical ranges are provided herein, the ranges are understood to include the endpoints of the range, as well as any number between the endpoints of the defined range. For example, "1 to 10" includes any number between 1 and 10, as well as the numbers 1 and 10.

[0029] When the term "about" is used in reference to a numerical value, it is understood to mean plus or minus 10%. For example, "about 100" includes 90-110.

[0030] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0031] Any tobacco plant or part thereof provided herein is specifically contemplated for use with any of the methods provided herein. Similarly, any modified tobacco plant or part thereof is specifically contemplated for use with any of the methods provided herein. Any nucleic acid sequence, amino acid sequence, or other composition provided herein is specifically contemplated for use with any of the methods provided herein.

[0032] Tobacco plants typically produce alkaloids at levels of 2% to 4% of their total dry weight. Nicotine is the major alkaloid compound in tobacco plants, often accounting for approximately 95% of the plant's total alkaloid content. The remaining pool of alkaloids primarily comprises other structurally related alkaloids, such as anabasine, anatabine, and nornicotine.

[0033] Nicotine is synthesized in the roots via one of two main metabolic pathways: the pyridine nicotine biosynthetic pathway and the pyrrolidine nicotine biosynthetic pathway.

[0034] In the pyridine nicotine biosynthetic pathway, quinolinate synthetase (QS) produces quinolinic acid, and the conversion of quinolinic acid to nicotinic acid mononucleotide (NAMN) is catalyzed by quinolinate phosphoribosyltransferase (QPT). NAMN can be converted to nicotinic acid either directly by NAMN glycohydrolase or through a multistep process involving the synthesis and degradation of nicotine adenine dinucleotide (NAD). Further reduction of nicotinic acid subsequently forms 3,6-dihydronicotinic acid.

[0035] In the pyrrolidine-nicotine biosynthetic pathway, decarboxylation of an amino acid (e.g., arginine) is required to form putrescine, and the methylation of putrescine to N-methylputrescine is catalyzed by N-methyltransferase (PMT). N-methylputrescine is then oxidized by diamine oxidase to form N-methyl-Δ 1 -pyrrolinium cation. 1 The -pyrrolinium cation couples with 3,6-dihydronicotinic acid or another nicotinic acid intermediate to form nicotine, however, the exact substrates and enzymes involved in these reactions remain unknown.

[0036] Provided herein are novel genes involved in the biosynthesis of nicotine or other alkaloids in tobacco.

[0037] In one aspect, the disclosure provides a modified tobacco plant or portion thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a modified tobacco plant or portion thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-58. In another aspect, the disclosure provides a modified tobacco plant or portion thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 59-116. In another aspect, the disclosure provides a modified tobacco plant or portion thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116.

[0038] In one aspect, the disclosure provides a modified tobacco plant, or portion thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a modified tobacco plant, or portion thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing the expression of an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58. In yet another aspect, the disclosure provides a modified tobacco plant, or portion thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing the expression of an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116.

[0039] In one aspect, the disclosure provides a modified tobacco plant, or portion thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a modified tobacco plant, or portion thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58. In another aspect, the disclosure provides a modified tobacco plant, or portion thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116.

[0040] In one aspect, the disclosure provides a modified tobacco plant or portion thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence that regulates the expression or activity of a gene, wherein the gene encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a modified tobacco plant or portion thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence that regulates the expression or activity of a gene, wherein the gene encodes an RNA sequence at least 80% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In one aspect, the endogenous nucleic acid sequence encodes a transcription factor capable of binding to a nucleic acid sequence encoding a gene. As used herein, "transcription factor" refers to a protein that controls the rate of transcription of a gene from DNA to messenger RNA by binding to a specific DNA sequence of the gene. Without being limited to any scientific theory, when a transcription factor binds to a gene, it turns on or enhances transcription of the gene compared to transcription in the absence of the transcription factor. In another aspect, the endogenous nucleic acid sequence encodes a repressor capable of binding to a nucleic acid sequence encoding a gene. As used herein, "repressor" refers to a protein that inhibits transcription of a gene from DNA to messenger RNA by binding to a specific DNA sequence of the gene. Without being limited to any scientific theory, when a repressor binds to a gene, it turns off or reduces transcription of the gene compared to transcription in the absence of the repressor. In a further embodiment, the endogenous nucleic acid sequence encodes a protein that produces a precursor required for the function of the polypeptide encoded by the gene. As a non-limiting example of a precursor, quinolate synthase produces quinolinic acid, a precursor required for quinolinate phosphoribosyltransferase to function. In one embodiment, the gene comprises a nucleic acid sequence at least 80% identical or similar to SEQ ID NOs: 1-58.

[0041] In one aspect, the disclosure provides a modified tobacco plant or portion thereof comprising (a) a genetic modification in a gene or (b) a genetic modification targeted to a gene, wherein the genetic modification down-regulates expression or activity of the gene, and wherein the gene encodes an amino acid sequence having at least 80% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a modified tobacco plant or portion thereof comprising (a) a genetic modification in a gene or (b) a genetic modification targeted to a gene, wherein the genetic modification down-regulates expression or activity of the gene, and wherein the gene encodes a nucleic acid sequence having at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-58. In another aspect, the disclosure provides a modified tobacco plant or portion thereof comprising (a) a genetic modification in a gene or (b) a genetic modification targeted to a gene, wherein the genetic modification down-regulates expression or activity of the gene, and wherein the gene encodes a nucleic acid sequence having at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 59-116. In another aspect, the disclosure provides a modified tobacco plant or portion thereof comprising (a) a genetic modification in a gene, or (b) a genetic modification targeted to a gene, wherein the genetic modification downregulates expression or activity of the gene, and the gene encodes a nucleic acid sequence having at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-116.

[0042] As used herein, "genetic modification" refers to a change in the genetic makeup of a plant or plant genome. Genetic modifications can be introduced by methods including, but not limited to, mutagenesis, genome editing, genetic transformation, or a combination thereof. Genetic modifications include, for example, a mutation in a gene (e.g., a non-natural mutation) or a transgene targeting a gene (e.g., an arginine decarboxylase (ADC) transgene targets the ADC gene). As used herein, "targeting" refers to either directly upregulating or directly downregulating the expression or activity of a gene. As used herein, "directly," in the context of a transgene affecting gene expression or activity, refers to an effect on the gene through physical contact or chemical interaction between the gene (e.g., a promoter region or UTR region) or a product encoded therein (e.g., an mRNA molecule or polypeptide) and the product encoded by the transgene (e.g., a small RNA or a protein such as a transcription factor or dominant-negative polypeptide variant). In some aspects, the transgene affects the expression or activity of the target gene without involving a transcription factor (e.g., the transgene does not encode and / or repress the expression or activity of a transcription factor, which in turn regulates the target gene).

[0043] mutation As used herein, "modified" in the context of plants refers to a plant that contains genetic changes that have been introduced for a particular purpose and beyond natural polymorphisms. Without limitation, modified plants can contain non-natural mutations or recombinant DNA constructs. In one aspect, modified tobacco plants contain non-natural mutations. In another aspect, modified tobacco plants contain recombinant DNA constructs. In another aspect, modified tobacco plants contain genetic modifications.

[0044] As used herein, a "mutation" refers to an inherited genetic modification introduced into a gene to alter the expression or activity of a product encoded by the reference sequence of the gene. A mutation in a particular gene, for example, arginine decarboxylase (ADC), is referred to as an ADC mutant. Such modifications can be in any sequence region of the gene, for example, the promoter, 5' untranslated region (UTR), exon, intron, 3' UTR, or terminator region. In some aspects, the mutation reduces, inhibits, or eliminates the expression or activity of the gene product. In other aspects, the mutation increases, elevates, enhances, or increases the expression or activity of the gene product.

[0045] In some embodiments, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. In some embodiments, the mutation is a "non-natural" or "non-naturally occurring" mutation. As used herein, "non-natural" or "non-naturally occurring" mutation refers to a non-spontaneous mutation generated by human intervention, and does not correspond to a spontaneous mutation generated without human intervention. Non-limiting examples of human intervention include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis) and targeted genetic modification (e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods). Non-naturally occurring and non-naturally occurring mutations do not include spontaneous mutations that occur naturally (e.g., via aberrant DNA replication in the germ line of a plant).

[0046] In certain embodiments, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. It will be understood that when identifying a mutation, the reference DNA sequence should be from the same tobacco variety. For example, if the modified tobacco plant containing the mutation is from variety TN90, the endogenous reference sequence must be the endogenous TN90 sequence, rather than a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if the modified tobacco cell containing the mutation is a TN90 cell, the endogenous reference sequence must be the endogenous TN90 sequence, rather than a homologous sequence from a tobacco cell from a different tobacco variety (e.g., K326).

[0047] In some embodiments, the tobacco plant or portion thereof is homozygous for at least one non-naturally occurring mutation. In another embodiment, the tobacco plant or portion thereof is heterozygous for at least one non-naturally occurring mutation. In another embodiment, the tobacco plant or portion thereof is homozygous for the introduced recombinant DNA construct. In another embodiment, the tobacco plant or portion thereof is hemizygous for the introduced recombinant DNA construct. In a further embodiment, the tobacco plant or portion thereof is heterozygous for the introduced recombinant DNA construct.

[0048] In certain aspects, the mutations provided herein create a dominant allele at the mutated locus. A dominant allele is an allele that masks the contribution of a second allele at the same locus. A dominant allele can be a "dominant negative allele" or a "dominant positive allele." A dominant negative allele, or antimorph, is an allele that acts opposite to normal allele function. Dominant negative alleles typically do not function normally, either directly inhibiting the activity of the wild-type protein (e.g., through dimerization) or inhibiting the activity of a second protein (e.g., an activator or downstream component of a pathway) that is necessary for the normal function of the wild-type protein. For example, a dominant negative allele abrogates or reduces the normal function of the allele in a heterozygous or homozygous state. A dominant positive allele can increase normal gene function (e.g., a hypermorph) or provide a new function to a gene (e.g., a neomorph). A semidominant allele occurs when the penetrance of the linked phenotype in an individual heterozygous for the allele is less than that observed in an individual homozygous for the allele.

[0049] In one aspect, the mutations provided herein create a dominant negative allele at the mutated locus. In another aspect, the mutations provided herein create a dominant positive allele at the mutated locus.

[0050] As used herein, "inducing" mutations refers to generating mutations in a polynucleotide sequence through human intervention. Many suitable methods for inducing mutations in tobacco are known in the art. Non-limiting examples of such methods include the use of chemical mutagens, irradiation, nucleases, transposons, and Agrobacterium. In one aspect, inducing mutations comprises the use of an agent selected from the group consisting of chemical mutagens, irradiation, transposons, Agrobacterium, and nucleases.

[0051] In some embodiments, inducing mutations comprises the use of a chemical mutagen, hi some embodiments, the chemical mutagen comprises ethyl methanesulfonate (EMS).

[0052] In another embodiment, inducing mutations comprises the use of irradiation, hi some embodiments, the irradiation comprises gamma rays, X-rays, ionizing radiation, or fast neutrons.

[0053] In one embodiment, inducing mutations comprises the use of a transposon, hi another embodiment, inducing mutations comprises the use of Agrobacterium.

[0054] In further embodiments, inducing mutations comprises the use of a nuclease. In certain embodiments, the nuclease is selected from the group consisting of meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, CRISPR / Cas9 nucleases, CRISPR / Cpf1 nucleases, CRISPR / CasX nucleases, CRISPR / CasY nucleases, and Csm1 nucleases. In certain embodiments, inducing mutations comprises the use of CRISPR / Cas9 nucleases. In certain embodiments, inducing mutations comprises the use of CRISPR / Cpf1 nucleases. In certain embodiments, inducing mutations comprises the use of CRISPR / CasX nucleases. In certain embodiments, inducing mutations comprises the use of CRISPR / CasY nucleases. In certain embodiments, inducing mutations comprises the use of Csm1 nucleases.

[0055] Several types of mutations are known in the art. In one embodiment, a mutation includes an insertion. An "insertion" refers to the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another embodiment, a mutation includes a deletion. A "deletion" refers to the removal of one or more nucleotides or amino acids from a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another embodiment, a mutation includes a substitution. A "substitution" refers to the replacement of one or more nucleotides or amino acids from a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another embodiment, a mutation includes an inversion. An "inversion" refers to when a segment of a polynucleotide or amino acid sequence is inverted end-to-end. A "duplication" refers to when a segment of a polynucleotide or amino acid sequence is repeated. The repeated segment can immediately follow the original segment, or it can be separated from the original segment by one or more nucleotides or amino acids. In certain aspects, the mutations provided herein include mutations selected from the group consisting of insertions, deletions, substitutions, duplications, and inversions.

[0056] In one aspect, the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116.

[0057] In one aspect, the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to the endogenous nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0058] In certain embodiments, the non-naturally occurring mutation comprises one or more mutation types selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice site mutation, and any combination thereof. As used herein, a "nonsense mutation" refers to a mutation in a nucleic acid sequence that introduces a premature stop codon into the amino acid sequence encoded by the nucleic acid sequence. As used herein, a "missense mutation" refers to a mutation in a nucleic acid sequence that causes a substitution within the amino acid sequence encoded by the nucleic acid sequence. As used herein, a "frameshift mutation" refers to an insertion or deletion in a nucleic acid sequence that shifts the frame for translating the nucleic acid sequence into an amino acid sequence. A "splice site mutation" refers to a mutation in a nucleic acid sequence that retains an intron for protein translation, or alternatively, for an exon that is excluded from protein translation. A splice site mutation can cause a nonsense, missense, or frameshift mutation.

[0059] Mutations in the coding region of a gene (e.g., exon mutations) can result in a truncated protein or polypeptide when the mutated messenger RNA (mRNA) is translated into the protein or polypeptide. In certain aspects, the present disclosure provides mutations that result in truncated proteins or polypeptides. As used herein, a "truncated" protein or polypeptide contains at least one fewer amino acid compared to an endogenous control protein or polypeptide. For example, if endogenous protein A contains 100 amino acids, a truncated version of protein A can contain 1 to 99 amino acids. In certain aspects, non-naturally occurring mutations result in truncated polypeptides.

[0060] Without being limited to any scientific theory, one way to cause protein or polypeptide truncation is by the introduction of a premature stop codon in the mRNA transcript of an endogenous gene. In certain aspects, the present disclosure provides mutations that result in a premature stop codon in the mRNA transcript of an endogenous gene. As used herein, a "stop codon" refers to a nucleotide triplet within an mRNA transcript that signals the termination of protein translation. A "premature stop codon" refers to a stop codon that is positioned earlier (e.g., 5') than the normal stop codon position in the endogenous mRNA transcript. Several stop codons are known in the art, including, but not limited to, "UAG," "UAA," "UGA," "TAG," "TAA," and "TGA."

[0061] In some embodiments, the mutations provided herein include null mutations. As used herein, "null mutation" refers to a mutation that confers a complete loss of function for the protein encoded by the gene containing the mutation, or alternatively, a complete loss of function for the small RNA encoded by the genomic locus. A null mutation can cause a lack of mRNA transcript production, a lack of small RNA transcript production, a lack of protein function, or a combination thereof.

[0062] The mutations provided herein can be located in any portion of the endogenous gene. In one aspect, the mutations provided herein are located in an exon of the endogenous gene. In another aspect, the mutations provided herein are located in an intron of the endogenous gene. In a further aspect, the mutations provided herein are located in the 5'-UTR of the endogenous gene. In yet another aspect, the mutations provided herein are located in the 3'-UTR of the endogenous gene. In yet another aspect, the mutations provided herein are located in the promoter of the endogenous gene. In yet another aspect, the mutations provided herein are located in the terminator of the endogenous gene. In one aspect, the non-naturally occurring mutations provided herein include mutations in a sequence region selected from the group consisting of a promoter, a 5'-UTR, a 3'-UTR, an exon, an intron, and a terminator.

[0063] Screening and selection of mutagenized tobacco plants can be performed by any method known to those skilled in the art. Examples of screening and selection methods include, but are not limited to, Southern analysis, PCR amplification for detecting polynucleotides, Northern blot, RNase protection, primer extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, next-generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting enzymatic or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blot, immunoprecipitation, and enzyme-linked immunoassays 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 the cited techniques are all known.

[0064] Nucleic acids and amino acids As used herein, an "endogenous" nucleic acid sequence refers to a nucleic acid sequence that naturally occurs in the genome of an organism. An endogenous nucleic acid sequence does not include heterologous sequences inserted into the genome through deliberate human intervention. Similarly, an endogenous amino acid sequence is a sequence that naturally occurs through translation of an endogenous nucleic acid molecule. In some aspects, the nucleic acid sequences provided herein are endogenous nucleic acid sequences.

[0065] As used herein, "heterologous" refers to a sequence (nucleic acid or amino acid) that originates from a foreign species or, if from the same species, has been substantially altered in composition and / or genomic locus from its native form by deliberate human intervention. The term is also applicable to nucleic acid constructs, also referred to herein as "polynucleotide constructs" or "nucleotide constructs." Thus, a "heterologous" nucleic acid construct is intended to mean a construct that originates from a foreign species or, if from the same species, has been substantially altered in composition and / or genomic locus from its 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 breeding of the transgenic plant with another plant of interest.

[0066] As used herein, "gene" refers to a polynucleotide capable of producing a functional unit (such as, but not limited to, a protein or a small RNA). A gene can include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription termination site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. A "gene sequence" can include a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription termination site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. In one aspect, a gene encodes a small RNA or a precursor thereof. In another aspect, a gene encodes a protein.

[0067] The term "percent identity" or "percent identical" as used herein with respect to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotides or amino acids) over a comparison window (an "alignable" region), (ii) determining the number of positions where identical nucleic acid bases (in the case of nucleotide sequences) or amino acid residues (in the case of proteins and polypeptides) occur in both sequences to obtain the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the comparison window, and then (iv) multiplying this quotient by 100% to obtain the percent identity. When "percent identity" is calculated relative to a reference sequence without specifying a specific comparison window, the percent identity is determined by dividing the number of matched positions over the aligned region by the total length of the reference sequence. Thus, for purposes of this application, when two sequences (query and subject) are optimally aligned (taking into account gaps in their alignment), the "percent identity" of the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions of the query sequence over its length (or comparison window) multiplied by 100%.

[0068] When percentages of sequence identity are used in terms of amino acids, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions (amino acid residues are substituted with other amino acid residues that have similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the molecule). When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0069] For optimal alignment of sequences to calculate percent identity, various pairwise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST™), which can be used to compare sequence identity or similarity between two or more nucleotide or amino acid sequences. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the above-mentioned percent identity ranges) can be determined by the ClustalW algorithm, see, for example, Chenna et al., "Multiple sequence alignment with the Clustal series of programs," Nucleic Acids Research 31:3497-3500 (2003); Thompson et al., "Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice," Nucleic Acids Research 22:4673-4680 (1994); Larkin MA et al., "Clustal W and Clustal X version 2.0," Bioinformatics 23:2947-48 (2007); and Altschul et al. "Basic local alignment search tool." J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.

[0070] The term "percent complementarity" or "percent complementary" as used herein with respect to two nucleotide sequences is similar to the concept of percent identity, but refers to the percent of nucleotides in the query sequence that optimally base-pair or hybridize with the nucleotides of the subject sequence when the query and subject sequences are aligned linearly and optimally base-paired without secondary folding structures such as loops, stems, or hairpins. Such percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and an RNA strand. "Percent complementarity" can be calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences linearly and in a fully extended alignment (i.e., without folding or secondary structures) over the comparison window, (ii) determining the number of base-pair positions between the two sequences over the comparison window to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying this quotient by 100% to obtain the percent complementarity of the two sequences. The optimal base pairing of two sequences can be determined based on the known pairing of nucleotide bases such as GC, AT, and AU through hydrogen bonds. When "percent complementarity" is calculated relative to a reference sequence without specifying a specific comparison window, the percent identity is determined by dividing the number of complementary positions between two linear sequences by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and subject) are optimally base-paired (allowing for mismatched or non-base-paired nucleotides), the "percent complementarity" of a query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions of the query sequence over its length, multiplied by 100%.

[0071] The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules can include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. Polynucleotides of the present disclosure also encompass all forms of sequence, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem and loop structures, and the like. In one aspect, the nucleic acid molecules provided herein are DNA molecules. In another aspect, the nucleic acid molecules provided herein are RNA molecules. In one aspect, the nucleic acid molecules provided herein are single-stranded. In another aspect, the nucleic acid molecules provided herein are double-stranded. The nucleic acid molecules can encode polypeptides or small RNA molecules.

[0072] As used herein, "recombinant nucleic acid" refers to a nucleic acid molecule formed by laboratory methods of genetic recombination, including, but not limited to, molecular cloning. Similarly, "recombinant DNA construct" refers to a DNA molecule formed by laboratory methods of genetic recombination.

[0073] Nucleic acids can be isolated using techniques routine 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 a nucleic acid in an expression vector. In addition, purified polypeptides can be obtained by chemical synthesis. The degree of purity of a polypeptide can be measured using any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0074] In one aspect, the present disclosure provides a method for detecting recombinant nucleic acids and polypeptides in plant cells. Without limitation, nucleic acids can also be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0075] In one aspect, the nucleic acid sequences provided herein are at least 70% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 75% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 80% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 85% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 88% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 90% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 91% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 92% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 93% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 94% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 95% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 96% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 97% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 98% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the nucleic acid sequences provided herein are at least 99% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116.In another aspect, the nucleic acid sequences provided herein are 100% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116.

[0076] In one aspect, the endogenous nucleic acid sequences provided herein are at least 70% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 75% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 80% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 85% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 88% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 90% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 91% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 92% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 93% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 94% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 95% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 96% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 97% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are at least 98% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116.In another aspect, the endogenous nucleic acid sequences provided herein are at least 99% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116. In another aspect, the endogenous nucleic acid sequences provided herein are 100% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 116.

[0077] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide provided herein. A protein provided herein can be encoded by a nucleic acid molecule provided herein. A protein can include a polypeptide provided herein. As used herein, "protein" refers to a chain of amino acid residues that can provide structure or enzymatic activity to a cell.

[0078] 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 proteins provided herein can be generated 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.

[0079] 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 direct and indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0080] In one embodiment, the amino acid sequences provided herein are at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another embodiment, the amino acid sequences provided herein are at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another embodiment, the amino acid sequences provided herein are at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another embodiment, the amino acid sequences provided herein are at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another embodiment, the amino acid sequences provided herein are at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another embodiment, the amino acid sequences provided herein are at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another embodiment, the amino acid sequences provided herein are at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the amino acid sequences provided herein are at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the amino acid sequences provided herein are at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the amino acid sequences provided herein are at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the amino acid sequences provided herein are at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the amino acid sequences provided herein are at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the amino acid sequences provided herein are at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.In another aspect, the amino acid sequences provided herein are at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In another aspect, the amino acid sequences provided herein are 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.

[0081] In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0082] In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the endogenous nucleic acid sequences provided herein encode polypeptides comprising an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In one aspect, the endogenous nucleic acid sequences provided herein encode a polypeptide comprising an amino acid sequence that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0083] promoter As commonly understood in the art, the term "promoter" 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 can be synthetically produced, altered, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can also include chimeric promoters that contain a combination of two or more heterologous sequences. Thus, promoters of the present application can include variants of promoter sequences that are similar in composition to, but not identical to, other promoter sequence(s) known or provided herein.

[0084] Promoters that drive expression in all or most tissues of a plant are referred to as "constitutive" promoters. In one embodiment, the constitutive promoter is selected from the group consisting of the cauliflower mosaic virus 35S promoter, the ubiquitin promoter, the actin promoter, an opine promoter, and the alcohol dehydrogenase promoter.

[0085] Promoters that drive expression during certain periods or stages of development are referred to as "developmental" promoters.

[0086] A promoter that drives enhanced expression in certain tissues of an organism relative to other tissues of the organism is referred to as a "tissue-preferred" promoter. Thus, a "tissue-preferred" promoter causes relatively high or preferential expression in certain tissue(s) of a plant, but lower levels of expression in other tissue(s) of the plant. As a non-limiting example, a root tissue-preferred promoter exhibits higher activity in root tissue, but may also exhibit activity, albeit at lower levels, in additional tissues such as stem, leaf, and flower tissue. A "tissue-specific" promoter causes expression only in certain tissues. As a non-limiting example, a root tissue-specific promoter drives expression only in root tissue. In one aspect, the tissue-specific promoter is a root tissue-specific promoter. In another aspect, the tissue-preferred promoter is a root tissue-preferred promoter.

[0087] In one embodiment, the root tissue-preferred promoter is the Cassava Vein Mosaic Virus (CsVMV) promoter.

[0088] An "inducible" promoter is a promoter that initiates transcription in response to an environmental stimulus, such as heat, cold, drought, light, or other stimulus, such as wound or chemical application.

[0089] In some aspects, the promoters provided herein are constitutive promoters. In another aspect, the promoters provided herein are inducible promoters. In a further aspect, the promoters provided herein are developmental promoters. In another aspect, the promoter is a tissue-preferred or tissue-specific promoter. In a further aspect, the promoter is selected from the group consisting of a constitutive promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducible promoter.

[0090] In one aspect, the present disclosure provides a heterologous promoter. In another aspect, the present disclosure provides a promoter operably linked to a heterologous polynucleotide. In another aspect, the present disclosure provides a polynucleotide sequence operably linked to a heterologous promoter.

[0091] As used herein, "operably linked" refers to a functional linkage between two or more elements. For example, an operably linked sequence between a polynucleotide of interest and a control sequence (e.g., a promoter) is a functional linkage that allows for expression of the polynucleotide of interest. Operably linked elements can be contiguous or non-contiguous. In one aspect, a promoter provided herein is operably linked to a heterologous nucleic acid molecule.

[0092] small RNA In one embodiment, the nucleic acid molecules provided herein are small RNAs. In another embodiment, the nucleic acid molecules encode small RNAs.

[0093] As used herein, "small RNA" refers to a non-coding RNA molecule between 16 and 50 nucleotides in length. In one embodiment, the small RNA comprises between 16 and 40 nucleotides. In another embodiment, the small RNA comprises between 16 and 30 nucleotides. In another embodiment, the small RNA comprises between 18 and 50 nucleotides. In another embodiment, the small RNA comprises between 18 and 40 nucleotides. In another embodiment, the small RNA comprises between 18 and 30 nucleotides. In another embodiment, the small RNA comprises between 18 and 25 nucleotides. In another embodiment, the small RNA comprises between 20 and 28 nucleotides. In another embodiment, the small RNA comprises between 20 and 24 nucleotides. In another embodiment, the small RNA comprises between 21 and 23 nucleotides. In another embodiment, the small RNA comprises 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0094] In certain embodiments, the small RNA is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA, and microRNA (miRNA).

[0095] miRNAs are typically about 19 to 25 nucleotides long (generally about 20 to 24 nucleotides in plants) and induce the in-trans cleavage of target transcripts, negatively regulating the expression of genes involved in various regulatory and developmental pathways. In some cases, miRNAs function to induce in-phase processing of siRNA primary transcripts.

[0096] It is understood in the art that in plants, miRNAs and targeting nucleic acids often do not share perfect complementarity (although they can have perfect complementarity). miRNAs and their targets can have some mismatches between them, but still allow the miRNA to reduce the expression and / or function of the target gene. See, for example, Liu et al., Plant Cell, 26:741-753 (2014) and Wang et al., Curr. Opin. Plant Biol., 27:118-124 (2015).

[0097] In some embodiments, the small RNA comprises 100% complementarity to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58. In some embodiments, the small RNA comprises at least 95% complementarity over 21 contiguous nucleotides to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58. In some embodiments, the small RNA comprises at least 90% complementarity over 21 contiguous nucleotides to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58. In some embodiments, the small RNA comprises at least 85% complementarity over 21 contiguous nucleotides to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58. In some embodiments, the small RNA comprises at least 95% complementarity over 20 contiguous nucleotides to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58. In some embodiments, the small RNA comprises at least 90% complementarity over 20 contiguous nucleotides to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58. In some embodiments, the small RNA comprises at least 85% complementarity over 20 contiguous nucleotides to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58. In some embodiments, the small RNA comprises at least 95% complementarity over 19 contiguous nucleotides to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58. In some embodiments, the small RNA comprises at least 90% complementarity over 19 contiguous nucleotides to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58. In some embodiments, the small RNA comprises at least 85% complementarity over 19 contiguous nucleotides to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-58.

[0098] Many microRNA genes (MIR genes) have been identified and are publicly available in databases ("miRBase," available online at microrna[dot]sanger[dot]ac[dot]uk / sequences; see also Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to occur both isolated and in clusters within the genome, in intergenic regions, but can also be located wholly or partially within introns of other genes (both protein-coding and non-protein-coding). For a review of miRNA biogenesis, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385. Transcription of MIR genes may, at least in some cases, be under the activatory control of the MIR gene's own promoter. The primary transcript, termed the "pri-miRNA," can be very large (several kilobases), may be polycistronic, and contains one or more pre-miRNAs (folded structures containing stem-loop arrangements that are processed into mature miRNAs), as well as the usual 5' "cap" and polyadenylated tail of an mRNA.

[0099] The maturation of mature miRNAs from their corresponding precursors (pri-miRNA and pre-miRNA) differs significantly between animals and plants. For example, in plant cells, microRNA precursor molecules are thought to be mostly completely processed into mature miRNAs in the nucleus, whereas in animal cells, pri-miRNA transcripts are processed in the nucleus by the animal-specific enzyme Drosha, followed by transport of the pre-miRNA into the cytoplasm, where it is further processed into mature miRNAs. Mature miRNAs in plants are typically 21 nucleotides in length.

[0100] Transgenic expression of miRNAs (whether naturally occurring or artificial) can be used to control the expression of the miRNA's target gene or genes. The inclusion of miRNA recognition sites in transgenically expressed transcripts is also useful for controlling transcript expression. Recognition sites for miRNAs have been validated in all regions of mRNAs, including the 5' untranslated, coding, and 3' untranslated regions, demonstrating 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 to induce such specific transcription of DNA sequences to which they are operably linked. The various utilities of miRNAs, their precursors, their recognition sites, and their promoters are described in detail in U.S. Patent Application Publication No. 2006 / 0200878A1, incorporated herein by reference. Non-limiting examples of these utilities include: (1) expression of natural 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, where the transgene is repressed when the mature miRNA is expressed; and (4) expression of transgenes driven by miRNA promoters.

[0101] Designing an artificial miRNA sequence can be as simple as substituting a sequence complementary to the intended target for nucleotides within the miRNA stem region of a miRNA precursor, as shown by Zeng et al. (2002) Mol. Cell, 9:1327-1333. 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, for example, in both tobacco cDNA and genomic DNA databases, by using a sequence alignment tool such as BLAST (see, e.g., Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402), to identify any potential matches to orthologs of the target transcript 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 87:101-102); Biotechnol., 22:326-330), and functional asymmetry (".DELTA..DELTA.G" or "ΔΔG") characterized by a negative difference in free energy (see Khvorova et al. (2003) Cell, 115:209-216). Preferably, 19-mers with all or most of the following characteristics are selected: (1) Reynolds score >4, (2) about 40% to about 60% GC content, (3) a negative ΔΔG, (4) a terminal adenosine, (5) lack of consecutive runs of four or more identical nucleotides, (6) location near the 3' end of the target gene, and (7) minimal difference from the miRNA precursor transcript.The position of every third nucleotide in an siRNA has been reported to be particularly important in influencing RNAi efficacy, and the algorithm "siExplorer" is publicly available at rna[dot]chem[dot]t[dot]u-tokyo[dot]ac[dot]jp / siexplorer.htm (see Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093 / nar / gkl1120); (c) determining the reverse complement of the selected 19-mer for use in generating 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 be either unpaired to prevent spread of silencing on the target transcript or paired with the target sequence to promote spread of silencing on the target transcript; and (d) transforming the artificial miRNA into a plant.

[0102] Without being limited to any scientific theory, it is understood in the art that RNAi knockdown of a candidate gene (e.g., by using artificial miRNA or siRNA) and mutations (e.g., missense or nonsense mutations) in the same candidate gene can both cause reduced expression and / or decreased protein activity, resulting in the same or similar phenotypes in plants. See, e.g., Agrawal et al., Microbiology and Molecular Biology Reviews, 67:657-685 (2003).

[0103] In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 75% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 80% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 85% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 90% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 95% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 96% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In certain aspects, the small RNAs provided herein comprise a nucleic acid sequence at least 97% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In certain aspects, the small RNAs provided herein comprise a nucleic acid sequence at least 98% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In certain aspects, the small RNAs provided herein comprise a nucleic acid sequence at least 99% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In certain aspects, the small RNAs provided herein comprise a nucleic acid sequence 100% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116.

[0104] In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 75% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 80% complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 85% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 90% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 95% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In certain embodiments, the small RNAs provided herein comprise a nucleic acid sequence at least 96% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In certain aspects, the small RNAs provided herein comprise a nucleic acid sequence at least 97% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In certain aspects, the small RNAs provided herein comprise a nucleic acid sequence at least 98% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In certain aspects, the small RNAs provided herein comprise a nucleic acid sequence at least 99% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In certain aspects, the small RNAs provided herein comprise a nucleic acid sequence 100% identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116.

[0105] In one aspect, the small RNAs provided herein comprise a nucleic acid sequence at least 88.7% identical to or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence at least 94.3% identical to or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is 100% identical to or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is at least 85% identical to or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is at least 90% identical to or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is at least 95% identical to or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116. In another aspect, the small RNAs provided herein comprise a nucleic acid sequence that is 100% identical to or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116.

[0106] In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In certain aspects, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide that is at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174. In certain aspects, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide that is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117 to 174.

[0107] In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 75% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 88% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain aspects, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain aspects, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 91% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain aspects, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 92% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain aspects, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 93% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom.In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 94% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain embodiments, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, or an RNA transcribed therefrom. In certain aspects, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116, or an RNA transcribed therefrom. In certain aspects, the small RNAs provided herein are capable of binding to and reducing the expression of a nucleic acid sequence 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 116, or an RNA transcribed therefrom.

[0108] As used herein, "capable of binding" is synonymous with "capable of hybridizing to." In certain embodiments, a first nucleic acid molecule capable of binding to a second nucleic acid molecule binds to the second nucleic acid molecule. As used herein, a first nucleic acid molecule can "hybridize" to a second nucleic acid molecule through non-covalent interactions (e.g., Watson-Crick base pairing) in a sequence-specific antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base pairing includes adenine pairing with thymine, adenine pairing with uracil, and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, for hybridization between two RNA molecules (e.g., dsRNA), it is also known in the art that guanine bases pair with uracil. For example, G / U base pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anticodon base pairing with codons within mRNA. In the context of the present disclosure, a guanine in the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil, and vice versa. As such, if G / U base pairing can be made at a given nucleotide position in the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, this position is not considered non-complementary, but instead is considered complementary.

[0109] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein, and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0110] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridization between nucleic acids with short stretches of complementarity (e.g., complementarity over 35 nucleotides or less), the location of mismatches becomes important (see Sambrook et al.). Typically, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Exemplary minimum lengths for a hybridizable nucleic acid are at least about 15 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 25 nucleotides, and at least about 30 nucleotides. Furthermore, those skilled in the art will recognize that the temperature and salt concentration of the wash solution can be adjusted as necessary according to factors such as the length of the complementary region and the degree of complementarity.

[0111] It is understood in the art that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable. Furthermore, a polynucleotide can hybridize across one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or a hairpin structure). For example, an antisense nucleic acid in which 18 of 20 nucleotides of an antisense compound are complementary to a target region and therefore specifically hybridizes represents 90 percent complementarity. In this example, the remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleotides and do not need to be contiguous with each other or with complementary nucleotides. The percent complementarity between particular stretches of nucleic acid sequences within a nucleic acid can be routinely determined using the BLAST® program (basic local alignment search tool) and the PowerBLAST program known in the art (see Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0112] Reduced expression / activity In one embodiment, the small RNA reduces the expression of any nucleic acid sequence to which it can bind. In another embodiment, the non-naturally occurring mutations provided herein reduce the expression of the mutated nucleic acid sequence compared to the non-mutated nucleic acid sequence in a control plant grown under equivalent conditions.

[0113] The reduced expression of endogenous nucleic acid sequences can be measured using any suitable method known in the art. Non-limiting examples of measuring expression include quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), RNA blot (e.g., Northern blot), and RNA sequencing. The difference in expression can be described as absolute or relative quantification. See, for example, Livak and Schmittgen, Methods, 25:402-408 (2001). If the endogenous nucleic acid sequence encodes a protein, changes in expression can be inferred by examining the accumulation of the encoded protein. Non-limiting examples of measuring protein accumulation include Western blot and enzyme-linked immunosorbent assay (ELISA).

[0114] In one embodiment, the reduction in expression is measured using qRT-PCR. In another embodiment, the reduction in expression is measured using RNA blot. In another embodiment, the reduction in expression is measured using RNA sequencing. In a further embodiment, the reduction in expression is measured using Western blot. In yet a further embodiment, the reduction in expression is measured using ELISA.

[0115] In some embodiments, non-naturally occurring mutations in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116 result in reduced levels of expression of the nucleic acid sequence compared to a nucleic acid sequence lacking the non-naturally occurring mutation in a control plant grown under equivalent conditions. In some embodiments, non-naturally occurring mutations in a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174 result in reduced levels of expression of the nucleic acid sequence compared to a nucleic acid sequence lacking the non-naturally occurring mutation in a control plant grown under equivalent conditions.

[0116] In some embodiments, the reduced expression comprises at least a 1% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduced expression comprises at least a 5% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduced expression comprises at least a 10% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduced expression comprises at least a 25% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduced expression comprises at least a 50% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduced expression comprises at least a 75% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduced expression comprises at least a 90% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In another embodiment, the reduction in expression comprises at least a 95% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions.

[0117] In some embodiments, the reduction in expression comprises a 1% to 99% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduction in expression comprises a 1% to 90% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduction in expression comprises a 1% to 75% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduction in expression comprises a 1% to 50% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduction in expression comprises a 1% to 25% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduction in expression comprises a 25% to 90% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In other embodiments, the reduction in expression comprises a 50% to 90% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions. In another embodiment, the reduction in expression comprises a 25% to 75% reduction compared to expression in the same tissue of a control plant grown under equivalent conditions.

[0118] In some embodiments, the reduction in expression comprises a statistically significant reduction compared to the expression in the same tissue of a control plant grown under the same conditions.Those skilled in the art will recognize that any level of reduction is envisioned, as long as the level of reduction is determined to be statistically significant using an acceptable statistical hypothesis test.As a non-limiting example, Student's t-test is one statistical hypothesis test that can be used to determine whether the reduction in expression between modified plants and control plants is statistically significant.As used herein, "statistically significant" refers to a p-value of 0.05 or less.

[0119] In one aspect, the non-naturally occurring mutation results in a reduced level of activity by a protein or polypeptide encoded by a nucleic acid sequence provided herein, compared to the activity in a control plant grown under equivalent conditions. In another aspect, the non-naturally occurring mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116 reduces the level of activity by the protein or polypeptide encoded by the nucleic acid sequence, when grown under equivalent conditions, compared to the activity of the protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco plant, wherein the nucleic acid sequence lacks the non-naturally occurring mutation in the control tobacco plant. In another aspect, the non-naturally occurring mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, reduces the level of activity by the protein or polypeptide encoded by the nucleic acid sequence, when grown under equivalent conditions, compared to the activity of the protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco plant, wherein the nucleic acid sequence lacks the non-naturally occurring mutation in the control tobacco plant.

[0120] As used herein, when referring to a protein or polypeptide, "activity" refers to the ability to perform an enzymatic function.

[0121] Increased expression / activity In some embodiments, the non-naturally occurring mutation results in increased expression of the nucleic acid sequence, ie, the non-naturally occurring mutation results in increased levels of expression of the nucleic acid sequence compared to expression of the nucleic acid sequence in the same tissue of a control tobacco plant when grown under equivalent conditions, wherein the nucleic acid sequence lacks at least one non-naturally occurring mutation in the control tobacco plant.

[0122] In one embodiment, the increased level of expression comprises at least a 5% increase compared to expression in the same tissue of a control plant grown under equivalent conditions. In another embodiment, the increased level of expression comprises at least a 10% increase compared to expression in the same tissue of a control plant grown under equivalent conditions. In another embodiment, the increased level of expression comprises at least a 25% increase compared to expression in the same tissue of a control plant grown under equivalent conditions. In another embodiment, the increased level of expression comprises at least a 50% increase compared to expression in the same tissue of a control plant grown under equivalent conditions. In another embodiment, the increased level of expression comprises at least a 75% increase compared to expression in the same tissue of a control plant grown under equivalent conditions. In another embodiment, the increased level of expression comprises at least a 100% increase compared to expression in the same tissue of a control plant grown under equivalent conditions. In another embodiment, the increased level of expression comprises at least a 200% increase compared to expression in the same tissue of a control plant grown under equivalent conditions. In another embodiment, the increased level of expression comprises at least a 500% increase compared to expression in the same tissue of a control plant grown under equivalent conditions.

[0123] In one aspect, the non-naturally occurring mutation results in an increased level of activity by the protein or polypeptide encoded by the nucleic acid sequence compared to the activity of the protein or polypeptide encoded by the nucleic acid sequence in a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks at least one non-naturally occurring mutation in the control tobacco plant.

[0124] alkaloid As used herein, "alkaloid" refers to complex nitrogen-containing compounds that occur naturally in tobacco and have pharmacological effects in humans or other animals. Non-limiting examples of alkaloids include nicotine, anabasine, anatabine, and nornicotine.

[0125] Nicotine is the major naturally occurring alkaloid in commercial cigarette tobacco, accounting for approximately 90% of the alkaloid content in Nicotiana tabacum. Other major alkaloids in tobacco include, but are not limited to, cotinine, nornicotine, myosmine, nicotyrine, anabasine, and anatabine. Minor tobacco alkaloids include, but are not limited to, nicotine-n-oxide, N-methylanatabine, N-methylanabasine, pseudooxynicotine, 2,3-dipyridyl, and others.

[0126] In some embodiments, the alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine.

[0127] Alkaloid levels can be assayed by methods known in the art, for example, by gas-liquid chromatography, high-performance liquid chromatography, radioimmunoassay, enzyme-linked immunosorbent assay, and mass spectrometry-based quantification. For example, nicotinic alkaloid levels can be measured by a GC-FID method based on CORESTA Recommended Method No. 7, 1987 and ISO standard (ISO TC126N 394 E). See also Hibi et al., Plant Physiology 100:826-35 (1992), for a method using gas-liquid chromatography equipped with a capillary column and an FID detector.

[0128] Alkaloid levels can be assayed from any plant tissue. Non-limiting examples include leaf and hairy root cultures. In addition, alkaloid levels can be assayed in dried or undried plant material.

[0129] Alternatively, total alkaloids in tobacco can be measured using a segmented-flow colorimetric method developed for the analysis of tobacco samples, adapted by Skalar Instrument Co. (West Chester, PA) and described by Collins et al., Tobacco Science 13:79-81 (1969). Briefly, tobacco samples are dried, crushed, and extracted prior to analysis of total alkaloids and reducing sugars. The method then uses acetic acid / methanol / water extraction and charcoal for decolorization. Total alkaloid determination is based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of aromatic amines to form a colored complex that is measured at 460 nm.

[0130] In some embodiments, the levels of individual alkaloids are measured on freeze-dried dry leaf samples using liquid chromatography with tandem mass spectrometry (LC / MS / MS). In some embodiments, the levels of individual alkaloids are measured on freeze-dried hairy root culture samples using liquid chromatography with tandem mass spectrometry (LC / MS / MS).

[0131] Unless otherwise specified, nicotine or alkaloid levels (or another leaf chemistry or characteristic) of tobacco plants are measured after topping in pooled leaf samples collected from leaf numbers 3, 4, and 5 after topping. As used herein, whenever a comparison between leaves from two plants (e.g., mutant plants vs. control plants) is mentioned, leaves from the same or equivalent leaf position(s) and developmental stage(s) are intended, from which effects due to genotypic differences, rather than other factors, can be demonstrated. As a non-limiting example, leaf 3 of the control plant is intended as a reference point for comparison with leaf 3 of a modified plant containing a non-natural mutation or recombinant DNA construct.

[0132] As used herein, leaf numbering is based on the leaf's position on the tobacco stem, with leaf number 1 being the youngest leaf (top) after pruning and the highest leaf number being assigned to the oldest leaf (bottom).

[0133] As used herein, "deflowering" refers to the removal of the shoot apical meristem, the flower, and the apical end of the shoot, including up to several adjacent leaves, when the tobacco plant is near vegetative maturity and at the beginning of reproductive growth. Typically, tobacco plants are deflowered at the button stage (shortly after the flowers begin to appear). For example, greenhouse- or field-grown tobacco plants can be deflowered when 50% of the plants have at least one flower. Deflowering tobacco plants results in the loss of apical dominance and also induces increased alkaloid production.

[0134] Typically, alkaloid levels (or another leaf chemistry or property characterization; e.g., polyamines) of tobacco plants are measured about 2 weeks after flower thinning. Other time points can also be used. In one embodiment, alkaloid levels (or another leaf chemistry or property characterization) of tobacco plants are measured about 1, 2, 3, 4, or 5 weeks after flower thinning. In another embodiment, nicotine, alkaloid, or polyamine levels (or another leaf chemistry or property characterization; e.g., polyamines) of tobacco plants are measured about 3, 5, 7, 10, 12, 14, 17, 19, or 21 days after flower thinning.

[0135] In certain aspects, a modified tobacco plant or portion thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid produces at least one leaf comprising a reduced amount of at least one alkaloid compared to the amount of alkaloid in a control tobacco plant lacking the at least one non-naturally occurring mutation in an endogenous nucleic acid when grown under equivalent conditions.

[0136] In certain aspects, a modified tobacco plant or portion thereof comprising a recombinant DNA construct provided herein produces at least one leaf that comprises a reduced amount of at least one alkaloid compared to the amount of alkaloid in a control tobacco plant lacking the recombinant DNA construct when grown under equivalent conditions.

[0137] In some embodiments, the reduced level of at least one alkaloid comprises a reduction of at least 0.5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of at least 1% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of at least 2% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of at least 3% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of at least 4% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of at least 5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of at least 10% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises at least a 15% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises at least a 20% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises at least a 25% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises at least a 35% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises at least a 50% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises at least a 75% reduction compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises at least a 90% reduction compared to a control tobacco plant when grown under equivalent conditions.

[0138] In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 99% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 90% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 80% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 70% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 60% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 50% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 40% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 30% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 20% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 10% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 5% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 10% to 75% reduction compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 10% to 50% reduction compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises a 10% to 25% reduction compared to a control tobacco plant when grown under equivalent conditions.

[0139] In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 0.5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 1% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 2% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 3% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 4% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 10% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 15% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 20% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 25% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 35% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine of at least 50% compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine by at least 75% compared to a control tobacco plant when grown under equivalent conditions, hi some embodiments, the reduced level of at least one alkaloid comprises a reduction in nicotine by at least 90% compared to a control tobacco plant when grown under equivalent conditions.

[0140] In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 99% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 90% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 80% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 70% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 60% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 50% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 40% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 30% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 20% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 10% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 5% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 10% to 75% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises a 10% to 50% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions, hi some embodiments, the reduced level of at least one alkaloid comprises a 10% to 25% reduction in nicotine compared to a control tobacco plant when grown under equivalent conditions.

[0141] In some embodiments, the modified tobacco plants comprise a nicotine level of 17 milligrams per gram of dry weight (mg / g) or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 16 mg / g or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 15 mg / g or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 14 mg / g or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 13 mg / g or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 12 mg / g or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 11 mg / g or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 10 mg / g or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 9 mg / g or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 8 mg / g or less. In some embodiments, the modified tobacco plants comprise a nicotine level of 7 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise a nicotine level of 6 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise a nicotine level of 5 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise a nicotine level of 4 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise a nicotine level of 3 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise a nicotine level of 2 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise a nicotine level of 1.5 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise a nicotine level of 1 mg / g or less of dry weight.

[0142] In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 0.5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 1% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 2% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 3% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 4% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 10% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 15% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 20% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 25% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 35% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anatabine by at least 50% compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anatabine by at least 75% compared to a control tobacco plant when grown under equivalent conditions, hi some embodiments, the reduced level of at least one alkaloid comprises a reduction of anatabine by at least 90% compared to a control tobacco plant when grown under equivalent conditions.

[0143] In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 99% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 90% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 80% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 70% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 60% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 50% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 40% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 30% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 20% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 10% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 5% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 10% to 75% reduction in anatabine compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anatabine by 10% to 50% relative to a control tobacco plant when grown under equivalent conditions, hi some embodiments, the reduced level of at least one alkaloid comprises a reduction of anatabine by 10% to 25% relative to a control tobacco plant when grown under equivalent conditions.

[0144] In some embodiments, the modified tobacco plants comprise anatabine levels of 1.5 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 1.4 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 1.3 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 1.2 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 1.1 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 1.0 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 0.9 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 0.8 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 0.7 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 0.6 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 0.5 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 0.4 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 0.3 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 0.25 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anatabine levels of 0.2 mg / g or less of dry weight.

[0145] In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by at least 0.5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by at least 1% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by at least 2% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by at least 3% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by at least 4% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by at least 5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anabasine by at least 10% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anabasine by at least 15% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anabasine by at least 20% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anabasine by at least 25% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anabasine by at least 35% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in anabasine by at least 50% compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by at least 75% compared to a control tobacco plant when grown under equivalent conditions, hi some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by at least 90% compared to a control tobacco plant when grown under equivalent conditions.

[0146] In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 99% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 90% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 80% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 70% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 60% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 50% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 40% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 30% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 20% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 10% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 5% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 10% to 75% reduction in anabasine compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by 10% to 50% relative to a control tobacco plant when grown under equivalent conditions, hi some embodiments, the reduced level of at least one alkaloid comprises a reduction of anabasine by 10% to 25% relative to a control tobacco plant when grown under equivalent conditions.

[0147] In some embodiments, the modified tobacco plants comprise anabasine levels of 0.6 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anabasine levels of 0.5 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anabasine levels of 0.4 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anabasine levels of 0.3 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anabasine levels of 0.25 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anabasine levels of 0.2 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anabasine levels of 0.15 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise anabasine levels of 0.1 mg / g or less of dry weight.

[0148] In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 0.5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 1% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 2% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 3% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 4% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 5% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 10% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 15% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 20% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 25% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 35% compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 50% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 75% compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a reduction in nornicotine by at least 90% compared to a control tobacco plant when grown under equivalent conditions.

[0149] In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 99% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 90% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 80% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 70% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 60% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 50% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 40% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 30% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 20% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 10% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 1% to 5% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions. In some embodiments, the reduced level of at least one alkaloid comprises a 10% to 75% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions.In some embodiments, the reduced level of at least one alkaloid comprises a 10% to 50% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions, hi some embodiments, the reduced level of at least one alkaloid comprises a 10% to 25% reduction in nornicotine compared to a control tobacco plant when grown under equivalent conditions.

[0150] In some embodiments, the modified tobacco plants comprise nornicotine levels of 1.0 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise nornicotine levels of 0.9 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise nornicotine levels of 0.8 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise nornicotine levels of 0.7 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise nornicotine levels of 0.6 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise nornicotine levels of 0.5 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise nornicotine levels of 0.4 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise nornicotine levels of 0.3 mg / g or less of dry weight. In some embodiments, the modified tobacco plants comprise nornicotine levels of 0.2 mg / g or less of dry weight.

[0151] plant As used herein, tobacco plants include Nicotiana tabacum, Nicotiana amplexicaulis PI 271989, Nicotiana benthamiana PI 555478, Nicotiana bigelovii PI 555485, Nicotiana debneyi, Nicotiana excelsior PI 224063, Nicotiana glutinosa PI 555507, Nicotiana goodspeedii PI 241012, Nicotiana gossei PI 230953, Nicotiana hesperis PI 555485, Nicotiana tabacum, Nicotiana amplexicaulis PI 271989, Nicotiana benthamiana PI 555478, Nicotiana bigelovii PI 555485, Nicotiana debneyi, Nicotiana excelsior PI 224063, Nicotiana glutinosa PI 555507, Nicotiana goodspeedii PI 241012, Nicotiana gossei PI 230953, Nicotiana hesperis PI 555485, Nicotiana hesperis PI271991, Nicotiana knightiana PI555527, Nicotiana maritima PI555535, Nicotiana megalosiphon PI555536, Nicotiana nudicaulis PI555540, Nicotiana paniculata PI555545, Nicotiana plumbaginifolia PI555548, Nicotiana repanda PI555552, Nicotiana rustica, Nicotiana suaveolens PI555553 The plant may be from any plant in the genus Nicotiana, including, but not limited to, Nicotiana suaveolens PI230960, Nicotiana sylvestris PI555569, Nicotiana tomentosa PI266379, Nicotiana tomentosiformis, and Nicotiana trigonophylla PI555572.In some aspects, the tobacco plants described herein are Nicotiana tabacum plants.

[0152] In some aspects, provided tobacco parts include, but are not limited to, leaves, stems, roots, trichomes, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, pods, embryos, endosperm, explants, callus, tissue cultures, shoots, cells, and protoplasts. In some aspects, provided tobacco parts do not include seeds. In some aspects, the present disclosure provides tobacco plant cells, tissues, and organs that are not reproductive material and do not mediate natural plant reproduction. In another aspect, the present disclosure also provides tobacco plant cells, tissues, and organs that are reproductive material and mediate natural plant reproduction. 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 tobacco plant cells. Somatic cells, as opposed to germline cells, do not mediate plant reproduction.

[0153] The cells, tissues, and organs can be from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperms, roots, shoots, stems, trichomes, 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 chloroplasts of tobacco plants. In a further aspect, the present disclosure provides epidermal cells, stomatal cells, leaves or root hairs, storage roots, or tubers. In another aspect, the present disclosure provides tobacco protoplasts.

[0154] Those skilled in the art understand that tobacco plants reproduce naturally through seeds, rather than through asexual or vegetative propagation. In one aspect, the present disclosure provides tobacco endosperm.

[0155] The present disclosure provides cells from the tobacco plants provided herein.

[0156] As used herein, a "progeny tobacco plant" or "progeny tobacco seed" can be from any hybrid generation, e.g., F1, F2, F3, F4, F5, F6, F7, etc.

[0157] In some embodiments, the tobacco plant or portion thereof is of a tobacco variety selected from the group consisting of flue-cured, bright, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish. In one embodiment, the modified tobacco plant or portion thereof provided herein is of a tobacco variety selected from the group consisting of flue-cured, light, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish.

[0158] In some aspects, the tobacco cells are of a tobacco variety selected from the group consisting of flue-cured, light, burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish. In some aspects, the modified tobacco cells are of a tobacco variety selected from the group consisting of flue-cured, light, burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish.

[0159] In some embodiments, the tobacco leaf is of a tobacco variety selected from the group consisting of flue-cured, light, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish.

[0160] In some embodiments, the cured tobacco leaves or plant parts are of a tobacco variety selected from the group consisting of flue-cured, light, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish. Those skilled in the art will further understand that cured tobacco is not a living organism and is incapable of growth or reproduction.

[0161] Flue-cured tobacco (also known as "Virginia" or "light" tobacco) represents approximately 40% of the world's tobacco production. Flue-cured tobacco is often referred to as "light" tobacco because it turns from a golden to deep orange color during curing. Flue-cured tobacco has a light, distinctive aroma and flavor. 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 tobacco plants or seeds, or modified tobacco plants or seeds provided herein are of a flue-cured tobacco variety selected from the group consisting of the varieties listed in Table 2 and any variety essentially derived from any one of the aforementioned varieties. See WO 2004 / 041006 A1. In a further aspect, the modified tobacco plants or seeds provided herein are of a flue-cured variety selected from the group consisting of K326, K346, and NC196.

[0162] Table 2: Flue-cured tobacco varieties TIFF2025525933000006.tif145155TIFF2025525933000007.tif159155

[0163] Air-cured tobaccos include "burley," "Maryland," and "dark" tobaccos. A common factor linking air-cured tobaccos is that the curing is primarily done without artificial sources of heat and humidity. Burley tobacco is light to dark brown in color, high in oil, and low in sugar. Burley tobacco is typically air-cured in barns. Major burley-growing countries include Argentina, Brazil, Italy, Malawi, and the United States.

[0164] Maryland tobacco is very fluffy, has good burning characteristics, low nicotine, and a neutral aroma. Major Maryland growing countries include the United States and Italy.

[0165] In one aspect, the tobacco plant or seed, or modified tobacco plant or seed provided herein is of a burley tobacco variety selected from the group consisting of the tobacco varieties listed in Table 3 and any variety essentially derived from any one of the foregoing varieties. In a further aspect, the modified tobacco plant or seed provided herein is of a burley variety selected from the group consisting of TN90, KT209, KT206, KT212, and HB4488.

[0166] (Table 3) Burley tobacco varieties TIFF2025525933000008.tif193170

[0167] In another aspect, the tobacco plant or seed, or modified tobacco plant or seed provided herein is of a Maryland tobacco variety selected from the group consisting of the tobacco varieties listed in Table 4 and any variety essentially derived from any one of the foregoing varieties.

[0168] Table 4. Maryland Tobacco Varieties TIFF2025525933000009.tif70128

[0169] Dark air-cured tobacco is distinguished from other tobacco types primarily by its curing process, which gives it a medium to dark brown color and a distinctive aroma. 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 variety selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One Sucker, Green River, Virginia sun-cured, and Paraguan Passado, and any variety essentially derived from any one of the foregoing varieties.

[0170] Dark fire-cured tobacco is generally dried over a low-burning fire on the floor of an enclosed drying barn. Dark fire-cured tobacco is typically used to make pipe blends, cigarettes, chewing tobacco, snuff, and strong-flavored cigars. The primary growing regions for dark fire-cured tobacco are Tennessee, Kentucky, and Virginia in the United States. In one aspect, the tobacco plants or seeds, or modified tobacco plants or seeds provided herein are of a dark fire-cured tobacco variety selected from the group consisting of the tobacco varieties listed in Table 5 and any variety essentially derived from any one of the foregoing varieties.

[0171] Table 5. Dark Tobacco Varieties TIFF2025525933000010.tif154157

[0172] Oriental tobacco is also referred to as Greek tobacco, aromatic tobacco, and Turkish tobacco due to the fact that it is typically grown in the Eastern Mediterranean region, such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. The small plant size, small leaf size, and unique aroma characteristics of Oriental tobacco varieties are the result of their adaptation to the poor soil and stressful climatic conditions in which they are grown. In one aspect, the tobacco plant or seed, or modified tobacco plant or seed provided herein is of an Oriental tobacco variety selected from the group consisting of the tobacco varieties listed in Table 6 and any variety essentially derived from any one of the foregoing varieties.

[0173] Table 6. Oriental Tobacco Varieties TIFF2025525933000011.tif105149

[0174] In certain aspects, the tobacco plants or seeds, or modified tobacco plants or seeds provided herein are of a cigar tobacco variety selected from the group consisting of the tobacco varieties listed in Table 7 and any variety essentially derived from any one of the foregoing varieties.

[0175] (Table 7) Cigar Tobacco Varieties TIFF2025525933000012.tif50167

[0176] In certain aspects, the tobacco plants or seeds, or modified tobacco plants or seeds provided herein are of a tobacco variety selected from the group consisting of the tobacco varieties listed in Table 8 and any variety essentially derived from any one of the foregoing varieties.

[0177] (Table 8) Other tobacco varieties TIFF2025525933000013.tif37128

[0178] In one embodiment, the tobacco plant or portion thereof is from a variety selected from the group consisting of tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In another embodiment, the tobacco plant or portion thereof is from a variety listed in Table 2. In another embodiment, the tobacco plant or portion thereof is from a variety listed in Table 3. In another embodiment, the tobacco plant or portion thereof is from a variety listed in Table 4. In another embodiment, the tobacco plant or portion thereof is from a variety listed in Table 5. In another embodiment, the tobacco plant or portion thereof is from a variety listed in Table 6. In another embodiment, the tobacco plant or portion thereof is from a variety listed in Table 7. In another embodiment, the tobacco plant or portion thereof is from a variety listed in Table 8.

[0179] In some embodiments, the modified tobacco plant or portion thereof is from a variety selected from the group consisting of tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In some embodiments, the modified tobacco plant or portion thereof is from a variety listed in Table 2. In another embodiment, the modified tobacco plant or portion thereof is from a variety listed in Table 3. In another embodiment, the modified tobacco plant or portion thereof is from a variety listed in Table 4. In another embodiment, the modified tobacco plant or portion thereof is from a variety listed in Table 5. In another embodiment, the modified tobacco plant or portion thereof is from a variety listed in Table 6. In another embodiment, the modified tobacco plant or portion thereof is from a variety listed in Table 7. In another embodiment, the modified tobacco plant or portion thereof is from a variety listed in Table 8.

[0180] In one embodiment, the tobacco seeds are from a variety selected from the group consisting of tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In another embodiment, the tobacco seeds are from a variety listed in Table 2. In another embodiment, the tobacco seeds are from a variety listed in Table 3. In another embodiment, the tobacco seeds are from a variety listed in Table 4. In another embodiment, the tobacco seeds are from a variety listed in Table 5. In another embodiment, the tobacco seeds are from a variety listed in Table 6. In another embodiment, the tobacco seeds are from a variety listed in Table 7. In another embodiment, the tobacco seeds are from a variety listed in Table 8.

[0181] In one embodiment, the tobacco cell is from a variety selected from the group consisting of tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In another embodiment, the tobacco cell is from a variety listed in Table 2. In another embodiment, the tobacco cell is from a variety listed in Table 3. In another embodiment, the tobacco cell is from a variety listed in Table 4. In another embodiment, the tobacco cell is from a variety listed in Table 5. In another embodiment, the tobacco cell is from a variety listed in Table 6. In another embodiment, the tobacco cell is from a variety listed in Table 7. In another embodiment, the tobacco cell is from a variety listed in Table 8.

[0182] All of the above-mentioned specific varieties of air-cured, dark air-cured, burley, Maryland, dark flame-cured, cigar, or Oriental type are listed for illustrative purposes only, and any additional air-cured, dark air-cured, burley, Maryland, dark flame-cured, cigar, or Oriental varieties are also contemplated in this application.

[0183] In some aspects, the tobacco plants or varieties provided herein are inbred tobacco plants or varieties. As used herein, an "inbred" tobacco variety is a variety that has been bred for genetic homogeneity.

[0184] As used herein, a "hybrid" is produced by crossing two plants from different varieties or species such that the progeny contain genetic material from each parent. One of skill in the art will recognize that higher hybrids can be produced as well. For example, a first hybrid can be produced by crossing variety C with variety D to produce a CxD hybrid, and a second hybrid can be produced by crossing variety F with variety E to produce an ExF hybrid. The first and second hybrids can be further crossed to produce a higher hybrid (CxD)x(ExF) that contains genetic information from all four parent varieties. In certain aspects, the modified tobacco plants provided herein are hybrid tobacco plants. In another aspect, the modified tobacco seeds provided herein are hybrid tobacco seeds. In certain aspects, the tobacco plants or varieties provided herein are hybrid tobacco plants or varieties. In another aspect, the modified tobacco plants provided herein are hybrid tobacco plants.

[0185] In certain aspects, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, wherein the non-naturally occurring mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one tobacco seed or plant germinated therefrom comprises the non-naturally occurring mutation. In another aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58, wherein the non-naturally occurring mutation is not present in an endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one tobacco seed or plant germinated therefrom comprises the non-naturally occurring mutation.In another aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116, wherein the non-naturally occurring mutation is not present in an endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one tobacco seed or plant germinated therefrom comprises the non-naturally occurring mutation. In another aspect, the present disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-116, where the non-naturally occurring mutation is not present in an endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one tobacco seed or plant germinated therefrom comprises the non-naturally occurring mutation. In one aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are the same tobacco variety. In another aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are two different tobacco varieties.

[0186] In certain aspects, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, wherein the recombinant DNA construct is absent from a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein the at least one progeny tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct. In another aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence at least 80% identical or similar to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58, wherein the recombinant DNA construct is absent in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein the at least one progeny tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct.In another aspect, the present disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence at least 80% identical or similar to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116, wherein the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one progeny tobacco seed or plant germinated therefrom comprises the recombinant DNA construct. In one aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are the same tobacco variety. In another aspect, in any of the preceding methods, the first tobacco variety and the second tobacco variety are two different tobacco varieties.

[0187] In certain aspects, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, and the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein the at least one progeny tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct. In another aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58, wherein the recombinant DNA construct is absent in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or a plant germinated therefrom, wherein the at least one progeny tobacco seed or a plant germinated therefrom comprises the recombinant DNA construct.In another aspect, the present disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116, and the recombinant DNA construct is absent in a control tobacco plant of the first tobacco variety; and (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one progeny tobacco seed or plant germinated therefrom comprises the recombinant DNA construct. In one aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are the same tobacco variety. In another aspect, in any of the foregoing methods, the first tobacco variety and the second tobacco variety are two different tobacco varieties.

[0188] As used herein, the term "crossing" refers to the intentional mating of two plants. In some embodiments, crossing includes pollination and / or fertilization of a first tobacco plant with a second tobacco plant. The two tobacco plants being crossed can be distantly related, closely related, or identical. In some embodiments, both of the two tobacco plants being crossed are modified tobacco plants. In some embodiments, the two tobacco plants being crossed are of the same tobacco variety. In some embodiments, the two tobacco plants being crossed are of two different tobacco varieties. In some embodiments, one of the two tobacco plants being crossed is male sterile. In some embodiments, one of the two tobacco plants being crossed is female sterile. In some embodiments, at least one of the two tobacco plants being crossed is a hybrid tobacco plant. In some embodiments, at least one of the two tobacco plants being crossed is a modified tobacco plant.

[0189] In some aspects, the tobacco plants or varieties provided herein are male sterile. In other aspects, the tobacco plants or varieties provided herein are cytoplasmic male sterile (CMS). In some aspects, the modified tobacco plants or varieties provided herein are male sterile. In other aspects, the modified tobacco plants or varieties provided herein are cytoplasmic male sterile (CMS). Male sterile tobacco plants can 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. W.H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, NY 761 pp.

[0190] In another aspect, the tobacco plants or varieties provided herein are female sterile. In another aspect, the modified tobacco plants or varieties provided herein are female sterile. As a non-limiting example, female sterile plants can be produced by mutating the STIG1 gene. See, e.g., Goldman et al. 1994, EMBO Journal 13:2976-2984. In one aspect, the modified tobacco plants provided herein are female sterile.

[0191] Unless otherwise specified, all comparisons to control plants require similar or equivalent growth conditions for the two plants being compared. As used herein, "grown under equivalent conditions," "similar growth conditions," or "equivalent growth conditions" refer to similar environmental conditions and / or agronomic practices for growing and meaningfully comparing two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices contribute to or explain any 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, undercutting, transplanting, flower thinning, and offshoot removal. See Chapters 4B and 4C in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp. 70-103. As used herein, a "control plant" refers to a plant of identical or nearly identical genetic make-up to the modified plant being compared, except for any non-naturally occurring mutations or recombinant DNA constructs provided herein that have been introduced into the modified plant.

[0192] In some embodiments, the modified tobacco plants described herein are low-alkaloid varieties or plants. As a non-limiting example, LA Burley 21 (LA BU21) is a low-alkaloid variety of tobacco. LA BU21 was created by incorporating low-alkaloid gene(s) from the Cuban Cigar variety into Burley 21 through several backcrosses. It has about 0.2% total alkaloids (dry weight), compared to about 3.5% (dry weight) of its parent, Burley 21. LA BU21 exhibits leaf grades significantly below commercially acceptable standards. LA BU21 also exhibits other undesirable leaf phenotypes characterized by lower yield, delayed maturation and senescence, higher susceptibility to insect feeding, and poor end-product quality after curing. LA BU21 leaves also exhibit traits such as higher polyamine content, higher chlorophyll content, and more mesophyll cells per unit leaf area. For further characterization of the leaf phenotype of LA BU21, see US2019 / 0271000.

[0193] In certain aspects, the present disclosure provides tobacco plants or portions thereof that contain a low-nicotine or low-alkaloid conferring mutation or transgene (e.g., a genetic modification in or targeting one or more ADC, AO, or ODC) and are capable of producing leaves containing equivalent levels of one or more polyamines relative to equivalent leaves of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent levels of one or more polyamines are within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the levels in equivalent leaves of a control plant that does not contain the same mutation or transgene. In some embodiments, the equivalent level of one or more polyamines is 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, or 19% to 20% of the level in an equivalent leaf of a control plant not containing the same mutation or transgene. In further embodiments, the equivalent level of one or more polyamines is 0.5% to 5%, 5% to 10%, or 10% to 20% of the level in an equivalent leaf of a control plant not containing the same mutation or transgene.

[0194] In certain aspects, the present disclosure provides an ADC mutant or transgenic tobacco plant or portion thereof, an AO mutant or transgenic tobacco plant or portion thereof, or an ODC mutant or transgenic tobacco plant or portion thereof that is capable of producing leaves containing equivalent chlorophyll levels relative to equivalent leaves of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent chlorophyll level is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene. In some embodiments, the equivalent chlorophyll level is 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, or 19% to 20% of the level in an equivalent leaf of a control plant that does not contain the same mutation or transgene. In further embodiments, the equivalent chlorophyll level is 0.5% to 5%, 5% to 10%, or 10% to 20% of the level in an equivalent leaf of a control plant that does not contain the same mutation or transgene.

[0195] In certain aspects, the present disclosure provides an ADC mutant or transgenic tobacco plant or part thereof, an AO mutant or transgenic tobacco plant or part thereof, or an ODC mutant or transgenic tobacco plant or part thereof that is capable of producing leaves that contain an equivalent number of mesophyll cells per unit of leaf area relative to an equivalent leaf of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent number of mesophyll cells per unit of leaf area is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in an equivalent leaf of a control plant that does not contain the same mutation or transgene. In some embodiments, the equivalent number of mesophyll cells per unit of leaf area is 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, or 19% to 20% of the level in an equivalent leaf of a control plant that does not contain the same mutation or transgene. In further embodiments, the equivalent number of mesophyll cells per unit of leaf area is 0.5% to 5%, 5% to 10%, or 10% to 20% of the level in an equivalent leaf of a control plant that does not contain the same mutation or transgene.

[0196] In certain aspects, the present disclosure provides an ADC mutant or transgenic tobacco plant or portion thereof, an AO mutant or transgenic tobacco plant or portion thereof, or an ODC mutant or transgenic tobacco plant or portion thereof that is capable of producing leaves that comprise an epidermal cell size equivalent to an equivalent leaf of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent epidermal cell size is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in an equivalent leaf of a control plant that does not contain the same mutation or transgene. In some embodiments, the equivalent epidermal cell size is 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, or 19% to 20% of the level in an equivalent leaf of a control plant not containing the same mutation or transgene. In further embodiments, the equivalent epidermal cell size is 0.5% to 5%, 5% to 10%, or 10% to 20% of the level in an equivalent leaf of a control plant not containing the same mutation or transgene.

[0197] In certain aspects, the present disclosure provides an ADC mutant or transgenic tobacco plant or portion thereof, an AO mutant or transgenic tobacco plant or portion thereof, or an ODC mutant or transgenic tobacco plant or portion thereof that is capable of producing leaves with an equivalent leaf yield relative to an equivalent leaf of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent leaf yield is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in an equivalent leaf of a control plant that does not contain the same mutation or transgene. In certain embodiments, the equivalent leaf yield is 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, or 19% to 20% of the level in an equivalent leaf of a control plant not containing the same mutation or transgene. In further embodiments, the equivalent leaf yield is 0.5% to 5%, 5% to 10%, or 10% to 20% of the level in an equivalent leaf of a control plant not containing the same mutation or transgene.

[0198] In certain aspects, the present disclosure provides an ADC mutant or transgenic tobacco plant or part thereof, an AO mutant or transgenic tobacco plant or part thereof, or an ODC mutant or transgenic tobacco plant or part thereof that exhibits an equivalent susceptibility to insect feeding relative to an equivalent leaf of a control plant that does not contain the same mutation or transgene. In one aspect, the equivalent susceptibility to insect feeding is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in an equivalent leaf of a control plant that does not contain the same mutation or transgene. In certain embodiments, the equivalent insect feeding susceptibility is 0.5% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, or 19% to 20% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene. In further embodiments, the equivalent insect feeding susceptibility is 0.5% to 5%, 5% to 10%, or 10% to 20% of the level in equivalent leaves of a control plant that does not contain the same mutation or transgene.

[0199] Any of the genetic modifications provided herein can be introduced into any ADC mutant or transgenic tobacco plant or part thereof, AO mutant or transgenic tobacco plant or part thereof, or ODC mutant or transgenic tobacco plant or part thereof.

[0200] The level of insect susceptibility can be assayed by methods known in the art, for example, in an insect feeding assay. Briefly, a quarter-inch layer of 0.7% agar in water is added to a 100 mm Petri dish and allowed to solidify. Leaf discs are cut from the Petri dish lid, placed on the plate, and gently pressed into the agar. Leaf discs are collected from plants at the 4-5 leaf stage. Discs are collected only from the leaf blade to exclude the major midrib. A single disc is collected from each of the four largest leaves of the plant, generating four replicates per plant. Four plants are sampled for a total of 16 biological replicate test lines. One second-instar caterpillar (e.g., Heliothis sp., Helicoverpa sp.) is added to the leaf and allowed to feed for 48 hours at ambient temperature. After 48 hours, the caterpillar larvae are weighed, and the final larval weight is recorded.

[0201] In some embodiments, a tobacco plant or portion thereof comprises a first genomic modification (e.g., in or targeting one or more ADC, AO, or ODC genes) that provides a lower level of nicotine or total alkaloids, and a second genomic modification that provides an equivalent level of one or more traits selected from the group consisting of total leaf polyamine levels, total root polyamine levels, total leaf chlorophyll levels, mesophyll cell number per unit leaf area, and leaf epidermal cell size, relative to a control tobacco plant, wherein the control plant does not have both the first and second genomic modifications. In one embodiment, a tobacco plant or portion thereof comprises a first genomic modification (e.g., in or targeting one or more ADC, AO, or ODC genes) that provides a lower level of nicotine or total alkaloids, and a second genomic modification that provides an equivalent level of total leaf polyamine levels, relative to a control tobacco plant, wherein the control plant does not have both the first and second genomic modifications. In some embodiments, a tobacco plant or portion thereof comprises a first genomic modification (e.g., in or targeting one or more ADC, AO, or ODC genes) that provides a lower level of nicotine or total alkaloids and a second genomic modification that provides a comparable level of total root polyamine levels relative to a control tobacco plant, wherein the control plant does not have both the first and second genomic modifications. In one embodiment, a tobacco plant or portion thereof comprises a first genomic modification (e.g., in or targeting one or more ADC, AO, or ODC genes) that provides a lower level of nicotine or total alkaloids and a second genomic modification that provides a comparable level of total leaf chlorophyll levels relative to a control tobacco plant, wherein the control plant does not have both the first and second genomic modifications. In some aspects, a tobacco plant or portion thereof comprises a first genomic modification (e.g., in or targeting one or more ADC, AO, or ODC genes) that provides a lower level of nicotine or total alkaloids, and a second genomic modification that provides an equivalent level of mesophyll cell number per unit leaf area relative to a control tobacco plant, wherein the control plant does not have both the first and second genomic modifications.In one aspect, a tobacco plant or portion thereof comprises a first genomic modification (e.g., in or targeted to one or more ADC, AO, or ODC genes) that provides a lower level of nicotine or total alkaloids and a second genomic modification that provides a comparable level of leaf epidermal cell size relative to a control tobacco plant, wherein the control plant does not have both the first and second genomic modifications. In some aspects, the second genomic modification is in or targeted to an ADC, AO, or ODC gene.

[0202] In some embodiments, the first genomic modification, the second genomic modification, or both, comprise a transgene, a mutation, or both. In some embodiments, the genomic modification, the second genomic modification, or both, comprise a transgene. In some embodiments, the first genomic modification, the second genomic modification, or both, comprise a mutation. In some embodiments, the first genomic modification, the second genomic modification, or both, are not transgene-based. In some embodiments, the first genomic modification, the second genomic modification, or both, are not mutation-based.

[0203] In some aspects, the tobacco plants provided herein comprise a reduced amount of total conjugated polyamines in their leaves relative to control tobacco plants. In one aspect, the tobacco plants provided herein comprise a reduced amount of total conjugated polyamines in their roots relative to control tobacco plants. As used herein, conjugated polyamines include, but are not limited to, soluble conjugated polyamines such as phenolamides comprising a backbone consisting of a free polyamine (e.g., putrescine, spermine, and / or spermidine) conjugated with one or more phenylpropanoids, such as ferulic acid, caffeic acid, and coumaric acid. Conjugated polyamines also include, but are not limited to, insoluble conjugated polyamines incorporated into structural polymers such as lignin. In some aspects, the tobacco plants provided herein comprise a reduced amount of total free polyamines (e.g., putrescine, spermine, and spermidine) in their leaves relative to control tobacco plants. In one aspect, the tobacco plants provided herein comprise a reduced amount of total conjugated polyamines in the roots relative to a control tobacco plant. In an aspect, the tobacco plants provided herein comprise a reduced amount of total conjugated forms of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in the leaves relative to a control tobacco plant. In an aspect, the tobacco plants provided herein comprise a reduced amount of total conjugated forms of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in the roots relative to a control tobacco plant. In an aspect, the tobacco plants provided herein comprise a reduced amount of total free forms of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in the leaves relative to a control tobacco plant. In an aspect, the tobacco plants provided herein comprise a reduced amount of total conjugated forms of one or more polyamines selected from the group consisting of putrescine, spermidine, and spermine in the roots relative to a control tobacco plant.

[0204] In some embodiments, the characteristics or traits of the tobacco plants described herein are measured at a time selected from the group consisting of just before flowering, at flower thinning, 1 week after flower thinning (WPT), 2 WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7 WPT, 8 WPT, and at harvest. In one embodiment, the tobacco plants provided herein comprising the first and second genomic modifications are capable of producing leaves having a leaf grade equivalent to that of a leaf from a control plant. In some embodiments, the tobacco plants provided herein comprising the first and second genomic modifications have a total leaf yield equivalent to that of a control plant.

[0205] In one aspect, a tobacco plant of the present disclosure comprises a nic1 mutation, a nic2 mutation, or both.

[0206] In certain aspects, the modified tobacco plants provided herein are characterized in that they contain a number of transcription factors, including agmatine deiminase (AIC), arginase, diamine oxidase, methylputrescine oxidase (MPO), NADH dehydrogenase, phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthase (SAMS), A622, NBBl, berberine bridging enzyme-like (BBL), MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factors, nicotine uptake permease (nicotine uptake permease), and / or methyltransferase (MPO). The present invention further includes transgenes or mutations that directly suppress the expression or activity of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, or seventeen or more genes or loci encoding proteins selected from the group consisting of: nucleotide permeases (NUPs), and MATE transporters. See Dewey and Xie, Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum, Phytochemistry 94 (2013) 10-27.

[0207] In some aspects, the modified tobacco plants provided herein further comprise a mutation in an ERF gene at the Nic2 locus (Nic2_ERF). In some aspects, the modified tobacco plants provided herein further comprise one or more mutations in one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten genes selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. See Shoji et al., Plant Cell, (10):3390-409 (2010), and Kajikawa et al., Plant physiol. 2017, 174:999-1011. In one aspect, the modified tobacco plants provided herein further comprise one or more mutations in ERF189, ERF115, or both. In certain aspects, the modified tobacco plants provided herein further comprise one or more transgenes that target and silence genes encoding one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten proteins selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

[0208] In some aspects, the modified tobacco plants provided herein further comprise a mutation in an ERF gene at the Nic1 locus (Nic1_ERF) (or the Nic1b locus as in WO / 2019 / 140297). See also WO / 2018 / 237107. In some aspects, the modified tobacco plants provided herein further comprise one or more mutations in two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. See WO / 2019 / 140297 and Kajikawa et al., Plant physiol. 2017, 174:999-1011. In certain aspects, the modified tobacco plants provided herein further comprise one or more mutations in one or more, two or more, three or more, four or more, five or more, or all six genes selected from the group consisting of ERFnew, ERF199, ERF19, ERF29, ERF210, and ERF91L2. In certain aspects, the modified tobacco plants provided herein further comprise one or more transgenes that target and silence genes encoding one or more, two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2.

[0209] In certain aspects, the modified tobacco plants provided herein contain at least one of the following enzymes: aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), and S-adenosyl-methyltransferase (SMT). The plant further comprises a first genetic modification comprising a mutation in a gene or locus encoding a protein selected from the group consisting of thionin synthase (SAMS), A622, NBBl, BBL, MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factor, nicotine uptake permease (NUP), and MATE transporter, and further comprises a second genetic modification targeting one or more amino acid sequences at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In one aspect, a modified tobacco plant provided herein comprises a first genetic modification to encode an aspartate oxidase, an agmatine deiminase (AIC), an arginase, a diamine oxidase, an arginine decarboxylase (ADC), a methylputrescine oxidase (MPO), an NADH dehydrogenase, an ornithine decarboxylase (ODC), a phosphoribosylanthranilate isomerase (PRAI), a putrescine N-methyltransferase (PMT), a quinolate phosphoribosyltransferase (QPT), a methyltransferase (MPO), a putrescine N-methyltransferase (PMT), a quinolate phosphoribosyltransferase (QPT), a putrescine N-methyltransferase (PMT ... and a transgene that targets and represses a gene or locus encoding a protein selected from the group consisting of S-adenosyl-methionine synthase (SAMS), A622, NBBl, BBL, MYC2, Nic1, Nic2, an ethylene response factor (ERF) transcription factor, a nicotine uptake permease (NUP), and a MATE transporter, and further comprises a second genetic modification that targets one or more amino acid sequences at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

[0210] Flue-cured tobacco / tobacco products In one aspect, the disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58. In another aspect, the disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116.

[0211] In one aspect, the disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58. In another aspect, the disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116.

[0212] In one aspect, the disclosure provides a method comprising preparing a tobacco plant using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a method comprising preparing a tobacco plant using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58. In another aspect, the disclosure provides a method comprising preparing a tobacco plant using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116.

[0213] "Curing" is a aging process that reduces moisture, results in the breakdown of chlorophyll, gives tobacco leaves a golden color, and converts starch to sugars. Thus, cured tobacco has a higher reducing sugar content and a lower starch content compared to harvested green leaves. In one aspect, the tobacco plants or plant components provided herein can be dried using conventional means, such as hot air curing, barn drying, fire-curing, air drying, or sun drying. For a description of different types of curing methods, see, for example, Chapter 1 in Tso (1999, Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco is typically aged in compressed conditions in wooden drums (e.g., hogsheads) or cardboard boxes for several years (e.g., 2-5 years), resulting in 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, including the introduction of steam at various temperatures, pasteurization, and conditioning the tobacco under vacuum with or without fermentation.

[0214] Information regarding the harvesting of burley and dark tobacco varieties can be found in the 2019-2020 Burley and Dark Tobacco Production Guide (December 2018), published by the University of Kentucky, the University of Tennessee, Virginia Polytechnic Institute and University, and North Carolina State University, which is incorporated herein by reference in its entirety.

[0215] In some aspects, the present disclosure provides a cured tobacco material derived from any tobacco plant or part thereof provided herein. In some aspects, the present disclosure provides a cured tobacco material derived from any modified tobacco plant or part thereof provided herein.

[0216] In one aspect, the dried tobacco material comprises a tobacco material selected from the group consisting of dried leaf material, dried stem material, dried bud material, dried flower material, and dried root material. In another aspect, the dried tobacco material comprises dried leaf material, dried stem material, or both. In a further aspect, the dried tobacco material comprises dried leaf material. In yet another aspect, the dried tobacco material comprises dried stem material.

[0217] In some embodiments, the cured tobacco material comprises flue-cured tobacco material. In another embodiment, the cured tobacco material comprises air-cured tobacco material. In another embodiment, the cured tobacco material comprises flame-cured tobacco material. In another embodiment, the cured tobacco material comprises sun-cured tobacco material. In another embodiment, the cured tobacco material provided herein is selected from the group consisting of air-cured tobacco material, flame-cured tobacco material, sun-cured tobacco material, and flue-cured tobacco material. In another embodiment, the cured tobacco material is from a tobacco variety selected from the group consisting of flue-cured, light, Burley, Virginia, Maryland, dark, Oriental, and Turkish varieties.

[0218] In some embodiments, the cured tobacco leaves provided herein are selected from the group consisting of air-cured, flue-cured, sun-cured, and flue-cured tobacco leaves. In some embodiments, the cured tobacco leaves are from a tobacco variety selected from the group consisting of flue-cured, light, burley, Virginia, Maryland, dark, Oriental, and Turkish varieties.

[0219] Fermentation is typically characterized by an initial high moisture content, the generation of heat, and a 10-20% loss in dry weight. See, e.g., U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, 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 be further processed (e.g., cut, shredded, expanded, or blended). See, e.g., U.S. Patent Nos. 4,528,993, 4,660,577, and 4,987,907. In certain aspects, the present disclosure provides fermented tobacco materials derived from any tobacco plant or portion thereof provided herein. In another aspect, the present disclosure provides a fermented tobacco material derived from any of the modified tobacco plants or parts thereof provided herein.

[0220] Tobacco materials obtained from the tobacco strains, varieties, or hybrids of the present disclosure can be used to make tobacco products. As used herein, "tobacco product" is defined as any product made from or derived from tobacco intended for human use or consumption. In one aspect, the present disclosure provides tobacco products comprising plant material from a tobacco plant provided herein. In another aspect, the present disclosure provides tobacco products comprising plant material from a modified tobacco plant provided herein. In another aspect, the present disclosure provides tobacco products comprising dried tobacco material. In another aspect, the present disclosure provides tobacco products comprising fermented tobacco material. In another aspect, the present disclosure provides tobacco products comprising tobacco blends.

[0221] Tobacco products include, but are not limited to, cigarette products (e.g., cigarettes and bidi cigarettes), cigar products (e.g., cigar wrapping tobacco and cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, oral nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, molded parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, and the like. See, e.g., U.S. Patent Publication No. 2006 / 0191548.

[0222] As used herein, a "cigarette" refers to a tobacco product having a "rod" and a "filler." The cigarette "rod" 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. "Filler" includes, but is not limited to, (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 within cigarette paper), (3) casing, (4) flavorings, and (5) all other additives (mixed with the tobacco and substitutes and wrapped within the cigarette).

[0223] In one aspect, the tobacco product comprises reconstituted tobacco. In another aspect, the present disclosure provides reconstituted tobacco comprising dried tobacco material. As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco dust and other tobacco scrap materials, processed into a sheet form, and cut into strips to resemble tobacco. In addition to cost savings, reconstituted tobacco is of great importance because it contributes to the taste of cigarettes from a flavor-developing process using a reaction between ammonia and sugars.

[0224] In some embodiments, the tobacco product comprises expanded tobacco. As used herein, "expanded tobacco" refers to a portion of tobacco filler that has been processed through expansion with a suitable gas so that the tobacco is "filled," resulting in a reduced density and increased fill capacity, thereby reducing the weight of the tobacco used in the cigarette.

[0225] Plant-derived tobacco products of the present disclosure also include cigarettes and other smoking articles, particularly smoking articles comprising a filter element, wherein the rod of smokable material comprises cured tobacco within a tobacco blend. In certain aspects, the tobacco product of the present disclosure is selected from the group consisting of cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco. In another aspect, the tobacco product of the present disclosure is selected from the group consisting of cigarettes, heated tobacco products, kreteks, bidi cigarettes, cigars, cigarillos, non-ventilated cigarettes, ventilated recess filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco products, gum, tablets, lozenges, and dissolvable strips.

[0226] In another aspect, the tobacco product of the present disclosure is a smokeless tobacco product. In some aspects, the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.

[0227] 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 for plugging or twisting. Moist smokeless tobacco is moist, finely divided tobacco, provided in loose form or in pouches, typically packaged in round cans and used as pinches or pouches by adult tobacco consumers, placed between the cheek and gum. Snus is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco that is taken orally or nasally.

[0228] In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of an electronically heated cigarette, an electronic cigarette, an electronic vaporization device, and an inhalable nicotine product.

[0229] In some aspects, the tobacco products of the present disclosure may be blended tobacco products.

[0230] In another aspect, the present disclosure provides a tobacco blend comprising a dried tobacco material, which may include any combination of dried, never-dried, fermented, unfermented, expanded, and reconstituted tobacco.

[0231] In some embodiments, the tobacco blend comprises at least 5% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 10% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 15% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 20% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 25% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 30% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 35% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 40% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 45% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 50% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 55% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 60% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 65% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 70% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 75% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 80% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 85% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 90% by weight of cured tobacco. In some embodiments, the tobacco blend comprises at least 95% by weight of cured tobacco.

[0232] In some embodiments, the tobacco blend comprises at least 5% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 10% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 15% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 20% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 25% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 30% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 35% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 40% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 45% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 50% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 55% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 60% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 65% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 70% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 75% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 80% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 85% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 90% by volume of cured tobacco. In some embodiments, the tobacco blend comprises at least 95% by volume of cured tobacco.

[0233] transformation In one aspect, the disclosure provides a method of producing a modified tobacco plant, the method comprising: (a) selecting a non-naturally occurring mutation in at least one tobacco cell in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, (b) selecting at least one tobacco cell comprising the non-naturally occurring mutation from step (a), and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, the disclosure provides a method of producing a modified tobacco plant, the method comprising: (a) selecting a non-naturally occurring mutation in at least one tobacco cell in an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58, (b) selecting at least one tobacco cell comprising the non-naturally occurring mutation from step (a), and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, the disclosure provides a method of producing a modified tobacco plant, the method comprising: (a) selecting a non-naturally occurring mutation in at least one tobacco cell in an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:59-116; (b) selecting at least one tobacco cell comprising the non-naturally occurring mutation from step (a); and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, the disclosure provides a method of producing a modified tobacco plant, the method comprising: (a) selecting a non-naturally occurring mutation in at least one tobacco cell in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-116; (b) selecting at least one tobacco cell comprising the non-naturally occurring mutation from step (a); and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In certain aspects, any of the foregoing methods further comprises (d) growing the modified tobacco plant regenerated in step (c).In another aspect, any of the foregoing methods further comprises (e) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the cross in step (e).

[0234] In one aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58; (b) selecting at least one tobacco cell that comprises the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In certain aspects, any of the foregoing methods further comprises (d) growing the modified tobacco plant regenerated in step (c).In another aspect, any of the foregoing methods further comprises (e) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; and (f) obtaining at least one seed from the cross in step (e).

[0235] In one aspect, the disclosure provides a method for producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, the disclosure provides a method of producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In another aspect, the disclosure provides a method of producing a modified tobacco plant, the method comprising: (a) introducing a recombinant DNA construct into at least one tobacco cell, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116; (b) selecting at least one tobacco cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b). In certain embodiments, any of the foregoing methods further comprises (d) growing the modified tobacco plant regenerated in step (c). In other embodiments, any of the foregoing methods further comprises (e) crossing the modified tobacco plant grown in step (d) with a second tobacco plant, and (f) obtaining at least one seed from the cross in step (e).

[0236] In one aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing the expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing the expression of an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58. In another aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing the expression of an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In another aspect, the method further comprises regenerating an altered tobacco plant from the transformed tobacco cell.

[0237] In one aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. In another aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-58. In another aspect, the disclosure provides a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59-116. In another aspect, the aforementioned method further comprises regenerating an altered tobacco plant from the transformed tobacco cell.

[0238] Numerous methods for introducing recombinant DNA constructs into plant cells are known in the art and can be used to generate transgenic plant cells and plants according to the methods of the present application. Any suitable method or technique for transforming plant cells known in the art can be used according to the present methods. Effective methods for plant transformation include bacterial-mediated transformation, such as Agrobacterium- or Rhizobium-mediated transformation, and biolistic transformation. Various methods are known in the art for regenerating or developing transgenic plants, such as transforming explants with transformation vectors via bacterial-mediated transformation or biolistic transformation, and then subsequently culturing the explants. Other methods for plant transformation, such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, and polyethylene glycol (PEG)-mediated transformation, are also known in the art. Transgenic plants produced by these transformation methods can be chimeric or non-chimeric with respect to the transformation event, depending on the method and explant used.

[0239] Methods for transforming plant cells are well known to those skilled in the art. For example, specific instructions for transforming plant cells with particles coated with recombinant DNA by microprojectile bombardment (e.g., biolistic transformation) can be found in U.S. Patent Nos. 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,153,812, and Agrobacterium-mediated transformation is described in U.S. Patent Nos. 5,159,135, 5,824,877, 5,591,616, 6,384,301, 5,750,871, 5,463,174, and 5,188,958, all of which are incorporated herein by reference. Additional methods for transforming plants can be found, for example, in Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any suitable method known to those of skill in the art can be used to transform tobacco cells with any of the nucleic acid molecules provided herein.

[0240] In one embodiment, the method of introducing a nucleic acid molecule into a tobacco cell comprises Agrobacterium-mediated transformation. In another embodiment, the method of introducing a nucleic acid molecule into a cell comprises PEG-mediated transformation. In another embodiment, the method of introducing a nucleic acid molecule into a cell comprises biolistic transformation. In another embodiment, the method of introducing a nucleic acid molecule into a cell comprises liposome-mediated transformation (lipofection). In another embodiment, the method of introducing a nucleic acid molecule into a cell comprises lentiviral transfection.

[0241] Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described in WO91 / 17424 and WO91 / 16024. Delivery can be to cells (in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration).

[0242] Any tobacco cell capable of regenerating a fertile tobacco plant is contemplated as a useful recipient cell for practicing the present disclosure. In some embodiments, a recombinant DNA construct is introduced into a tobacco cell. In some embodiments, a recombinant DNA construct is introduced into a tobacco protoplast cell. In other embodiments, a recombinant DNA construct is introduced into a tobacco callus cell. In some embodiments, a recombinant DNA construct is introduced into a tobacco cell selected from the group consisting of seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem cells, embryo cells, endosperm cells, root cells, shoot cells, stem cells, flower cells, inflorescence cells, stalk cells, pedicel cells, style cells, stigma cells, receptacle cells, petal cells, sepal cells, pollen cells, anther cells, filament cells, ovary cells, ovule cells, pericarp cells, and phloem cells.

[0243] Callus can be initiated from a variety of tissue sources, including, but not limited to, immature embryos or parts of embryos, apical meristems of seedlings, microspores, etc. Those cells that can be propagated as callus can serve as recipient cells for transformation. Practical transformation methods and materials for producing the transgenic plants of the present disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Patent Nos. 6,194,636 and 6,232,526, and U.S. Patent Application Publication No. 2004 / 0216189, all of which are incorporated herein by reference.

[0244] leaf grade As used herein, "USDA Leaf Grade Index" refers to the subdivision of leaf types by group, quality, and color. In one aspect, the USDA Grade Quality Score is quantified as a numerical representation of a grade from 0 to 100 determined by a certified tobacco leaf grader and is a weighted average of all stem positions. The higher the grade index, the higher the quality. As used below, "point" refers to each integer numerical representation of the USDA Leaf Grade Score. For example, the difference between a USDA Leaf Grade Index score of 90 and a score of 85 is 5 points.

[0245] Alternatively, leaf grade can be determined via hyperspectral imaging, see, e.g., WO2011 / 027315 (published March 10, 2011, and incorporated by reference in its entirety).

[0246] As used herein, a "certified tobacco grader" refers to a person trained to grade tobacco leaf according to the USDA official standard grades as defined by the United States Department of Agriculture (USDA), Agricultural Marketing Systems, published at 7 CFR § 29. USDA grade index scores may be assigned by employees, former employees, or individuals otherwise trained to grade tobacco leaf according to the USDA official standard grades. An exemplary process for standard operations for a commercial inspection service begins with a grower releasing tobacco to market, after which the tobacco is arranged in flat baskets as lots. Each lot is weighed and then inspected by a certified tobacco grader. After inspection, the grader assigns each lot a grade, which becomes a grade certificate indicating group, quality, and color. The process for grading experimental lots is similar; however, the experimental tobacco is not marketed or otherwise used for commercial purposes.

[0247] Tobacco grades are evaluated based on factors including, but not limited to, leaf position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, gloss (related to leaf strength and color depth and brilliance), hygroscopicity (the ability of tobacco leaves to absorb and retain ambient moisture), and green hue or color cast. Leaf grades can be determined, for example, using official standard grades promulgated by the U.S. Department of Agriculture's Agricultural Marketing Service (7 U.S.C. § 511). For example, the Official Standard Grades for Burley Tobacco (U.S. Type 31 and Foreign Type 93) (55 F.R. 40645), effective November 5, 1990; the Official Standard Grades for Flue-Cured Tobacco (U.S. Types 11, 12, 13, 14, and Foreign Type 92), effective March 27, 1989 (54 F.R. 7925); the Official Standard Grades for Pennsylvania Seedleaf Tobacco (U.S. Type 41), effective January 8, 1965 (29 F.R. 16854); the Official Standard Grades for Ohio Cigar-Leaf Tobacco (U.S. Type 41), effective December 8, 1963 (29 F.R. 16854); Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061), effective November 20, 1969; Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061), effective November 20, 1969; Official Standard Grades for Georgia and Florida Shade-Grown Cigar-Wrapper Tobacco (U.S. Type 62), effective April 1971. USDA grading index values may be determined according to industry-recognized grading indexes.See, e.g., Bowman et al., Tobacco Science, 32:39-40 (1988), Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index), and Miller et al., 1990, Tobacco Intern., 192:55-57 (all of the foregoing references are incorporated by reference in their entirety).

[0248] Unless otherwise specified, the leaf grade index value, alkaloid, or nicotine level measurements referred to herein for a tobacco plant, variety, cultivar, or strain refer to an average measurement, including, for example, the average of multiple leaves from a single plant, or the average measurement from a population of tobacco plants from a single variety, cultivar, or strain. The population of tobacco plants or collection of tobacco leaves for determining the average measurement (e.g., leaf grade or alkaloid or nicotine level) can be of any size, for example, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, or more. A population of at least five or more tobacco plants is used to determine the standard deviation. The average measurement or grade index value is determined according to standard protocols recognized in the industry.

[0249] As used herein, a "USDA grading group," "group," or "group" is a type of division covering closely related grades based on certain characteristics related to stem position, body, or general quality. Group is the primary factor in USDA grade. Group determination is part of the grading procedure and is assigned by certified tobacco leaf graders.

[0250] In one embodiment, the modified tobacco plant comprising the non-naturally occurring mutation comprises a USDA leaf grade index that is equal to or higher than a control tobacco plant lacking the non-naturally occurring mutation when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprising the recombinant DNA construct comprises a USDA leaf grade index that is equal to or higher than a control tobacco plant lacking the recombinant DNA construct when grown under equivalent conditions.

[0251] As used herein, an "equivalent" USDA Leaf Grade Index refers to within 15%. For example, if a control plant has a USDA Leaf Grade Index of 100, an equivalent USDA Leaf Grade Index is 85-100.

[0252] In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is at least 1% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 5% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 10% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 20% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 30% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 40% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 50% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 75% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 100% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions.

[0253] In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 1% to 100% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 1% to 75% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 1% to 50% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 1% to 40% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 1% to 30% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 1% to 20% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 1% to 10% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 10% to 75% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 10% to 50% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is 1% to 30% higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions.

[0254] In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is at least 1 point higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 2 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 3 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 4 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 5 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is at least 6 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 7 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 8 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 9 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 10 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 11 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 12 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions.In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 13 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 14 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 15 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 16 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 17 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 18 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 19 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 20 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 25 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 30 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 35 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 40 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions.In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is at least 50 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions.

[0255] In some embodiments, the modified tobacco plants comprise a USDA leaf grade index that is between 1 point and 100 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is between 1 point and 75 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is between 1 point and 50 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is between 1 point and 25 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plants comprise a USDA leaf grade index that is between 1 point and 10 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is between 1 point and 5 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is between 10 points and 50 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions. In another embodiment, the modified tobacco plant comprises a USDA leaf grade index that is between 10 points and 25 points higher than the USDA leaf grade index of a control tobacco plant when grown under equivalent conditions.

[0256] In some embodiments, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 1 point. In other embodiments, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 2 points. In other embodiments, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 3 points. In other embodiments, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 4 points. In other embodiments, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 5 points. In other embodiments, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 6 points. In other embodiments, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 7 points. In another embodiment, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 8 points. In another embodiment, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 9 points. In another embodiment, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 10 points. In another embodiment, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 11 points. In another embodiment, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 12 points. In another embodiment, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 13 points. In another embodiment, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 14 points. In another embodiment, the equivalent USDA leaf grade index between modified and control tobacco plants grown under equivalent conditions is within 15 points.

[0257] Aroma / Flavor In some aspects, the modified tobacco plants provided herein contain similar levels of one or more tobacco aroma compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, labdenoids, cembrenoids, sugar esters, and reducing sugars compared to a control tobacco plant when grown under equivalent conditions. As used herein, "similar" levels refer to within 20%.

[0258] As used herein, "tobacco aroma compounds" refer to compounds associated with the flavor and aroma of tobacco smoke. These compounds include, but are not limited to, 3-methylvaleric acid, valeric acid, isovaleric acid, cembrenoids and labdenoids, diterpenes, and sugar esters. The concentration of tobacco aroma compounds can be measured by any known metabolite profiling method in the art, including, but not limited to, gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry. See The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013).

[0259] As used herein, "reducing sugar(s)" refers to any sugar (monosaccharide or polysaccharide) that has a free or potentially free aldehyde or ketone group. Glucose and fructose act as nicotine buffers in cigarette smoke by lowering the pH of the smoke and effectively reducing the amount of "free" unprotonated nicotine. Reducing sugars balances the flavor of the smoke, for example, by modifying the sensory impact of nicotine and other tobacco alkaloids. An inverse relationship between sugar content and alkaloid content has been reported between tobacco varieties, within the same variety, and within the same plant strain due to growing conditions. Reduction of sugar levels can be measured using a segmented-flow colorimetric method developed for the analysis of tobacco samples, adapted by Skalar Instrument Co. (West Chester, PA) and described in Davis, Tobacco Science 20:139-144 (1976). For example, the sample is dialyzed against a sodium carbonate solution. Copper neocuproine is added to the sample, and the solution is heated. The copper neocuproine chelate is reduced in the presence of sugars to give a colored complex that is measured at 460 nm.

[0260] TSNA In some embodiments, provided modified tobacco plants further comprise one or more mutations at one or more loci encoding nicotine demethylases (e.g., CYP82E4, CYP82E5, CYP82E10) that confer a reduced amount of nornicotine when grown under equivalent conditions compared to control tobacco plants lacking one or more mutations at one or more loci encoding nicotine demethylases (see U.S. Patent Nos. 8,319,011, 8,124,851, 9,187,759, 9,228,194, 9,228,195, and 9,247,706). In some embodiments, the described modified tobacco plants further comprise reduced nicotine demethylase activity when grown and cured under equivalent conditions compared to control plants. In further aspects, provided tobacco plants further comprise one or more mutations or transgenes that provide elevated levels of one or more antioxidants (see U.S. Patent Application Publication Nos. 2018 / 0119163 and WO2018 / 067985). In other aspects, provided tobacco plants further comprise one or more mutations or transgenes that provide reduced levels of one or more tobacco-specific nitrosamines (TSNAs). In some aspects, the TSNA is selected from the group consisting of N'-nitrosonornicotine (NNN), 4-methylnitrosamino-l-(3-pyridyl)-l-butanone (NNK), N'-nitrosoanatabine (NAT), and N'-nitrosoanabasine (NAB).

[0261] Embodiment The following non-limiting embodiments are contemplated. 1. A modified tobacco plant or part thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. 2. A modified tobacco plant or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. 3. A modified tobacco plant or part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. 4. The modified tobacco plant or part thereof of embodiment 1, wherein the tobacco plant produces at least one leaf comprising a reduced amount of at least one alkaloid compared to the amount of alkaloid in a control tobacco plant lacking the at least one non-naturally occurring mutation in the endogenous nucleic acid sequence when grown under equivalent conditions. 5. The modified tobacco plant or part thereof of embodiment 2 or 3, wherein the tobacco plant produces at least one leaf comprising a reduced amount of at least one alkaloid compared to the amount of the alkaloid in a control tobacco plant lacking the recombinant DNA construct when grown under equivalent conditions. 6. The modified tobacco plant or part thereof of any one of embodiments 1, 2, or 4, wherein the endogenous nucleic acid sequence comprises a nucleic acid sequence that is at least 80% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-58. 7. The modified tobacco plant or part thereof of embodiment 3, wherein the nucleic acid sequence comprises a nucleic acid sequence that is at least 80% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-58. 8. The modified tobacco plant or part thereof of any one of embodiments 1, 2, or 4, wherein the endogenous nucleic acid sequence comprises a nucleic acid sequence that is at least 80% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 59-116. 9. The modified tobacco plant or part thereof of embodiment 3, wherein the nucleic acid sequence comprises a nucleic acid sequence that is at least 80% identical to or complementary to a sequence selected from the group consisting of SEQ ID NOs: 59 to 116. 10. The modified tobacco plant or part thereof of embodiment 1 or 4, wherein the tobacco plant is homozygous for the at least one non-naturally occurring mutation. 11. The modified tobacco plant or part thereof of embodiment 1 or 4, wherein the tobacco plant is heterozygous for the at least one non-naturally occurring mutation. 12. The modified tobacco plant or part thereof of any one of embodiments 4-6, 8, 10, or 11, wherein the at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine. 13. The modified tobacco plant or part thereof of any one of embodiments 4-6, 8, or 10-12, wherein the reduced amount of at least one alkaloid comprises a reduction of at least 1%. 14. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, or 11, wherein the at least one non-naturally occurring mutation comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, a duplication, and an inversion. 15. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, or 11, wherein the at least one non-naturally occurring mutation comprises at least one mutation selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, and a splice site mutation. 16. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, 11, 14, or 15, wherein the at least one non-naturally occurring mutation comprises a null mutation. 17. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, 11, or 14-16, wherein the at least one non-naturally occurring mutation results in truncation of the polypeptide. 18. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, 11, or 14-17, wherein the at least one non-naturally occurring mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5'-untranslated region (UTR), an exon, an intron, a 3'-UTR, and a terminator. 19. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, 11, or 14-18, wherein the at least one non-naturally occurring mutation results in a reduced level of expression of the nucleic acid sequence compared to expression of the nucleic acid sequence in the same tissue of a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks the at least one non-naturally occurring mutation in the control tobacco plant. 20. The modified tobacco plant or part thereof of any one of embodiments 1, 4, 10, 11, or 14-19, wherein the at least one non-naturally occurring mutation results in a reduced level of activity by the protein or polypeptide encoded by the nucleic acid sequence compared to the activity of the protein or polypeptide encoded by the nucleic acid sequence in a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks the at least one non-naturally occurring mutation in the control tobacco plant. 21. The modified tobacco plant or part thereof of any one of embodiments 2, 3, 5, 7, or 9, wherein the promoter comprises a promoter selected from the group consisting of a constitutive promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducible promoter. 22. The modified tobacco plant or part thereof of embodiment 21, wherein the tissue-preferred promoter comprises a root-preferred promoter. 23. The modified tobacco plant or part thereof of embodiment 21, wherein the tissue-specific promoter comprises a root-specific promoter. 24. The modified tobacco plant or part thereof according to embodiment 21, wherein the constitutive promoter is selected from the group consisting of a cauliflower mosaic virus (CaMV) 35S promoter, a ubiquitin promoter, an actin promoter, an opine promoter, and an alcohol dehydrogenase promoter. 25. The modified tobacco plant or part thereof of embodiment 2, wherein the at least one small RNA is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA, and microRNA. 26. The modified tobacco plant or part thereof of embodiment 2, wherein the at least one small RNA comprises 18 to 30 nucleotides. 27. The modified tobacco plant or part thereof of embodiment 2, wherein the at least one small RNA comprises a nucleic acid sequence that is at least 90% complementary to a sequence selected from the group consisting of SEQ ID NOs: 59-116. 28. The modified tobacco plant or part thereof of any one of embodiments 1-27, wherein the modified tobacco plant is of a tobacco variety selected from the group consisting of flue-cured, light, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish. 29. The modified tobacco plant or part thereof of any one of embodiments 1-116, wherein the modified tobacco plant is of a variety selected from the group consisting of the tobacco varieties listed in Tables 2-8. 30. The modified tobacco plant or part thereof of any one of embodiments 1-29, wherein the modified tobacco plant is a hybrid. 31. The modified tobacco plant or part thereof of any one of embodiments 1-30, wherein the modified tobacco plant is male sterile or cytoplasmic male sterile. 32. The modified tobacco plant or part thereof of any one of embodiments 1-30, wherein the modified tobacco plant is female sterile. 33. A cured tobacco material derived from the modified tobacco plant or part thereof according to any one of embodiments 1-32. 34. The dried tobacco material of embodiment 33, wherein the dried tobacco material comprises dried leaf material, dried stem material, or both. 35. The cured tobacco material of embodiment 33 or 34, wherein the cured tobacco material comprises flue-cured tobacco material, air-cured tobacco material, flame-cured tobacco material, and sun-cured tobacco material. 36. A tobacco blend comprising the dried tobacco material of any one of embodiments 33-35. 37. The tobacco blend of embodiment 36, wherein the tobacco blend comprises at least 10% by weight of cured tobacco. 38. The tobacco blend of embodiment 36, wherein the tobacco blend comprises at least 10% by volume of cured tobacco. 39. A tobacco product comprising a tobacco blend according to any one of embodiments 36 to 38. 40. A tobacco product comprising the dried tobacco material of any one of embodiments 33 to 35. 41. The tobacco product of embodiment 39 or 40, wherein the tobacco product is selected from the group consisting of cigarettes, heated tobacco products, kreteks, bidi cigarettes, cigars, cigarillos, non-ventilated cigarettes, ventilated recess filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouch chewing tobacco products, gum, tablets, lozenges, and dissolvable strips. 42. The tobacco product of embodiment 39 or 40, wherein the tobacco product is a smokeless tobacco product. 43. The tobacco product of embodiment 42, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus. 44. Reconstituted tobacco comprising a dried tobacco material according to any one of embodiments 33-35. 45. A method for producing a modified tobacco plant, comprising: (a) selecting a non-naturally occurring mutation in at least one tobacco cell in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell containing the non-naturally occurring mutation from step (a); (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b); A method comprising: 46. A method for producing a modified tobacco plant, comprising: (a) introducing into at least one tobacco cell a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell containing the recombinant DNA construct; (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b); A method comprising: 47. A method for producing a modified tobacco plant, comprising: (a) introducing into at least one tobacco cell a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell containing the recombinant DNA construct; (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b); A method comprising: 48. The method of embodiment 45, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the mutation when grown under equivalent conditions. 49. The method of embodiment 46 or 47, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the recombinant DNA construct when grown under equivalent conditions. 50. The method of any one of embodiments 45, 46, 48, or 49, wherein the endogenous nucleic acid sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-58. 51. The method of embodiment 47 or 49, wherein the nucleic acid sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 58. 52. The method of any one of embodiments 45, 46, 48, or 49, wherein the endogenous nucleic acid sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 59-116. 53. The method of embodiment 47 or 49, wherein the nucleic acid sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 59 to 116. 54. The method of any one of embodiments 48-53, wherein the at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine. 55. The method of any one of embodiments 48-53, wherein said reduced amount of at least one alkaloid comprises a reduction of at least 1%. 56. The method of any one of embodiments 45, 48, 50, 52, 54, or 55, wherein the non-naturally occurring mutation comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, a duplication, and an inversion. 57. The method of any one of embodiments 45, 48, 50, 52, or 54-56, wherein the non-naturally occurring mutation comprises a mutation selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, and a splice site mutation. 58. The method of any one of embodiments 45, 48, 50, 52, or 54-57, wherein the non-naturally occurring mutation comprises a null mutation. 59. The method of any one of embodiments 45, 48, 50, 52, or 54-58, wherein the non-naturally occurring mutation results in truncation of the polypeptide. 60. The method of any one of embodiments 45, 48, 50, 52, or 54-59, wherein the non-naturally occurring mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5'-untranslated region (UTR), an exon, an intron, a 3'-UTR, and a terminator. 61. The method of any one of embodiments 45, 48, 50, 52, or 54-60, wherein said inducing comprises the use of an agent selected from the group consisting of a chemical mutagen, irradiation, a transposon, Agrobacterium, and a nuclease. 62. The method of embodiment 61, wherein the nuclease is selected from the group consisting of meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, CRISPR / Cas9 nucleases, CRISPR / Cpf1 nucleases, CRISPR / CasX nucleases, CRISPR / CasY nucleases, Csm1 nucleases, or any combination thereof. 63. The method of embodiment 61, wherein the chemical mutagen comprises ethyl methanesulfonate. 64. The method of embodiment 61, wherein the irradiation comprises gamma rays, X-rays, ionizing radiation, or fast neutrons. 65. The method of embodiment 46, wherein the small RNA is selected from the group consisting of double-stranded RNA, small interfering RNA (siRNA), trans-acting siRNA, and microRNA. 66. The method of embodiment 46, wherein the at least one small RNA comprises 18 to 30 nucleotides. 67. The method of embodiment 46, wherein the at least one small RNA comprises a nucleic acid sequence that is at least 90% complementary to a sequence selected from the group consisting of SEQ ID NOs: 59-116. 68. The method of any one of embodiments 46, 47, or 49-53, wherein the promoter comprises a promoter selected from the group consisting of a constitutive promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducible promoter. 69. The method of embodiment 68, wherein the tissue-preferred promoter comprises a root-preferred promoter. 70. The method of embodiment 68, wherein the tissue-specific promoter comprises a root-specific promoter. 71. The method of embodiment 68, wherein the constitutive promoter is selected from the group consisting of a cauliflower mosaic virus (CaMV) 35S promoter, a ubiquitin promoter, an actin promoter, an opine promoter, and an alcohol dehydrogenase promoter. 72. The method of any one of embodiments 45 to 71, wherein the at least one tobacco cell is a tobacco protoplast cell. 73. The method of any one of embodiments 45-71, wherein the at least one tobacco cell is a tobacco callus cell. 74. The method of any one of embodiments 45-71, wherein the at least one tobacco cell is selected from the group consisting of a seed cell, a fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a meristem cell, an embryo cell, an endosperm cell, a root cell, a shoot cell, a stem cell, a flower cell, an inflorescence cell, a stalk cell, a pedicel cell, a style cell, a stigma cell, a receptacle cell, a petal cell, a sepal cell, a pollen cell, an anther cell, a filament cell, an ovary cell, an ovule cell, a pericarp cell, and a phloem cell. 75. (d) growing the modified tobacco plants regenerated in step (c). 48. The method of any one of embodiments 45 to 47, further comprising: 76. (e) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; (f) obtaining at least one seed from the cross in step (e); 76. The method of embodiment 75, further comprising: 77. The method of embodiment 45 or 48, wherein at least one non-naturally occurring mutation results in a reduced level of expression of the nucleic acid sequence compared to the expression of the nucleic acid sequence in the same tissue of a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks at least one non-naturally occurring mutation in the control tobacco plant. 78. The method of embodiment 77, wherein the reduced level of expression comprises at least a 5% reduction. 79. The method of embodiment 45 or 48, wherein the at least one non-naturally occurring mutation results in an increased level of expression of the nucleic acid sequence compared to expression of the nucleic acid sequence in the same tissue of a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks the at least one non-naturally occurring mutation in the control tobacco plant. 80. The method of embodiment 79, wherein the increased level of expression comprises an increase of at least 5%. 81. The method of embodiment 45 or 48, wherein the at least one non-naturally occurring mutation results in a reduced level of activity by the protein or polypeptide encoded by the nucleic acid sequence compared to the activity of the protein or polypeptide encoded by the nucleic acid sequence in a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks the at least one non-naturally occurring mutation in the control tobacco plant. 82. The method of embodiment 45 or 48, wherein the at least one non-naturally occurring mutation confers an increased level of activity by the protein or polypeptide encoded by the nucleic acid sequence compared to the activity of the protein or polypeptide encoded by the nucleic acid sequence in a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks the at least one non-naturally occurring mutation in the control tobacco plant. 83. The method of any one of embodiments 45-82, wherein the modified tobacco plant is of a tobacco variety selected from the group consisting of flue-cured, light, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish. 84. The method of any one of embodiments 45-83, wherein the modified tobacco plant is of a variety selected from the group consisting of the varieties listed in Tables 2-8. 85. The method of any one of embodiments 45-84, wherein the modified tobacco plant is a hybrid. 86. The method of any one of embodiments 45-85, wherein the modified tobacco plant is male sterile or cytoplasmic male sterile. 87. The method of any one of embodiments 45-85, wherein the modified tobacco plant is female sterile. 88. The method of embodiment 45 or 48, wherein the modified tobacco plant comprises an equivalent or higher USDA leaf grade index when grown under equivalent conditions compared to a control tobacco plant lacking the non-naturally occurring mutation. 89. The method of embodiment 46 or 47, wherein the modified tobacco plant comprises an equivalent or higher USDA leaf grade index compared to a control tobacco plant lacking the recombinant DNA construct when grown under equivalent conditions. 90. A method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence, and the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. 91. A method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. 92. A method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. 93. The method of any one of embodiments 90-92, wherein the dried tobacco material comprises dried leaf material, dried stem material, or both. 94. The method of any one of embodiments 90-92, wherein the cured tobacco material comprises flue-cured tobacco material, air-cured tobacco material, flame-cured tobacco material, and sun-cured tobacco material. 95. The method of any one of embodiments 90-92, wherein the tobacco product is selected from the group consisting of cigarettes, kreteks, bidi cigarettes, cigars, cigarillos, non-vented cigarettes, ventilated recess filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouch chewing tobacco products, gum, tablets, lozenges, and dissolvable strips. 96. The method of any one of embodiments 90-92, wherein the tobacco product is a smokeless tobacco product. 97. The method of embodiment 96, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus. 98. The method of any one of embodiments 90-92, wherein the cured tobacco material is of a tobacco variety selected from the group consisting of flue-cured, light, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish varieties. 99. The method of any one of embodiments 90-98, wherein the endogenous nucleic acid sequence comprises a sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-116. 100. The method of any one of embodiments 92-98, wherein the nucleic acid sequence comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-116. 101. A method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. 102. A method comprising transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174. 103. A method for producing a modified tobacco plant, comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, and the non-naturally occurring mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one progeny tobacco seed or plant germinated therefrom comprises the non-naturally occurring mutation; and A method comprising: 104. A method for producing a modified tobacco plant, comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, wherein the recombinant DNA construct is not present in the endogenous nucleic acid sequence in a control tobacco plant of the same variety; (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one tobacco seed or plant germinated therefrom comprises the recombinant DNA construct; and A method comprising: 105. A method for producing a modified tobacco plant, comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, wherein the recombinant DNA construct is not present in the nucleic acid sequence in a control tobacco plant of the first tobacco variety; (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein the at least one tobacco seed or plant germinated therefrom comprises the recombinant DNA construct; and A method comprising: 106. The method of any one of embodiments 103-105, wherein the plants germinated in step (b) comprise a reduced amount of at least one alkaloid compared to the control tobacco plants when grown under equivalent condi...

Claims

1. 1. A modified tobacco plant or part thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

2. 1. A modified tobacco plant or part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

3. 1. A modified tobacco plant or part thereof comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

4. 2. The modified tobacco plant or part thereof of claim 1, wherein the tobacco plant produces at least one leaf comprising a reduced amount of at least one alkaloid compared to the amount of alkaloid in a control tobacco plant lacking the at least one non-naturally occurring mutation in the endogenous nucleic acid sequence when grown under equivalent conditions.

5. 4. The modified tobacco plant or part thereof of claim 2 or 3, wherein the tobacco plant produces at least one leaf comprising a reduced amount of at least one alkaloid compared to the amount of the alkaloid in a control tobacco plant lacking the recombinant DNA construct when grown under equivalent conditions.

6. A dried tobacco material derived from the modified tobacco plant or part thereof according to any one of claims 1 to 5.

7. The dried tobacco material of claim 6 , comprising dried leaf material, dried stem material, or both.

8. 7. The cured tobacco material of claim 6, including flue-cured tobacco material, air-cured tobacco material, fire-cured tobacco material, and sun-cured tobacco material.

9. A tobacco blend comprising the dried tobacco material of any one of claims 6 to 8.

10. 10. The tobacco blend of claim 9, comprising at least 10% by weight of dried tobacco.

11. 10. The tobacco blend of claim 9, comprising at least 10% by volume of dried tobacco.

12. A tobacco product comprising a tobacco blend according to any one of claims 9 to 11.

13. A tobacco product comprising the dried tobacco material according to any one of claims 6 to 8.

14. 14. The tobacco product of claim 12 or 13, wherein the tobacco product is selected from the group consisting of cigarettes, heated tobacco products, kreteks, bidi cigarettes, cigars, cigarillos, non-ventilated cigarettes, vented recess filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco products, gum, tablets, lozenges, and dissolvable strips.

15. 14. The tobacco product of claim 12 or 13, which is a smokeless tobacco product.

16. 16. The tobacco product of claim 15, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.

17. A reconstituted tobacco comprising the dried tobacco material of any one of claims 6 to 8.

18. 1. A method for producing a modified tobacco plant, comprising: (a) inducing a non-naturally occurring mutation in at least one tobacco cell in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell containing the non-naturally occurring mutation from step (a); (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b); A method comprising:

19. 1. A method for producing a modified tobacco plant, comprising: (a) introducing into at least one tobacco cell a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell containing the recombinant DNA construct; (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b); A method comprising:

20. 1. A method for producing a modified tobacco plant, comprising: (a) introducing into at least one tobacco cell a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174; (b) selecting at least one tobacco cell containing the recombinant DNA construct; (c) regenerating at least one modified tobacco plant from the at least one tobacco cell selected in step (b); A method comprising:

21. 20. The method of claim 18, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the mutation when grown under equivalent conditions.

22. 21. The method of claim 19 or 20, wherein the at least one modified tobacco plant comprises a reduced amount of at least one alkaloid compared to a control tobacco plant lacking the recombinant DNA construct when grown under equivalent conditions.

23. (e) growing the modified tobacco plants regenerated in step (c). The method of any one of claims 18 to 20, further comprising:

24. (g) crossing the modified tobacco plant grown in step (d) with a second tobacco plant; (h) obtaining at least one seed from the cross in step (e); 24. The method of claim 23, further comprising:

25. 20. The method of claim 18, wherein the at least one non-naturally occurring mutation results in a reduced level of expression of the nucleic acid sequence compared to expression of the nucleic acid sequence in the same tissue of a control tobacco plant when grown under equivalent conditions, and the nucleic acid sequence lacks the at least one non-naturally occurring mutation in the control tobacco plant.

26. 1. A method comprising: preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence, and the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs:117-174.

27. 1. A method comprising: preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

28. 1. A method comprising: preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein the modified tobacco plant comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs:117-174.

29. 29. The method of any one of claims 26 to 28, wherein the dried tobacco material comprises dried leaf material, dried stem material, or both.

30. 29. The method of any one of claims 26 to 28, wherein the cured tobacco material comprises flue-cured tobacco material, air-cured tobacco material, flame-cured tobacco material, and sun-cured tobacco material.

31. 29. The method of any one of claims 26 to 28, wherein the tobacco product is selected from the group consisting of cigarettes, kreteks, bidi cigarettes, cigars, cigarillos, non-ventilated cigarettes, ventilated recess filter cigarettes, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouch chewing tobacco products, gum, tablets, lozenges, and dissolvable strips.

32. The method of any one of claims 26 to 28, wherein the tobacco product is a smokeless tobacco product.

33. 33. The method of claim 32, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.

34. 29. The method of any one of claims 26 to 28, wherein the cured tobacco material is a cured tobacco material of a tobacco variety selected from the group consisting of flue-cured, bright, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish varieties.

35. 28. The method of claim 26 or 27, wherein the endogenous nucleic acid sequence comprises a sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-116.

36. 29. The method of claim 28, wherein the nucleic acid sequence comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-116.

37. 1. A method comprising: transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

38. 1. A method comprising: transforming a tobacco cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

39. 1. A method for producing a modified tobacco plant, comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence, the endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, and the non-naturally occurring mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein said at least one tobacco seed or plant germinated therefrom comprises said non-naturally occurring mutation; and A method comprising:

40. 1. A method for producing a modified tobacco plant, comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA capable of binding to and reducing expression of an endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, wherein the recombinant DNA construct is not present in the endogenous nucleic acid sequence in a control tobacco plant of the same variety; (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein said at least one tobacco seed or plant germinated therefrom comprises said recombinant DNA construct; and A method comprising:

41. 1. A method for producing a modified tobacco plant, comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, wherein the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, the recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174, and the recombinant DNA construct is not present in the nucleic acid sequence in a control tobacco plant of the first tobacco variety; (b) selecting at least one progeny tobacco seed or plant germinated therefrom, wherein said at least one tobacco seed or plant germinated therefrom comprises said recombinant DNA construct; and A method comprising:

42. 42. The method of any one of claims 39 to 41, wherein the plants germinated in step (b) comprise a reduced amount of at least one alkaloid compared to the control tobacco plants when grown under equivalent conditions.

43. 1. A modified tobacco plant or part thereof comprising at least one non-naturally occurring mutation in an endogenous nucleic acid sequence that regulates the expression or functional activity of a gene, wherein said gene encodes a polypeptide comprising an amino acid sequence that is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-174.

44. 44. A cured tobacco material derived from the modified tobacco plant or part thereof of claim 43.

45. 45. A tobacco blend comprising the dried tobacco material of claim 44.

46. 46. A tobacco product comprising the tobacco blend of claim 45.

47. A tobacco product comprising the dried tobacco material of claim 44.

48. 45. A reconstituted tobacco comprising the dried tobacco material of claim 44.

49. 1. A modified tobacco plant or part thereof comprising: (a) a genetic modification in a gene; or (b) a genetic modification targeted to said gene, wherein said genetic modification downregulates the expression or activity of said gene, and said gene encodes a nucleic acid sequence having at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-116.

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